Performance-Dependent Consolidation of Learned Vocal Changes in Adult Songbirds.

Motor skills learned through practice are consolidated at later time, which can include nighttime, but the time course of motor memory consolidation and its underlying mechanisms remain poorly understood. We investigated neural substrates underlying motor memory consolidation of learned changes in birdsong, a tractable model system for studying neural basis of motor skill learning. Previous studies in male zebra finches and Bengalese finches have demonstrated that adaptive changes in adult song structure learned through a reinforcement paradigm are initially driven by a cortical-basal ganglia circuit, and subsequently consolidated into downstream cortical motor circuitry. However, the time course of the consolidation process, including whether it occurs offline during nighttime or online during daytime, remains unclear and even controversial. Here, we provide in both species experimental evidence of virtually no consolidation of learned vocal changes during nighttime. We demonstrate instead that the consolidation occurs during daytime and the amount of consolidation is strongly correlated with the amount of learning, suggesting online, performance-dependent mechanisms of consolidation of learned vocal changes. Moreover, by using computer simulations based on our experimental results, we demonstrate that such online, performance-dependent consolidation can account for the contradicting conclusions concerning the time course of consolidation process reached by previous studies. These results thus reconcile a controversy in the study of vocal motor consolidation in songbirds, and illustrate the neural substrates through which newly learned motor skills initially implemented by cortical-basal ganglia circuits become encoded in the cortical motor circuitry.SIGNIFICANCE STATEMENT Motor skills learned through repetitive practice become stable and are consolidated into cortical motor circuits. We investigate neural substrates of this "motor memory consolidation" in adult songbirds, which produce songs that are complex motor skills learned and maintained through repetitive vocal practice. We demonstrate that learned changes in song acoustic structure are consolidated into the cortical motor circuits predominantly during daytime, but not during nighttime, depending on ongoing song performance. These consolidation mechanisms reconcile seemingly contradicting results of previous studies regarding the time course of vocal learning consolidation, and provide fundamental insights into the process through which learned performance of complex motor skills is consolidated and encoded in in motor circuits.

Measuring context dependency in birdsong using artificial neural networks.

Context dependency is a key feature in sequential structures of human language, which requires reference between words far apart in the produced sequence. Assessing how long the past context has an effect on the current status provides crucial information to understand the mechanism for complex sequential behaviors. Birdsongs serve as a representative model for studying the context dependency in sequential signals produced by non-human animals, while previous reports were upper-bounded by methodological limitations. Here, we newly estimated the context dependency in birdsongs in a more scalable way using a modern neural-network-based language model whose accessible context length is sufficiently long. The detected context dependency was beyond the order of traditional Markovian models of birdsong, but was consistent with previous experimental investigations. We also studied the relation between the assumed/auto-detected vocabulary size of birdsong (i.e., fine- vs. coarse-grained syllable classifications) and the context dependency. It turned out that the larger vocabulary (or the more fine-grained classification) is assumed, the shorter context dependency is detected.

Effects of background noise on acoustic characteristics of Bengalese finch songs.

Online regulation of vocalization in response to auditory feedback is one of the essential issues for vocal communication. One such audio-vocal interaction is the Lombard effect, an involuntary increase in vocal amplitude in response to the presence of background noise. Along with vocal amplitude, other acoustic characteristics, including fundamental frequency (F0), also change in some species. Bengalese finches (Lonchura striata var. domestica) are a suitable model for comparative, ethological, and neuroscientific studies on audio-vocal interaction because they require real-time auditory feedback of their own songs to maintain normal singing. Here, the changes in amplitude and F0 with a focus on the distinct song elements (i.e., notes) of Bengalese finches under noise presentation are demonstrated. To accurately analyze these acoustic characteristics, two different bandpass-filtered noises at two levels of sound intensity were used. The results confirmed that the Lombard effect occurs at the note level of Bengalese finch song. Further, individually specific modes of changes in F0 are shown. These behavioral changes suggested the vocal control mechanisms on which the auditory feedback is based have a predictable effect on amplitude, but complex spectral effects on individual note production.

Variability in the temporal parameters in the song of the Bengalese finch (Lonchura striata var. domestica).

Birdsong provides a unique model for studying the control mechanisms of complex sequential behaviors. The present study aimed to demonstrate that multiple factors affect temporal control in the song production. We analyzed the song of Bengalese finches in various time ranges to address factors that affected the duration of acoustic elements (notes) and silent intervals (gaps). The gaps showed more jitter across song renditions than did notes. Gaps had longer duration in branching points of song sequence than in stereotypic transitions, and the duration of a gap was correlated with the duration of the note that preceded the gap. When looking at the variation among song renditions, we found notable factors in three time ranges: within-day drift, within-bout changes, and local jitter. Note durations shortened over time from morning to evening. Within each song bout note durations lengthened as singing progressed, while gap durations lengthened only during the late part of song bout. Further analysis after removing these drift factors confirmed that the jitter remained in local song sequences. These results suggest distinct sources of temporal variability exist at multiple levels on the basis of this note-gap relationship, and that song comprised a mixture of these sources.

Multichannel instantaneous frequency analysis of ultrasound propagating in cancellous bone.

An ultrasonic pulse propagating in cancellous bone can be separated into two waves depending on the condition of the specimen. These two waves, which are called the fast wave and the slow wave, provide important information for the diagnosis of osteoporosis. The present study proposes to utilize a signal processing method that extracts the instantaneous frequency (IF) of waveforms from multiple spectral channels. The instantaneous frequency was expected to be able to show detailed time-frequency properties of ultrasonic waves being transmitted through cancellous bone. The employed method, termed the multichannel instantaneous frequency (MCIF) method, showed robustness against background noise as compared to the IF that was directly derived from the original waveform. The extracted IF revealed that the frequency of the fast wave was affected by both the propagation distance within the specimen and the bone density, independently. On the other hand, the alternation of the center frequency of the originally transmitted wave did not produce proportional changes in the extracted IF values of the fast waves, suggesting that the fast wave IF mainly reflected the thickness of the specimens. These findings may provide the possibility of obtaining a more precise diagnosis of osteoporosis.

Spectral consistency in sound sequence affects perceptual accuracy in discriminating subdivided rhythmic patterns.

Musical compositions are distinguished by their unique rhythmic patterns, determined by subtle differences in how regular beats are subdivided. Precise perception of these subdivisions is essential for discerning nuances in rhythmic patterns. While musical rhythm typically comprises sound elements with a variety of timbres or spectral cues, the impact of such spectral variations on the perception of rhythmic patterns remains unclear. Here, we show that consistency in spectral cues affects perceptual accuracy in discriminating subdivided rhythmic patterns. We conducted online experiments using rhythmic sound sequences consisting of band-passed noise bursts to measure discrimination accuracy. Participants were asked to discriminate between a swing-like rhythm sequence, characterized by a 2:1 interval ratio, and its more or less exaggerated version. This task was also performed under two additional rhythm conditions: inversed-swing rhythm (1:2 ratio) and regular subdivision (1:1 ratio). The center frequency of the band noises was either held constant or alternated between two values. Our results revealed a significant decrease in discrimination accuracy when the center frequency was alternated, irrespective of the rhythm ratio condition. This suggests that rhythm perception is shaped by temporal structure and affected by spectral properties.

Effective modulation from the ventral medial to the dorsal medial portion of the prefrontal cortex in memory confidence-based behavioral control.

Metacognition includes the ability to refer to one's own cognitive states, such as confidence, and adaptively control behavior based on this information. This ability is thought to allow us to predictably control our behavior without external feedback, for example, even before we take action. Many studies have suggested that metacognition requires a brain-wide network of multiple brain regions. However, the modulation of effective connectivity within this network during metacognitive tasks remains unclear. This study focused on medial prefrontal regions, which have recently been suggested to be particularly involved in metacognition. We examined whether modulation of effective connectivity specific to metacognitive behavioral control is observed using model-based network analysis and dynamic causal modeling (DCM). The results showed that negative modulation from the ventral medial prefrontal cortex to the dorsal medial prefrontal cortex was observed in situations that required metacognitive behavioral control but not in situations that did not require such metacognitive control. Furthermore, this modulation was particularly pronounced in the group of participants who could better use metacognition for behavioral control. These results imply hierarchical properties of metacognition-related brain networks.

Chronic exposure of female mice to selective serotonin reuptake inhibitors during lactation induces vocal behavior deficits in pre-weaned offspring.

Developmental factors for autism spectrum disorders (ASDs) have been an ongoing debate despite an increasing number of reports on genetic factors. Recent studies have suggested maternal intake of selective serotonin reuptake inhibitors (SSRIs) as a possible developmental factor elevating the risk for ASD in offspring. Here, we show that maternal exposure of mice to an SSRI, Fluoxetine (FLX), induces abnormal ultrasonic vocalizations (USVs), an indicator of ASD-related behavior. We tested the effect of FLX intake during pregnancy, lactation, or both. We found that the lactation and both conditions decreased the number of USVs emitted by offspring pups. An index for assessing the syllables' frequency modulation revealed that highly modulated syllables appeared to be inhibited only in both conditions. Furthermore, we found that the number of serotonergic neurons at adulthood was reduced in the progeny of mice treated with FLX in all conditions. In addition, maternal exposure to FLX through pregnancy and lactation induced a high death rate of early post-natal pups. These suggest that the maternal exposure to SSRIs affects early development of offsprings as well as the serotonergic system. Focusing on vocal communication, our results indicate that intake of an SSRI during lactation increases the risk of abnormal USVs in pups, and provides potential insights into the development of ASD.

Spontaneous variability predicts compensative motor response in vocal pitch control.

Our motor system uses sensory feedback to keep desired performance. From this view, motor fluctuation is not simply 'noise' inevitably caused in the nervous system but would play a role in generating variations to explore better outcomes via sensory feedback. Vocalization system offers a good model for studying such sensory-motor interactions since we regulate vocalization by hearing our own voice. This behavior is typically observed as compensatory responses in vocalized pitch, or fundamental frequency (fo), when artificial fo shifts were induced in the auditory feedback. However, the relationship between adaptive regulation and motor exploration in vocalization has remained unclear. Here we investigated behavioral variability in spontaneous vocal fo and compensatory responses against fo shifts in the feedback, and demonstrated that larger spontaneous fluctuation correlates with greater compensation in vocal fo. This correlation was found in slow components (≤ 5 Hz) of the spontaneous fluctuation but not in fast components (between 6 and 30 Hz), and the slow one was amplified during the compensatory responses. Furthermore, the compensatory ratio was reduced when large fo shifts were applied to the auditory feedback, as if reflecting the range of motor exploration. All these findings consistently suggest the functional role of motor variability in the exploration of better vocal outcomes.

Advanced paternal age diversifies individual trajectories of vocalization patterns in neonatal mice.

Infant crying is a communicative behavior impaired in neurodevelopmental disorders (NDDs). Because advanced paternal age is a risk factor for NDDs, we performed computational approaches to evaluate how paternal age affected vocal communication and body weight development in C57BL/6 mouse offspring from young and aged fathers. Analyses of ultrasonic vocalization (USV) consisting of syllables showed that advanced paternal age reduced the number and duration of syllables, altered the syllable composition, and caused lower body weight gain in pups. Pups born to young fathers had convergent vocal characteristics with a rich repertoire, whereas those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Additional analyses revealed that some pups from aged fathers displayed atypical USV trajectories. Thus, our study indicates that advanced paternal age has a significant effect on offspring's vocal development. Our computational analyses are effective in characterizing altered individual diversity.

Switching perception of musical meters by listening to different acoustic cues of biphasic sound stimulus.

Meter is one of the core features of music perception. It is the cognitive grouping of regular sound sequences, typically for every 2, 3, or 4 beats. Previous studies have suggested that one can not only passively perceive the meter from acoustic cues such as loudness, pitch, and duration of sound elements, but also actively perceive it by paying attention to isochronous sound events without any acoustic cues. Studying the interaction of top-down and bottom-up processing in meter perception leads to understanding the cognitive system's ability to perceive the entire structure of music. The present study aimed to demonstrate that meter perception requires the top-down process (which maintains and switches attention between cues) as well as the bottom-up process for discriminating acoustic cues. We created a "biphasic" sound stimulus, which consists of successive tone sequences designed to provide cues for both the triple and quadruple meters in different sound attributes, frequency, and duration. Participants were asked to focus on either frequency or duration of the stimulus, and to answer how they perceived meters on a five-point scale (ranged from "strongly triple" to "strongly quadruple"). As a result, we found that participants perceived different meters by switching their attention to specific cues. This result adds evidence to the idea that meter perception involves the interaction between top-down and bottom-up processes.

Neural correlates of vocal initiation in the VTA/SNc of juvenile male zebra finches.

Initiation and execution of complex learned vocalizations such as human speech and birdsong depend on multiple brain circuits. In songbirds, neurons in the motor cortices and basal ganglia circuitry exhibit preparatory activity before initiation of song, and that activity is thought to play an important role in successful song performance. However, it remains unknown where a start signal for song is represented in the brain and how such a signal would lead to appropriate vocal initiation. To test whether neurons in the midbrain ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) show activity related to song initiation, we carried out extracellular recordings of VTA/SNc single units in singing juvenile male zebra finches. We found that a subset of VTA/SNc units exhibit phasic activity precisely time-locked to the onset of the song bout, and that the activity occurred specifically at the beginning of song. These findings suggest that phasic activity in the VTA/SNc represents a start signal that triggers song vocalization.

Capturing the Effects of Domestication on Vocal Learning Complexity.

Domesticated and vocal learning species can serve as informative model organisms for the reduction of reactive aggression and emergence of speech in our lineage. Amidst mounting evidence that domestication modifies vocal repertoires across different species, we focus on the domesticated Bengalese finch, which has a more complex song than the wild-type white-rumped munia. Our explanation for this effect revolves around the glutamate neurotransmitter system. Glutamate signaling (i) is implicated in birdsong learning, (ii) controls dopamine activity in neural circuits crucial for vocal learning, (iii) is disproportionately targeted in the evolution of domesticates, and (iv) regulates stress responses and aggressive behaviors attenuated under domestication. We propose that attenuated excitation of stress-related neural circuits potentiates vocal learning via altered dopaminergic signaling.

Arousal State-Dependent Alterations in Neural Activity in the Zebra Finch VTA/SNc.

Sleep-wake behaviors are important for survival and highly conserved among animal species. A growing body of evidence indicates that the midbrain dopaminergic system is associated with sleep-wake regulation in mammals. Songbirds exhibit mammalian-like sleep structures, and neurons in the midbrain ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) possess physiological properties similar to those in mammals. However, it remains uncertain whether the neurons in the songbird VTA/SNc are associated with sleep-wake regulation. Here, we show that VTA/SNc neurons in zebra finches exhibit arousal state-dependent alterations in spontaneous neural activity. By recording extracellular single-unit activity from anesthetized or freely behaving zebra finches, we found that VTA/SNc neurons exhibited increased firing rates during wakefulness, and the same population of neurons displayed reduced firing rates during anesthesia and slow-wave sleep. These results suggest that the songbird VTA/SNc is associated with the regulation of sleep and wakefulness along with other arousal regulatory systems. These findings raise the possibility that fundamental neural mechanisms of sleep-wake behaviors are evolutionarily conserved between birds and mammals.

Unconscious and Distinctive Control of Vocal Pitch and Timbre During Altered Auditory Feedback.

Vocal control plays a critical role in smooth social communication. Speakers constantly monitor auditory feedback (AF) and make adjustments when their voices deviate from their intentions. Previous studies have shown that when certain acoustic features of the AF are artificially altered, speakers compensate for this alteration in the opposite direction. However, little is known about how the vocal control system implements compensations for alterations of different acoustic features, and associates them with subjective consciousness. The present study investigated whether compensations for the fundamental frequency (F0), which corresponds to perceived pitch, and formants, which contribute to perceived timbre, can be performed unconsciously and independently. Forty native Japanese speakers received two types of altered AF during vowel production that involved shifts of either only the formant frequencies (formant modification; Fm) or both the pitch and formant frequencies (pitch + formant modification; PFm). For each type, three levels of shift (slight, medium, and severe) in both directions (increase or decrease) were used. After the experiment, participants were tested for whether they had perceived a change in the F0 and/or formants. The results showed that (i) only formants were compensated for in the Fm condition, while both the F0 and formants were compensated for in the PFm condition; (ii) the F0 compensation exhibited greater precision than the formant compensation in PFm; and (iii) compensation occurred even when participants misperceived or could not explicitly perceive the alteration in AF. These findings indicate that non-experts can compensate for both formant and F0 modifications in the AF during vocal production, even when the modifications are not explicitly or correctly perceived, which provides further evidence for a dissociation between conscious perception and action in vocal control. We propose that such unconscious control of voice production may enhance rapid adaptation to changing speech environments and facilitate mutual communication.

USVSEG: A robust method for segmentation of ultrasonic vocalizations in rodents.

Rodents' ultrasonic vocalizations (USVs) provide useful information for assessing their social behaviors. Despite previous efforts in classifying subcategories of time-frequency patterns of USV syllables to study their functional relevance, methods for detecting vocal elements from continuously recorded data have remained sub-optimal. Here, we propose a novel procedure for detecting USV segments in continuous sound data containing background noise recorded during the observation of social behavior. The proposed procedure utilizes a stable version of the sound spectrogram and additional signal processing for better separation of vocal signals by reducing the variation of the background noise. Our procedure also provides precise time tracking of spectral peaks within each syllable. We demonstrated that this procedure can be applied to a variety of USVs obtained from several rodent species. Performance tests showed this method had greater accuracy in detecting USV syllables than conventional detection methods.

Undirected singing rate as a non-invasive tool for welfare monitoring in isolated male zebra finches.

Research on the songbird zebra finch (Taeniopygia guttata) has advanced our behavioral, hormonal, neuronal, and genetic understanding of vocal learning. However, little is known about the impact of typical experimental manipulations on the welfare of these birds. Here we explore whether the undirected singing rate can be used as an indicator of welfare. We tested this idea by performing a post hoc analysis of singing behavior in isolated male zebra finches subjected to interactive white noise, to surgery, or to tethering. We find that the latter two experimental manipulations transiently but reliably decreased singing rates. By contraposition, we infer that a high-sustained singing rate is suggestive of successful coping or improved welfare in these experiments. Our analysis across more than 300 days of song data suggests that a singing rate above a threshold of several hundred song motifs per day implies an absence of an acute stressor or a successful coping with stress. Because singing rate can be measured in a completely automatic fashion, its observation can help to reduce experimenter bias in welfare monitoring. Because singing rate measurements are non-invasive, we expect this study to contribute to the refinement of the current welfare monitoring tools in zebra finches.

Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds.

Understanding the structure and function of neural circuits underlying speech and language is a vital step toward better treatments for diseases of these systems. Songbirds, among the few animal orders that share with humans the ability to learn vocalizations from a conspecific, have provided many insights into the neural mechanisms of vocal development. However, research into vocal learning circuits has been hindered by a lack of tools for rapid genetic targeting of specific neuron populations to meet the quick pace of developmental learning. Here, we present a viral tool that enables fast and efficient retrograde access to projection neuron populations. In zebra finches, Bengalese finches, canaries, and mice, we demonstrate fast retrograde labeling of cortical or dopaminergic neurons. We further demonstrate the suitability of our construct for detailed morphological analysis, for in vivo imaging of calcium activity, and for multi-color brainbow labeling.

Acoustical cues for perception of emotional vocalizations in rats.

The ultrasonic vocalizations of rats can transmit affective states to listeners. For example, rats typically produce shorter calls in a higher frequency range in social situations (pleasant call: PC), whereas they emit longer calls with lower frequency in distress situations (distress call: DC). Knowing what acoustical features contribute to auditory discrimination between these two calls will help to better characterize auditory perception of vocalized sounds in rats. In turn, this could lead to better estimation of models for processing vocalizations in sensory systems in general. Here, using an operant discrimination procedure, we examined the impact of various acoustical features on discriminating emotional ultrasonic vocalizations. We did this by systematically swapping three features (frequency range, time duration, and residual frequency-modulation pattern) between two emotional calls. After rats were trained to discriminate between PC and DC, we presented probe stimuli that were synthesized calls with one or two acoustical features swapped, and examined if the rats judged these calls as either PC or DC. The results revealed that all features were important for discrimination between the two call types, but frequency range provided the most information for discrimination. This supports the hypothesis that while rats utilize all acoustical features to perceive emotional vocalizations, they considerably rely on frequency cues.

Regulation of action selection based on metacognition in humans via a ventral and dorsal medial prefrontal cortical network.

Metacognition is defined as cognition about one's own cognitive state; it enables us to estimate our own performance during goal-directed actions and to select a suitable strategy based on that estimation. Identifying the neural mechanisms that underlie this process will contribute to our understanding of how we realize adaptive self-control in daily life. Here, we focused on the neural substrates that allow us to voluntarily utilize prospective metacognition to carry out such action selection. Participants were asked to bet on their recall of sound stimuli presented at an earlier time in a delayed match-to-sample task of rapidly changing sound stimuli. During the task, brain activity was measured using functional magnetic resonance imaging. We found that the brain network composed of the ventral and dorsal parts of the medial prefrontal cortex and the medial precuneus regulated the strategic selection of risk/return profiles based on metacognition. In particular, increments in functional connectivity between the ventral and dorsal medial prefrontal cortices during high-risk/return bets correlated with the adaptiveness of the bet (as measured by the correspondence between choosing high risk/return bets and high accuracy of task performance). This index is considered to reflect the degree of voluntary use of metacognition to bet. These findings suggest that the strong connectivity within the network involving the ventral and dorsal medial prefrontal cortices enables us to utilize metacognition to select actions for achieving a goal efficiently.