Respective Involvement of the Right Cerebellar Crus I and II in Syntactic and Semantic Processing for Comprehension of Language.

The right posterolateral portions of the cerebellum (crus-I/II) are involved in language processing. However, their functional role in language remains unknown. The cerebellum is hypothesized to acquire an internal model that is a functional copy of mental representations in the cerebrum and to contribute to cognitive function. In this research, based on the cerebellar internal model hypothesis, we conducted task-based and resting-state functional magnetic resonance imaging (fMRI) experiments to investigate the role of the cerebellum in the syntactic and semantic aspects of comprehension of sentences. In a syntactic task, participants read sentences with center-embedded hierarchical structures. The hierarchical level-dependent activity was found in the right crus-I as well as Broca's area (p < 0.05, voxel-based small volume correction (SVC)). In a semantic task, the participants read three types of sentences for investigation of sentence-level, phrase-level, and word-level semantic processing. The semantic level-dependent activity was found in the right crus-II as well as in the left anterior temporal lobe and the left angular gyrus (p < 0.05, voxel-based SVC). Moreover, the right crus-I/II showed significant activity when the cognitive load was high. Resting-state fMRI demonstrated intrinsic functional connectivity between the right crus-I/II and language-related regions in the left cerebrum (p < 0.05, voxel-based SVC). These findings suggest that the right crus-I and crus-II are involved, respectively, in the syntactic and semantic aspects of sentence processing. The cerebellum assists processing of language in the cerebrum when the cognitive load is high.

The effect of acquisition duration on cerebral blood flow-based resting-state functional connectivity.

Resting-state functional connectivity (rs-FC) is widely used to examine the functional architecture of the brain, and the blood-oxygenation-level-dependent (BOLD) signal is often utilized for determining rs-FC. However, the BOLD signal is susceptible to various factors that have less influence on the cerebral blood flow (CBF). Therefore, CBF could comprise an alternative for determining rs-FC. Since acquisition duration is one of the essential parameters for obtaining reliable rs-FC, we investigated the effect of acquisition duration on CBF-based rs-FC to examine the reliability of CBF-based rs-FC. Nineteen participants underwent CBF scanning for a total duration of 50 min. Variance of CBF-based rs-FC within the whole brain and 13 large-scale brain networks at various acquisition durations was compared to that with a 50-min duration using the Levene's test. Variance of CBF-based rs-FC at any durations did not differ from that at a 50-min duration (p > .05). Regarding variance of rs-FC within each large-scale brain network, the acquisition duration required to obtain reliable estimates of CBF-based rs-FC was shorter than 10 min and varied across large-scale brain networks. Altogether, an acquisition duration of at least 10 min is required to obtain reliable CBF-based rs-FC. These results indicate that CBF-based resting-state functional magnetic resonance imaging (rs-fMRI) with more than 10 min of total acquisition duration could be an alternative method to BOLD-based rs-fMRI to obtain reliable rs-FC.

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.

Nutrient infusion evoked magnetic resonance imaging signal in the human hypothalamus.

BACKGROUND: The hypothalamus receives ingested nutrient information via ascending gut-related projections and plays a significant role in the regulation of food intake. Human neuroimaging studies have observed changes in the activity or connectivity of the hypothalamus in response to nutrient ingestion. However, previous neuroimaging studies have not yet assessed differences in temporal changes of hypothalamic responses to various nutrients in humans. Thus a repeated measures functional magnetic resonance imaging (fMRI) study using 30-min scans was designed to examine differences in hypothalamic responses to various nutrients. METHODS: In this study, 18 healthy adults (mean age, 22.4 years; standard deviation, 4.8; age range, 19-39 years; 11 males and seven females) underwent fMRI sessions. On the day of each session, one of the four solutions (200 ml of monosodium glutamate, glucose, safflower oil emulsion, or saline) was administered to participants while fMRI scanning. RESULTS: Infused amino acid and glucose, but not lipid emulsion, increased lateral hypothalamic responses as compared to a saline infusion ([x, y, z] = [4, -4, -10], z = 2.96). In addition, only hypothalamic responses to saline, but not those to the infusion of other nutrients, elicited a subjective sensation of hunger. CONCLUSION: These findings suggest that lateral hypothalamic responses to ingested nutrients may mediate homeostatic sensations in humans.

Corticobasal ganglia projecting neurons are required for juvenile vocal learning but not for adult vocal plasticity in songbirds.

Birdsong, like human speech, consists of a sequence of temporally precise movements acquired through vocal learning. The learning of such sequential vocalizations depends on the neural function of the motor cortex and basal ganglia. However, it is unknown how the connections between cortical and basal ganglia components contribute to vocal motor skill learning, as mammalian motor cortices serve multiple types of motor action and most experimentally tractable animals do not exhibit vocal learning. Here, we leveraged the zebra finch, a songbird, as an animal model to explore the function of the connectivity between cortex-like (HVC) and basal ganglia (area X), connected by HVC(X) projection neurons with temporally precise firing during singing. By specifically ablating HVC(X) neurons, juvenile zebra finches failed to copy tutored syllable acoustics and developed temporally unstable songs with less sequence consistency. In contrast, HVC(X)-ablated adults did not alter their learned song structure, but generated acoustic fluctuations and responded to auditory feedback disruption by the introduction of song deterioration, as did normal adults. These results indicate that the corticobasal ganglia input is important for learning the acoustic and temporal aspects of song structure, but not for generating vocal fluctuations that contribute to the maintenance of an already learned vocal pattern.

Comparison of traveling-subject and ComBat harmonization methods for assessing structural brain characteristics.

Multisite magnetic resonance imaging (MRI) is increasingly used in clinical research and development. Measurement biases-caused by site differences in scanner/image-acquisition protocols-negatively influence the reliability and reproducibility of image-analysis methods. Harmonization can reduce bias and improve the reproducibility of multisite datasets. Herein, a traveling-subject (TS) dataset including 56 T1-weighted MRI scans of 20 healthy participants in three different MRI procedures-20, 19, and 17 subjects in Procedures 1, 2, and 3, respectively-was considered to compare the reproducibility of TS-GLM, ComBat, and TS-ComBat harmonization methods. The minimum participant count required for harmonization was determined, and the Cohen's d between different MRI procedures was evaluated as a measurement-bias indicator. The measurement-bias reduction realized with different methods was evaluated by comparing test-retest scans for 20 healthy participants. Moreover, the minimum subject count for harmonization was determined by comparing test-retest datasets. The results revealed that TS-GLM and TS-ComBat reduced measurement bias by up to 85 and 81.3%, respectively. Meanwhile, ComBat showed a reduction of only 59.0%. At least 6 TSs were required to harmonize data obtained from different MRI scanners, complying with the imaging protocol predetermined for multisite investigations and operated with similar scan parameters. The results indicate that TS-based harmonization outperforms ComBat for measurement-bias reduction and is optimal for MRI data in well-prepared multisite investigations. One drawback is the small sample size used, potentially limiting the applicability of ComBat. Investigation on the number of subjects needed for a large-scale study is an interesting future problem.

Production of regular rhythm induced by external stimuli in rats.

Rhythmic ability is important for locomotion, communication, and coordination between group members during the daily life of animals. We aimed to examine the rhythm perception and production abilities in rats within the range of a subsecond to a few seconds. We trained rats to respond to audio-visual stimuli presented in regular, isochronous rhythms at six time-intervals (0.5-2 s). Five out of six rats successfully learned to respond to the sequential stimuli. All subjects showed periodic actions. The actions to regular stimuli were faster than randomly presented stimuli in the medium-tempo conditions. In slower and faster tempo conditions, the actions of some subjects were not periodic or phase-matched to the stimuli. The asynchrony regarding the stimulus onset became larger or smaller when the last stimulus of the sequence was presented at deviated timings. Thus, the actions of the rats were tempo matched to the regular rhythm, but not completely anticipative. We also compared the extent of phase-matching and variability of rhythm production among the interval conditions. In interval conditions longer than 1.5 s, variability tended to be larger. In conclusion, rats showed a tempo matching ability to regular rhythms to a certain degree, but maintenance of a constant tempo to slower rhythm conditions was difficult. Our findings suggest that non-vocal learning mammals have the potential to produce flexible rhythms in subsecond timing.

Impact of endogenous melatonin on rhythmic behaviors, reproduction, and survival revealed in melatonin-proficient C57BL/6J congenic mice.

The hormone melatonin is synthesized from serotonin by two enzymatic reactions (AANAT and ASMT/HIOMT) in the pineal gland following a circadian rhythm with low levels during the day and high levels at night. The robust nightly peak of melatonin secretion is an output signal of the circadian clock to the whole organism. However, so far the regulatory roles of endogenous melatonin in mammalian biological rhythms and physiology processes are poorly understood. Here, we establish congenic mouse lines (>N10 generations) that are proficient or deficient in melatonin synthesis (AH+/+ or AH-/- mice, respectively) on the C57BL/6J genetic background by crossing melatonin-proficient MSM/Ms with C57BL/6J. AH+/+ mice displayed robust nightly peak of melatonin secretion and had significantly higher levels of pineal and plasma melatonin vs AH-/- mice. Using this mice model, we investigated the role of endogenous melatonin in regulating multiple biological rhythms, physiological processes, and rhythmic behaviors. In the melatonin-proficient (AH+/+) mice, the rate of re-entrainment of wheel-running activity was accelerated following a 6-hour phase advance of dark onset when comparted with AH-/- mice, suggesting a role of endogenous melatonin in facilitating clock adjustment. Further in the AH+/+ mice, there was a significant decrease in body weight, gonadal weight and reproductive performance, and a significant increase in daily torpor (a hypothermic and hypometabolic state lasting only hours during adverse conditions). Endogenous melatonin, however, had no effect in the modulation of the diurnal rhythm of 2-[125 I]-iodomelatonin receptor expression in the SCN, free-running wheel behavior in constant darkness, life span, spontaneous homecage behaviors, and various types of social-emotional behaviors. The findings also shed light on the role of endogenous melatonin in mice domestication and provide new insights into melatonin's action in reducing energy expenditure during a food shortage. In summary, the congenic mice model generated in this study offers a significant advantage toward understanding of the role of endogenous melatonin in regulating melatonin receptor-mediated rhythm behaviors and physiological functions.

Different Reactions of Zebra Finches and Bengalese Finches to a Three-Component Mixture of Anesthetics.

Kawai et al. (2011) recently introduced a mixture of three anesthetic agents (here called MMB) that has an effect similar to ketamine/xylazine in mice, which might allow more effective reaction to changes in the animal condition, as an antagonist is available, and which can be used without license for handling narcotic drugs. Using Kawai's study as a baseline, we tested whether this anesthesia and its antagonist can also be used in avian studies. In the present study, we used two species, the zebra finch and the Bengalese finch, of the avian family Estrildidae. In zebra finches, anesthesia effects similar to the use of ketamine/xylazine and to those obtained in mice can be reached by the use of MMB if a higher dose is applied. MMB leads to more variable anesthesia, but has the advantage of a longer time window of deep anesthesia. An antagonist to one component of MMB reduced the awaking time, but was not as effective as in mice. For Bengalese finches, MMB cannot be generally recommended because of difficult handling and high mortality rate when used without antagonist, but could be used for perfusions instead of pentobarbital.

Stepwise acquisition of vocal combinatorial capacity in songbirds and human infants.

Human language, as well as birdsong, relies on the ability to arrange vocal elements in new sequences. However, little is known about the ontogenetic origin of this capacity. Here we track the development of vocal combinatorial capacity in three species of vocal learners, combining an experimental approach in zebra finches (Taeniopygia guttata) with an analysis of natural development of vocal transitions in Bengalese finches (Lonchura striata domestica) and pre-lingual human infants. We find a common, stepwise pattern of acquiring vocal transitions across species. In our first study, juvenile zebra finches were trained to perform one song and then the training target was altered, prompting the birds to swap syllable order, or insert a new syllable into a string. All birds solved these permutation tasks in a series of steps, gradually approximating the target sequence by acquiring new pairwise syllable transitions, sometimes too slowly to accomplish the task fully. Similarly, in the more complex songs of Bengalese finches, branching points and bidirectional transitions in song syntax were acquired in a stepwise fashion, starting from a more restrictive set of vocal transitions. The babbling of pre-lingual human infants showed a similar pattern: instead of a single developmental shift from reduplicated to variegated babbling (that is, from repetitive to diverse sequences), we observed multiple shifts, where each new syllable type slowly acquired a diversity of pairwise transitions, asynchronously over development. Collectively, these results point to a common generative process that is conserved across species, suggesting that the long-noted gap between perceptual versus motor combinatorial capabilities in human infants may arise partly from the challenges in constructing new pairwise vocal transitions.

Auditory-vocal coupling in the naked mole-rat, a mammal with poor auditory thresholds.

Naked mole-rats are extremely social and extremely vocal rodents, displaying a wide range of functionally distinct call types and vocalizing almost continuously. Their vocalizations are low frequency, and a behavioral audiogram has shown that naked mole-rats, like other subterranean mammals, hear only low frequencies. Hence, the frequency range of their hearing and vocalizations appears to be well matched. However, even at low frequencies, naked mole-rats show very poor auditory thresholds, suggesting vocal communication may be effective only over short distances. However, in a tunnel environment where low frequency sounds propagate well and background noise is low, it may be that vocalizations travel considerable distances at suprathreshold intensities. Here, we confirmed hearing sensitivity using the auditory brainstem response; we characterized signature and alarm calls in intensity and frequency domains and we measured the effects of propagation through tubes with the diameter of naked mole-rat tunnels. Signature calls-used for intimate communication-could travel 3-8 m at suprathreshold intensities, and alarm calls (lower frequency and higher intensity), could travel up to 15 m. Despite this species' poor hearing sensitivity, the naked mole-rat displays a functional, coupled auditory-vocal communication system-a hallmark principle of acoustic communication systems across taxa.

Fast voltage-sensitive dye imaging of excitatory and inhibitory synaptic transmission in the rat granular retrosplenial cortex.

Rodent granular retrosplenial cortex (GRS) has dense connections between the anterior thalamic nuclei (ATN) and hippocampal formation. GRS superficial pyramidal neurons exhibit distinctive late spiking (LS) firing property and form patchy clusters with prominent apical dendritic bundles. The aim of this study was to investigate spatiotemporal dynamics of signal transduction in the GRS induced by ATN afferent stimulation by using fast voltage-sensitive dye imaging in rat brain slices. In coronal slices, layer 1a stimulation, which presumably activated thalamic fibers, evoked propagation of excitatory synaptic signals from layers 2-4 to layers 5-6 in a direction perpendicular to the layer axis, followed by transverse signal propagation within each layer. In the presence of ionotropic glutamate receptor antagonists, inhibitory responses were observed in superficial layers, induced by direct activation of inhibitory interneurons in layer 1. In horizontal slices, excitatory signals in deep layers propagated transversely mainly from posterior to anterior via superficial layers. Cortical inhibitory responses upon layer 1a stimulation in horizontal slices were weaker than those in the coronal slices. Observed differences between coronal and horizontal planes suggest anisotropy of the intracortical circuitry. In conclusion, ATN inputs are processed differently in coronal and horizontal planes of the GRS and then conveyed to other cortical areas. In both planes, GRS superficial layers play an important role in signal propagation, which suggests that superficial neuronal cascade is crucial in the integration of multiple information sources.NEW & NOTEWORTHY Superficial neurons in the rat granular retrosplenial cortex (GRS) show distinctive late-spiking (LS) firing property. However, little is known about spatiotemporal dynamics of signal transduction in the GRS. We demonstrated LS neuron network relaying thalamic inputs to deep layers and anisotropic distribution of inhibition between coronal and horizontal planes. Since deep layers of the GRS receive inputs from the subiculum, GRS circuits may work as an integrator of multiple sources such as sensory and memory information.

Chick Development and Asynchroneous Hatching in the Zebra Finch (Taeniopygia guttata castanotis).

The mode of hatching in birds has important impacts on both parents and chicks, including the costs and risks of breeding for parents, and sibling competition in a clutch. Birds with multiple eggs in a single clutch often begin incubating when most eggs are laid, thereby reducing time of incubation, nursing burden, and sibling competition. In some songbirds and some other species, however, incubation starts immediately after the first egg is laid, and the chicks thus hatch asynchronously. This may result in differences in parental care and in sibling competition based on body size differences among older and younger chicks, which in turn might produce asynchronous development among siblings favoring the first hatchling, and further affect the development and fitness of the chicks after fledging. To determine whether such processes in fact occur in the zebra finch, we observed chick development in 18 clutches of zebra finches. We found that there were effects of asynchronous hatching, but these were smaller than expected and mostly not significant. Our observations suggest that the amount of care given to each chick may be equated with such factors as a camouflage effect of the down feathers, and that the low illumination within the nest also complicates the determination of the hatching order by the parents.

Hormonal Responses to a Potential Mate in Male Birds.

Social interactions rapidly modulate circulating hormone levels and behavioral patterns in most male animals. In male birds, sexual interaction or visual exposure to a conspecific female usually causes an increase in the levels of peripheral reproductive hormones, such as gonadotropins and androgens. Although the perception of a female presence is processed in the brain and peripheral hormonal levels are regulated by the hypothalamus-pituitary-gonadal (HPG) axis, the specific neural circuitry and neurochemical systems that translate social signals into reproductive physiology in male birds were not well understood until 2008. Today, there is growing evidence that two neuropeptides localized in the hypothalamus, gonadotropin-releasing hormone and gonadotropin-inhibitory hormone, are responsive to social information. These two neuropeptides have thus begun to be regarded as modulators translating social stimuli into changes in the levels of peripheral reproductive hormones. Here, we review previous studies that investigated the male responses of the HPG axis to the mere presence of a female or to sexual interaction, and describe the neurochemical pathways linking visual perception of a potential mate to rapid peripheral hormonal changes via the brain-pituitary endocrine system in sexually mature male Japanese quail.

Hierarchical emergence of sequence sensitivity in the songbird auditory forebrain.

Bengalese finches (Lonchura striata var. domestica) generate more complex sequences in their songs than zebra finches. Because of this, we chose this species to explore the signal processing of sound sequence in the primary auditory forebrain area, field L, and in a secondary area, the caudomedial nidopallium (NCM). We simultaneously recorded activity from multiple single units in urethane-anesthetized birds. We successfully replicated the results of a previous study in awake zebra finches examining stimulus-specific habituation of NCM neurons to conspecific songs. Then, we used an oddball paradigm and compared the neural response to deviant sounds that were presented infrequently, with the response to standard sounds, which were presented frequently. In a single sound oddball task, two different song elements were assigned for the deviant and standard sounds. The response bias to deviant elements was larger in NCM than in field L. In a triplet sequence oddball task, two triplet sequences containing elements ABC and ACB were assigned as the deviant and standard. Only neurons in NCM that displayed broad-shaped spike waveforms had sensitivity to the difference in element order. Our results suggest the hierarchical processing of complex sound sequences in the songbird auditory forebrain.

Auditory Responses to Vocal Sounds in the Songbird Nucleus Taeniae of the Amygdala and the Adjacent Arcopallium.

Many species of animals communicate with others through vocalizations. Over time, these species have evolved mechanisms to respond to biologically relevant vocal sounds via adaptive behaviors. Songbirds provide a good opportunity to search for the neural basis of this adaptation, because they interact with others through a variety of vocalizations in complex social relationships. The nucleus taeniae of the amygdala (TnA) is a structure located in the ventromedial arcopallium, which is akin to the mammalian medial amygdala. Studies on the anatomy and function of this nucleus have led to the speculation that the TnA is one of the possible neural substrates that represents the relevance of acoustic stimuli related to behavior. However, neural responses in this nucleus to auditory stimuli have not been studied in depth. To give a detailed description about auditory responses of the TnA in the songbird, we conducted neural recordings from the TnA and the adjacent arcopallium in adult male and female Bengalese finches under anesthesia. The birds were exposed to auditory stimuli including natural vocalizations as well as synthesized noise. We demonstrated that a substantial population of neurons in the TnA and the adjacent arcopallium responded to vocal sounds and that some neurons were selectively activated to specific stimuli. Proportions of responsive cells and stimulus-selective cells were larger in males than in females. In addition, a larger ratio of selective cells was observed in the arcopallium compared to the TnA. These findings support the idea that neuronal activity in the TnA and the neighboring area represents behavioral relevance of sounds. Further studies in electrophysiology combined with evidence from other fields, such as region-specific gene expression patterns, are required to fully understand the functions of the TnA as well as the evolution of the amygdala in songbirds and vertebrate animals.

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.

Neural basis of decision making guided by emotional outcomes.

Emotional events resulting from a choice influence an individual's subsequent decision making. Although the relationship between emotion and decision making has been widely discussed, previous studies have mainly investigated decision outcomes that can easily be mapped to reward and punishment, including monetary gain/loss, gustatory stimuli, and pain. These studies regard emotion as a modulator of decision making that can be made rationally in the absence of emotions. In our daily lives, however, we often encounter various emotional events that affect decisions by themselves, and mapping the events to a reward or punishment is often not straightforward. In this study, we investigated the neural substrates of how such emotional decision outcomes affect subsequent decision making. By using functional magnetic resonance imaging (fMRI), we measured brain activities of humans during a stochastic decision-making task in which various emotional pictures were presented as decision outcomes. We found that pleasant pictures differentially activated the midbrain, fusiform gyrus, and parahippocampal gyrus, whereas unpleasant pictures differentially activated the ventral striatum, compared with neutral pictures. We assumed that the emotional decision outcomes affect the subsequent decision by updating the value of the options, a process modeled by reinforcement learning models, and that the brain regions representing the prediction error that drives the reinforcement learning are involved in guiding subsequent decisions. We found that some regions of the striatum and the insula were separately correlated with the prediction error for either pleasant pictures or unpleasant pictures, whereas the precuneus was correlated with prediction errors for both pleasant and unpleasant pictures.

Twitter evolution: converging mechanisms in birdsong and human speech.

Vocal imitation in human infants and in some orders of birds relies on auditory-guided motor learning during a sensitive period of development. It proceeds from 'babbling' (in humans) and 'subsong' (in birds) through distinct phases towards the full-fledged communication system. Language development and birdsong learning have parallels at the behavioural, neural and genetic levels. Different orders of birds have evolved networks of brain regions for song learning and production that have a surprisingly similar gross anatomy, with analogies to human cortical regions and basal ganglia. Comparisons between different songbird species and humans point towards both general and species-specific principles of vocal learning and have identified common neural and molecular substrates, including the forkhead box P2 (FOXP2) gene.

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.