Awake Birds Research Articles

Responses to Song Playback Differ in Sleeping versus Anesthetized Songbirds.

Vocal learning in songbirds is mediated by a highly localized system of interconnected forebrain regions, including recurrent loops that traverse the cortex, basal ganglia, and thalamus. This brain-behavior system provides a powerful model for elucidating mechanisms of vocal learning, with implications for learning speech in human infants, as well as for advancing our understanding of skill learning in general. A long history of experiments in this area has tested neural responses to playback of different song stimuli in anesthetized birds at different stages of vocal development. These studies have demonstrated selectivity for different song types that provide neural signatures of learning. In contrast to the ease of obtaining responses to song playback in anesthetized birds, song-evoked responses in awake birds are greatly reduced or absent, indicating that behavioral state is an important determinant of neural responsivity. Song-evoked responses can be elicited during sleep as well as anesthesia, and the selectivity of responses to song playback in adult birds is highly similar between anesthetized and sleeping states, encouraging the idea that anesthesia and sleep are similar. In contrast to that idea, we report evidence that cortical responses to song playback in juvenile zebra finches (Taeniopygia guttata) differ greatly between sleep and urethane anesthesia. This finding indicates that behavioral states differ in sleep versus anesthesia and raises questions about relationships between developmental changes in sleep activity, selectivity for different song types, and the neural substrate for vocal learning.

Distinct timescales for the neuronal encoding of vocal signals in a high-order auditory area

The ability of the auditory system to selectively recognize natural sound categories while maintaining a certain degree of tolerance towards variations within these categories, which may have functional roles, is thought to be crucial for vocal communication. To date, it is still largely unknown how the balance between tolerance and sensitivity to variations in acoustic signals is coded at a neuronal level. Here, we investigate whether neurons in a high-order auditory area in zebra finches, a songbird species, are sensitive to natural variations in vocal signals by recording their responses to repeated exposures to identical and variant sound sequences. We used the songs of male birds which tend to be highly repetitive with only subtle variations between renditions. When playing these songs to both anesthetized and awake birds, we found that variations between songs did not affect the neuron firing rate but the temporal reliability of responses. This suggests that auditory processing operates on a range of distinct timescales, namely a short one to detect variations in vocal signals, and longer ones that allow the birds to tolerate variations in vocal signal structure and to encode the global context.

Neuronal Encoding in a High-Level Auditory Area: From Sequential Order of Elements to Grammatical Structure.

Sensitivity to the sequential structure of communication sounds is fundamental not only for language comprehension in humans but also for song recognition in songbirds. By quantifying single-unit responses, we first assessed whether the sequential order of song elements, called syllables, in conspecific songs is encoded in a secondary auditory cortex-like region of the zebra finch brain. Based on a habituation/dishabituation paradigm, we show that, after multiple repetitions of the same conspecific song, rearranging syllable order reinstated strong responses. A large proportion of neurons showed sensitivity to song context in which syllables occurred providing support for the nonlinear processing of syllable sequences. Sensitivity to the temporal order of items within a sequence should enable learning its underlying structure, an ability considered a core mechanism of the human language faculty. We show that repetitions of songs that were ordered according to a specific grammatical structure (i.e., ABAB or AABB structures; A and B denoting song syllables) led to different responses in both anesthetized and awake birds. Once responses were decreased due to song repetitions, the transition from one structure to the other could affect the firing rates and/or the spike patterns. Our results suggest that detection was based on local differences rather than encoding of the global song structure as a whole. Our study demonstrates that a high-level auditory region provides neuronal mechanisms to help discriminate stimuli that differ in their sequential structure.SIGNIFICANCE STATEMENT Sequence processing has been proposed as a potential precursor of language syntax. As a sequencing operation, the encoding of the temporal order of items within a sequence may help in recognition of relationships between adjacent items and in learning the underlying structure. Taking advantage of the stimulus-specific adaptation phenomenon observed in a high-level auditory region of the zebra finch brain, we addressed this question at the neuronal level. Reordering elements within conspecific songs reinstated robust responses. Neurons also detected changes in the structure of artificial songs, and this detection depended on local transitions between adjacent or nonadjacent syllables. These findings establish the songbird as a model system for deciphering the mechanisms underlying sequence processing at the single-cell level.

Midbrain Synchrony to Envelope Structure Supports Behavioral Sensitivity to Single-Formant Vowel-Like Sounds in Noise.

Vowels make a strong contribution to speech perception under natural conditions. Vowels are encoded in the auditory nerve primarily through neural synchrony to temporal fine structure and to envelope fluctuations rather than through average discharge rate. Neural synchrony is thought to contribute less to vowel coding in central auditory nuclei, consistent with more limited synchronization to fine structure and the emergence of average-rate coding of envelope fluctuations. However, this hypothesis is largely unexplored, especially in background noise. The present study examined coding mechanisms at the level of the midbrain that support behavioral sensitivity to simple vowel-like sounds using neurophysiological recordings and matched behavioral experiments in the budgerigar. Stimuli were harmonic tone complexes with energy concentrated at one spectral peak, or formant frequency, presented in quiet and in noise. Behavioral thresholds for formant-frequency discrimination decreased with increasing amplitude of stimulus envelope fluctuations, increased in noise, and were similar between budgerigars and humans. Multiunit recordings in awake birds showed that the midbrain encodes vowel-like sounds both through response synchrony to envelope structure and through average rate. Whereas neural discrimination thresholds based on either coding scheme were sufficient to support behavioral thresholds in quiet, only synchrony-based neural thresholds could account for behavioral thresholds in background noise. These results reveal an incomplete transformation to average-rate coding of vowel-like sounds in the midbrain. Model simulations suggest that this transformation emerges due to modulation tuning, which is shared between birds and mammals. Furthermore, the results underscore the behavioral relevance of envelope synchrony in the midbrain for detection of small differences in vowel formant frequency under real-world listening conditions.

Role of intracranial cavities in avian directional hearing

Whereas it is clear from anatomical studies that all birds have complex interaural canals connecting their middle ears, the effect of interaural coupling on directional hearing has been disputed. A reason for conflicting results in earlier studies may have been that the function of the tympanic ear and hence of the interaural coupling is sensitive to variations in the intracranial air pressure. In awake birds, the middle ears and connected cavities are vented actively through the pharyngotympanic tube. This venting reflex seems to be suppressed in anesthetized birds, leading to increasingly lower pressure in the interaural cavities, stiffening the eardrums, and displacing them medially. This causes the sensitivity, as well as the interaural coupling, to drop. Conversely, when the middle ears are properly vented, robust directional eardrum responses, most likely caused by internal coupling, have been reported. The anatomical basis of this coupling is the 'interaural canal,' which turns out to be a highly complex canal and cavity system, which we describe for the zebra finch. Surprisingly, given the complexity of the interaural canals, simple models of pipe-coupled middle ears fit the eardrum directionality data quite well, but future models taking the complex anatomy into consideration should be developed.

Electrophysiological mismatch response recorded in awake pigeons from the avian functional equivalent of the primary auditory cortex.

The neural response to occasional variations in acoustic stimuli in a regular sequence of sounds generates an N-methyl-D-aspartate receptor-modulated event-related potential in primates and rodents in the primary auditory cortex known as mismatch negativity (MMN). The current study investigated MMN in pigeons (Columba livia L) through intracranial recordings from Field L of the caudomedial nidopallium, the avian functional equivalent of the mammalian primary auditory cortex. Auditory evoked field potentials were recorded from awake birds using a low-frequency (800 Hz) and high-frequency (1400 Hz) deviant auditory oddball procedure with deviant-as-standard (flip-flop design) and multiple-standard control conditions. An MMN-like field potential was recorded and blocked with systemic 5 mg/kg ketamine administration. Our results are similar to human and rodent findings of an MMN-like event-related potential in birds suggestive of similar auditory sensory memory mechanisms in birds and mammals that are homologue from a common ancestor 300 million years ago or resulted from convergent evolution.

Functional MRI and functional connectivity of the visual system of awake pigeons

At present, functional MRI (fMRI) is increasingly used in animal research but the disadvantage is that the majority of the imaging is applied in anaesthetized animals. Only a few articles present results obtained in awake rodents. In this study both traditional fMRI and resting state (rsfMRI) were applied to four pigeons, that were trained to remain still while being imaged, removing the need for anesthesia. This is the first time functional connectivity measurements are performed in a non-mammalian species. Since the visual system of pigeons is a well-known model for brain asymmetry, the focus of the study was on the neural substrate of the visual system. For fMRI a visual stimulus was used and functional connectivity measurements were done with the entopallium (E; analog for the primary visual cortex) as a seed region. Interestingly in awake pigeons the left E was significantly functionally connected to the right E. Moreover we compared connectivity maps for a seed region in both hemispheres resulting in a stronger bilateral connectivity starting from left E then from right E. These results could be used as a starting point for further imaging studies in awake birds and also provide a new window into the analysis of hemispheric dominance in the pigeon.

Hypothermic abilities of migratory songbirds at a stopover site

Migratory birds use stopover sites for resting and feeding. Breaks after prolonged flights may allow cooling down the muscles and save energy through hypothermia. We measured hypothermic abilities in two long-distance migratory songbirds (garden warbler, Sylvia borin, and icterine warbler, Hippolais polyglotta) at a stopover site during spring migration selecting (1) birds just arrived after a prolonged flight (morning) (2) birds that had been resting in the vegetation for several hours (afternoon) and (3) birds caught in the late afternoon and kept overnight in standard conditions (night). In the first two samples, body temperature was measured immediately, 30 and 180 min after capture. In the night sample, it was measured at 1 a.m. in birds awake and asleep, and at 7 a.m., when all birds were awake. Morning birds showed the highest values (39.7°C), dropping of about 3°C after 180 min. In the afternoon birds (2), the time course was similar, but values (38.5°C) were lower than that of morning birds of about 1°C. At night, asleep birds had lower temperature than awake birds, reaching up to 10°C less than daytime values, but recovering in the early morning. Such remarkable plasticity in thermogenic abilities during migration could allow small migrants to economize energy in emergency situations.

Neural adaptation to tone sequences in the songbird forebrain: patterns, determinants, and relation to the build-up of auditory streaming

Neural responses to tones in the mammalian primary auditory cortex (A1) exhibit adaptation over the course of several seconds. Important questions remain about the taxonomic distribution of multi-second adaptation and its possible roles in hearing. It has been hypothesized that neural adaptation could explain the gradual "build-up" of auditory stream segregation. We investigated the influence of several stimulus-related factors on neural adaptation in the avian homologue of mammalian A1 (field L2) in starlings (Sturnus vulgaris). We presented awake birds with sequences of repeated triplets of two interleaved tones (ABA-ABA-...) in which we varied the frequency separation between the A and B tones (DeltaF), the stimulus onset asynchrony (time from tone onset to onset within a triplet), and tone duration. We found that stimulus onset asynchrony generally had larger effects on adaptation compared with DeltaF and tone duration over the parameter range tested. Using a simple model, we show how time-dependent changes in neural responses can be transformed into neurometric functions that make testable predictions about the dependence of the build-up of stream segregation on various spectral and temporal stimulus properties.

Estradiol Shapes Auditory Processing in the Adult Brain by Regulating Inhibitory Transmission and Plasticity-Associated Gene Expression

Estradiol impacts a wide variety of brain processes, including sex differentiation, mood, and learning. Here we show that estradiol regulates auditory processing of acoustic signals in the vertebrate brain, more specifically in the caudomedial nidopallium (NCM), the songbird analog of the mammalian auditory association cortex. Multielectrode recordings coupled with local pharmacological manipulations in awake animals reveal that both exogenous and locally generated estradiol increase auditory-evoked activity in NCM. This enhancement in neuronal responses is mediated by suppression of local inhibitory transmission. Surprisingly, we also found that estradiol is both necessary and sufficient for the induction of multiple mitogen-activated protein kinase (MAPK)-dependent genes thought to be required for synaptic plasticity and memorization of birdsong. Specifically, we show that local blockade of estrogen receptors or aromatase activity in awake birds decrease song-induced MAPK-dependent gene expression. Infusions of estradiol in acoustically isolated birds induce transcriptional activation of these genes to levels comparable with song-stimulated animals. Our results reveal acute and rapid nongenomic functions for estradiol in central auditory physiology and suggest that such roles may be ubiquitously expressed across sensory systems.

Response Properties of the Auditory Telencephalon in Songbirds Change with Recent Experience and Season

The caudomedial nidopallium (NCM) is a telencephalic auditory area that is selectively activated by conspecific vocalizations in zebra finches and canaries. We recently demonstrated that temporal and spectral dynamics of auditory tuning in NCM differ between these species [1]. In order to determine whether these differences reflect recent experience, we exposed separate groups of each species and sex to different housing conditions. Adult birds were housed either in an aviary with conspecifics (NORM), with heterospecifics (canary subjects in a zebra finch aviary, and vice versa: (CROSS)), or in isolation (ISO) for 9 days prior to testing. We then recorded extracellular multi-unit electrophysiological responses to simple pure tone stimuli (250–5000 Hz) in awake birds from each group and analyzed auditory tuning width using methods from our earlier studies. Relative to NORM birds, tuning was narrower in CROSS birds, and wider in ISO birds. The trend was greater in canaries, especially females. The date of recording was also included as a covariate in ANCOVAs that analyzed a larger set of the canary data, including data from birds tested outside of the breeding season, and treated housing condition and sex as independent variables. These tests show that tuning width was narrower early in the year and broader later. This effect was most pronounced in CROSS males. The degree of the short-term neural plasticity described here differs across sexes and species, and may reflect differences in NCM's anatomical and functional organization related to species differences in song characteristics, adult plasticity and/or social factors. More generally, NCM tuning is labile and may be modulated by recent experience to reflect the auditory processing required for behavioral adaptation to the current acoustic, social or seasonal context.

Inhibitory network interactions shape the auditory processing of natural communication signals in the songbird auditory forebrain.

The role of GABA in the central processing of complex auditory signals is not fully understood. We have studied the involvement of GABA A-mediated inhibition in the processing of birdsong, a learned vocal communication signal requiring intact hearing for its development and maintenance. We focused on caudomedial nidopallium (NCM), an area analogous to parts of the mammalian auditory cortex with selective responses to birdsong. We present evidence that GABA A-mediated inhibition plays a pronounced role in NCM's auditory processing of birdsong. Using immunocytochemistry, we show that approximately half of NCM's neurons are GABAergic. Whole cell patch-clamp recordings in a slice preparation demonstrate that, at rest, spontaneously active GABAergic synapses inhibit excitatory inputs onto NCM neurons via GABA A receptors. Multi-electrode electrophysiological recordings in awake birds show that local blockade of GABA A-mediated inhibition in NCM markedly affects the temporal pattern of song-evoked responses in NCM without modifications in frequency tuning. Surprisingly, this blockade increases the phasic and largely suppresses the tonic response component, reflecting dynamic relationships of inhibitory networks that could include disinhibition. Thus processing of learned natural communication sounds in songbirds, and possibly other vocal learners, may depend on complex interactions of inhibitory networks.

Species differences in auditory processing dynamics in songbird auditory telencephalon

The caudomedial nidopallium (NCM) of songbirds is a telencephalic area involved in the auditory processing and memorization of complex vocal communication signals. We used pure tone stimuli and multiunit electrophysiological recordings in awake birds to investigate whether the basic properties of song-responding circuits in NCM differ between canaries and zebra finches, two species whose songs are markedly different in their spectral and temporal organization. We found that the responses in zebra finch NCM are characterized by broad tuning and sustained responses that may facilitate the integration of zebra finch song syllables and call notes that are of long duration and have a broad harmonic structure. In contrast, we found that the responses in canary NCM show narrower tuning and less sustained responses over the time periods analyzed. These characteristics may contribute to enhanced processing of the narrow-band whistles, rapid trills, and steep frequency modulations that are prominent features of canary song. These species differences are much less pronounced in field L2, the direct thalamorecipient region that represents a preceding station in the central avian auditory pathway. NCM responses did not differ across sexes of either species, but field L2 did show wider tuning in zebra finch females relative to males. In sum, species differences in the response properties of NCM likely reflect selectivity for the acoustic elements of each species' vocal repertoire.

Distributed and Selective Auditory Representation of Song Repertoires in the Avian Song System

For many songbirds, the vocal repertoire constitutes acoustically distinct songs that are flexibly used in various behavioral contexts. To investigate how these different vocalizations are represented in the song neural system, we presented multiple song stimuli while performing extracellular recording in nucleus HVC in adult male song sparrows Melospiza melodia, a species known for its complex vocal repertoire and territorial use of song. We observed robust auditory responses to natural song stimuli in both awake and anesthetized animals. Auditory responses were selective for multiple songs of the bird's own repertoire (BOR) over acoustically modified versions of these stimuli. Selectivity was evident in both awake and anesthetized HVC, in contrast to auditory selectivity in zebra finch HVC, which is apparent only under anesthesia. Presentation of multiple song stimuli at different recording locations demonstrated that stimulus acoustic features and local neuronal tuning both contribute to auditory responsiveness. HVC auditory responsiveness was broadly distributed and nontopographic. Variance in auditory responsiveness was greater among than within HVC recording locations in both anesthetized and awake birds, in contrast to the global nature of auditory representation within zebra finch HVC. To assess the spatial consistency of auditory representation within HVC, we measured the repeatability with which ensembles of BOR songs were represented across the nucleus. Auditory response ranks to different songs were more consistent across recording locations in awake than in anesthetized animals. This spatial reliability of auditory responsiveness suggests that sound stimulus acoustic features contribute relatively more to auditory responsiveness in awake than in anesthetized animals.

State‐dependent hemispheric specialization in the songbird brain

Lateralization of brain functions is a widespread phenomenon in vertebrates. With the well-known lateralization in the processing of human speech and the parallels that exist between birdsong and language, songbirds are interesting for addressing such questions. In the present study, we investigated the central processing of communicative and artificial sounds in starlings, in an integrative part of the song system: the HVC. Neuronal responses to acoustic stimuli were systematically recorded in both hemispheres while the birds were awake, and then anesthetized, allowing quantitative comparisons of the responses obtained in each situation. The total proportion of responsive sites in the HVC of the left and right hemispheres of all birds revealed a significant predominance of the HVC of the right hemisphere when the birds were awake, whereas a high interindividual variability appeared when the birds were anesthetized. When neuronal responses as a whole were further examined, the responses to each type of stimulus appeared to be nonrandomly distributed over the different situations, and some specialization may appear. The results suggest a complex and state-dependent hemispheric specialization toward behaviorally relevant classes of stimuli. In awake birds, the HVC of the left hemisphere may be more involved in the processing of songs that are used in individual recognition at distance, whereas the HVC of the right hemisphere may deal with long and complex sequences of a song that is involved in short-distance communication, especially between males and females. With birds under anesthesia, this pattern is strongly modified.

Auditory responses in multiple sensorimotor song system nuclei are co-modulated by behavioral state.

Auditory responsiveness in nucleus HVC, a high-order sensorimotor area of the avian song system, is modulated by changes in behavioral state. Modulation is not observed in the primary thalamo-recipient auditory area Field L, the indirect source of auditory input to HVC. In this study, we show that auditory responsiveness in nucleus interfacialis (NIf), the immediate auditory afferent to HVC, is modulated by behavioral state. While auditory responsiveness is generally greater in NIf during wakefulness and in HVC during sedation, simultaneous recordings reveal a co-variation of auditory response magnitude. This co-variation is observed both in awake birds, where responses are spontaneously variable, and in sedated birds during manipulations of arousal levels. Auditory responses in NIf and HVC, which are selective for the bird's own song (BOS) during sedation, become predominantly unselective during wakefulness. This loss of selectivity is accompanied by a decrease in the similarity of NIf and HVC response patterns. To explore the role of NIf in shaping HVC auditory responses, we pharmacologically manipulated NIf while recording in HVC. Injection of the GABA(A) agonist muscimol into NIf eliminated most spontaneous activity and all auditory responses in the ipsilateral HVC, while injections of the GABA(A) antagonist bicuculline increased HVC auditory responsiveness and selectivity. These findings indicate that HVC is not the initial site of behavioral state-dependent modulation in the song system. Together with the suppression of HVC auditory responses by muscimol in NIf, these results suggest that NIf plays an important role in the flow of auditory information to HVC.

Offline learning and the role of autogenous speech: new suggestions from birdsong research

Vocal learning in humans is isolated in evolution, nevertheless the comparative approach can help give insight into mechanisms of speech. Birdsong and speech learning share similar computational problems and may share similar neural mechanisms. In birds, there is a well-known sensory representation of the individual’s own song. A similar representation is postulated for speech, and the implications are explored. Auditory responses to own song are observed in an anterior forebrain pathway that is implicated in song recognition. By analogy, speech perception may be referenced to an internal representation of own voice. This also predicts sex differences in speech perception. In awake birds, auditory responses are reduced or absent in motor pathways, which argues against a theory of perception by reference to production. Recent evidence demonstrates that auditory responses in birds are much stronger while animals sleep, and that during sleep there is replay of song motor patterns. If humans exhibit similar phenomena, then there should be circadian patterns in speech production, and possibly in speech perception. Humans should exhibit strong responses (e.g., ERP) to playback of speech during sleep when playback is matched to the perception of own speech. Sleep should be important for speech maintenance, and learning.

Typology of activity bouts and effect of fearfulness on behaviour in meat-type chickens

Increased physical exercise is known to prevent leg problems in meat-type chickens. Our aims were to study in detail the organisation of general and locomotor activity, to determine how physical exercise could be promoted and to investigate the effects of tonic immobility (TI) duration on spontaneous activity. Chicks were allocated to two groups according to their TI duration at 3 and 20 days (fearful birds, HF, n=11; non-fearful birds, LF, n=14). The behaviour patterns expressed by awake birds were recorded as ‘activity bouts’. Activity bouts were then classified into five classes according to the most long-lasting activity in the bout. The behaviour patterns were similar in both groups. The FORAGING class contained long-lasting bouts with high levels of locomotor activity which decreased considerably with age, when bouts of EATING and DRINKING classes became more and more numerous. Bouts in the PREENING and NONE classes contained low activity. In conclusion, the level of activity in home pens was poorly related to TI. Categorisation of bouts into five classes is valuable to understand the general organisation of activity in chickens. The FORAGING class included bouts with high levels of locomotion and it could thus be used as a general indicator of activity.

Singing-Related Neural Activity in a Dorsal Forebrain–Basal Ganglia Circuit of Adult Zebra Finches

The anterior forebrain pathway (AFP) of songbirds, a specialized dorsal forebrain-basal ganglia circuit, is crucial for song learning but has a less clear function in adults. We report here that neurons in two nuclei of the AFP, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and Area X, show marked changes in neurophysiological activity before and during singing in adult zebra finches. The presence of modulation before song output suggests that singing-related AFP activity originates, at least in part, in motor control nuclei. Some neurons in LMAN of awake birds also responded selectively to playback of the bird's own song, but neural activity during singing did not completely depend on auditory feedback in the short term, because neither the level nor the pattern of this activity was strongly affected by deafening. The singing-related activity of neurons in AFP nuclei of songbirds is consistent with a role of the AFP in adult singing or song maintenance, possibly related to the function of this circuit during initial song learning.

Gating of auditory responses in the vocal control system of awake songbirds.

Nucleus HVc of the avian song system is a forebrain structure critical in song production, perception and learning. Here we show that most HVc neurons that respond to auditory stimuli under anesthesia show no responses to the same stimulus in the awake, unrestrained bird. This suppression of auditory responses in awake birds does not occur in the forebrain field L complex, which is one of the auditory input stages for HVc. Gating of auditory input at the junction between the auditory and vocal control system may be essential for regulating auditory feedback signals necessary for song learning and maintenance.