Vernal growth of vocal control nucleus Area X, but not HVC, precedes gonadal recrudescence in wild black-capped chickadees (Poecile atricapillus).

In temperate-zone songbirds, the neuroanatomical changes which occur in advance of breeding, including the growth of nuclei of the vocal control system, are believed to occur downstream of gonadal recrudescence. However, evidence from wild birds is mixed. Here, we captured black-capped chickadees from the wild in early spring (March-April), summer (August-September), and winter (December-January); in addition to measuring the volumes of two vocal control nuclei (Area X and HVC), we also quantified two indicators of reproductive state (gonads and circulating gonadal steroids). Most birds captured in early spring had regressed gonads and low levels of circulating gonadal steroids, indicating these birds were not yet in full breeding condition. However, these early spring birds still had a significantly larger Area X than winter birds, while HVC did not differ in size across groups. Using data from a previously published seasonal study of black-capped chickadees (Phillmore et al., Developmental Neurobiology, 2015;75:203-216), we then compared Area X and HVC volumes from our early spring group to a breeding group of chickadees captured 3-4 weeks later in the spring. While Area X volume did not differ between the studies, breeding males in Phillmore et al. (2015) had a significantly larger HVC. Taken together, this suggests that the vernal growth of Area X occurs ahead of HVC in black-capped chickadees, and that the overall vernal changes in the vocal control system occur at least partially in advance of the breeding-associated upregulation of the hypothalamic-pituitary-gonadal axis.

Effect of feed-time duration on discrimination of vocalizations in a go/no-go operant paradigm.

Refining and modifying experimental procedures play a vital role in improving methodology while also reducing animal distress. In this study, we asked if an increase in feed time duration affects discrimination in an operant go/no-go task. Specifically, we used zebra finches' sexually dimorphic distance calls as acoustic stimuli to test whether there were any significant differences in performance on an operant discrimination task requiring zebra finches to classify calls according to the sex of the producer when a key experimental parameter, feed time duration, was increased from 1 s to 2 s. We found no differences in learning speed (trials to criterion) between birds that were given 1 s or 2 s of food access following a correct go response. Our results indicate doubling food access duration did not impact the speed of acquisition of distance call discrimination in zebra finches. These findings suggest that we can provide twice as much time for zebra finches to access food, potentially improving animal welfare, with no impact on experimental outcomes.

Comparing methodologies for classification of zebra finch distance calls.

Bioacoustic analysis has been used for a variety of purposes including classifying vocalizations for biodiversity monitoring and understanding mechanisms of cognitive processes. A wide range of statistical methods, including various automated methods, have been used to successfully classify vocalizations based on species, sex, geography, and individual. A comprehensive approach focusing on identifying acoustic features putatively involved in classification is required for the prediction of features necessary for discrimination in the real world. Here, we used several classification techniques, namely discriminant function analyses (DFAs), support vector machines (SVMs), and artificial neural networks (ANNs), for sex-based classification of zebra finch (Taeniopygia guttata) distance calls using acoustic features measured from spectrograms. We found that all three methods (DFAs, SVMs, and ANNs) correctly classified the calls to respective sex-based categories with high accuracy between 92 and 96%. Frequency modulation of ascending frequency, total duration, and end frequency of the distance call were the most predictive features underlying this classification in all of our models. Our results corroborate evidence of the importance of total call duration and frequency modulation in the classification of male and female distance calls. Moreover, we provide a methodological approach for bioacoustic classification problems using multiple statistical analyses.

Understanding hippocampal neural plasticity in captivity: Unique contributions of spatial specialists.

Neural plasticity in the hippocampus has been studied in a wide variety of model systems, including in avian species where the hippocampus underlies specialized spatial behaviours. Examples of such behaviours include navigating to a home roost over long distances by homing pigeons or returning to a potential nest site for egg deposit by brood parasites. The best studied example, however, is food storing in parids and the interaction between this behaviour and changes in hippocampus volume and neurogenesis. However, understanding the interaction between brain and behaviour necessitates research that includes studies with at least some form of captivity, which may itself affect hippocampal plasticity. Captivity might particularly affect spatial specialists where free-ranging movement on a large scale is especially important in daily, and seasonal, behaviours. This review examines how captivity might affect hippocampal plasticity in avian spatial specialists and specifically food-storing parids, and also considers how the effects of captivity may be mitigated by researchers studying hippocampal plasticity when the goal is understanding the relationship between behaviour and hippocampal change.

The puzzle of spontaneous alternation and inhibition of return: How they might fit together.

Two isolated spatial phenomena share a similar "been there; done that" effect on spatial behavior. Originally discovered in rodent learning experiments, spontaneous alternation is a tendency for the organism to visit a different arm in a T-maze on subsequent trials. Originally discovered in human studies of attention, inhibition of return is a tendency for the organism to orient away from a previously attended location. Whereas spontaneous alternation was identified by O'Keefe & Nadel as dependent on an intact hippocampus, inhibition of return is dependent on neural structures that participate in oculomotor control (the superior colliculus, parietal and frontal cortex). Despite the isolated literatures, each phenomenon has been assumed to reflect a basic novelty-seeking process, avoiding places previously visited or locations attended. In this commentary, we explore and compare the behavioral manifestations and neural underpinnings of these two phenomena, and suggest what is still needed to determine whether they operate in parallel or serial.

Operant discrimination of relative frequency ratios in black-capped chickadee song.

The two-note fee bee song of the black-capped chickadee (Poecile atricapillus) is sung at many different absolute frequencies, but the relative frequencies, or "pitch ratios", between the start and end of the fee note (glissando) and the fee and the bee notes (inter-note interval) are preserved with each pitch-shift. Ability to perceive these ratios and their relative salience varies with sex of the bird and setting: while both sexes appear to perceive changes in the inter-note interval, males appear to attend to the glissando in the field, and females appear to attend to both ratios. In this study, we compared directly whether male and female chickadees could discriminate between normal fee bee songs and songs that had one or both of the pitch ratios altered, and whether birds attended to one type of alteration over another. Both sexes learned to discriminate normal from altered songs; songs lacking an inter-note interval were more easily discriminated than songs with only the glissando removed. Females performed slightly better than males, including in the most difficult task with the stimuli lacking the glissando. Our study illustrates the value of using perceptual tasks to directly compare performance between the sexes and to demonstrate the difference between perception of and attention to acoustic features of vocal communication.

Vocal production and playback of altered song do not affect ZENK expression in black-capped chickadees (Poecile atricapillus).

The two-note fee bee song of the black-capped chickadee (Poecile atricapillus) is sung at many different absolute frequencies, but the relative frequencies between the start and end of the fee note (the glissando) and between the fee and the bee notes (the inter-note ratio) are preserved regardless of absolute frequency. If these relative frequencies are experimentally manipulated, birds exhibit reduced behavioural responses to playback of altered songs both in field studies and laboratory studies. Interestingly, males appear to be sensitive to alterations in the glissando, while females appear to be sensitive to alterations in both the glissando and the inter-note ratio. In this study, we sought to determine whether the behaviour of male and female chickadees corresponds to differences in zenk protein immunoreactivity (ZENK-ir) in auditory perceptual regions following playback of fee bee songs with typical and altered pitch ratios. Overall, there was a small but significant sex difference in ZENK-ir (females>males), but altering relative frequencies did not reduce ZENK-ir compared to typical song. Birds did vocalize less in response to playback of songs that lacked an inter-note interval, but amount of singing fee bee song, chick-a-dee calls, or gargles was not correlated with ZENK-ir in perceptual regions (caudomedial nidopallium, NCM and caudomedial mesopallium, CMM) or in HVC, which is part of the song system. Our results confirm that ZENK-ir in NCM and CMM is not involved in fine-grain perceptual discrimination, however it did not support the idea that increased vocalizing increases ZENK-ir in HVC.

Effects of sex and seasonality on the song control system and FoxP2 protein expression in black-capped chickadees (Poecile atricapillus).

Plasticity in behavior is mirrored by corresponding plasticity in the brain in many songbird species. In some species, song system nuclei (Phillmore et al. [2006]: J Neurobiol 66:1002-1010) are larger in birds in breeding condition than birds in nonbreeding condition, possibly due to increased vocal output in spring. FOXP2, a transcription factor associated with language expression and comprehension in humans and song learning in songbirds, also shows plasticity. FoxP2 expression in songbird Area X, a region important for sensorimotor integration, is related to developmental and adult vocal plasticity (Teramitsu et al. [2010]: J Neurosci 24:3152-3163, Chen et al. [2013], J Exp Biol 216:3682-3692). In this study, we examined whether sex and breeding condition affects both song control system volume (HVC, X) and FoxP2 protein expression in black-capped chickadees (Poecile atricapillus). HVC volume was larger in males in breeding condition than males in nonbreeding condition, but there were no sex differences. In contrast, Area X volume was larger in males than females, regardless of breeding condition, likely reflecting that male and female chickadees produce learned chick-a-dee calls year round, but output of the learned song increases in breeding males. FoxP2 protein levels did not differ between sexes or breeding condition when calculated as a ratio of labeled cells in Area X to labeled cells in the surrounding striato-pallium, however, absolute density of FoxP2 in both regions was higher in males than in females. This may indicate that chickadees maintain a level of FoxP2 necessary for plasticity year-round, but males have greater potential for plasticity compared to females.

Zenk expression in auditory regions changes with breeding condition in male Black-capped chickadees (Poecile atricapillus).

Black-capped chickadees (Poecile atricapillus) produce different vocalizations at different times of year: the fee-bee song is produced by males primarily in spring, whereas the chick-a-dee call is produced year-round but most frequently in the fall and winter. We wanted to determine if neural response to playback in auditory regions of the songbird brain varied with season in parallel to production. We captured adult male black-capped chickadees from the wild in either breeding condition or non-breeding condition and within 24-48 h of bringing them into the laboratory setting, played them recordings of either conspecific vocalizations (fee-bee songs or chick-a-dee calls), heterospecific vocalizations (Song Sparrow, Melospiza melodia, song), or silence. We then measured ZENK protein immunoreactivity (Zenk-ir) in caudomedial nidopallium (NCM) and caudomedial mesopallium (CMM), two regions important for perception of conspecific vocalizations. We found that, overall, non-breeding birds had greater Zenk-ir than breeding birds. In addition, we found that birds in non-breeding condition had significantly greater Zenk-ir to heterospecific song than birds in breeding condition, but this difference was not seen in birds that heard conspecific songs or calls. Finally, in NCMd chickadees had greater response to playback of conspecific vocalizations (when combining song and call groups) than playback of heterospecific vocalizations but only while in breeding condition. Our results qualify the claim that Zenk-ir is biased toward conspecific vocalizations, and indicate that specificity of neural response varies with season. Variation could be a result of increased production and perceptual demand in spring, or hormonal changes in breeding birds, possibly because chickadees display vocal plasticity in chick-a-dee calls in the fall, requiring a degree of neural plasticity across seasons.

Discrimination: from behaviour to brain.

Discrimination is a skill needed by many organisms for survival: decisions about food, shelter, and mate selection all require the ability to distinguish among stimuli. This article reviews the how and why of discrimination and how researchers may exploit this natural skill in the laboratory to learn more about what features of stimuli animals use to discriminate. The paper then discusses the possible neurophysiological basis of discrimination and proposes a model, based on one of stimulus-association put forth by Beninger and Gerdjikov (2004) [Beninger, R.J., Gerdjikov, T.V., 2004. The role of signaling molecules in reward-related incentive learning. Neurotox. Res., 6, 91-104], to account for the role of dopamine in how an animal learns to discriminate rewarded from non-rewarded stimuli.

Effects of learning on song preferences and Zenk expression in female songbirds.

Male songbirds learn to produce their songs, and females attend to these songs during mate choice. The evidence that female song preferences are learned early in life, however, is mixed. Here we review studies that have found effects of early song learning on adult song preferences, and those that have not. In at least some species, early experience with song can modify adult song preferences. Whether this learning needs to occur during an early sensitive phase, akin to male imitative vocal learning, or not remains an open question. Studies of the neural bases for female song preferences highlight activity (as measured by immediate-early gene induction) in regions of the auditory forebrain as often, but not always, being associated with song preferences. Immediate-early gene induction in these regions, however, is not specific to songs experienced early in life. On the whole, inherited factors, early experience, and adult experience all appear to play a role in shaping female songbirds preferences for male songs.

Annual cycle of the black-capped chickadee: seasonality of food-storing and the hippocampus.

Previous research presents a mixed picture of seasonal variation in the hippocampus of food-storing black-capped chickadees. One field study has shown an October peak in hippocampus volume, although laboratory studies conducted to determine whether photoperiod regulates this seasonal growth have failed to find changes in the size of the hippocampus. To resolve the discrepancy between field and lab reports we examined caching activity, hippocampal volume, and neurogenesis in adult male black-capped chickadees at four times over the annual cycle: October, January, April and July. We found that more birds stored food in October than at other times of year, but did not observe a significant change in the size of the hippocampus over the annual cycle. Telencephalon volume, however, was larger in October than in July. Hippocampal neuronal recruitment showed a significant peak in January, but there was no seasonal change in neuronal recruitment in the adjacent hyperpallium apicale. These results indicate that there might be seasonal variation in the recruitment of new neurons into the hippocampus of chickadees without overall seasonal change in hippocampal size.

Annual cycle of the black-capped chickadee: seasonality of singing rates and vocal-control brain regions.

Black-capped chickadees have a rich vocal repertoire including learned calls and the learned fee-bee song. However, the neural regions underlying these vocalizations, such as HVC, area X, and RA (robust nucleus of arcopallium), remain understudied. Here, we document seasonal changes in fee-bee song production and show a marked peak in singing rate during March through May. Despite this, we found only minimal seasonal plasticity in vocal control regions of the brain in males. There was no significant effect of time of year on the size of HVC, X, or RA in birds collected in January, April, July, and October. We then pooled birds into two groups, those with large testes (breeding condition) and those with small testes (nonbreeding), regardless of time of year. Breeding birds had slightly larger RA, but not HVC or X, than nonbreeding birds. Breeding birds had slightly larger HVC and RA, but not X, as a proportion of telencephalon volume than did nonbreeding birds. Birds collected in July had heavier brains than birds at other times of year, and had the greatest loss in brain mass during cryoprotection. The absence of any overall seasonal change in the vocal-control regions of chickadees likely results from a combination of individual differences in the timing of breeding phenology and demands on the vocal-control regions to produce learned calls year-round.

Immediate early gene expression following exposure to acoustic and visual components of courtship in zebra finches.

Sensory driven immediate early gene expression (IEG) has been a key tool to explore auditory perceptual areas in the avian brain. Most work on IEG expression in songbirds such as zebra finches has focused on playback of acoustic stimuli and its effect on auditory processing areas such as caudal medial mesopallium (CMM) caudal medial nidopallium (NCM). However, in a natural setting, the courtship displays of songbirds (including zebra finches) include visual as well as acoustic components. To determine whether the visual stimulus of a courting male modifies song-induced expression of the IEG ZENK in the auditory forebrain we exposed male and female zebra finches to acoustic (song) and visual (dancing) components of courtship. Birds were played digital movies with either combined audio and video, audio only, video only, or neither audio nor video (control). We found significantly increased levels of Zenk response in the auditory region CMM in the two treatment groups exposed to acoustic stimuli compared to the control group. The video only group had an intermediate response, suggesting potential effect of visual input on activity in these auditory brain regions. Finally, we unexpectedly found a lateralization of Zenk response that was independent of sex, brain region, or treatment condition, such that Zenk immunoreactivity was consistently higher in the left hemisphere than in the right and the majority of individual birds were left-hemisphere dominant.

An examination of avian hippocampal neurogenesis in relationship to photoperiod.

More neurons are recruited into the hippocampus of adult black-capped chickadees in the fall than at other times of year. To determine whether declining photoperiod and photorefractoriness are cues stimulating this neuronal recruitment; we examined three groups of chickadees receiving bromodeoxyuridine injections 10 days before being killed: one group was photostimulated with long days (15L), a second group was held on 15L until photorefractory and another group was held on 15L until photorefractory, then moved to short days (8L). Measures of neurogenesis revealed no significant differences in hippocampal neuronal recruitment. However, neuronal recruitment was increased in the hyperpallium apicale of photostimulated birds. These results suggest that declining photoperiod per se is not a major factor regulating enhanced neuronal recruitment to the hippocampus in the fall.

Effects of songs and calls on ZENK expression in the auditory telencephalon of field- and isolate-reared black capped chickadees.

We examined the effects of hearing two different conspecific vocalizations on expression of the immediate-early gene ZENK in the caudomedial neostriatum (NCM) and the caudomedial portion of the ventral hyperstriatum (cmHV) in male and female black-capped chickadees (Poecile atricapilla). Both the fee-bee song and the chick-a-dee call induced Zenk protein expression in NCM and in cmHV, however, patterns of expression to songs and calls varied across brain region. In the dorsal region of NCM, fee-bee songs induced more Zenk expression than chick-a-dee calls. In ventral NCM and cmHV, Zenk expression did not differ between songs and calls. We found that sex of the listener also affected Zenk expression: there was more robust ZENK response in males than in females. Finally, we compared field- and isolate-reared chickadees and found similar Zenk expression to fee-bee song in each group. These findings indicated that the type of conspecific vocalization, as well as the sex of the listener, appear to modulate IEG expression in the songbird ascending auditory pathway.

Open-ended categorization of chick-a-dee calls by black-capped chickadees (Poecile atricapilla).

The authors trained black-capped chickadees (Poecile atricapilla) in an operant discrimination with exemplars of black-capped and Carolina chick-a-dee calls, with the goal of determining whether the birds memorized the calls of conspecifics and heterospecifics or classified the calls by species. Black-capped calls served as both rewarded (S+) and unrewarded (S-) stimuli (the within-category discrimination), whereas Carolina chick-a-dee calls served as S-s (the between-category discrimination) in the black-capped chick-a-dee call S+ group. The Carolina call S+ group had Carolina calls as S+s and S-s (within-category) and black-capped calls as S-s (between-category). Both groups discriminated between call categories faster than within a call category. In 2 subsequent experiments, both S+ groups showed transfer to novel calls and propagation back to between-category calls. The results favor the hypothesis that the acoustically similar social calls of the 2 species constitute separate open-ended categories.

Discrimination of individual vocalizations by black-capped chickadees (Poecile atricapilla).

The auditory perceptual abilities of male black-capped chickadees (Poecile atricapilla) were examined using an operant go/no-go discrimination among 16 individual vocalizations recorded at 5 m. The birds learned to discriminate about equally well among eight male chickadee fee-bee songs and eight female zebra finch (Taeniopygia guttata) distance calls. These results do not indicate that chickadees have a species-specific advantage in individual recognition for conspecific over heterospecific vocalizations. We then transferred the chickadees to a discrimination of the same songs and calls rerecorded at a moderate distance. These results showed accurate transfer of discrimination from 16 vocalizations recorded at 5 m to novel versions of the same 16 songs and calls rerecorded at 25 m. That is, chickadees recognized individual songs and calls despite degradation produced by rerecording at 25 m. Identifying individual vocalizations despite their transformation by distance cues is here described as a biologically important example of perceptual constancy.