Hemispheric dominance in HVC is experience-dependent in juvenile male zebra finches.

Juvenile male zebra finches (Taeniopygia guttata) must be exposed to an adult tutor during a sensitive period to develop normal adult song. The pre-motor nucleus HVC (acronym used as a proper name), plays a critical role in song learning and production (cf. Broca's area in humans). In the human brain, left-side hemispheric dominance in some language regions is positively correlated with proficiency in linguistic skills. However, it is unclear whether this pattern depends upon language learning, develops with normal maturation of the brain, or is the result of pre-existing functional asymmetries. In juvenile zebra finches, even though both left and right HVC contribute to song production, baseline molecular activity in HVC is left-dominant. To test if HVC exhibits hemispheric dominance prior to song learning, we raised juvenile males in isolation from adult song and measured neuronal activity in the left and right HVC upon first exposure to an auditory stimulus. Activity in the HVC was measured using the immediate early gene (IEG) zenk (acronym for zif-268, egr-1, NGFI-a, and krox-24) as a marker for neuronal activity. We found that neuronal activity in the HVC of juvenile male zebra finches is not lateralized when raised in the absence of adult song, while normally-reared juvenile birds are left-dominant. These findings show that there is no pre-existing asymmetry in the HVC prior to song exposure, suggesting that lateralization of the song system depends on learning through early exposure to adult song and subsequent song-imitation practice.

Effect of estradiol and predator cues on behavior and brain responses of captive female house sparrows (Passer domesticus).

The presence of predators can cause major changes in animal behavior, but how this interacts with hormonal state and brain activity is poorly understood. We gave female house sparrows (Passer domesticus) in post-molt condition an estradiol (n = 17) or empty implant (n = 16) for 1 week. Four weeks after implant removal, a time when female sparrows show large differences in neuronal activity to conspecific vs. heterospecific song, we exposed birds to either 30 min of conspecific song or predator calls, and video recorded their behavior. Females were then euthanized, and we examined neuronal activity using the expression of the immediate early gene (IEG) ZENK to identify how the acoustic stimuli affected neuronal activation. We predicted that if female sparrows with estradiol implants reduce neuronal activity in response to predator calls as they do to neutral tones and non-predatory heterospecifics, they would show less fear behavior and a decreased ZENK response in brain regions involved in auditory (e.g., caudomedial mesopallium) and threat perception functions (e.g., medial ventral arcopallium) compared to controls. Conversely, we predicted that if females maintain auditory and/or brain sensitivity towards predator calls, then female sparrows exposed to estradiol would not show any differences in ZENK response regardless of playback type. We found that female sparrows were less active during predator playbacks independent of hormone treatment and spent more time feeding during conspecific playback if they had previously been exposed to estradiol. We observed no effect of hormone or sound treatment on ZENK response in any region of interest. Our results suggest that female songbirds maintain vigilance towards predators even when in breeding condition.

Song system neuroanatomy, and immediate early gene expression in a finch species with extensive male and female song.

Birdsong is a relatively well-studied behavior, both due to its importance as a model for vocal production learning and as an intriguing complex social behavior. Until the last few decades, work on birdsong focused almost exclusively on males. However, it is now widely accepted that female song not only exists, but is fairly common throughout the oscine passerines. Despite this, and the large number of researchers who have begun exploring female song in the field, researchers in the lab have been slow to adopt model species with female song. Studying female song in the lab is critical for our understanding of sex-specific factors in the physiology controlling this fascinating behavior. Additionally, as a model for vocal production learning in humans, understanding the mechanistic and neuroendocrine control of female song is clearly important. In this study, we examined the red-cheeked cordon bleu (RCCB), an Estrildid finch species with extensive female song. Specifically, we found that there were no significant sex differences in circulating levels of testosterone and progesterone, nor in song production rate. There were no significant differences in cell densities in the three nuclei of the song control system we examined. Additionally, the volume of the robust nucleus of the arcopallium was not significantly different and we report the smallest sex difference in HVC yet published in a songbird. Finally, we demonstrated similar levels of motor driven immediate early gene expression in both males and females after song production.

Dim artificial light at night alters immediate early gene expression throughout the avian brain.

Artificial light at night (ALAN) is a pervasive pollutant that alters physiology and behavior. However, the underlying mechanisms triggering these alterations are unknown, as previous work shows that dim levels of ALAN may have a masking effect, bypassing the central clock. Light stimulates neuronal activity in numerous brain regions which could in turn activate downstream effectors regulating physiological response. In the present study, taking advantage of immediate early gene (IEG) expression as a proxy for neuronal activity, we determined the brain regions activated in response to ALAN. We exposed zebra finches to dim ALAN (1.5 lux) and analyzed 24 regions throughout the brain. We found that the overall expression of two different IEGs, cFos and ZENK, in birds exposed to ALAN were significantly different from birds inactive at night. Additionally, we found that ALAN-exposed birds had significantly different IEG expression from birds inactive at night and active during the day in several brain areas associated with vision, movement, learning and memory, pain processing, and hormone regulation. These results give insight into the mechanistic pathways responding to ALAN that underlie downstream, well-documented behavioral and physiological changes.

Experience-Dependent Egr1 Expression in the Hippocampus of Japanese Quail.

The hippocampal formation (HF) is a structure critical to navigation and many forms of memory. In mammals, the firing of place cells is widely regarded as the fundamental unit of HF information processing. Supporting homology between the avian and mammalian HF, context-specific patterns of Egr1 have been reported in birds that are comparable to those produced by place cell firing in mammals. Recent electrophysiological data, however, suggest that many avian species lack place cells, potentially undermining the correspondence between Egr1 and place cell-related firing in the avian brain. To clarify this, the current study examines Egr1 expression in Japanese quail under conditions known to elicit only weakly spatially modulated firing patterns and report robust context-dependent Egr1 expression. These data confirm that context-dependent expression of Egr1 is not dependent on precise place fields and provide insight into how these birds are able to perform complex spatial tasks despite lacking mammalian-like place cells.

Neuroendocrine and behavioral response to testosterone-induced female song in canaries (Serinus canaria).

Male song in songbirds is a critical and elaborate signal for mate attraction. In many species female listeners respond to male song both behaviorally and physiologically (e.g. copulation solicitation displays and production of the immediate early gene ZENK in auditory regions). It is becoming increasingly well known that females in many species also sing. However, in common lab species, such as canaries (Serinus canaria), female song is limited and has been primarily studied in the context of administering of exogenous testosterone (T) to increase song rate and length. In this study we addressed to what extent female canary songs are masculinized by the administration of exogenous T based on the behavioral and physiological responses of avian receivers. Specifically, are T induced female songs sufficient to elicit courtship behaviors and auditory ZENK expression in female listeners? We found that female songs after 3 weeks of exogenous T were significantly longer and more complex than female songs after 12 weeks of exogenous T. Additionally, we found that playback of 3-week T song significantly increased sexual response behaviors and the expression of ZENK in the auditory brain regions of female listeners. Finally, we conclude that extended periods of T do not necessarily maintain the masculinization of female song.

Novel objects alter immediate early gene expression globally for ZENK and regionally for c-Fos in neophobic and non-neophobic house sparrows.

Neophobia - an animal's reluctance to approach novel objects, try new foods, or explore unfamiliar environments - affects whether animals can adapt to new environments and exploit novel resources. However, despite its importance, the neurobiological mechanisms underlying this personality trait are poorly understood. In this study, we examined regional brain activity using the expression of two immediate early genes (IEGs), ZENK and c-Fos, in response to novel objects or control conditions in captive house sparrows (Passer domesticus, n = 22). When exposed to novel objects, we predicted that we would see differential IEG activity in brain regions involved in regulating stress and emotion (hippocampus, medial ventral arcopallium, lateral septum), reward and learning (striatum), and executive function (NCL) between neophobic and non-neophobic individuals. To classify birds by phenotype, we used behavior trials that tested willingness to approach a food dish in the presence of several different novel objects, habituation to one novel object, and willingness to try several different novel foods. We then exposed birds to a new novel object or a control condition and assessed protein expression of two IEGs in neophobic vs non-neophobic individuals after this final exposure. An analysis of average sparrow feeding times in the presence of novel objects showed a bimodal distribution of neophobia behavior. There was also high repeatability of individual novel object responses, and average responses to all three trial types (novel object, novel food, and habituation to a novel object) were significantly correlated. Although we saw no differences between neophobic and non-neophobic birds in IEG expression in response to novel objects in any of the 6 brain regions examined, there was a significant global decrease in ZENK expression and a significant increase in c-Fos expression in the medial ventral arcopallium and the caudal hippocampus in response to novel objects compared to controls, suggesting that these two regions may be important in novelty detection and threat perception. Additionally, there was no object effect in the rostral hippocampus, which supports the hypothesis that the avian hippocampus may have a rostrocaudal functional gradient similar to the septotemporal gradient in mammals.

Differential immediate early gene activity revealed by playback of male and female incomplete chick-a-dee calls.

In both humans and animals, biological differences between males and females has long been a topic of research. In songbirds, sexual dimorphisms can be seen in many species' plumage and heard in some species' songs. However, not all songbirds have such overt phenotypic sexual differences, leading to the question: are all vocalizations dimorphic? One of the most used and versatile vocalizations of the black-capped chickadee (Poecile atricapillus) is their namesake chick-a-dee call, that is produced by both sexes. This call is composed of four note types: A, B, C (together chick-a), and D (dee). Previous research has found that A notes contain information regarding the sex of the caller. However, chickadees do not categorize full chick-a-dee calls, or altered chick-a calls, based on the sex of the caller. Here we presented both male and female chickadees with altered chick-a calls (dee portion removed) of both sexes and measured the number of ZENK labeled cells in auditory nuclei. We found that calls produced by males and females had more ZENK labeled cells than the control condition; however, there was no significant difference in ZENK labeled cells between male and female listeners. Overall, our results suggest that black-capped chickadees do not perceive sexual differences in the production of chick-a calls.

Abcam Monoclonal Egr-1 ab133695 is an effective primary antibody replacement for Santa Cruz sc-189 polyclonal Egr-1 in songbirds.

BACKGROUND: The immediate early gene ZENK (acronym zif268, Egr-1, NGFI-A, krox24) has been used extensively in songbird research (Mello et al., 1992; Jarvis and Nottebohm, 1997), as well as other research areas. ZENK has been used in assessing learning and memory, measuring neural activation, and identifying the cellular and molecular substrates involved in the first stages of memory formation (Watson and Clements, 1980). Previous songbird research has found that neurons located within the areas involved in auditory perception, namely the caudomedial nidopallium and caudomedial mesopallium, exhibit high levels of ZENK protein expression in response to conspecific songs and calls (Mello and Ribeiro, 1998; Avey et al., 2011). NEW METHOD: In large part due to its neuronal-specific labeling of ZENK protein, Santa Cruz Egr-1 sc-189 has been widely accepted as the standard primary antibody in songbird research. However, Santa Cruz Biotechnology Egr-1 no longer specifically labels and has also discontinued production of Egr-1 sc-189. Thus, the current study is focused on analyzing the effectiveness of alternative primary antibodies: Abcam polyclonal c-Fos, Abcam monoclonal ab133695 Egr-1, and Proteintech polyclonal Egr-1. RESULTS: Abcam monoclonal Egr-1 was successful in specifically labeling ZENK positive cells in the songbird auditory nuclei. Abcam polyclonal c-Fos and Proteintech polyclonal Egr-1 were found to have non-specific labeling. COMPARISON WITH EXISTING METHODS: Abcam monoclonal Egr-1 ab133695 was found to produce differential and specific labeling in the targeted auditory nuclei similar to previous studies successfully using Santa Cruz polyclonal Egr-1 (i.e. Mello and Ribeiro, 1998). CONCLUSIONS: Abcam monoclonal Egr-1 effectively labels ZENK in the songbird auditory nuclei, making it a suitable primary antibody replacement for Santa Cruz polyclonal Egr-1.

Retrieval of contextual aversive memory and induction of Zenk expression in the hippocampus of pigeons.

The hippocampus has a fundamental role in many learning and memory processes, which include the formation and retrieval of context-fear associations, as evidenced by studies in rodents and birds. The present paper has analyzed contextual memory and Zenk expression in the hippocampus of the pigeon after fear conditioning. Pigeons were trained under four conditions: with 3 tone-shock associations (Paired), with shock and tone presented randomly (Unpaired), with exposure to the experimental chamber without stimulation (Control) and with only daily handling (Naive). The testing was conducted 24 h after training. All sessions were digitally recorded. The level of freezing expressed by the Paired and Unpaired groups differed significantly from that of the control group during both training and test sessions. Pigeons from the Paired group revealed a significantly greater density of Zenk positive nuclei in the ventromedial region of the hippocampus than did the Unpaired, Control and Naive groups. These data suggest that Zenk-mediated processes of synaptic plasticity in the hippocampus are induced during the retrieval of conditioned fear memory in the pigeon.

Neural activity associated with rhythmicity of song in juvenile male and female zebra finches.

Rhythm is an important aspect of both human speech and birdsong. Adult zebra finches show increased neural activity following exposure to arrhythmic compared to rhythmic song in regions similar to the mammalian auditory association cortex and amygdala. This pattern may indicate that birds are detecting errors in the arrhythmic song relative to their learned song template or to more general expectations of song structure. Here we exposed juvenile zebra finches to natural conspecific song (rhythmic) or song with altered inter-syllable intervals (arrhythmic) prior to or during template formation, or afterward as males are matching vocal production to a memorized song template (sensorimotor integration). Before template formation, expression of the immediate early gene ZENK was increased in the caudomedial nidopallium (NCM) of birds exposed to rhythmic relative to arrhythmic song. During template formation, ZENK expression was increased in the caudomedial mesopallium (CMM) of birds exposed to arrhythmic relative to rhythmic song. These results suggest that the youngest birds may be predisposed to respond to a more natural stimulus, and a template may be required for arrhythmic song to elicit increased neural activity. It also appears that functional development across the brain regions investigated continues to maturity.

ZENK expression in the auditory pathway of black-capped chickadees (Poecile atricapillus) as a function of D note number and duty cycle of chick-a-dee calls.

Black-capped chickadees (Poecile atricapillus) use their namesake chick-a-dee call for multiple functions, altering the features of the call depending on context. For example, duty cycle (the proportion of time filled by vocalizations) and fine structure traits (e.g., number of D notes) can encode contextual factors, such as predator size and food quality. Wilson and Mennill (2011) found that chickadees show stronger behavioral responses to playback of chick-a-dee calls with higher duty cycles, but not to the number of D notes. That is, independent of the number of D notes in a call, but dependent on the overall proportion of time filled with vocalization, birds responded more to higher duty cycle playback compared to lower duty cycle playback. Here we presented chickadees with chick-a-dee calls that contained either two D (referred to hereafter as 2 D) notes with a low duty cycle, 2 D notes with a high duty cycle, 10 D notes with a high duty cycle, or 2 D notes with a high duty cycle but played in reverse (a non-signaling control). We then measured ZENK expression in the auditory nuclei where perceptual discrimination is thought to occur. Based on the behavioral results of Wilson and Mennill, 2011, we predicted we would observe the highest ZENK expression in response to forward-playing calls with high duty cycles; we predicted we would observe no significant difference in ZENK expression between forward-playing high duty cycle playbacks (2 D or 10 D). We found no significant difference between forward-playing 2 D and 10 D high duty cycle playbacks. However, contrary to our predictions, we did not find any effects of altering the duty cycle or note number presented.

Simulated viral infection in early-life alters brain morphology, activity and behavior in zebra finches (Taeniopygia guttata).

Early-life immune challenges (ELIC) have long-term effects on adult behavior and brain development. ELIC studies on birds are still few, but they are epidemiologically crucial since birds are important hosts of many mosquito-borne viruses. In this study, we administered a viral infection mimicking agent, Polyinosinic: polycytidylic acid (Poly I:C), to nestling zebra finches on post-hatch day 14. When birds became sexually mature, their general activity (i.e., hopping, feeding behavior) and mosquito defense behaviors (i.e., hops, head movements, pecks, wing movements, foot movements, and scratches) were measured. Following behavioral trials, brains of male birds were collected for anatomical and histochemical analyses. Poly I:C challenge had sex-dependent effects on general activity and mosquito defense behaviors. When compared to control females, Poly I:C challenged females hopped and fed less often in their general activities, but hopped more often in the presence of mosquitoes. Poly I:C challenged males did not differ from control males in any behaviors. Brain analysis revealed that the nucleus taeniae of the amygdala (TnA) of Poly I:C challenged males were smaller in volume yet had more neurons expressing immediate-early gene proteins compared with controls, suggesting a more active TnA. These results suggest that immune challenges early in the life could have long-term effects on behaviors and brains of zebra finches, which may influence disease spread and fitness of individual birds.

Activation of immediate early genes by nicotine after chronic neonatal nicotine exposure in brain areas involved in stress and anxiety responses.

Maternal smoking has negative long-term consequences on affective behaviors, and in rodents, chronic neonatal nicotine exposure (CNN) results in increased anxiety. In rat pups, acute nicotine stimulation activates brain regions associated with stress and anxiety, but chronic nicotine exposure could desensitize of nicotinic acetylcholine receptors, the molecular target of nicotine. Here, we determined whether CNN affected neuronal activation by an acute nicotine challenge. Using in situ hybridization, we analyzed mRNA expression of the immediate-early genes (IEGs) c-Fos, Arc, Egr-1 and Npas4, which are markers for neuronal activation and implicated in synaptic plasticity. Following CNN (6 mg/kg/day) or control treatment from postnatal day (P)1 to P7, an acute i.p. nicotine (0.7 mg/kg) or saline injection (control) was administered on P8, and brains collected after 30 min. In drug-naive pups, acute nicotine stimulated IEGs expression specifically in brain areas associated with innate anxiety including the paraventricular hypothalamic nucleus, central nucleus of the amygdala (CeA), and locus coeruleus (LC). Following CNN, acute nicotine stimulated IEG expression in all three areas, but activation was significantly reduced in the LC (c-Fos, Egr-1, Npas4), and CeA (c-Fos). Notably, nicotine-induced Npas4 expression was greatly diminished in the LC, which may affect inhibitory synapse formation in noradrenergic neurons. Thus, after CNN, neurons located in areas associated with anxiety brain circuitry maintained responsiveness to nicotine, but tolerance differentially developed to nicotine. In the developing brain, repeated activation by nicotine of areas related to limbic pathways could alter circuit connectivity and increase responsiveness to stress and anxiety later in life.

ZENK expression following conspecific and heterospecific playback in the zebra finch auditory forebrain.

Zebra finches (Taeniopygia guttata) are sexually dimorphic songbirds, not only in appearance but also in vocal production: while males produce both calls and songs, females only produce calls. This dimorphism provides a means to contrast the auditory perception of vocalizations produced by songbird species of varying degrees of relatedness in a dimorphic species to that of a monomorphic species, species in which both males and females produce calls and songs (e.g., black-capped chickadees, Poecile atricapillus). In the current study, we examined neuronal expression after playback of acoustically similar hetero- and conspecific calls produced by species of differing phylogenetic relatedness to our subject species, zebra finch. We measured the immediate early gene (IEG) ZENK in two auditory areas of the forebrain (caudomedial mesopallium, CMM, and caudomedial nidopallium, NCM). We found no significant differences in ZENK expression in either male or female zebra finches regardless of playback condition. We also discuss comparisons between our results and the results of a previous study conducted by Avey et al. [1] on black-capped chickadees that used similar stimulus types. These results are consistent with the previous study which also found no significant differences in expression following playback of calls produced by various heterospecific species and conspecifics [1]. Our results suggest that, similar to black-capped chickadees, IEG expression in zebra finch CMM and NCM is tied to the acoustic similarity of vocalizations and not the phylogenetic relatedness of the species producing the vocalizations.

Vasoactive intestinal peptide, a promising agent for myopia?

AIM: To investigate the role of vasoactive intestinal peptide (VIP) in form-deprivation myopia (FDM). METHODS: FDM was created in three groups of eight chicks by placing a translucent diffuser on their right eyes. Intravitreal injections of saline and VIP were applied once a day into the occluded eyes of groups 2 and 3, respectively. Retinoscopy and axial length (AL) measurements were performed on the first and 8th days of diffuser wear. The retina mRNA levels of the VIP receptors and the ZENK protein in right eyes of the three groups and left eyes of the first group on day 8 were determined using real time polymerase chain reaction (PCR). RESULTS: The median final refraction (D) in right eyes were -13.75 (-16.00, -12.00), -11.50 (-12.50, -7.50), and -1.50 (-4.75, -0.75) in groups 1, 2, and 3, respectively (P<0.001). The median AL (mm) in right eyes were 10.65 (10.00, 11.10), 9.90 (9.70, 10.00), and 9.20 (9.15, 9.25) in groups 1, 2, and 3, respectively (P<0.001). The median delta-delta cycle threshold (CT) values for the VIP2 receptors were 1.07 (0.82, 1.43), 1.22 (0.98, 1.65), 0.29 (0.22, 0.45) in right eyes of groups 1, 2, and 3, and 1.18 (0.90, 1.37) in left eyes of group 1, respectively (P=0.001). The median delta-delta CT values for the ZENK protein were 1.07 (0.63, 5.03), 3.55 (2.20, 5.55), undetectable in right eyes of groups 1, 2, and 3 and 1.89 (0.21, 4.73) in left eyes of group 1, respectively (P=0.001). CONCLUSION: VIP has potential inhibitory effects in the development of FDM.

Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions in response to cognitive performance in Indian house crows exposed to abrupt light environment.

Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment.

Changes in dopamine and ZENK during suppression of myopia in chicks by intense illuminance.

High ambient illuminances have been found to slow the development of deprivation myopia in several animal models. Almost complete inhibition of myopia was observed in chickens when intermittent episodes of high illuminance were alternated with standard office illuminance (50% duty cycle, alternate periods of 1 min 15,000 lux and 1 min 500 lux, continued for 10 h per day), or when illuminances were increased to 40,000 lux. Since the mechanisms by which bright light suppresses myopia are poorly understood, we have studied the roles of two well-established signaling molecules in myopia, dopamine and ZENK, in the chicken. In line with previous studies, we found that retinal dopamine release (as reflected by vitreal DOPAC content) was severely reduced during development of deprivation myopia. We found that illuminance of 15,000 lux, provided by quartz-halogen lamps, partially rescued the drop in retinal dopamine release. The finding is in line with the assumption that dopamine is involved in the light-induced inhibition of myopia. No differences in vitreal DOPAC were found when bright light was provided continuously or with 1:1 min alternating exposure with 500 lux. As previously described by others, wearing diffusers suppressed the expression of ZENK protein in glucagonergic amacrine cells (GACs) but neither continuous nor 1:1 min alternating bright to normal light could rescue the suppression of ZENK in GACs. While it is well known that light increases global retinal ZENK mRNA and protein levels, the changes of ZENK protein induced specifically in GACs by diffuser wear appear independent of light levels.

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.

Annual life-history dependent seasonal differences in neural activity of the olfactory system between non-migratory and migratory songbirds.

Present study investigated seasonal plasticity in neural activity of the olfactory system, and assessed whether this was influenced by differences in seasonal life-history states (LHSs) between the non-migratory and migratory birds. Brains of non-migratory Indian weaver birds and migratory redheaded buntings were processed for ZENK immunohistochemistry, a marker of neuronal activation, at the times of equinoxes (March, September) and solstices (June, December), which correspond with the periods of different seasonal LHSs during the year. Immunoreactivity was quantified in brain regions comprising the olfactory system viz. olfactory bulb (OB), anterior olfactory nucleus (AON), prepiriform cortex (CPP), lateral olfactory tract (LOT) and olfactory cortex (piriform cortex, CPI; lateral olfactory cortex, LOC). In weaver birds, ZENK-like immunoreactive (ZENK-lir) cells were significantly higher in all the brain areas during post-breeding season (September) than during the other seasons; OBs had higher neuronal activity in the breeding season (June), however. A similar neural activity pattern but at enhanced levels was found in migratory buntings almost all the year. These results for the first time show LHS-associated differences in the seasonal plasticity of a sensory system between the non-migratory and migratory songbirds.