Early Gene ZENK Research Articles

Independent effects of song quality and experience with photostimulation on expression of the immediate, early gene ZENK (EGR‐1) in the auditory telencephalon of female European starlings

Age influences behavioral decisions such as reproductive timing and effort. In photoperiodic species, such age effects may be mediated, in part, by the individual's age-accrued experience with photostimulation. In female European starlings (Sturnus vulgaris) that do not differ in age, experimental manipulation of photostimulation experience (photoexperience) affects hypothalamic, pituitary, and gonadal activity associated with reproductive development. Does photoexperience also affect activity in forebrain regions involved in processing a social cue, the song of males, which can influence mate choice and reproductive timing in females? Female starlings prefer long songs over short songs in a mate-choice context, and, like that in other songbird species, their auditory telencephalon plays a major role in processing these signals. We manipulated the photoexperience of female starlings, photostimulated them, briefly exposed them to either long or short songs, and quantified the expression of the immediate-early gene ZENK (EGR-1) in the caudomedial nidopallium as a measure of activity in the auditory telencephalon. Using an information theoretic approach, we found higher ZENK immunoreactivity in females with prior photostimulation experience than in females experiencing photostimulation for the first time. We also found that long songs elicited greater ZENK immunoreactivity than short songs did. We did not find an effect of the interaction between photoexperience and song length, suggesting that photoexperience does not affect forebrain ZENK-responsiveness to song quality. Thus, photoexperience affects activity in an area of the forebrain that processes social signals, an effect that we hypothesize mediates, in part, the effects of age on reproductive decisions in photoperiodic songbirds.

Pair bonding in the female zebra finch: A potential role for the nucleus taeniae

Male and female zebra finches are highly social and form pair bonds typically associated with reproduction. To determine how these bonds affect a female's behavioral response to future interactions, females were paired with a male for 2 weeks, separated for 48 h, and then exposed to the same or a novel male. Control females were left unpaired and introduced to a novel male. Behaviors, as well as neural ZENK expression, were quantified. Females displayed higher levels of behaviors associated with pair bonds (clumping and preening) toward their mates than novel males, and display of these behaviors was correlated with expression of the immediate early gene ZENK in the nucleus taeniae of one group of females, those interacting with their mates. Behaviors of the stimulus males were largely unaffected, but those interacting with an unpaired female attempted to mount more than those interacting with their mates. The results indicate that the nucleus taeniae may play some role in the maintenance of pair bonds in this species. Additionally, females may provide some signal to influence elements of the behavior of males.

Female stimuli trigger gene expression in male pigeons

The immediate early gene zenk encodes transcriptional regulators and is believed to be one of the first steps in the formation of long-term memories associated with a given stimulus. In this study, we investigated the expression of ZENK protein product in brain regions that are likely involved in the processing of social stimuli, such as a potential mate. Male pigeons (Columba livia) were exposed to one of the following: (1) a live female pigeon, (2) a video playback of a female pigeon, (3) a video playback of a female cockatoo, or (4) an empty stimulus chamber. The brains were then processed immunohistochemically using an antibody against ZENK protein. When the stimulus was a live pigeon compared to all other stimuli, there were more numerous and more darkly-stained ZENK-positive cells in three “association” regions of the telencephalon: the hyperpallium apicale, the lateral portion of the intermediate nidopallium, and the medial nidopallium. There were no significant differences among the video-playback stimuli. The results suggest that the level of ZENK expression in these “association” regions was influenced not only by the visual presence of a potential mate, but by the presence of non-visual signals, the quality of the image, and the real-time interaction with the stimulus.

Independent effects of song quality and experience with photostimulation on expression of the immediate, early gene ZENK (EGR-1) in the auditory telencephalon of female European starlings

Independent effects of song quality and experience with photostimulation on expression of the immediate, early gene ZENK (EGR-1) in the auditory telencephalon of female European starlings

Sound-induced brain activity depends on stimulus subjective salience in female zebra finches

A key point in the study of acoustic perception is whether brain responsiveness to sounds depends on sound acoustic structure or sound perceptive salience. Songbirds provide some evidence that higher auditory regions are sensitive to the subjective importance of the stimulus for the subject. In the present paper, we compare brain activation elicited by mate versus non-mate calls in female zebra finches Taeniopygia guttata. Using playback, we examined the responsiveness of the caudal telencephalon by measuring the evoked expression of the immediate early gene ZENK. Our results show that mate calls elicit a significantly higher ZENK expression than the calls of another male in hippocampus, but not in auditory areas. Using a hierarchical ascending classification, we show that this difference in brain activation is not explained by call acoustic structure, but relies on call identity. Thus, these results give evidence for a genomic response to calls in hippocampus that differentiate between call identity, and not between call structure. Our study gives further insight into the implication of the hippocampus in sound recognition in female songbirds. To cite this article: C. Vignal et al., C. R. Biologies 331 (2008).

Estradiol modulates brainstem catecholaminergic cell groups and projections to the auditory forebrain in a female songbird

In songbirds, hearing conspecific song induces robust expression of the immediate early gene zenk in the auditory forebrain. This genomic response to song is well characterized in males and females of many species, and is highly selective for behaviorally relevant song. In white-throated sparrows, the selectivity of the zenk response requires breeding levels of estradiol; we previously showed that in non-breeding females with low levels of plasma estradiol, the zenk response to hearing song is no different than the response to hearing frequency-matched tones. Here, we investigated the role of brainstem catecholaminergic cells groups, which project to the forebrain, in estradiol-dependent selectivity. First, we hypothesized that estradiol treatment affects catecholaminergic innervation of the auditory forebrain as well as its possible sources in the brainstem. Immunohistochemical staining of tyrosine hydroxylase revealed that estradiol treatment significantly increased the density of catecholaminergic innervation of the auditory forebrain as well as the number of catecholaminergic cells in the locus coeruleus (A6) and the ventral tegmental area (A10), both of which are known to contain estrogen receptors in songbirds. Second, we hypothesized that during song perception, catecholaminergic cell groups of the brainstem actively participate in auditory selectivity via estrogen-dependent changes in activity. We found that hearing songs did not induce the expression of zenk, a putative marker of activity, within catecholaminergic neurons in any of the cell groups quantified. Together, our results suggest that estradiol induces changes in brainstem catecholaminergic cell groups that may play a neuromodulatory role in behavioral and auditory selectivity.

FnTm2, a novel brain‐specific transcript, is dynamically expressed in the song learning circuit of the zebra finch

Zebra finch males learn their song by imitation, a process influenced by social variables. The neural pathways for acquisition and production of learned song are known, but the cellular and molecular underpinnings are not. Here we describe a novel gene named "FnTm2" ("Phantom 2") that is predicted to encode a small protein (220 aa) with a single fibronectin type III domain and a single transmembrane domain. This gene shows great variability in its expression in song system neurons of the anterior forebrain pathway (AFP), a circuit that influences song discrimination and is necessary for normal song acquisition. AFP nuclei that express FnTm2 include the nucleus HVC (its Area X-projecting neurons), Area X, and LMAN (core and shell). FnTm2 expression does not correlate with singing behavior like the immediate early gene ZENK. It is expressed variably during sleeping hours and is not dependent on an intact song circuit. FnTm2's expression is sensitive to hearing, because in deafened birds its expression is substantially reduced in the core of LMAN. Furthermore, a comparison of FnTm2 expression between mice and zebra finches revealed a conserved pattern of expression in the "limbic system." We suggest that FnTm2 may be sensitive to affective and/or attentional states and thus may provide insights on how social variables influence the production and discrimination of learned vocalizations.

Neural orchestration of mate-choice plasticity in songbirds

Females may choose between prospective mates based on signals that reflect male quality. In songbirds, variation in male song can be such a signal. Females can adjust their choosiness according to the prevalence of preferred song, presumably because environmental variation can influence the availability of preferred phenotypes. Studies using induction of the immediate early gene ZENK (also called zif-268, egr-1, NGFI-A, and Krox-24) as an indicator of neuronal activity have revealed that the caudomedial mesopallium (CMM) and caudomedial nidopallium (NCM) of the female auditory forebrain are sensitive to song quality and respond proportionally to the strength of the female’s choosiness. In European starlings (Sturnus vulgaris), long songs are preferred by females and induce higher ZENK expression in the CMM and NCM compared to short songs. Experimentally reducing the perceived prevalence of long songs in the environment reduces this forebrain sensitivity to song length, raising the possibility that the songbird auditory forebrain integrates information about the social environment that enables the female to adjust her choosiness according to the availability of preferred phenotypes. In some songbird species, the noradrenergic system modulates mate-choice behavior, modulates song-induced auditory forebrain activity, and itself is modulated by the prevailing song environment, suggesting a possible role for this system in mediating the effects of social environment on forebrain and behavioral sensitivity to mate-choice cues. The presence of such a system would seem beneficial when the availability of preferred mates varies.

Rapid contact call-driven induction of NR2A and NR2B NMDA subunit mRNAs in the auditory thalamus of the budgerigar ( Melopsittacus undulatus)

In situ hybridization histochemistry was used to assess the effect of auditory stimulation with natural contact calls on expression of NR2A and NR2B NMDA subunit mRNAs in neurons of the thalamic auditory relay nucleus ovoidalis (Ov) of a vocal learning parrot species, the budgerigar ( Melopsittacus undulatus). The results showed that both the core (Ov) and ventromedial shell subdivisions (Ovm) of ovoidalis contained neurons expressing NR2A and NR2B mRNA in no-stimulation control subjects and that the distributions of neurons expressing these subunit mRNAs were very similar in both the core and shell of Ov. Contact call stimulation (5, 30 and 180 min) resulted in substantial increases of 50–60% in the number of neurons expressing NR2A and NR2B mRNAs in both the core and shell. Staining intensity, as measured by the optical density of stained somata approximately doubled compared to controls for both NR2 subunits in the 5 and 30 min conditions, but declined from 30 to 180 min. In all conditions, the density, but not staining intensity, of neurons expressing NR2B exceeded NR2A expression. Furthermore, the density of neurons expressing both subunit mRNAs in call stimulation conditions was greater in the core than in the shell despite the fact that total neuronal density was approximately 20% higher in the shell. Previous experiments have shown that call stimulation is more effective at inducing expression of the immediate early gene zenk in the Ov shell than core; however the present results do not indicate that either NR2A or NR2B mRNA expression mediates this effect since neither subunit exhibits greater expression in Ovm. Ca ++ release is needed for immediate early gene expression, however and, notably, Ovm contains large numbers of neurons containing CGRP, a peptide which has been shown to increase cytosolic Ca ++ levels.

A muscarinic cholinergic antagonist and a dopamine agonist rapidly increase ZENK mRNA expression in the form-deprived chicken retina

Increases in the expression of the immediate early gene ZENK in the retina, measured by changes in the levels of mRNA and protein immunoreactivity, are amongst the most rapid responses so far measured to conditions that decrease the rate of eye growth in chickens. Our aim was to determine whether atropine, a muscarinic cholinergic antagonist, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide, a dopamine agonist, which are known to block excessive eye growth, produce similar changes in ZENK expression. Form-deprivation resulted in significant down-regulation of the expression of retinal ZENK mRNA within 1 h of fitting the diffusers, whereas removal of the diffusers from the eyes of chickens that had developed form-deprivation myopia resulted in significant up-regulation of retinal ZENK expression within 1 h. When atropine (10 μL of 25 mM solution) and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (10 μL of a 10 mM solution) were injected intravitreally, just prior to fitting the diffusers, the down-regulation of retinal ZENK mRNA caused by form-deprivation was reversed. This resulted in levels of ZENK mRNA higher than in control or contralateral control eyes. The doses were chosen because they are known to block the excessive axial elongation induced by form-deprivation, without affecting the growth of control eyes. Neither agent had any effect on retinal ZENK expression within this time period when injected into control eyes. These results suggest that both muscarinic acetylcholine antagonists and dopamine agonists act early in the signal cascade controlling eye growth, possibly within the retina itself.

Evidence that norepinephrine influences responses to male courtship song and activity within song control regions and the ventromedial nucleus of the hypothalamus in female European starlings

Little is known about the neural regulation of female responses to male courtship displays. Female European starlings use attributes of male song to assess potential mates and can be highly motivated to approach nest boxes broadcasting male song. The catecholamine norepinephrine (NE) regulates attention and arousal and has recently been implicated in motivated/goal-directed behaviors, suggesting a role for NE in female responses to male song. In the present study, treating females with the noradrenergic neurotoxin N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) increased the number of times females were observed on or near a nest box broadcasting sexually motivated male song, suggesting an inhibitory role for norepinephrine in the regulation of female responses to male song. DSP-4 treatment reduced immunolabeling for the immediate early gene ZENK within area X, a region involved in song learning and possibly discrimination, and the ventromedial nucleus of the hypothalamus, a region involved in female sexual behavior. Furthermore, female responses to male song and numbers of ZENK-labeled cells within area X related positively in DSP-4-treated birds but not controls. Female responses to male song and the density of dopamine-β-hydroxylase in area X and another song nucleus LMAN related positively exclusively in DSP-4-treated birds. Similar trends were consistently observed for the ventromedial nucleus of the hypothalamus. These data suggest roles for area X, LMAN, and the ventromedial nucleus of the hypothalamus in female responses to male song and highlight norepinephrine as an important neuromodulator of this behavior.

Neuronal activation related to auditory memory in parrot brain

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.

Sexual dimorphism in song-induced ZENK expression in the medial striatum of juvenile zebra finches

In the brains of male zebra finches ( Taeniopygia guttata), the nuclei that direct song learning and production are larger than the corresponding regions in females, who do not sing. The dimorphism in Area X of the medial striatum (MSt), an area important for song learning, is even more dramatic in that it is identifiable in males but not females by Nissl stain. In the present study, conspecific song, but not other auditory stimuli, induced expression of the immediate early gene ZENK in the MSt surrounding but not within Area X in juvenile males (30 and 45 days post-hatch). ZENK immunoreactivity following conspecific songs was homogeneous throughout the MSt of females at the same ages. Little to no FOS immunoreactivity was observed in Area X or the rest of the MSt, and levels were not influenced by the type of auditory stimulus presented. Thus, the clear morphological difference in the lateral MSt (Area X) of males and females is mirrored by a specific functional one, and the data suggest a role for ZENK expression in the MSt outside of Area X in responding to relevant song stimuli.

Social context modulates behavioural and brain immediate early gene responses to sound in male songbird

Although it is well known that brain sensory information processing is a highly modulated phenomenon, how this brain function is shaped by experience and social context remains a question to explore. In this paper, we present the first attempt to investigate this problem using a songbird acoustic communication paradigm. Social context is well known to influence acoustic communicating behaviours in birds. The present paper investigates whether brain processing of auditory inputs can be modulated by this 'audience effect'. Given that call-based communication is known to be highly context-dependent, we focused on the response of male zebra finches (Taeniopygia guttata) to female calls. We tested to see if the current social context surrounding the hearing bird can modify a sound-induced immediate early gene (IEG) activation in the specific region of the caudomedial nidopallium (NCM), a songbird brain analogous to the superficial layers of the mammalian primary auditory cortex. Our results show that the expression of the sound-induced immediate early gene ZENK in the NCM is considerably enhanced when the hearing bird is in the presence of conspecifics, compared to when he is alone. This context-dependent increase of a sound-induced immediate early gene expression can be correlated with the differential behavioural response of males to the playback of the same acoustic stimulus as a function of social context.

FOS and ZENK responses in 45-day-old zebra finches vary with auditory stimulus and brain region, but not sex

Male zebra finches ( Taeniopygia guttata) begin to sing around 45 days posthatch (d45) and tune their songs to match learned templates. Females never develop song, but they use male conspecific vocalizations for mate choice. While auditory perception is critical for both sexes, the responses of the immediate early genes (IEGs) ZENK and FOS differ in auditory brain areas of d30 males and females. The present study examined expression of these IEGs in the caudomedial nidopallium (NCM), caudomedial mesopallium (CMM; formerly cHV), and the hippocampus (HP) in both sexes at d45 in response to conspecific, heterospecific, or no songs. Overall, zebra finch song presentations resulted in the highest density of ZENK and FOS cells in each region analyzed, but expression varied across brain areas. Contrary to d30 birds, the IEG response patterns did not differ between the sexes. ZENK-immunoreactivity was significantly increased following exposure to conspecific songs compared to no songs, and zebra finch song presentations produced more FOS-immunoreactive nuclei than both heterospecific and no songs. While the pattern was consistent, significant effects of stimulus type were seen only in the NCM when the brain regions were analyzed separately. Furthermore, levels of FOS- and ZENK-immunoreactive neurons were higher in the lateral than medial NCM in both sexes. Along with previous work from our lab and others, these data suggest that at d45 neuronal responses within perceptual regions are still maturing on some levels, but IEG expression has acquired a number of adult characteristics.

Neuronal activation related to auditory perception in the brain of a non-songbird, the ring dove

In songbirds, parrots, and hummingbirds, which need to learn their songs, exposure to conspecific song leads to increased expression of the immediate early gene (IEG) ZENK in a number of forebrain regions, including the caudomedial nidopallium (NCM) and the caudomedial mesopallium (CMM). Here we investigated the pattern of IEG expression in response to auditory stimulation in the brain of the ring dove (Streptopelia risoria), a non-songbird that does not need to learn its vocalizations. Ring dove males were exposed to conspecific vocalizations (coos), to heterospecific vocalizations (zebra finch song) or they were kept in silence. IEG expression was investigated by means of immunocytochemical analysis of the distribution of the ZENK protein product Zenk. In all three groups there was Zenk expression in several forebrain regions including the NCM and the CMM, similar to earlier findings in song-learning species. Quantitative analysis of the NCM, the CMM, and the hippocampus revealed significantly greater Zenk expression in birds exposed to conspecific vocalizations than in birds kept in silence, in the CMM only. These results show that there is substantial Zenk expression in the forebrain of a non-song-learning bird, comparable to that in avian song-learning taxa. These findings suggest that there is IEG expression specific to conspecific auditory stimulation in the CMM in both songbirds and non-songbirds.

Immediate early gene expression associated with induction of brooding behavior in Japanese quail

Certain species can be induced to foster infant or neonatal animals through the process of sensitization. We induced brooding behavior in adult Japanese quail through repeated exposure to foster chicks across five 20-min trials. Brooding behavior was characterized by a bird allowing chicks to approach and remain underneath its wings while assuming a distinctive stationary crouching posture, preening, and feather fluffing. Birds who did not show brooding behavior actively avoided chicks. Among the birds that brooded chicks, females brooded chicks for longer durations compared to males. Brooding females continued a regular daily egg laying pattern; males showed no significant changes in testosterone levels after exposure to chicks. In a second experiment, we measured expression of two immediate early gene (IEG) protein products, ZENK and Fos, to identify the brain regions activated or inhibited by brooding behavior in females. ZENK and Fos expression in brooding or sensitized females (SF) were compared with expression in nonmaternal females with chicks (NMF) and with females without chicks and with blocks as control objects (BL). There was a reduced density of ZENK-like immunoreactive (ZENK-lir) cells in the medial preoptic nucleus (POM) in NMF birds. In SF birds, the density of Fos-like immunoreactive (Fos-lir) cells was elevated in the bed nucleus stria terminalis, medial portion (BSTm), and ectostriatum (E). These experiments begin to define the neural circuitry underlying brooding behavior in Japanese quail, and establish a model for future studies of the neural mechanisms of avian parental behavior.

Differential expression of the immediate early genes FOS and ZENK following auditory stimulation in the juvenile male and female zebra finch

The brains of adult zebra finches ( Taeniopygia guttata) are tuned to the songs of conspecifics. In adult males, the caudomedial neostriatum (NCM) responds to zebra finch song, and in adult females the NCM and hippocampus (HP) are active following exposure to zebra finch song more than other auditory stimuli. The caudal hyperstriatum ventrale (cHV) in both sexes also responds to song, but in females not as selectively as the NCM and HP. While much is known about the adult perceptual circuit, less is known about its development. The present study exposed d30 male and female zebra finches to conspecific or heterospecific song, tones or silence, and examined the densities of FOS- and ZENK-immunoreactive nuclei in the NCM, cHV and HP. Significant interactions existed between sex and auditory stimulus condition for both immediate early genes, but they were in opposite directions. That is, across the three regions, FOS-immunoreactive neurons were increased in females following presentation of conspecific songs; males did not show an effect of stimulus exposure. In contrast, the density of ZENK-positive neurons was increased in males, but not females, following zebra finch song exposure. The FOS results demonstrate that some neural responses required for song perception may develop earlier in females than males; data on ZENK induction suggest the opposite. Overall, differences in juvenile immediate early gene activation suggest either that males and females employ divergent neural mechanisms for song perception or that the developmental trajectories leading to common neural responses differ.

Immediate early gene response to hearing song correlates with receptive behavior and depends on dialect in a female songbird.

Stimulus-induced expression of the immediate early gene ZENK (egr-1) in the songbird's auditory forebrain presumably depends on the behavioral significance of the stimulus. Few studies, however, have quantified both the ZENK and behavioral responses to a stimulus in the same individuals. We played conspecific male song of either hatch (local) or foreign dialect to female white-crowned sparrows (Zonotrichia leucophrys oriantha) and quantified both the auditory ZENK response and their behavioral response, which is known to depend on dialect. Birds hearing hatch dialect showed greater ZENK induction in the caudomedial hyperstriatum ventrale and the dorsal portion of the caudomedial neostriatum than birds hearing foreign dialect, supporting previous work showing a relationship between ZENK and salience of the stimulus. In the dorsal portion of the caudomedial neostriatum, ZENK induction was correlated with the amount of non-vocal courtship behavior; however, in the caudomedial hyperstriatum ventrale, ZENK induction was more highly correlated with the females' own vocal behavior and thus may have been partly self-induced. Some females sang and showed a male-like pattern of ZENK induction in their song systems. This study provides the first evidence that the ZENK response in a sensory area to a social stimulus is proportional to the animal's preference for the stimulus.

Sexual imprinting leads to lateralized and non-lateralized expression of the immediate early gene zenk in the zebra finch brain

Sexual imprinting is an early learning process by which young birds acquire the features of a potential sexual partner. The physiological basis of this learning process is an irreversible reduction of spine densities in two forebrain areas, the lateral neo- and hyperstriatum (LNH) and the medial neo- and hyperstriatum (MNH). The aim of the present study was to investigate whether the immediate early gene zenk, which has been shown frequently to play a role in plastic processes in the song system of zebra finches, may also be involved in the structural changes observed in these areas. The first exposure to a female after an isolation period enhances zenk expression in a variety of brain areas including LNH, MNH, and optic tectum. In contrast to earlier results, it was only the neostriatal part of LNH which showed an enhancement on first courtship, while exposure to a nestbox enhanced the label within the entire LNH area. Unexpectedly, the IEG expression was clearly lateralized in some layers of the optic tectum. Because lateralization occurred independent of the experimental condition, our study adds to recent results which also support the idea of a lateralized organization of the avian visual system.