Zebra Finch Song System Research Articles

Developmental Changes in the Sexually Dimorphic Expression of Secretory Carrier Membrane Protein 1 and Its Co-Localisation with Androgen Receptor Protein in the Zebra Finch Song System

The song system of zebra finches differs dramatically between the sexes in terms of both structure and function. Only males sing and the brain regions regulating the learning and production of this behaviour are far more developed in males than females. Mechanisms regulating sexual differentiation likely include both direct genetic and hormonal processes. Expression of both mRNA and the protein product for secretory carrier membrane protein 1 (SCAMP1), a sex chromosome gene, are increased in the brains of juvenile males compared to females. Here we investigated developmental changes in SCAMP1 containing cells in song nuclei and co-localisation with androgen receptor (AR) protein from post-hatching day 25 through adulthood. Almost all SCAMP1 cells co-expressed AR and approximately half of the AR cells expressed SCAMP1 in the HVC and robust nucleus in the arcopallium (RA) of both sexes and in the Area X of males (which could not be clearly defined in females). In HVC and RA, more single and double-labelled cells were detected in males than females overall, and the sex differences increased as animals matured. The results suggest the potential for interaction of these two proteins in regulating development of brain and/or behaviour.

Food for song: expression of c-Fos and ZENK in the zebra finch song nuclei during food aversion learning.

BackgroundSpecialized neural pathways, the song system, are required for acquiring, producing, and perceiving learned avian vocalizations. Birds that do not learn to produce their vocalizations lack telencephalic song system components. It is not known whether the song system forebrain regions are exclusively evolved for song or whether they also process information not related to song that might reflect their ‘evolutionary history’.Methodology/Principal FindingsTo address this question we monitored the induction of two immediate-early genes (IEGs) c-Fos and ZENK in various regions of the song system in zebra finches (Taeniopygia guttata) in response to an aversive food learning paradigm; this involves the association of a food item with a noxious stimulus that affects the oropharyngeal-esophageal cavity and tongue, causing subsequent avoidance of that food item. The motor response results in beak and head movements but not vocalizations. IEGs have been extensively used to map neuro-molecular correlates of song motor production and auditory processing. As previously reported, neurons in two pallial vocal motor regions, HVC and RA, expressed IEGs after singing. Surprisingly, c-Fos was induced equivalently also after food aversion learning in the absence of singing. The density of c-Fos positive neurons was significantly higher than that of birds in control conditions. This was not the case in two other pallial song nuclei important for vocal plasticity, LMAN and Area X, although singing did induce IEGs in these structures, as reported previously.Conclusions/SignificanceOur results are consistent with the possibility that some of the song nuclei may participate in non-vocal learning and the populations of neurons involved in the two tasks show partial overlap. These findings underscore the previously advanced notion that the specialized forebrain pre-motor nuclei controlling song evolved from circuits involved in behaviors related to feeding.

Using Digital Images of the Zebra Finch Song System as a Tool to Teach Organizational Effects of Steroid Hormones: A Free Downloadable Module

Zebra finch song behavior is sexually dimorphic: males sing and females do not. The neural system underlying this behavior is sexually dimorphic, and this sex difference is easy to quantify. During development, the zebra finch song system can be altered by steroid hormones, specifically estradiol, which actually masculinizes it. Because of the ease of quantification and experimental manipulation, the zebra finch song system has great potential for use in undergraduate labs. Unfortunately, the underlying costs prohibit use of this system in undergraduate labs. Further, the time required to perform a developmental study renders such undertakings unrealistic within a single academic term. We have overcome these barriers by creating digital tools, including an image library of song nuclei from zebra finch brains. Students using this library replicate and extend a published experiment examining the dose of estradiol required to masculinize the female zebra finch brain. We have used this library for several terms, and students not only obtain significant experimental results but also make gains in understanding content, experimental controls, and inferential statistics (analysis of variance and post hoc tests). We have provided free access to these digital tools at the following website: http://mdcune.psych.ucla.edu/modules/birdsong.

The effects of estradiol on 17β-hydroxysteroid dehydrogenase type IV and androgen receptor expression in the developing zebra finch song system

Recent work in zebra finches suggests that genes and hormones may act together to masculinize the brain. This study tested the effects of exogenous estradiol (E2) on 17β-hydroxysteroid dehydrogenase type IV (HSD17B4) and the co-localization of HSD17B4 and androgen receptor (AR) mRNA. We asked three primary questions: First, how does post-hatching E2 treatment affect HSD17B4 mRNA expression in males and females? Second, is this gene expressed in the same cells as AR. Third, if so does E2 modulate co-expression? Female finches implanted with 50 μg of E2 on the third day post-hatching showed a significant increase in the density of cells expressing HSD17B4 and AR in HVC at day 25. Co-localization of AR cells that also expressed HSD17B4 was high across groups (> 81%). We found significant sex differences in co-localization in both the HVC and Area X of control animals, with males showing a higher percentage of cells expressing AR mRNA that also expressed HSD17B4 in comparison to females. However, although E2 treatments significantly increased the number of cells expressing HSD17B4 mRNA and AR mRNA in the HVC of females, the percentage of HSD17B4 cells co-expressing AR was reduced in HVC and Area X in E2-treated animals. These results lend support to the hypothesis that genes and hormones may act in concert to modulate the sexually differentiation of the zebra finch song system. Further, the data suggest that a single hormonal mechanism cannot mimic the complex development of male singing behavior and associated song nuclei.

Small molecule analysis and imaging of fatty acids in the zebra finch song system using time-of-flight-secondary ion mass spectrometry.

Fatty acids are central to brain metabolism and signaling, but their distributions within complex brain circuits have been difficult to study. Here we applied an emerging technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS), to image specific fatty acids in a favorable model system for chemical analyses of brain circuits, the zebra finch (Taeniopygia guttata). The zebra finch, a songbird, produces complex learned vocalizations under the control of an interconnected set of discrete, dedicated brain nuclei 'song nuclei'. Using ToF-SIMS, the major song nuclei were visualized by virtue of differences in their content of essential and non-essential fatty acids. Essential fatty acids (arachidonic acid and docosahexaenoic acid) showed distinctive distributions across the song nuclei, and the 18-carbon fatty acids stearate and oleate discriminated the different core and shell subregions of the lateral magnocellular nucleus of the anterior nidopallium. Principal component analysis of the spectral data set provided further evidence of chemical distinctions between the song nuclei. By analyzing the robust nucleus of the arcopallium at three different ages during juvenile song learning, we obtain the first direct evidence of changes in lipid content that correlate with progression of song learning. The results demonstrate the value of ToF-SIMS to study lipids in a favorable model system for probing the function of lipids in brain organization, development and function.

Sex- and age-related differences in ribosomal proteins L17 and L37, as well as androgen receptor protein, in the song control system of zebra finches

The zebra finch song system is sexually dimorphic—only males sing, and the morphology of forebrain regions controlling the learning and production of this song is greatly enhanced in males compared to females. Masculinization appears to involve effects of steroid hormones as well as other factors, perhaps including the expression of sex chromosome genes (males: ZZ, females: ZW). The present study investigated three proteins—two encoded by Z-linked genes, ribosomal proteins L17 and L37 (RPL17 and RPL37), including their co-localization with androgen receptor (AR), from post-hatching day 25 to adulthood. Extensive co-expression of AR with the ribosomal proteins was detected in the three song nuclei investigated (HVC, robust nucleus of the arcopallium (RA), and Area X) across these ages. In general, more cells expressed each of these proteins in males compared to females, and the sex differences increased as animals matured. Specific patterns differed across regions and between RPL17 and RPL37, which suggest potential roles of one or both of these proteins in the incorporation and/or differentiation of song system cells.

Co-localization of Sorting Nexin 2 and androgen receptor in the song system of juvenile zebra finches

Mechanisms regulating sexual differentiation of the zebra finch song system appear to include both genetic and hormonal factors. Sorting Nexin 2 (SNX2), which is involved in trafficking proteins between cellular membranes, and androgen receptor (AR) mRNA are both increased in song control nuclei of juvenile males compared to females. Here, in situ hybridization for SNX2 and immunohistochemistry for AR were used to evaluate these sexual dimorphisms in more detail. Estimates of the total number of HVC cells expressing SNX2 and AR, individually as well as together, were greater in 25-day-old males compared to females. The densities of these types of cells were generally also increased in males compared to females in HVC and Area X (or the equivalent portion of the medial striatum in females). On average, more than half of the AR+ cells co-expressed SNX2 in both brain regions. The potential, therefore, exists for both AR and SNX2 to be involved in masculinization of these two brain regions. One possibility is that they, either separately or in conjunction, enhance the action of trophic factors within the brain.

Neurocalcin protein labeling reveals a dimorphism within the developing zebra finch brain

The exact mechanism(s) responsible for sexual differentiation of the zebra finch song system remains unknown but likely involve a combination of hormonal and genetic factors. One product that may play a role is the calcium binding protein, neurocalcin. A previous study indicated that neurocalcin mRNA was widely distributed throughout the zebra finch telencephalon, overlapping with song control regions. Because it was not clear how much of that expression was functionally relevant, we labeled tissue with a specific antibody. At the three age ranges that were examined, there were region-specific fluctuations in the number of neurocalcin immunoreactive cells. During post-hatching developmental ages (P) 18–23, quantitative analyses indicated that females had significantly more neurons containing neurocalcin protein in HVC and RA than males. This difference was not detected at P10 or in adults. Labeling in LMAN did not reveal a sex difference at any of the ages. In other areas of the brain, semi-quantitative analyses of the relative number of cells immunopositive for neurocalcin varied across specific regions, but with the possible exception of the lateral striatum, no obvious sex differences were apparent. Taken together, these data are consistent with the idea that neurocalcin may be important for regulating sexual dimorphisms within the neural song system at a specific developmental period. In addition, because of variations in events related to post-transcriptional modification, a thorough study of a gene's role in development and/or behavior should include investigations of its transcription as well as translation.

The selective estrogen receptor-alpha coactivator, RPL7, and sexual differentiation of the songbird brain

The brain and behavior of the Australian zebra finch (Taeniopygia guttata) are sexually dimorphic. Only males sing courtship songs and the regions of the brain involved in the learning and production of song are significantly larger in males than females. Therefore the zebra finch serves as an excellent model for studying the mechanisms that influence brain sexual differentiation, and the majority of past research on this system has focused on the actions of steroid hormones in the development of these sex differences. Coregulators, such as coactivators and corepressors, are proteins and RNA activators that work by enhancing or depressing the transcriptional activity of the nuclear steroid receptor with which they associate, and thereby modulating the development of sex-specific brain morphologies and behaviors. The actions of these proteins may help elucidate the hormonal mechanisms that underlie song nuclei development. Research described in this review focus on the role of estrogen receptor coactivators in the avian brain; more specifically we will focus on the role of RPL7 (ribosomal protein L7; also known as L7/SPA) on sexual differentiation of the zebra finch song system. Collectively, these studies provide information about the role of steroid receptor coactivators on development of the zebra finch song system as well as on sexual differentiation of brain.

Effects of estradiol on incorporation of new cells in the developing zebra finch song system: Potential relationship to expression of ribosomal proteins L17 and L37

Mechanisms regulating masculinization of the zebra finch song system are unclear; both estradiol and sex-specific genes may be important. This study was designed to investigate relationships between estrogen and ribosomal proteins (RPL17 and RPL37; sex-linked genes) that exhibit greater expression in song control nuclei in juvenile males than females. Four studies on zebra finches were conducted using bromodeoxyuridine (BrdU) injections on posthatching days 6-10 with immunohistochemistry for the ribosomal proteins and the neuronal marker HuC/D at day 25. Volumes of brain regions were also assessed in Nissl-stained tissue. Most BrdU+ cells expressed RPL17 and RPL37. The density and percentage of cells co-expressing BrdU and HuC/D was greatest in Area X. The density of BrdU+ cells in Area X (or its equivalent) and the percentage of these cells that were neurons were greater in males than females. In RA and HVC, total BrdU+ cells were increased in males. A variety of effects of estradiol were also detected, including inducing an Area X in females with a masculine total number of BrdU+ cells, and increasing the volume and percentage of new neurons in the HVC of females. The same manipulation in males decreased the density of BrdU+ cells in Area X, total number of BrdU+ cells in RA, and density of new neurons in HVC and RA. These data are consistent with the idea that RPL17, RPL37, and estradiol might all influence sexual differentiation, perhaps with the hormone and proteins interacting, such that an appropriate balance is required for normal development.

Sexual differentiation of the zebra finch song system: potential roles for sex chromosome genes

BackgroundRecent evidence suggests that some sex differences in brain and behavior might result from direct genetic effects, and not solely the result of the organizational effects of steroid hormones. The present study examined the potential role for sex-biased gene expression during development of sexually dimorphic singing behavior and associated song nuclei in juvenile zebra finches.ResultsA microarray screen revealed more than 2400 putative genes (with a false discovery rate less than 0.05) exhibiting sex differences in the telencephalon of developing zebra finches. Increased expression in males was confirmed in 12 of 20 by qPCR using cDNA from the whole telencephalon; all of these appeared to be located on the Z sex chromosome. Six of the genes also showed increased expression in one or more of the song control nuclei of males at post-hatching day 25. Although the function of half of the genes is presently unknown, we have identified three as: 17-beta-hydroxysteroid dehydrogenase type IV, methylcrotonyl-CoA carboxylase, and sorting nexin 2.ConclusionThe data suggest potential influences of these genes in song learning and/or masculinization of song system morphology, both of which are occurring at this developmental stage.

Expression of the GABA A receptor γ4-subunit gene in discrete nuclei within the zebra finch song system

The acquisition, production and maintenance of song by oscine birds is a form of audition-dependent learning that, in many ways, resembles the process by which humans learn to speak. In songbirds, the generation of structured song is determined by the activity of two interconnected neuronal pathways (the anterior forebrain pathway and the vocal motor pathway), each of which contains a number of discrete nuclei that together form the song system. It is becoming increasingly evident that inhibitory GABAergic mechanisms are indispensable in counterbalancing the excitatory actions of glutamate and, thus, likely shape the neuronal firing patterns of neurons within this network. Furthermore, there is compelling evidence for the involvement of GABA A receptors, although the molecular composition of these has, to date, remained elusive. Here we describe the isolation of a complementary DNA for the zebra finch GABA A receptor γ4 subunit, and map the expression pattern of the corresponding gene within the zebra finch ( Taeniopygia guttata) brain. Our findings show, remarkably, that the γ4-subunit transcript is highly enriched in the major nuclei of the song system, including the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the medial magnocellular nucleus of the anterior nidopallium (MMAN), Area X, the robust nucleus of the arcopallium (RA) and the HVC (used as the proper name), as well as Field L, which innervates the area surrounding HVC. In summary, we have demonstrated the presence of the mRNA for the γ4 subunit of the GABA A receptor, the major inhibitory receptor in brain, in most of the nuclei of the two neural circuits that mediate song production in the zebra finch. This not only marks the beginning of the characterization of the GABA A receptor subtype(s) that mediates the actions of GABA in the song system but it also provides a robust molecular marker with which to distinguish song system-specific brain structures.

A dose–response study of estradiol's effects on the developing zebra finch song system

To gauge the sensitivity of the female zebra finch song system to estradiol (E2), we used subcutaneous implants to administer various doses of E2 to hatchling female zebra finches. Four different doses of E2 were administered: 50, 15, 5 and 0-μg via subcutaneous silicon “ropes” at hatching, and the brains were examined in adulthood. Further, we examined whether masculinization was all-or-none once a threshold was reached or if the morphology of the song system would show a graded response to the various doses of E2. Finally, we asked if the various dependent measures – volume of song nuclei, neuron size, and neuron number – would show differential sensitivity to E2. Fifteen micrograms was sufficient to masculinize many aspects of the song system and was often as effective as 50-μg, causing a dramatic difference relative to the 0-μg group. Different aspects of the song system seemed differentially sensitive to the effects of E2: volumes of song control nuclei, the size of RA neurons, and the number of HVC neurons were significantly masculinized by 15-μg E2, but the number of RA neurons and HVC and lMAN soma sizes required 50-μg. The results suggest that several developmental processes are influenced by E2, possibly because of multiple sites of action or multiple processes that respond to E2.

Post-hatch oral estrogen exposure reduces oviduct and egg mass and alters nest-building behavior in adult zebra finches ( Taeniopygia guttata)

In addition to well recognized effects on the zebra finch song system, we previously showed that post-hatch oral estrogen and xenoestrogen exposure disrupts reproduction by increasing eggshell breakage in females and decreasing fertility in males. Here we show that post-hatch exposure to estradiol benzoate (by oral gavage on days 5 to 11) at a 100 nmol EB per g body mass dose (EB100) also reduces adult oviduct mass. Further, EB100 and doses two orders of magnitude lower (EB10, EB1) reduce egg mass and length. Similar to the induction of song-control nuclei in females, dosing with EB10 and EB100 increased and masculinized another highly differentiated behavior: nest-building. Zebra finches orally exposed as chicks were observed during reproductive trials in communal breeding cages for 4 or 6weeks duration. EB100 males and females and EB10 males showed increased nest-building behaviors. Further, EB10 and EB100 birds had larger nests than canola oil-treated controls, and EB100 birds had faster rates of nest-building than controls, while EB1 birds had significantly slower rates of nest-building than controls. Additionally, EB100 males and females also showed an increased preference for a coarser male-typical nest-building material (jute) over a finer, female-typical material (wool), suggesting a masculinization of nest-building behavior at the higher doses. The change in zebra finch nest-building behavior induced by early EB exposure suggests that nest size and quality, in addition to egg mass and length, may provide new endpoints for assessing avian exposure to xeno- and phyto-estrogens in wild birds.

Effects of long-term flutamide treatment during development in zebra finches

The molecular mechanisms responsible for the sexual differentiation of the zebra finch song system remain mysterious. Androgen receptors are expressed in a sexually dimorphic fashion in the zebra finch song system: males have more cells expressing androgen receptors, and this sex difference appears very early in development (day 9 posthatch). Estrogen administration to hatchling females up-regulates androgen receptor expression in their song system and profoundly masculinizes their song system's morphology. Co-administering flutamide, an androgen receptor blocker, with estrogen impedes estrogen's masculinizing effects on the song system, suggesting that androgens are required for masculine development. Accordingly, to investigate further the role of androgens in the sexual differentiation of the zebra finch song system, we sought to block androgen activity in males by administering large, sustained doses of flutamide from just before androgen receptors are expressed in the song system (day 7) through to the day of sacrifice (days 61–63). Flutamide profoundly reduced the size of the testes, demonstrating that this drug and mode of administration could have a large impact on tissues. In contrast, flutamide had only a minor impact on the song system: the number of RA neurons was slightly reduced, and the corrected HVC volume showed a trend toward demasculinization. Other brain measures (uncorrected HVC, and corrected and uncorrected volumes of Area X, lMAN, RA, and Rotundus; neuron size in lMAN, HVC, and RA; and number of HVC and LMAN neurons) were not significantly affected. The present results do not support an important role for androgen in masculinizing the song circuit after posthatch day 7.

Sexually dimorphic SCAMP1 expression in the forebrain motor pathway for song production of juvenile zebra finches

Mechanisms regulating sexual differentiation of the zebra finch song system are not well understood. The present study was designed to more fully characterize secretory carrier membrane protein 1 (SCAMP1), which was identified in a cDNA microarray screen as showing increased expression in the forebrains of developing male compared with female zebra finches. We completed the sequence of the open reading frame and used in situ hybridization to compare mRNA in song control regions of juvenile (25-day-old) individuals. Expression was significantly greater in the HVC (used as a proper name) and robust nucleus of the arcopallium (RA) in males than in females. Immunohistochemistry revealed that SCAMP1 protein is also expressed in these two brain regions, and qualitatively appears greater in males. Western analysis confirmed that the protein is increased in the telencephalon of males when compared with females at 25 days of age. These results are consistent with the idea that SCAMP1 is involved in masculinization of these brain areas, perhaps facilitating the survival of cells within them.

Sexually dimorphic expression of the genes encoding ribosomal proteins L17 and L37 in the song control nuclei of juvenile zebra finches

Studies evaluating the role of steroid hormones in sexual differentiation of the zebra finch song system have produced complicated and at times paradoxical results, and indicate that additional factors may be critical. Therefore, in a previous study we initiated a screen for differential gene expression in the telencephalon of developing male and female zebra finches. The use of cDNA microarrays and real-time quantitative PCR revealed increased expression of the genes encoding ribosomal proteins L17 and L37 (RPL17 and RPL37) in the male forebrain as a whole. Preliminary in situ hybridization data then indicated enhanced expression of both these genes in song control regions. Two experiments in the present study quantified the mRNA expression. The first utilized 25-day-old male and female zebra finches. The second compared a separate set of juveniles to adults of both sexes to both re-confirm enhanced expression in juvenile males and to determine whether it is limited to developing animals. In Experiment 1, males exhibited increased expression of both RPL17 and RPL37 compared to females in Area X, the robust nucleus of the arcopallium (RA), and the ventral ventricular zone (VVZ), which may provide neurons to Area X. Experiment 2 replicated the sexually dimorphic expression of these genes at post-hatching day 25, and documented that the sex differences are eliminated or greatly reduced in adults. The results are consistent with the idea that these ribosomal proteins may influence sexual differentiation of Area X and RA, potentially regulating the genesis and/or survival of neurons.

Widespread Capacity for Steroid Synthesis in the Avian Brain and Song System

Steroids exert powerful effects on the brains and behavior of many species, but measures and manipulations of endocrine physiology in songbirds often reveal unexplained connections between steroids and the brain. The zebra finch song system, a sensorimotor neural circuit sensitive to steroids throughout life, organizes and functions largely in apparent independence from gonadally derived steroids. We tested the hypothesis that the zebra finch brain has the capacity for de novo steroidogenesis and that neurally synthesized steroids, neurosteroids, may impact the song system. Using multiple techniques, we demonstrate that the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A1), and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase, the first three factors in the steroidogenic pathway, are expressed in both developing and adult zebra finch brain. Detailed expression mapping at posthatch d 20 (P20) and adult reveals widespread area-specific expression and coexpression patterns for steroidogenic acute regulatory protein, CYP11A1, and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase, which suggest neurosteroids may modulate multiple brain functions, including sensory and motor systems. Notably, whereas expression of other steroidogenic genes such as aromatase has been essentially absent from the song system, each of the major song nuclei express at least a subset of steroidogenic genes described here, establishing the song system as a potential steroidogenic circuit.

Enhanced gene expression in the forebrain of hatchling and juvenile male zebra finches

The molecular mechanisms regulating sexual differentiation of the brain are largely unknown, although progress is being made, particularly in some mammalian systems. To uncover more of the key factors, a screen was conducted for genes involved in sexually dimorphic development of the neural song system in zebra finches. cDNA microarrays were initially used to compare gene expression in the telencephalons of hatchling and juvenile males and females. Then, real-time quantitative polymerase chain reaction (PCR) was employed to confirm sex differences, and the brain regions expressing the cDNAs of interest were localized using in situ hybridization. Several genes, including those likely to encode two ribosomal proteins (RPL17 and RPL37), SCAMP1, ZNF216, and a COBW-domain containing protein, showed enhanced expression in the telencephalon of males compared to females. In several cases, expression in the song control nuclei specifically was detected only in males. Interestingly, the sequences of some of these cDNAs shared substantial homology with regions of the chicken Z chromosome (male birds are ZZ, females ZW). Thus, we have identified genes likely to be involved in masculinization of the structure and/or function of the song circuit, some of which could be initial triggers for the sexual differentiation process.

Nicotine-mediated plasticity in robust nucleus of the archistriatum of the adult zebra finch

Activation of neuronal nicotinic acetylcholine receptors (nAChRs) modulates the induction of long-term potentiation (LTP), a possible cellular mechanism for learning. This study was undertaken to determine the effects of activation of nAChRs by nicotine on long-term plasticity in the songbird zebra finch, which is a valuable model to study synaptic plasticity and its implications to behavioral learning. Electrophysiological recordings in the robust nucleus of the archistriatum (RA) in adult zebra finch brain slices reveal that tetanic stimulation alone does not produce LTP. However, LTP is induced by such stimulation in the presence of nicotine. The nicotine-mediated LTP is blocked by dihydro-β-erythroidine (DHβE, 1 μM), an antagonist having a greater effect against nAChRs containing the alpha 4 subunit. In the presence of methyllcaconitine (MLA, 10 nM), an antagonist of nAChRs containing the alpha 7 subunit, a long-term depression (LTD) is unmasked, implicating a bi-directional type of plasticity in the zebra finch RA, which is modulated by differential activation of nAChR subtypes. Intracellular recordings from single neurons show a depression of the afterhyperpolarization (AHP) and an increase in frequency of evoked and spontaneous action potentials in the presence of nicotine. These results suggest that nicotinic cholinergic mechanisms may play a critical role in synaptic plasticity in the zebra finch song system and thereby influence song learning and plasticity.