Sex differences in structure and expression of the sex chromosome genes CHD1Z and CHD1W in zebra finches

Genes on the sex chromosomes are unique because of their sex-specific inheritance. One question is whether homologous gene pairs on the sex chromosomes, which have diverged in their sequence, have acquired different functions. We have analyzed the first homologous pair of genes (CHD1Z and CHD1W) discovered on the avian Z and W sex chromosomes of the zebra finch (Taeniopygia guttata) to examine whether functional differences may have evolved. Sequence analysis revealed that the two genes maintained a high degree of similarity especially within the C, H, and D domains, but outside of these regions larger differences were observed. Expression studies showed that CHD1W was unique to females and has the potential to produce a protein that CHD1Z does not. CHD1Z mRNA was expressed at a higher level in the male brain than in the female brain at various post-hatch ages. Reporter constructs containing the 5' flanking regions of each gene showed they had the ability to drive reporter expression in primary cell cultures. The 5' flanking region sequence of CHD1Z and CHD1W exhibited little homology, and differences in putative promoter elements were apparent. These differences between CHD1Z and CHD1W suggest that the two proteins may have diverged in their function.     

Dynamic gene expression in the song system of zebra finches during the song learning period

The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post‐hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1315–1338, 2015     

Effects of corticosterone and DHEA on doublecortin immunoreactivity in the song control system and hippocampus of adult song sparrows

Adult neuroplasticity is strongly influenced by steroids. In particular, corticosterone (CORT) and dehydroepiandrosterone (DHEA) can have opposing effects, where CORT reduces while DHEA increases neurogenesis and neuron recruitment. It has been previously shown that in adult male song sparrows, DHEA treatment increases neuron recruitment throughout the telencephalon, including the lateral ventricular zone, while the effect of CORT treatment is restricted to HVC, one of the song control regions. These data suggest that the two steroids may differentially affect proliferation, migration, differentiation, and/or survival of new neurons. To determine if CORT or DHEA alters the migration and differentiation of young neurons, we examined an endogenous marker of migrating immature neurons, doublecortin (DCX), in HVC and hippocampus of adult male song sparrows that were treated with CORT and/or DHEA for 28 days. In HVC, DHEA increased the number of DCX‐labeled round cells, while CORT had no main effect on the number of DCX‐labeled cells. Furthermore, DHEA increased the area covered by DCX immunoreactivity in HVC, regardless of CORT treatment. In the hippocampus, neither DHEA nor CORT affected DCX immunoreactivity. These results suggest that DHEA enhances migration and differentiation of young neurons into HVC while CORT does not affect the process, whether in the presence of DHEA or not. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 52–62, 2014     

The distribution of tyrosine hydroxylase immunoreactivity in the brains of male and female zebra finches

A system of brain nuclei controls song learning and behavior in zebra finches (Poephila guttata). The size of song-control nuclei are much larger in males, which sing, than in females, which do not sing. This study examined the distribution of fibers, terminals, and cell bodies that are immunoreactive for tyrosine hydroxylase (TH) (the rate-limiting enzyme in the synthesis of catecholamines) in song-control nuclei of adult males and females and juvenile males. In addition, the broad pattern of TH staining throughout the brain was described. There was a sex difference in TH immunoreactivity within song-control nuclei: males had light to moderate staining in all three cortical nuclei examined, whereas females had little or no label in corresponding areas [lateral magnocellular nucleus of the anterior neostriatum (IMAN), higher vocal center (HVC), and robust nucleus of the archistriatum (RA)]. The song-control nucleus area X (X), located in the striatum of avian basal ganglia, was more darkly stained than the surrounding striatum only in males; X was not defined by more intense immunoreactivity in females and hence could not be visualized. There were no apparent differences in TH staining in males ranging in age from 50 days to adulthood (>90 days). Outside of the song-control system there were no substantive differences as a function of sex or age in the pattern or intensity of TH labeling. Major areas of telencephalic staining included the striatal region of basal ganglia, which was covered with dense, fine-grained label, and the septum, where cell bodies were encircled by extremely well-labeled thick processes. In the diencephalon, the preoptic area and hypothalamus included a complex pattern of darkly stained somata and fiber and terminal labeling. Darkly stained somata surrounded the pretectal nucleus, and labeled processes ramified throughout the superficial layers of the optic tectum. The midbrain and hindbrain contained a dense plexus of extremely dark cell bodies corresponding to mammalian substantia nigra, adjacent tegmental areas, and locus ceruleus. Labeled hindbrain cells were also seen in the pontine region, around nucleus solitarius, and in the ventrolateral medulla. © 1993 John Wiley & Sons, Inc.     

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.     

Intracranial administration of the G‐protein coupled estrogen receptor 1 antagonist,G‐15, selectively affects dimorphic characteristics of the song system in zebra finches (Taeniopygia guttata)

In zebra finches (Taeniopygia guttata), estradiol contributes to sexual differentiation of the song system but the receptor(s) underlying its action are not exactly known. Whereas mRNA and/or protein for nuclear estrogen receptors ERα and ERβ are minimally expressed, G‐protein coupled estrogen receptor 1 (GPER1) has a much greater distribution within neural song regions and the syrinx. At present, however, it is unclear if this receptor contributes to dimorphic development of the song system. To test this, the specific GPER1 antagonist, G‐15, was intracranially administered to zebra finches for 25 days beginning on the day of hatching. In males, G‐15 significantly decreased nuclear volumes of HVC and Area X. It also decreased the muscle fiber sizes of ventralis and dorsalis in the syrinx. In females, G‐15 had no effect on measures within the brain, but did increase fiber sizes of both muscle groups. In sum, these data suggest that GPER1 can have selective and opposing influences on dimorphisms within the song system, but since not all features were affected additional factors are likely involved. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

Conspecific and heterospecific song discrimination in male zebra finches with lesions in the anterior forebrain pathway

Adult zebra finches can produce normal song in the absence of Area X, lMAN, or DLM, nuclei that constitute the anterior forebrain pathway of songbirds. Here, we address whether lesions involving Area X and lMAN affect adult male zebra finches' ability to discriminate between conspecific or heterospecific songs. Intact birds and lesioned birds were trained on an operant GO/NOGO conditioning paradigm to discriminate between hetero- or conspecific songs. Both lesioned and intact birds were able to learn all discriminations. Lesioned and intact birds performed equivalently on canary song discriminations. In contrast, discriminations involving bird's own song took significantly more trials to learn for lesioned birds than for intact birds. Discrimination between conspecific songs in general also took longer in the lesioned birds, but missed significance level. Birds with control lesions medial to Area X did not show any differences from intact animals. Our results suggest that an intact anterior forebrain pathway is not required to discriminate between heterospecific songs. In contrast, Area X and lMAN contribute to a male zebra finch's ability to discriminate between its own song and that of other zebra finches. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 81–90, 1998     

Hormonal and environmental control of song control region growth and new neuron addition in adult male house finches, Carpodacus mexicanus

In songbirds, testosterone (T) mediates seasonal changes in the sizes and neuroanatomical characteristics of brain regions that control singing (song control regions; SCRs). One model explaining the mechanisms of the growth of one SCR, the HVC, postulates that in the spring increasing photoperiod and circulating T concentrations enhance new neuron survival, thus increasing total neuron number. However, most research investigating the effects of T on new neuron survival has been done in autumn. The present study investigated the effects of photoperiod and T treatment on SCR growth and new neuron survival in the HVC in photosensitive adult male House Finches, Carpodacus mexicanus, under simulated spring-like conditions. Birds were castrated, given T-filled or empty Silastic capsules and maintained on short days (SD; 8L:16D) or long days (LD; 16L:8D). To mark new cells, birds received bromodeoxyuridine injections 11 days after experimental manipulations began and were sacrificed 28 days later. Testosterone treatment increased the sizes of two SCRs, the HVC and Robust nucleus of the arcopallium (RA). Exposure to LD did not affect HVC volume, but did increase RA volume. Testosterone treatment increased the total number of HVC neurons, but did not affect the number of new HVC neurons. Thus, T initiates SCR growth and increases neuron survival, but effects of T on new neuron incorporation may be limited in photosensitive birds under spring-like conditions. These results provide new insight into the effects of photoperiod and T treatment on vernal SCR growth and new neuron incorporation and support current models explaining this growth.     

Enhanced expression of tubulin-specific chaperone protein A, mitochondrial ribosomal protein S27, and the DNA excision repair protein XPACCH in the song system of juvenile male zebra finches

Recent evidence suggests that sexual dimorphisms in the zebra finch song system and behavior arise due to factors intrinsic to the brain, rather than being solely organized by circulating steroid hormones. The present study examined expression of 10 sex chromosome genes in the song system of 25-day-old zebra finches in an attempt to further elucidate these factors. Increased expression in males was confirmed for nine of the genes by real-time qPCR using cDNA from individual whole telecephalons. In situ hybridization at the same age revealed specific, male-enhanced mRNA for three of the nine genes in one or more song control nuclei. These genes encode tubulin-specific chaperone A, mitochondrial ribosomal protein S27, and a DNA repair protein XPACCH. Based on what is currently known about these proteins' functions and their localization to particular components of the song circuit, we hypothesize that they each may be involved in specific aspects of masculinization.     

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. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Right-side dominance for song control in the zebra finch

Adult male zebra finches underwent unilateral denervation of the syrinx or unilateral lesion of the forebrain nucleus HVC known to be important for song control. Disruptive effects of song were greater after right-side than after left-side operations. After denervation of the right half of the syrinx, the fundamental frequencies of all syllables within a song converged on a value near 500 Hz, and nearly all syllables were altered in type. In contrast, the syllables produced after denervation of the left side of the syrinx largely maintained their preoperative frequencies, and fewer syllables changed in type. Unlike nerve sections, HVC lesions did not result in strikingly lateralized effects on syllable phonology; however, HVC lesions did affect the temporal patterning of a bird's song, whereas nerve sections did not, and changes in temporal patterning were more marked after right than after left HVC lesions. Right-side dominance for zebra finch song control is the reverse of that described in other songbird species with lateral asymmetry for vocal communication. We suggest that the need for a dominant side is more important than the side of dominance. © 1992 John Wiley & Sons, Inc.     

Sexually dimorphic expression and estradiol mediated up‐regulation of a sex‐linked ribosomal gene,RPS6, in the zebra finch brain

Sex‐linked genes are considered to be a major contributor to neural sex differences in zebra finches. While several candidates have been identified, additional ones are continuously being discovered. Here we report on a novel Z‐linked ribosomal gene (rpS6) that is enhanced in the male brain as compared to the female's throughout life. In both sexes, expression of rpS6 is highest at P3 and P8 (just before the onset of morphologically detectable sex differences), decreases around P15, and then remains decreased through adulthood. Analysis of rpS6 mRNA revealed widespread distribution throughout the brain. However, within song regions HVC and RA, mRNA containing cells were greater in males as compared to females. Hormones are also involved in the development of neural dimorphisms, so we additionally investigated whether rpS6 might interact with estradiol (E₂). An up‐regulation of rpS6 gene was observed in both sexes following treatment with E₂ and the effect was approximately twice as large in males as compared with females. These data suggest that rpS6 may be involved in sexual differentiation of the zebra finch brain, and that the effect is facilitated by E₂. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 599–608, 2013     

Characterization and localization of D1 dopamine receptors in the sexually dimorphic vocal control nucleus,area X,and the basal ganglia of european starlings

D1 dopamine receptors were pharmacologically characterized and localized by quantitative autoradiography in the basal ganglia of male and female European starlings (Sturnus vulgaris). The D1 selective antagonist SCH 23390 was used to label this receptor subtype. Starlings are songbirds and possess a neural circuit implicated in the learning and production of song. This circuit includes a sexually dimorphic nucleus, area X, that is a subregion of the parolfactory lobe of the basal ganglia and is known from work on zebra finches to receive dopaminergic input from the area ventralis of Tsai. We focused our investigation on the D1-like receptor subtype because they are abundant in the basal ganglia. Competition studies indicate that a variety of dopaminergic ligands compete with [³H] SCH 23390 for the binding site in an order of potency characteristic of a D1-like receptor. Autoradiographic studies of the basal ganglia revealed high D1 receptor densities in the avian homologues of the caudate-putamen and relatively low-receptor densities were observed in the avian homologue of the globus pallidus. In male starlings, area X could be reliably discerned on the autoradiograms by the higher density of D1 receptors compared to the surrounding parolfactory lobe (LPO). This was also true for females, though nt as reliably as in males. When we compared the mean D1 receptor density in area X for males and females we did not find a significant sex difference. However, we also analyzed the data by comparing sex differences in the degree to which area X has a higher receptor density in comparison with the surrounding LPO. When we normalized D1 receptor density in area X relative to the LPO, we did find a significant sex difference. This sex difference in relative receptor density represents another neural sex difference in the song circuit that may mediate sex differences in the learning and production of song in starlings and other songbirds. 1994 John Wiley & Sons, Inc.     

Development of the catecholaminergic innervation of the song system of the male zebra finch

Catecholamines (CA) have been proposed to have neuromodulatory actions, particularly on attention and learning, in a number of neural systems. Because several of the interconnected brain nuclei that mediate song learning and production in the adult male zebra finch (Taeniopygia guttata) contain these neurotransmitters, we investigated the appearance of the catecholaminergic innervation of the song nuclei of male zebra finches during posthatch development, specifically during the period in which song learning occurs. We studied the development of immunoreactivity for tyrosine hydroxylase (TH) in the song nuclei HVc, RA, NIf, LMAN, and Area X in young males aged 20, 35, and 60 days as well as in adults (>90 days). We also visualized catecholamines directly in Area X using CA histofluorescence. Both TH immunoreactivity and CA histofluorescence were initially low in Area X relative to their levels in the surrounding parolfactory lobe (LPO), and then increased during development to become more intense than in LPO by days 60–90. Similarly, TH immunoreactivity in HVc was initially low relative to that in the surrounding neostriatum, then increased during development to become more intense than that in the surround by day 60. TH immunostaining also increased markedly in NIf, RA, and LMAN over the same period. These results show that the levels of catecholamines and their major synthetic enzyme increase in song nuclei during development and thus raise the possibility that these transmitters contribute to the development of the song system or to song learning. © 1996 John Wiley & Sons, Inc.     

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.