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Keywords:

  • evolution;
  • mineralocorticoid receptor;
  • parrot;
  • quail;
  • songbird

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Mineralocorticoid receptor is the receptor for corticosteroids such as corticosterone or aldosterone. Previously, we found that mineralocorticoid receptor was highly expressed in song nuclei of a songbird, Bengalese finch (Lonchura striata var. domestica). Here, to examine the relationship between mineralocorticoid receptor expression and avian vocal learning, we analyzed mineralocorticoid receptor expression in the developing brain of another vocal learner, budgerigar (Melopsittacus undulatus) and non-vocal learners, quail (Coturnix japonica) and ring dove (Streptopelia capicola). Mineralocorticoid receptor showed vocal control area-related expressions in budgerigars as Bengalese finches, whereas no such mineralocorticoid receptor expressions were seen in the telencephalon of non-vocal learners. Thus, these results suggest the possibility that mineralocorticoid receptor plays a role in vocal development of parrots as songbirds and that the acquisition of mineralocorticoid receptor expression is involved in the evolution of avian vocal learning.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Vocal learning is the ability to acquire vocalization through imitation. Among avian species, three families of birds, songbirds, parrots and hummingbirds have this ability (Jarvis 2004). Because these birds are taxonomically distantly related, it has been suggested that they acquired this ability independently. In the brains of vocal learners, there is a series of nuclei and neural circuit specialized for vocal learning and production (Fig. 1; Nottebohm et al. 1976, 1982; Brauth et al. 1994; Striedter 1994; Durand et al. 1997; Gahr 2000; Jarvis & Mello 2000; Jarvis et al. 2000; Jarvis 2004; Bolhuis & Gahr 2006; Bolhuis et al. 2010). By contrast, birds such as chicken and pigeon have no such neural circuit. Because of these structure-related behavioral differences, the avian vocal system is a good model for studying brain evolution from a morphological and functional perspective (Matsunaga & Okanoya 2009b). Although it has been demonstrated that various genes show vocal control area-related expressions at the developmental stage (Denisenko-Nehrbass et al. 2000; Wade 2000; Haesler et al. 2004; Kim et al. 2004; Chen et al. 2005; Li et al. 2007; Matsunaga & Okanoya 2008a,b, 2009a, 2011; Matsunaga et al. 2008) as well as at the adult stage (Denisenko-Nehrbass et al. 2000; Haesler et al. 2004; Wada et al. 2004; Gahr 2007; Lovell et al. 2008; Matsunaga & Okanoya 2008a,b, 2009a; Matsunaga et al. 2008; Kato & Okanoya 2010), it still remains unknown what molecular mechanisms establish neural circuits of the vocal control system.

image

Figure 1.  Phylogenetic tree of avian species and schematic representation of the vocal system. (A) Phylogenetic relationship based on Hackett et al. (2008). In this study, we compared Bengalese finch, budgerigar, ring dove and quail. Although Tyrannidae is more closely related to songbirds, it is difficult to analyze these birds, because they have been designated special natural treasures. (B–D) Sagittal view of a Bengalese finch (B), budgerigar (C) and quail and ring dove (D) brain. The avian vocal system is composed of the telencephalic vocal learning pathway and the general vocal production pathway in the brainstem. The telencephalic vocal pathway is found only in vocal learning species. Red and blue lines indicate anterior and posterior pathways for vocal learning, respectively. The yellow line indicates the general vocalization pathway seen in the brainstem of all avian species. AAC, central nucleus of the anterior arcopallium; DLM, dorsal lateral nucleus of the thalamus; DM, dorsal medial nucleus of the midbrain; DMm, magnocellular nucleus of the dorsal thalamus; LMAN, lateral magnocellular nucleus of the anterior nidopallium; MO, oval nucleus of the mesopallium; MSt, medial striatum; NAO, oval nucleus of the anterior nidopallium; NLC, central nucleus of the lateral nidopallium; RA, robust nucleus of the arcopallium; RAm, nucleus retroambigualis; nXIIts, nucleus XII, tracheosyringeal part. We used the terminology of Reiner et al. (2004).

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Previously, we analyzed various gene expressions in a songbird, Bengalese finch (Lonchura striata var. domestica), and found that mineralocorticoid receptor (MR) showed vocal control area-related expressions (Suzuki et al. 2011). MR is the receptor for corticosteroid such as corticosterone or aldosterone. Glucocorticoid hormones (GCs; cortisol in fish and many mammals, and corticosterone in birds, reptiles, amphibians, and many rodents) are steroid hormones released when the hypothalamic–pituitary–adrenal axis is activated in response to stressful stimuli (Romero 2004; Cockrem 2007). GCs regulate downstream gene expressions and lead to adaptive responses to stress by binding to two types of receptor: mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). MR is a high-affinity receptor for corticosterone and responds to basal levels of corticosterone under normal conditions, whereas GR is a low-affinity receptor and is activated only by high levels of corticosterone during stressful conditions (Reul & de Kloet 1985; de Kloet & Reul 1987; Arriza et al. 1988; de Kloet et al. 1990; de Kloet 1991; Oitzl et al. 2010). Originally, corticosterone and its receptors were thought to be involved in adaptive response to stressful stimuli (de Kloet et al. 1998). However, many studies in rodents have suggested that corticosterone and its receptors play multiple roles in various neural processes such as neurogenesis (Gould et al. 1992; Karishma & Herbert 2002; Wong & Herbert 2004, 2005, 2006), neuronal plasticity (Pavlides et al. 1995, 1996; de Kloet et al. 1999), learning, memory, and emotion (Oitzl & de Kloet 1992; Sandi & Rose 1994; Brinks et al. 2007). In bird studies, it has been demonstrated that developmental stress or chronic corticosterone administration in juvenile birds decreases the size of song nuclei, with a reduction in song complexity (Nowicki et al. 2002; Spencer et al. 2003, 2004; Buchanan et al. 2004; MacDonald et al. 2006), suggesting that corticosterone negatively regulates vocal development. On the other hand, rodent studies revealed that functional blockage of MR or disruption of MR induces impaired neurogenesis and degeneration of hippocampal neurons (Sloviter et al. 1993; Sousa et al. 1997; Gass et al. 2000). Moderate levels of stress hormone block apoptotic cell death and enhance long-term potentiation in hippocampal neurons by activating MR signaling (Woolley et al. 1991; Diamond et al. 1992; Pavlides et al. 1995). Furthermore, MR counteracts GR-mediated neuronal cell death in hippocampal neurons (Sousa et al. 1999; Crochemore et al. 2005), suggesting that these two receptors function in an opposite manner. Thus, it is suspected that high dose of corticosterone negatively regulate vocal development via GR, whereas low dose of corticosterone positively regulates vocal development via MR.

Previously, we performed in situ hybridization analysis, and found that MR showed strong vocal control area-related expressions in juvenile and adult Bengalese finches whereas GR showed strong expression in the hypothalamic areas (Suzuki et al. 2011). Since MR expression is more tightly linked to vocal system than GR, to examine the relationship between corticosterone and the evolution of avian vocal learning system, here, we analyzed MR expression in the developing brain of other vocal learner, budgerigar (Melopsittacus undulatus) and non-vocal learners, quail (Coturnix japonica) and ring dove (Streptopelia capicola) and compared these four species.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Animals and sample preparation

The research protocols were approved by the Animal Care and Use Committee of RIKEN and conformed to National Institutes of Health (NIH) Guidelines (No. H20-2-231). We used three male 30-day postnatal (P30) budgerigars (Melopsittacus undulatus) and three P30 male Japanese quails (Coturnix japonica), two P24 and one P45 male ring dove (Streptopelia capicola) bred at our lab facilities (we also used three male P14 budgerigar and three male P14 quail and got similar results). All birds were deeply anesthetized with an intramuscular injection of sodium pentobarbital (50 mg/kg) and then killed. After decapitation, their brains were embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek) and frozen on dry ice for cryosectioning. Frozen sections for in situ hybridization or thionine staining for neuroanatomical reference were cut serially in 20 μm thicknesses using a cryostat (Leica). To extract total RNA, brain tissues were dissected and placed in Qiazol Lysis reagent (Qiagen), and the RNA was purified using an RNeasy Lipid Tissue Mini kit (Qiagen). The sex of the birds was determined by extracting genomic DNA from a portion of a digit with a DNeasy tissue kit (Qiagen) and performing a polymerase chain reaction (PCR) with primers that amplify the chromo-helicase-DNA binding gene (Ellegren 1996) or verified sex organs.

DNA isolation and probe preparation

We isolated budgerigar MR (AB649451) and quail MR (AB649452) cDNAs by reverse transcription (RT)-PCR. The primers we used were 5′-AATTACCTGTGTGCGGGAAG-3′ and 5′- CCTGAGACTCTCGGAAGGTG-3′. The cDNAs that we isolated corresponded to the region encoding the terminal region of the DNA binding domain and almost all of the ligand binding domain was used as probes (Fig. 2A). The nucleotide sequence similarities of budgerigar and quail MR cDNAs to Bengalese finch cDNAs are 94.7% and 92.2%, respectively (at the amino acid level, 99.2% and 99.6%, respectively) (Fig. 2B). Each cDNA fragment was inserted in the pGEM-T Easy vectors (Promega). The plasmids were linearized with ApaI enzyme to release the fragment, and probes were synthesized using SP6 RNA polymerase (Roche Diagnosticswith digoxigenin (DIG)-labeling mix (Roche Diagnostics). For ring dove, we used the quail probe.

image

Figure 2.  Schematic drawing of the domain structure of mineralocorticoid receptor (MR) and the position of probes used for in situ hybridization (A) and sequence similarities of MR cDNA at the amino acid level among Bengalese finch, budgerigar and quail (B). Asterisks indicate conserved amino acids among the three species. Sequences surrounded by gray boxes indicate the sequence encoding DNA-binding domain and ligand-binding domain, respectively.

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In situ hybridization

In situ hybridization of all tissue sections was performed using the method described by Matsunaga & Okanoya (2008a). The sections were post-fixed for 10 min with phosphate buffered saline (PBS) solution with 4% paraformaldehyde (PFA), and then washed three times in PBS for 3 min. The slides were delipidated with acetone, acetylated, and washed in PBS with 1% Triton-X100 (Wako Pure Chemical). The slides were then incubated at room temperature with hybridization buffer containing 50% formamide (Wako Pure Chemicals), 5× standard sodium citrate (SSC), 5× Denhardt’s solution (Sigma), 250 μg/mL yeast transfer RNA (Roche Diagnostics), and 500 μg/mL DNA (Roche Diagnostics). Sections were hybridized at 72°C overnight in hybridization buffer with RNA probes. They were then rinsed in 0.2× SSC for 2 h and then blocked for 2 h in a solution of 0.1 mol/L Tris (pH 7.5) and 0.15 mol/L NaCl with 10% sheep serum. The slides were incubated overnight with alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche Diagnostics). After washing, AP activity was detected by adding 337.5 mg/mL nitroblue tetrazolium chloride and 175 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics). Since we used probes derived from quail cDNA as probes for ring dove MR, staining level was relatively low in the brain sections of ring doves. All images were captured using an ORCA-Flash2.8 digital camera (Hamamatsu Photonics) under a BX-50 microscope (Olympus, Tokyo, Japan). Photoshop software (ver. CS5; Adobe Systems) was used to crop unnecessary areas, juxtapose panels, and enhance contrast and brightness, as required.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Mineralocorticoid receptor expression in vocal control areas of the budgerigar brain

First, we examined MR expression in the developing budgerigar brain. As in the case of Bengalese finch (Figs 3A–C, 4A–C; Suzuki et al. 2011), MR was expressed in most vocal control areas such as the central nucleus of the lateral nidopallium (NLC), central nucleus of the anterior arcopallium (AAC), magnocellular nucleus of the dorsal thalamus (DMm), nucleus XII, tracheosyringeal part (nXIIts), oval nucleus of the mesopallium (MO) and oval nucleus of the anterior nidopallium (NAO), corresponding areas to vocal control nuclei of songbirds such as the HVC, robust nucleus of the arcopallium (RA), dorsal lateral nucleus of the thalamus (DLM), nXIIts and lateral magnocellular nucleus of the anterior nidopallium (LMAN) (Figs 3D,F, 4D,F). However, in contrast to Bengalese finch, only faint expression was seen in the dorsal medial nucleus of the midbrain (DM) and medial striatum (MSt) (Figs 3E, 4E). Thus, although there are some differences, MR expression was seen in most vocal control areas of the parrot (budgerigar) brain, like songbird (Bengalese finch).

image

Figure 3. In situ hybridization of transverse section of P30 male Bengalese finch (A–C), P30 male budgerigar (D–F), P30 male quail (G–I) and P24 ring dove (J–L) of the telencephalon. St, striatum. Scale bars are 1 mm, except for (B), (C) (500 μm).

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image

Figure 4. In situ hybridization of transverse section of P30 male Bengalese finch (A–C), P30 male budgerigar brain (D–F), P30 male quail brain (G–I) and P24 ring dove brain (J–L) of the thalamus and brainstem. B′, H′′ and K′′ are high magnification images of dorsal lateral nucleus of the mesencephalon (MLd) in B, H and K, respectively. E′, H′ and K′ are high magnification images of dorsal medial nucleus of the midbrain (DM) in B, H and K, respectively. DT, dorsal thalamus; Ov, nucleus ovoidalis. Scale bars are 1 mm (A, B, D–H, J, K), 500 μm (C, I, L) and 50 μm (B′, E′, E′′, H′, H′′, K′, K′′).

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Mineralocorticoid receptor expression in corresponding areas of the non-vocal learner

Next, we examined MR expression in corresponding areas to vocal control areas in the brain of non-vocal learners, quail and ring dove. In the frontal region of nidopallium (NF), the corresponding areas including the LMAN in Bengalese finch and the NAO in parrot, respectively, only sparse MR expressions were seen in both species (Fig. 3G,J). Only sparse expressions were seen in the striatum (Fig. 3G,J), the corresponding areas including the Area X in Bengalese finch and MSt in budgerigar. In the caudolateral nidopallium (NCL) and the intermediate arcopallium (Ai), corresponding areas including the HVC, RA in Bengalese finch and the NLC, AAC in budgerigar (it is thought that HVC and NLC is the part of NCL and RA and AAC is the part of Ai), there was no strong MR expression (Fig. 3H,I,K,L), although a house keeping gene such as β-tubulin expression was similar between vocal learner and non-vocal leaner (Fig. S1). However, in contrast to telencephalic vocal control areas, strong MR expression was seen in the dorsal thalamus (DT) including the corresponding area of thalamic vocal nucleus, and nXIIts, as Bengalese finch and budgerigar (Fig. 4G,I,J,L), and faint expression was seen in the DM as budgerigar (Fig. 4H,K). Thus, MR expression differs between vocal learners and non-vocal learners particularly in the telencephalon.

Similar expression pattern of mineralocorticoid receptor in non-vocal control areas between vocal learners and non-vocal learners

To examine whether difference of MR expression between vocal learners and non-vocal learners is vocal control area-specific, we analyzed MR expression in non-vocal control areas.

Secondary auditory areas such as the caudomedial mesopallium (CMM) and caudal medial nidopallium (NCM), connected to vocal areas, are known to play essential roles in sensory learning of vocalization (Bolhuis & Gahr 2006). However, even though chicken and pigeon are not able to learn vocalizations, they also have these areas in their brain (Terpstra et al. 2005). Actually, MR expression was mutually seen in these areas of budgerigar, quail and ring dove brain (Fig. 5D,G,J), as in songbird brain (Fig. 5A).

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Figure 5.  Similar mineralocorticoid receptor (MR) expressions in non-vocal areas. In situ hybridization of transverse section of P30 male Bengalese finch (A–C), P30 male budgerigar brain (D–F), P30 male quail brain (G–I) and P24 ring dove brain (J–L). (A, D, G, J) Hippocampal region (Hp), caudomedial mesopallium (CMM) and caudal medial nidopallium (NCM), (B, E, H, K) nucleus taeniae of the amygdala (TnA), (C, F, I, L) nucleus rotundus (Rt) and dorsal lateral geniculate nucleus (GLd). J′ is the high magnification image of NCM. Note that MR expressions in these areas are similar between vocal learners (Bengalese finch and budgerigar) and non-vocal learner (quail, ring dove). Scale bars are 1 mm, except for K (500 μm) and 50 μm (J′).

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In Bengalese finch, strong MR expression is detected in other telencephalic areas such as the hippocampal region (Hp) and nucleus taeniae of the amygdala (TnA), and thalamic visual areas such as nucleus rotundus (Rt) and dorsal lateral geniculate nucleus (GLd), and auditory areas such as nucleus ovoidalis (Ov) and dorsal lateral nucleus of the mesencephalon (MLd) (Figs 4A,B, 5A–C; Suzuki et al. 2011). In three other species, MR was also expressed in these areas (Figs 4D,E,G,H,J,K, 5D–L). These results suggest that MR expression in non-vocal areas is highly conserved among these three species in contrast to the vocal areas.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Since songbirds (Bengalese finch, zebra finch, canary) and parrots (budgerigar) are distantly related to each other on the evolutionary lineage, they might have evolved their vocal learning systems independently. Indeed, some gene expressions are highly diverse between these two species (Wada et al. 2004; Matsunaga & Okanoya 2011). By contrast, in this study, we found that MR expression was seen in most of the vocal control areas of budgerigar as Bengalese finch, suggesting that MR expression in vocal control areas might be highly constrained among various vocal learning species.

Exceptionally, MR expression in the striatal vocal control area is different between Bengalese finch and budgerigar. Interestingly, such difference is also seen in androgen receptor (AR) expression. Though AR expressions are detected in almost all vocal control nuclei of songbirds (Gahr 2007; Matsunaga & Okanoya 2008a) and budgerigar (Matsunaga & Okanoya 2008b), its expression is lacking in budgerigar MSt. AR and MR may be regulated by the same gene cascade that is necessary for vocal control system in both species.

Many ecological studies in songbirds have suggested that birdsongs are used as an indicator of male quality during mate selection (Kroodsma 1976; Catchpole & Slater 1995; Lampe & Saetre 1995; Hasselquist et al. 1996; Searcy & Yasukawa 1996; Okanoya 2004a,b). Birds are subjected to various stresses (e.g., food limitations, parasitic infections, or sibling competition) during the developmental stage. Since developmental stress affects song development and later successful mate attraction (Nowicki et al. 2002; Buchanan et al. 2003; Spencer et al. 2003, 2004, 2005a,b; Soma et al. 2006; Zann & Cash 2008), corticosterone may be involved in generating vocal diversity among individuals. In this study, we found that MR expression is seen in various vocal control areas of the developing budgerigar brain. Therefore, corticosterone may work in budgerigar brains in a similar manner as in songbirds to give diversity in vocal behaviors among individuals.

Mineralocorticoid receptor expressions are not seen in telencephalic corresponding regions of vocal control areas in the brain of non-vocal learners, quail and ring dove, though MR expressions in non-vocal areas such as the secondary auditory areas, Hp and TnA are similar among four species. In the thalamus and brainstem, MR expression is relatively similar among these species. Thus, noticeable differences are particularly seen in the telencephalic vocal region between vocal learners and non-vocal learners. Though structure of vocal control areas are not seen in non-vocal learners, anatomical and gene expression analyses reveal that some anatomical and molecular basis is shared between vocal learners and non-vocal learners. It is suggested that vocal control system might have diverged from pre-existing areas that both groups have (Matsunaga & Okanoya 2009b). Indeed, some cadherin expressions are similar among vocal control nuclei and their surrounding regions of vocal learners and their corresponding areas of non-learners (Matsunaga & Okanoya 2011). Immediate early gene expressions suggest that vocal control system might have diverged from the general motor pathway (Feenders et al. 2008). The vocal learning system might have been evolved directly by the acquisition of telencephalic MR expression in primordial regions that both vocal learners and non-vocal learners have. Otherwise, it might have been evolved cooperatively in parallel with the acquisition of telencephalic MR expression. MR expression might have enhanced differences among individuals in brain development and vocal behaviors, according to the environment. As a result, it might have increased the importance of vocalizations in reproductive competition and induced further evolution of vocal learning. To examine these hypotheses, further study is needed such as gene manipulation analysis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank the Support Unit for Bio-material Analysis, RIKEN BSI Research Resources Center, for DNA sequence analysis. E.M. was supported by RIKEN Special Postdoctoral Researchers Program, Grant-in-aid for young scientist (B) 21700365 from Japan Society of Promotion of Science.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Fig. S1. Immunostaining of the RA (Bengalese finch), AAC (budgerigar) and Ai (quail) for ß-tubulin.

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DGD_1302_sm_FigS1.pdf157KSupporting info item

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