Comparative analysis of mineralocorticoid receptor expression among vocal learners (Bengalese finch and budgerigar) and non-vocal learners (quail and ring dove) has implications for the evolution of avian vocal learning
Laboratory for Symbolic Cognitive Development, RIKEN Brain Science Institute, Wako 351-0198
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.
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
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.
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.
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).
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).
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.
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.
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.
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.