Fig 1.
Coronal brain sections at the level of song control areas.
(A) At the level of the anterior forebrain, song control areas LMAN and Area X are clearly visible at low magnification. Whereas LMAN is intensely labelled for δ-OR mRNA, Area X and the surrounding MSt have lower levels of staining compared to the surrounding nidopallium. (B) At the level of the posterior forebrain, HVC is clearly visible as a result of high levels of δ-OR mRNA expression. Whereas the boundaries of the vocal motor nucleus RA can also be clearly demarcated, the staining intensity was lower in this area. Scale bar, 200 μm. AId, Dorsal intermediate arcopallium; Cb, Cerebellum; HA, Hyperpallium apicale; HD, Hyperpallium dorsale; Hp, Hippocampus; LAD, Lamina arcopallialis dorsalis; LaM, Lamina mesopallialis; LFM, Lamina frontalis suprema; LFS, Lamina frontalis superior; LMAN, Lateral magnocellular nucleus of the anterior nidopallium; LPS, Lamina pallio-subpallialis; NCL, Caudolateral nidopallium; NCM, Caudomedial nidopallum; OT, Optic tectum; RA, Robust nucleus of arcopallium.
Fig 2.
The expression of δ-OR mRNA in song control areas.
(A) In the anterior forebrain pathway, magnocellular neurons in the central (core) region and parvicellular (shell) neurons of the nidopallial song control nucleus LMAN express high levels of the δ-OR mRNA (insets show staining in individual neurons in each area). (B) Area X demonstrated lower levels of staining compared to the overlying nidopallium. The level of δ-OR mRNA expression in the Area X(L) neuron (white arrowhead) was significantly higher than that in a smaller neuron [Area X(S); yellow arrowhead] in this region. (C) DLM, a thalamic nucleus which is also a part of the AFP and projects to LMAN, is weakly stained for δ-ORs. (D) and (E) Song control nuclei (RA and HVC) of the vocal motor pathway also demonstrate δ-OR mRNA expression. Whereas neurons in HVC are strongly labelled, those in RA express lower levels of staining. Scale bars, 100 μm for low magnification images and 50 μm for insets. DLM, Dorsolateral nucleus of the medial thalamus; LMAN, Lateral magnocellular nucleus of the anterior nidopallium; RA, Robust nucleus of arcopallium.
Fig 3.
Quantification of δ-OR mRNA in the zebra finch brain.
(A) A comparison of δ-OR mRNA levels across various regions in the brain. In the boxplots used here, boxes represent the interquartile range, whiskers represent the maximum and minimum values in the data and the outliers are represented by data points above and below the whiskers. Amongst the song control areas, neurons were most intensely labeled in HVC and LMAN whereas those in DLM, Area X and RA have lower levels of label for δ-OR mRNA (statistical comparison indicated by blue line). Auditory cortical areas (separated by a dashed line from other brain regions) also demonstrate comparably high levels of label at the level of CMM, NCM and NIf. Levels of label in these regions were significantly higher than those in the thalamic nucleus Ov and MLd (a midbrain nucleus), whereas the lowest levels of δ-OR mRNA were present in Field L. (B) In the anterior forebrain, areas of the avian basal ganglia, that is, MSt and Area X demonstrate weak labeling and the surrounding nidopallium is comparatively strongly labeled. At more caudal levels, neurons in GP were intensely labelled compared with those in LSt and the intermediate nidopallium (Int. Nido.). (C) At an intermediate level along the rostro-caudal extent of the brain, the auditory nucleus NIf demonstrates higher levels of label for δ-OR mRNA than the surrounding Int. Nido. whereas Field L demonstrates the least. (D) The song control nucleus HVC can be clearly demarcated from the surrounding nidopallium. (E) The lowest levels of label for δ-OR mRNA are present in the RA cup region compared to the surrounding arcopallium and the song control areas RA and AId. (F) Comparably low levels of δ-OR mRNA were present in the thalamic nuclei DLM and Ov. (G) Cells in the midbrain auditory nucleus MLd demonstrated lower staining intensity for δ-ORs than the surrounding subdivisions of ICo. ***, P<0.001; **, P<0.01; *, P<0.05. ANP, Anterior nidopallium; AId, dorsal intermediate arcopallium; CMM, caudomedial mesopallium; DLM, Dorsolateral nucleus of the medial thalamus; DM ICo, dorsomedial part of nucleus intercollicularis; LMAN, Lateral magnocellular nucleus of the anterior nidopallium; GP, Globus pallidus; LSt, Lateral striatum; MLd, Nucleus mesencephalicus lateralis, pars dorsalis; MSt, Medial Striatum; NCL, Caudolateral nidopallium; NCM, Caudomedial nidopallium; NIf, Nucleus interfaciallis nidopalli; Ov, Nucleus ovoidalis; RA, Robust nucleus of arcopallium.
Fig 4.
The expression of δ-OR mRNA at an intermediate plane along the rostro-caudal extent of the brain.
(A) At low magnification, parts of the basal ganglia (the LSt and GP) and the auditory area NIf are clearly visible based on higher levels of δ-OR mRNA expression. (B) The auditory nucleus NIf can be clearly demarcated from the surrounding intermediate nidopallium (INP). (C) Field L is clearly visible as a highly myelinated area but with low levels of δ-OR mRNA. (D) The thalamic nuclei DLM (a part of the song control system) and Ov (a part of the auditory system) are clearly visible based on the presence of low levels of δ-OR mRNA in these regions. (E) A high magnification image of DLM and Ov demonstrates that the boundaries of both thalamic nuclei are clearly visible and can be easily delineated from the surrounding thalamus. (F) The auditory nucleus (MLd) can be easily delineated from surrounding midbrain based on high levels of myelination. However, individual neurons in this region are weakly labelled, as can be seen in the inset at higher magnification. Scale bar for A, C, D, E and F, 200μm, scale bar for B, 100μm and scale bar for the inset in F, 20μm. DLM, Dorsolateral nucleus of the medial thalamus; DM ICo, dorsomedial part of nucleus intercollicularis; GP, Globus pallidus; IM, Nucleus isthmi, pars magnocellularis; IPc, Nucleus isthmus, pars pavocellularis; LSt, Lateral striatum; MLd, Nucleus mesencephalicus lateralis, pars dorsalis; NIf, Nucleus interfaciallis nidopallii; Ov, Nucleus ovoidalis; OT, Optic tectum; Pt, Nucleus pretectalis; Rt, Nucleus rotundus; SPL, Nucleus spiriform lateralis.
Fig 5.
Schematic demonstrating δ-OR mRNA expression in the zebra finch brain.
A summary of the pattern of δ-OR mRNA expression in various regions observed in our study. Dark blue indicates areas with the most prominent label, whereas light blue indicates regions with low staining intensity.