Role of the midbrain dopaminergic system in modulation of vocal brain activation by social context

Abstract

In a well-studied model of social behaviour, male zebra finches sing directed song to court females and undirected song, used possibly for practice or advertisement. Although the two song types are similar, the level of neural activity and expression of the immediate early gene egr-1 are higher during undirected than during directed singing in the lateral part of the basal ganglia song nucleus AreaX (LAreaX) and its efferent pallial song nuclei lateral magnocellular nucleus of the anterior nidopallium (LMAN) and the robust nucleus of the arcopallium (RA). As social interactions are dependent on brain motivation systems, here we test the hypothesis that the midbrain ventral tegmental area-substantia nigra pars compacta (VTA-SNc) complex, which provides a strong dopaminergic input to LAreaX, is a source of this modulation. Using egr-1 expression, we show that GABAergic interneurons in VTA-SNc are more active during directed courtship singing than during undirected singing. We also found that unilateral removal of VTA-SNc input reduced singing-dependent gene expression in ipsilateral LAreaX during both social contexts but it did not eliminate social context differences in LAreaX. In contrast, such lesions reduced and eliminated the social context differences in efferent nuclei LMAN and RA, respectively. These results suggest that VTA-SNc is not solely responsible for the social context gene regulation in LAreaX, but that VTA-SNc input to LAreaX enhances the singing-regulated gene expression in this nucleus and, either through LAreaX or through direct projections to LMAN and RA, VTA-SNc is necessary for context-dependent gene regulation in these efferent nuclei.

FIG. 1

Social context-dependent gene expression. (A) Schematic diagram of songbird brain showing the song system and VTA–SNc, which sends a strong dopaminergic projection to LAreaX and weaker projections to HVC and RA. Black solid arrows and yellow nuclei, vocal motor pathway; grey arrows and green nuclei, vocal pallial–basal ganglia–thalamic loop; dashed arrows, connections between the two vocal pathways. (B) Egr-1 mRNA expression (white, silver grains) in sagittal brain sections (counterstained with cresyl violet) from animals that sang similar amounts (50 and 70 motifs) of (a, c and e) UD and (b, d and f) FD song. Camera lucida drawings on the left highlight the VMN, VTA and SNc regions; there is no distinct boundary between VTA and SNc. Sections in (a)–(d) are cut in the sagittal plane, and (e) and (f) in the coronal plane. NIII, third cranial nerve. (C) Quantification of egr-1 expression. FD, n = 14; UD, n = 10; SF, n = 4; SA, n = 5. * P < 0.05, *** P < 0.0001, one-way anova, Holm–Sidak post hoc test. (D) Cell types with FD-driven egr-1 expression in VTA–SNc. Arrows, single-labelled cells; arrowheads, double-labelled cells. (a) Retrograde tracer backfilled from LAreaX to VTA neurons (red) and egr-1 mRNA expression (white silver grains). (b) Expression of TH (green-labelled cytoplasm) and egr-1 protein (red-labelled nuclei). Only one double-labelled cell can be seen in this image. (c) GABA (green-labelled cytoplasm) and egr-1 protein (red-labelled nuclei); inset in lower left shows a 3× higher magnification of a double-labelled neuron. (d) Percentage of egr-1 positive neurons that were double-labelled with tracer from LAreaX (n = 11 sections), TH (n = 10 sections) or GABA (n = 7 sections) in VTA–SNc [out of 45–314 neurons across all sections in all birds (n = 3–5) for each single-labelled category]. *** P < 0.0001, one-way anova, Holm–Sidak post hoc test. Error bars for all panels represent SEM. Scale bars, 250 µm (B), 50 µm (D).

FIG. 2

VTA–SNc requirements for singing-regulated gene expression in the song system. (A) Top panels, camera lucida drawing (left) and TH somata immunostaining (right, green) showing unilateral lesion of VTA–SNc. Bottom panels, camera lucida drawing and TH fibre immunostaining (green) showing ipsilateral removal of TH input to AreaX and surrounding striatum. Sections are frontal. (B) Egr-1 expression (white silver grains) in LAreaX after UD and FD of sham-operated (left panels) and unilateral VTA–SNc-lesioned (right panels) males, showing decreased expression in LAreaX and striatum on the lesioned side. The sham-operated bird that sang directed song has high expression in the medial striatum, which may be due to locomotor activity (G. Feenders, H. Mouritsen and E. D. Jarvis, personal communication). (C) Egr-1 expression in HVC and RA of VTA–SNc-lesioned animals, showing decreased expression in ipsilateral RA but not HVC. (D) Correlation of VTA–SNc lesion size ratio and TH fibre intensity ratios in ipsilateral : contralateral LAreaX (n = 24 sham and lesion surgery-treated animals; simple regression). (E) Correlation of ratios of TH fibre intensity and egr-1 expression in ipsilateral : contralateral LAreaX (n = 11 FD and n = 9 UD surgery treated animals; and n = 3 FD and n = 3 UD intact animals; simple regression). (F–K) Correlations of egr-1 expression in different brain regions of intact (n = 3 FD, n = 3 UD) and sham-operated (n = 5 FD, n = 4 UD) animals (solid lines and circles) vs. VTA–SNc-lesioned (n = 6 FD, n = 5 UD) animals (dashed lines and open circles). SA animals that received VTA–SNc lesions were included in the correlations as baseline nonsinging control egr-1 levels. In these graphs, expression levels increased in vocal nuclei of both hemispheres with increasing amounts of song production (no. of songs not shown), except for the striatum (St). The r², P, and slope (s) values (obtained from simple regressions) above the solid lines are for the intact + sham-operated animals; those below the dashed line are for the VTA–SNc-lesioned animals. The P-values between the solid and dashed lines (obtained from multiple regression) show significant differences between the curves of intact + sham-operated and lesioned groups in LAreaX, RA and striatum adjacent to LAreaX, but not in LMAN, HVC or DM. Equal expression between hemispheres results in a slope of 1. Scale bars, 0.25 mm (A, top panel), 0.5 mm (all others).

FIG. 3

Differential effects of VTA–SNc lesions alone and combined VTA–SNc and LoC lesions on singing behaviour. Song motifs of two birds before (pre) and after (post) lesions are available as Supplementary Audio S1–Supplementary Audio S4 (.wav files). (A) Unilateral VTA–SNc-lesioned animal with LoC intact bilaterally (DBH-stained neurons, green). Frontal section. (B) Song motif structure and syntax of the bird in (A) did not show major changes; pre- and post-lesion song motif sequence and syllable structure (a–d) were similar. (C) Unilateral VTA–SNc-lesioned animal with a unilateral LoC lesion (DBH-stained neurons, green). Frontal section. (D) Song structure and syntax of the bird in (C) showed major changes; song syllable structure was degraded (syllables a′, c′, e′ and f′) and motif sequence had deletions (syllable b) and additions (syllable g; Supplementary audio S4). Scale bar, 0.25 mm.

FIG. 4

Social context-dependent regulation of egr-1 in sham-operated and VTA–SNc-lesioned animals. (A) Expression ratios in vocal nuclei relative to HVC in sham-operated birds (FD, n = 5; UD, n = 4). (B) Expression ratios in vocal nuclei relative to HVC in VTA–SNc-lesioned birds (FD, n = 6; UD, n = 5). Ratios > 1.0 indicate expression levels higher than HVC; ratios < 1.0 indicate expression levels lower than HVC. * P < 0.05, ** P < 0.001, *** P < 0.0001, t-test between FD and UD groups.

FIG. 5

Quantification of singing rate before and after VTA–SNc lesions. Singing was recorded 1 day before and 5–7 days after VTA–SNc lesions. Birds with the large LoC lesions were not included in this analysis. * P < 0.05, paired t-test.