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. 2011 Feb 1;519(2):211-37.
doi: 10.1002/cne.22513.

Brain Expression and Song Regulation of the Cholecystokinin Gene in the Zebra Finch (Taeniopygia Guttata)

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Free PMC article

Brain Expression and Song Regulation of the Cholecystokinin Gene in the Zebra Finch (Taeniopygia Guttata)

Peter V Lovell et al. J Comp Neurol. .
Free PMC article

Abstract

The gene encoding cholecystokinin (Cck) is abundantly expressed in the mammalian brain and has been associated with such functions as feeding termination and satiety, locomotion and self-stimulation, the modulation of anxiety-like behaviors, and learning and memory. Here we describe the brain expression and song regulation of Cck in the brain of the adult male zebra finch (Taeniopygia guttata), a songbird species. Using in situ hybridization we demonstrate that Cck is highly expressed in several discrete brain regions, most prominently the caudalmost portion of the hippocampal formation, the caudodorsal nidopallial shelf and the caudomedial nidopallium (NCM), the core or shell regions of dorsal thalamic nuclei, dopaminergic cell groups in the mesencephalon and pons, the principal nucleus of the trigeminal nerve, and the dorsal raphe. Cck was largely absent in song control system, a group of nuclei required for vocal learning and song production in songbirds, although sparse labeling was detected throughout the striatum, including song nucleus area X. We also show that levels of Cck mRNA and the number of labeled cells increase in the NCM of males and females following auditory stimulation with conspecific song. Double labeling further reveals that the majority of Cck cells, excluding those in the reticular nucleus of the thalamus, are non-GABAergic. Together, these data provide the first comprehensive characterization of Cck expression in a songbird, and suggest a possible involvement of Cck regulation in important aspects of birdsong biology, such as perceptual processing, auditory memorization, and/or vocal-motor control of song production.

Figures

Figure 1
Figure 1
Camera lucida drawings of parasagittal brain sections from an adult male zebra finch at ~0.6 mm (A), and ~2.4 mm (B) from the midline. Rectangles in A and B indicate the positions of the photomicrographs in Figures 6 and 7D, respectively. A: Region over which optical densitometry for Cck mRNA and Cck cell counts were performed (presented in Figure 12) are depicted in grey for auditory NCM (region 1) and a non-auditory region of the medial striatum (region 2). B: The major nuclei of the song system that underlie vocal learning and singing behavior in songbirds are depicted in grey and black, respectively. The song system is composed of a direct motor pathway consisting of projections from HVC to RA, from RA to DM and motor neurons of the tracheosyringeal nucleus (nXIIts) of the medulla, or to brainstem nuclei involved in respiratory control (Bottjer and Arnold, 1984; Sohrabji et al., 1989; Vates et al., 1997; Wild et al., 1997), and an indirect pathway that includes a projection loop from Area X in the striatum to thalamic DLM (not depicted in Fig. 1B), from DLM to LMAN, and from LMAN back to area X (Bottjer et al., 1989; Luo et al., 2001; Vates et al., 1997). For abbreviations see Table 1.
Figure 2
Figure 2
The zebra finch Cck gene. A: The gene encoding Cck is represented by three exons (Ex1–3) that span a ~7 kb region of chromosome 2. At least three transcript variants of Cck (var1–3; 100% identical within their predicted peptide coding regions) are found in the zebra finch brain based on EST evidence; these differ according to the length of their 3’-untranslated region prior to a predicted poly-adenylation tag. A single clone representing Cck var1 (**; CK302967) was used to derive probes for all of the in situ hybridization data presented here; similar results were obtained using a clone representing the longer Cck var3 (CK314171). B: Alignment of the predicted amino acid sequence of zebra finch CCK protein with orthologs from chicken, human and mouse. Numbers on the right indicate the relative position of amino acid residues in each sequence; conserved amino acid residues for any position are in shaded in black. Arrows indicate the predicted cleavage sites for various CCK peptide products. A conserved tyrosine residue at position 122 of the zebra finch sequence, which is typically sulfanated, is indicated by an asterisk. The location of the carboxy-terminus of the peptide after final cleavage is indicated with an arrow.
Figure 3
Figure 3
Comparison of Cck mRNA expression in the rostromedial nidopallium as revealed by in situ hybridization of antisense (A, B) and sense (C, D) strand riboprobes targeting Cck variants 3 (A, C) and 1 (B, D) in serial parasagittal brain sections from an adult male zebra finch (~0.6 mm from the midline). Scale bar: 100µm.
Figure 4
Figure 4
Cck expression in serial parasagittal sections from an adult male zebra finch brain. Left panels in A–I are high-resolution photo-montaged images of in situ hybridization data in sections ~0.2 mm (A), ~0.6 mm (B), ~1.0 mm (C), ~1.4 mm (D), ~1.8 mm (E), ~2.2 mm (F), ~2.6 mm (G), ~3.2 mm (H), and ~3.6 mm (I) from the midline. The upper right panels in A–I are camera lucida drawings based on dark-field views of the sections shown in the left panels and superposition of cytoarchitectonic features seen on adjacent Nissl-stained sections. Rectangles in A–E indicate the relative positions of the drawing presented in Figures 8–11. Arrows in A and B denote the rostral tip of the lateral ventricle; asterisks in A and B indicate the possible location of an anterior olfactory area based on a comparison with the chick brain atlas. For anatomical abbreviations see Table 1. Scale bar: 1mm.
Figure 4
Figure 4
Cck expression in serial parasagittal sections from an adult male zebra finch brain. Left panels in A–I are high-resolution photo-montaged images of in situ hybridization data in sections ~0.2 mm (A), ~0.6 mm (B), ~1.0 mm (C), ~1.4 mm (D), ~1.8 mm (E), ~2.2 mm (F), ~2.6 mm (G), ~3.2 mm (H), and ~3.6 mm (I) from the midline. The upper right panels in A–I are camera lucida drawings based on dark-field views of the sections shown in the left panels and superposition of cytoarchitectonic features seen on adjacent Nissl-stained sections. Rectangles in A–E indicate the relative positions of the drawing presented in Figures 8–11. Arrows in A and B denote the rostral tip of the lateral ventricle; asterisks in A and B indicate the possible location of an anterior olfactory area based on a comparison with the chick brain atlas. For anatomical abbreviations see Table 1. Scale bar: 1mm.
Figure 4
Figure 4
Cck expression in serial parasagittal sections from an adult male zebra finch brain. Left panels in A–I are high-resolution photo-montaged images of in situ hybridization data in sections ~0.2 mm (A), ~0.6 mm (B), ~1.0 mm (C), ~1.4 mm (D), ~1.8 mm (E), ~2.2 mm (F), ~2.6 mm (G), ~3.2 mm (H), and ~3.6 mm (I) from the midline. The upper right panels in A–I are camera lucida drawings based on dark-field views of the sections shown in the left panels and superposition of cytoarchitectonic features seen on adjacent Nissl-stained sections. Rectangles in A–E indicate the relative positions of the drawing presented in Figures 8–11. Arrows in A and B denote the rostral tip of the lateral ventricle; asterisks in A and B indicate the possible location of an anterior olfactory area based on a comparison with the chick brain atlas. For anatomical abbreviations see Table 1. Scale bar: 1mm.
Figure 4
Figure 4
Cck expression in serial parasagittal sections from an adult male zebra finch brain. Left panels in A–I are high-resolution photo-montaged images of in situ hybridization data in sections ~0.2 mm (A), ~0.6 mm (B), ~1.0 mm (C), ~1.4 mm (D), ~1.8 mm (E), ~2.2 mm (F), ~2.6 mm (G), ~3.2 mm (H), and ~3.6 mm (I) from the midline. The upper right panels in A–I are camera lucida drawings based on dark-field views of the sections shown in the left panels and superposition of cytoarchitectonic features seen on adjacent Nissl-stained sections. Rectangles in A–E indicate the relative positions of the drawing presented in Figures 8–11. Arrows in A and B denote the rostral tip of the lateral ventricle; asterisks in A and B indicate the possible location of an anterior olfactory area based on a comparison with the chick brain atlas. For anatomical abbreviations see Table 1. Scale bar: 1mm.
Figure 5
Figure 5
Cck expression in the dorsolateral corticoid area and the piriform cortex. (A) Camera lucida drawing of a frontal section at the level of the caudal thalamus and hippocampus. Rectangles depict the locations of the photomicrographs shown in B and C. (B, C) High magnification dark-field views of emulsion autoradiography depicting Cck-labeled cells along the dorsolateral and ventrolateral telencephalon from the section depicted in (A). Based on a comparative analysis with the chick and pigeon brain atlases, these areas likely correspond to the external portion of the dorsolateral corticoid area (B) and the piriform cortex (C). Scale bars: 100 µm. For abbreviations see Table 1.
Figure 6
Figure 6
Cck expression in the hippocampal formation and periventricular stratum. (A) Camera lucida drawing of a parasagittal section depicting the caudomedial auditory telencephalon and hippocampal formation (location indicated in Fig. 1A). Rectangles indicate the location of the photomicrographs in B–D. (B) Detail view of Cck-labeled cells within the hippocampal formation in a parasagittal brain section; the dashed lines indicate the location of the ventricle and pial surface between the cerebellum and NCM. (C) Detail view of labeled cells along a narrow periventricular band beneath the ventral-most region of the parahippocampal area (APH, arrows) and the dorsal portion of the caudal mesopallium (CM, arrowheads); he approximate location of the ventricle is depicted by the dashed line. (D) Bright-field view of Nissl-stained cells in a parasagittal section immediately adjacent to that shown in C; arrows and arrowheads are at the same relative positions as the ones in C. Scale bars: 200 µm for B; 100 µm for C–D; For abbreviations see Table 1.
Figure 7
Figure 7
Cck expression in nidopallial and sub-pallial areas. (A) Camera lucida drawing of a parasagittal section through the telencephalon (similar level as in Fig. 4B). Rectangles indicate the locations of the photomicrographs in B and E. (B–G) Bright-field views of Cck positive cells within various portions of the nidopallium, and striatum in a parasagittal section. (B) Low power view of populations of Cck-expressing cells with high labeling intensity interspersed among cells with low intensity labeling in the medial intermediate nidopallium. (C) High power view of a cluster of small, round cells with high labeling intensity within the intermediate nidopallium. (D) High magnification view of labeled cells laterally within the nidopallial auditory shelf region ventral to HVC (location indicated in Fig. 4E); note cells are mostly absent within HVC. (E) Low magnification view of interspersed large and small cells with high labeling intensity within striatal song nucleus area X. Image was taken at the same magnification as the photomicrograph in B. (F–G) High magnification views of strongly-labeled small (F) and large (G) cells in area X. Scale bars: 100 µm for B and E, 100 µm for D, and 10 µm for C, F and G. For abbreviations see Table 1.
Figure 8
Figure 8
Cck expression in the thalamus. (A) Camera lucida drawing of a frontal section depicting some major nuclei in the thalamus (location indicated in the section on the top left inset). Rectangles denote the locations of the photomicrographs in B and D. (B) Dark-field view in a frontal section of emulsion autoradiography showing Cck expression restricted to the shell of DMA. Dashed lines denote the approximate boundary of the DMA core based on Nissl staining and define the midline. (C) High power bright-field view from a parasagittal section through the thalamus (location indicated in Fig. 4C) showing labeled cells within the shell of nucleus ovoidalis (Ov). The dotted lines denote the approximate boundaries of the Ovcore/Ovshell and tract (tOv), based on Nissl staining. (D) Dark-field view in a frontal section of emulsion autoradiography depicting Cck expression in nucleus rotundus (Rt). Scale bars: 100 µm. For other abbreviations see Table 1.
Figure 9
Figure 9
Cck expression within midbrain dopaminergic nuclei. (A, C) Camera lucida drawings from a sagittal brain section depicting dopaminergic cell groups in the midbrain (locations indicated in Fig. 4B and D, respectively). Rectangles indicate the location of the photomicrographs shown in B and D–E. (B, D–E) High magnification bright-field views depicting Cck-labeled cells in the ventral tegmental area (VTA; B), dopaminergic cell group A11 (D), and substantia nigra pars reticulata (SNr; E). Dashed lines depict the boundaries of oculomotor nucleus fibers (B) and of IsO (D). Scale bars: 100 µm. For other abbreviations see Table 1.
Figure 10
Figure 10
Cck expression in mesencephalic and metencephalic nuclei. (A, B) Camera lucida drawings of the midbrain from a parasagittal section (A, location indicated in Fig. 4A), a frontal section (B, location indicated by rectangle in the upper right inset). Rectangles indicate the locations of the photomicrographs shown in A’ and B’. (A’) Bright-field view in a frontal section of Cck-labeled cells in the dorsal raphe (DR). (B’) Dark-field view of emulsion autoradiography showing labeled cells within the median raphe nucleus (MnR). (C, C’) Low power bright-field views of parasagittal sections through the midbrain’s intercollicular complex (ICo) in adjacent Nissl-stained (C) and chromagen-reacted sections (C’) showing sparse Cck-labeled cells within shell-like regions outside of nuclei MLd and DM, but not within the core regions of these nuclei (core boundaries indicated by solid lines). (D) Camera lucida drawing of the pons from a parasagittal section (location indicated in Fig. 4E). Rectangle indicates the location of the photomicrograph shown in D’. (D’) Bright-field view of Cck-labeled cells in the principal trigeminal nerve nucleus (PrV). Scale bars: 100 µm. For abbreviations see Table 1.
Figure 11
Figure 11
Cck-expressing cells are non-GABAergic. (A–C) High power views of the nidopallium in a parasagittal section depicting Cck-positive cells (A; blue channel), Gad65-positive cells (B; green channel), and a merge of the two channels (C). Cell nuclei in A and B (red channel) were revealed by propidium iodide. (D) Camera lucida drawing of a parasagittal section (similar level as in Fig. 4E) showing detailed view of the thalamus and basal forebrain. Rectangle indicates the location of the photomicrographs shown in E–F. (E–G) Low power views of the reticular nucleus of the thalamus (Re) depicting Cck-labeled cells (E; blue channel), Gad65-positive cells (F; green channel), and a merge of the two channels (G). Arrowheads in G indicate examples of double-labeled cells. Scale bars: 10 µm for A-C; 100 µm for E–G. For abbreviations see Table 1.
Figure 12
Figure 12
Song stimulation induces Cck expression in NCM. (A) Schematic showing the design of the song stimulation experiment (see Materials and Methods for details); arrows indicate the time (in minutes) that females were sacrificed following the onset of song stimulation (see Materials and Methods for details). (B) Average optical density values from phosphorimager autoradiography representing Cck expression over NCM (top panel), and counts of Cck-expressing cells within NCM (bottom panel; black bars) and the striatum (white bars) measured 0, 30, 90, and 240 minutes after song stimulation onset. Asterisks denote values that were significantly different from the unstimulated control group (time 0) according to an ANOVA and post hoc t-tests. (C) Representative maps (from parasagittal sections at ~0.6 mm from the midline) showing Cck-expressing cells (black circles) that were detected in NCM by fluorescent in situ hybridization 0, 90, and 240 minutes after the onset of song stimulation.

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