Anatomical Organization of Urocortin 3-Synthesizing Neurons and Immunoreactive Terminals in the Central Nervous System of Non-Human Primates [ Sapajus spp.]

Abstract

Urocortin 3 (UCN3) is a neuropeptide member of the corticotropin-releasing factor (CRF) peptide family that acts as a selective endogenous ligand for the CRF, subtype 2 (CRF₂) receptor. Immunohistochemistry and in situ hybridization data from rodents revealed UCN3-containing neurons in discrete regions of the central nervous system (CNS), such as the medial preoptic nucleus, the rostral perifornical area (PFA), the medial nucleus of the amygdala and the superior paraolivary nucleus. UCN3-immunoreactive (UCN3-ir) terminals are distributed throughout regions that mostly overlap with regions of CRF₂ messenger RNA (mRNA) expression. Currently, no similar mapping exists for non-human primates. To better understand the role of this neuropeptide, we aimed to study the UCN3 distribution in the brains of New World monkeys of the Sapajus genus. To this end, we analyzed the gene and peptide sequences in these animals and performed immunohistochemistry and in situ hybridization to identify UCN3 synthesis sites and to determine the distribution of UCN3-ir terminals. The sequencing of the Sapajus spp. UCN3-coding gene revealed 88% and 65% identity to the human and rat counterparts, respectively. Additionally, using a probe generated from monkey cDNA and an antiserum raised against human UCN3, we found that labeled cells are mainly located in the hypothalamic and limbic regions. UCN3-ir axons and terminals are primarily distributed in the ventromedial hypothalamic nucleus (VMH) and the lateral septal nucleus (LS). Our results demonstrate that UCN3-producing neurons in the CNS of monkeys are phylogenetically conserved compared to those of the rodent brain, that the distribution of fibers agrees with the distribution of CRF₂ in other primates and that there is anatomical evidence for the participation of UCN3 in neuroendocrine control in primates.

Keywords: autonomic nervous system; corticotropin-releasing factor; corticotropin-releasing factor receptors; monkey brain; paraventricular nucleus of the hypothalamus; stress response.

Figure 1

Urocortin 3 immunoreactivity (UCN3-ir) fiber density scale. Bright field photomicrographs showing various UCN3-ir fiber and terminal projection densities: (A) low density [+], (B) moderate density [++], (C) high density [+++] and [−] indicating a complete absence of specific labeling. This scale was used as a reference to perform the fiber density evaluation presented in Table 3. Scale bar: 25 μm (A–C).

Figure 2

Sapajus prepro-UCN3 (ppUCN3) structure. (A) Nucleotide and amino acid (aa) sequences of the Sapajus prepro-hormone. Corresponding aa are shown below each codon. The numbers on the right indicate base position (normal text) or residue position (boldface). (B) Comparison of the primary structures of rat, monkey and human prepro-UCN3. Boxed residues indicate mismatches relative to the other two sequences. Sequences have been aligned for best fit. (C) Comparison of UCN3 residue sequences of Danio, Danio rerio; Fugu, Tetraodon nigroviridis; Xenopus, Xenopus laevis and tropicalis; Chicken, Gallus; Rat, Rattus norvegicus; Sapajus, Sapajus spp.; Human, Homo sapiens, Mouse, Mus musculus and Dog, Canis lupus. Boxed regions indicate residues that are shared among species.

Figure 3

Representative photomicrographs illustrating UCN3 immunostaining in the intermediate part of the lateral septal nucleus (LSI) and paraventricular hypothalamic nucleus, magnocellularpart, dorsal division (PaMD) regions of wild-type UCN3 (wt/UCN3) mice or “knockout” UCN3 (ko/UCN3) mice. (A) Bright field photomicrograph showing the LSI region of a wt/UCN3 mouse. (A′) Higher magnification of (A) UCN3-ir cells and fibers, indicated by black arrows. (B) Bright field photomicrograph showing the LSI region of a ko/UCN3 mouse. (B′) Higher magnification of the square area in (B). Note the absence of labeling. (C) Bright field photomicrograph revealing the PaMD region of a wt/UCN3 mouse. (C′) Higher magnification of the square area in (C) showing UCN3-ir cells and fibers, indicated by black arrows. (D) Bright field photomicrograph the PaMD region of a ko/UCN3 mouse. (D′) Higher magnification of the square area in (D). Note the absence of labeling. Scale bars: 100 μm (A,B); 50 μm (C,D); 20 μm (A′–D′).

Figure 4

Distribution of UCN3-ir cells in the central nervous system (CNS) of Sapajus spp. A series of bright field images showing representative regions that display the highest densities of UCN3-ir labeled cells. Nissl stained regions of (A) hypothalamus, (B) supraoptic nucleus, (C) amygdala. Dashed lines show nuclear boundaries. (A′,A″) Bright field photomicrographs demonstrating UCN3-ir neurons in the PaMD and juxtaparaventricular part of lateral hypothalamus (JPLH; B′,B″) in the SO and, (C′,C″) in the Me. Scale bars: (A,C) 200 μm; (B,A′,B′,C′) 100 μm; (A″–C″) 20 μm.

Figure 5

Localization of UCN3-ir and corticotropin-releasing factor (CRF)-ir labeled cells in the paraventricular nucleus of the hypothalamus (Pa) of Sapajus spp. (A) Confocal photomicrograph showing UCN3-ir cells located in the Sapajus Pa. (A′) Higher magnification of the square area in (A) demonstrating UCN3-ir labeled cell, indicated by the white arrow. (B) Confocal photomicrograph revealing CRF-ir cells in the region of Pa. (B′) Higher magnification of the square area in (B) showing CRF-ir labeled cells, indicated by the white arrowhead. Note the morphological difference between UCN3 (small neuron) and CRF (large neuron) labeled cells in the monkey Pa. Scale bars: (A,B) 50 μm; (A′,B′) 20 μm.

Figure 6

Distribution of UCN3 messenger RNA (mRNA) in the CNS of Sapajus spp. (A–C) A series of darkfield photomicrographs showing UCN3 mRNA expression (silver grains) in the median preoptic nucleus (MnPO), JPLH, PaMD, paraventricular hypothalamic nucleus,parvicellular part, dorsal division (PaPD) and medial amygdaloid nucleus (Me) regions; (A′–C′) bright field photomicrographs showing silver grains over Nissl-counterstained neurons at higher magnification. Scale bars: (A–C) 50 μm; (A′–C′) 10 μm.

Figure 7

UCN3 mRNA hybridization control in the CNS of Sapajus spp. A series of darkfield and bright field photomicrographs showing that when using a cRNA sense probe for prepro-UCN3, no labeled cells were hybridized. Scale bars: (A–C) 50 μm; (A′–C′) 10 μm.

Figure 8

Distribution of UCN3-ir fibers in the CNS of Sapajus spp. A series of bright field photomicrographs of selective innervation by UCN3-ir fibers in discrete regions of the monkey brain. (A–C) Are Nissl-stained sections of representative areas with overlays indicating the structure boundaries. (A′) Preoptic area; (B′) LSI (C′) ventromedial nucleus of the hypothalamus, dorsomedial part (VHMDM). (A″–C″) Are higher magnification photomicrographs of (A′–C′) showing UCN3-ir fibers and terminals. Please note UCN3-ir fibers and varicosities (black arrows) and the very dense amount of UCN3-ir fibers in LSI and VMHDM in (B′,C′). Scale bars: (A–C) 200 μm; (A′) 50 μm; (B′,C′) 100 μm; (A″–C″) 10 μm.

Figure 9

Distribution of UCN3-ir fibers in the hypophysis of Sapajus spp. (A) Photomontage of bright field photomicrographs of the entire Sapajus pituitary histology by H&E staining showing the clear distinction between the anterior lobe (AL), pars intermedia (PI) and posterior lobe (PL). (A′) Higher magnification of (A) demonstrating the boundaries between the AL, PI and PL. (B) Bright field photomicrograph showing UCN3-ir fibers in the posterior lobe. (B′) Higher magnification of the square area in (B) showing an UCN3-ir bundle of fibers, indicated by the black arrow; (C) Fluorescence photomicrograph of UCN3-ir fibers in the posterior lobe; (C′) higher magnification of the square area in (C) showing an UCN3-ir bundle of fibers, indicated by the white arrow. Scale bars: (A′,B) 100 μm; (B′,C′) 25 μm; (C) 200 μm.

Figure 10

Digital photomontage of the hypothalamo-neurohypophysial tract (hnt) of Sapajus spp. Note the UCN3-ir cells in the paraventricular nucleus of the hypothalamus and their stained UCN3 fibers leaving this group of cells in a descending pathway towards the median eminence (ME) and posterior hypophysis through the hypothalamic-pituitary tract, which is visualized bilaterally. Scale bar: 1000 μm.