Substance P immunoreactivity exhibits frequent colocalization with kisspeptin and neurokinin B in the human infundibular region - PubMed (original) (raw)

Substance P immunoreactivity exhibits frequent colocalization with kisspeptin and neurokinin B in the human infundibular region

Erik Hrabovszky et al. PLoS One. 2013.

Abstract

Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Overlapping distribution of Substance P, kisspeptin and neurokinin B immunoreactivities in the infundibular nucleus.

Histological sections from a 69-year-old man (A, E) and a 57-year-old postmenopausal woman (B, F) illustrate higher numbers and heavier labeling of substance P (SP)-immunoreactive (IR) neurons in the infundibular nucleus (Inf) of women vs. men. The comparative mapping of SP (B, F; rabbit SP antiserum), kisspeptin (KP; C, G) and neurokinin B (NKB; D, H) immunoreactivities using the ABC method and silver-gold-intensified nickel-diaminobenzidine chromogen on tissue samples of the same female individual reveals a considerable overlap in the distribution of the three neuronal phenotypes. Note that other brain regions including the internal globus pallidus (IGP; I-K), exhibit unique and often non-overlapping immunoreactivity patterns for the same three neuropeptides. This site contains a dense plexus of single-labeled SP-IR axons (I; rabbit antiserum) and thus, can be used in triple-immunofluorescent experiments as an internal method control for antibody specificity. In triple-immunofluorescent experiments, the IGP exhibited heavy SP (L; rat SP antibodies) and little KP (M) and NKB (N) signals, as expected from the similar results of immuno-peroxidase experiments (I–K). Scale bar in N corresponds to 180 µm in A-D, 42 µm in E-H, 77µm in I-K and 50µm in L-N.

Figure 2

Figure 2. Regional abundances of Substance P-immunoreactive perikarya in the Inf of men below (‘young’ group) and above (‘aged’ group) 50 years of age and of postmenopausal women.

The maximal number of immunoreactive cell bodies per 0.25mm2 counting area was determined with the aid of an ocular frame and used as the index of the regional neuron density. The abundance (regional density) of substance P-immunoreactive neurons defined this way is several-fold higher in postmenopausal women, compared with age-matched men, but does not differ significantly between men below (‘young’) and above (‘aged’) 50 years of age. *P<0.0005.

Figure 3

Figure 3. Triple-immunofluorescent localization of Substance P within kisspeptin-immunoreactive and neurokinin B-immunoreactive perikarya in the Inf.

Triple-immunofluorescent studies of Substance P (SP; green), kisspeptin (KP; red) and neurokinin B (NKB; blue) immunoreactivities in the human infundibular nucleus reveal a substantial degree of colocalization between the three neuropeptides. Examples of single-, double- and triple-labeled perikarya are indicated by arrowheads. For color-coding, see pie chart at the top of the figure. Low-power images (A–C) illustrate that varying subsets of immunoreactive somata (three-color arrowheads; whitish neuronal labeling) contain all three neuropeptides in the Inf of men (A, 64 ys) as well as women (B, 58 ys; C, 57 ys). Note the presence of many single- and double-labeled cell bodies, in addition to these triple-labeled perikarya. The pie chart illustrates the mean incidences of the different cell phenotypes (mean±SEM of 5 postmenopausal women; 777 labeled neurons). 16.5±6.9% of the immunolabeled somata (representing 25.1±7.7% of the NKB-IR and 30.6±8.8% of the KP-IR perikarya) are triple-labeled (white color). Note that the most abundant labeled cell phenotype (38.6±6.6%) co-contains KP and NKB and is devoid of SP (purple color). Scale bars=25µm.

Figure 4

Figure 4. Triple-immunofluorescent localization of Substance P within kisspeptin-immunoreactive and neurokinin B-immunoreactive fibers in the Inf and the infundibular stalk.

Representative confocal images (A, 83-year-old women; B, 69-year-old women; C, 64-year-old man) demonstrate that a subset of varicose peptidergic axons (indicated by the three-color arrows) in the Inf contain SP, KP and NKB, whereas the majority are single- or double-labeled (color-coded single and double arrows); arrowheads indicate labeled perikarya. The observation that colocalizations are only partial also provides an important endogenous method control for the triple-labeling technique. SP/KP fibers occur in yellow and SP/NKB fibers in turquoise color. High-power confocal images of quadruple-immunolabeled sections from the infundibular stalk (InfS; 83-year-old woman) in D-F illustrate a considerable degree of overlap between SP-IR, KP-IR and NKB-IR axon projections around portal capillary vessels. SP-IR axons often contain KP and NKB as well (D), suggesting the putative co-release of these neuropeptides into the hypophysial portal circulation. Orange pseudocolor in E labels GnRH-IR axons of the same field. Merged panel (F) reveals occasional appositions between SP-IR and GnRH-IR axons, which raise the possibility that SP influences GnRH neurosecretion at this site via axo-axonal interactions. In some of these appositions indicated by orange arrows the SP-IR axon is triple-labeled (SP/KP/NKB). For color-coding of the arrows and arrowheads, see pie chart at the top of Figure 3. Scale bars=25µm in A and 10µm in B-F.

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