Forebrain projections of arcuate neurokinin B neurons demonstrated by anterograde tract-tracing and monosodium glutamate lesions in the rat

Abstract

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor alpha (ERalpha). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERalpha and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH.

Figure 1

Line drawings of representative sections from control rats used for analysis of NKB-ir fiber density. Sections were matched to plates in a rat brain atlas listed at the top of each drawing (Swanson, 1992) and regions of interest (shaded areas) were outlined with the aid of the adjacent Nissl-stained sections. The distance from Bregma (mm) is shown in the lower left corner of each map. Scale bar in Plate 17 = 250 μm (applies to all).

Figure 2

Representative photomicrographs of adjacent sections in the mid-level of the arcuate nucleus in control (A, C, E) and MSG-treated (B, D, F) rats. (A, B), Methylene blue stain; (C, D, E, F), NKB-immunoreactivity with nickel intensified DAB. The boxes in C and D show the region illustrated in the high magnification photomicrographs of E and F. The Nissl stains from the MSG-treated rats revealed marked degeneration of neurons at most levels of the arcuate nucleus. Immunocytochemical stains confirmed the near-total loss of NKB-ir cells in the arcuate nucleus of MSG-treated animals. The high magnification photomicrographs illustrate loss of cell bodies and fibers in the arcuate nucleus and adjacent median eminence. The arrow in E points to an NKB-ir somata. Scale bar in A = 250 μm (applies to A and B), Scale bar in C = 200 μm (applies to C and D). Scale bar in E = 25 μm (applies to A-B).

Figure 3

Representative photomicrographs of NKB-immunoreactivity at the level of the premammillary arcuate nucleus in control (A) and MSG-treated (B) rats. Note the relative preservation of NKB-immunoreactive cell bodies at this level of the arcuate nucleus. Scale bar in A = 100 μm (applies to A and B).

Figure 4

Computer assisted drawings of NKB-ir neurons (filed circles) and fibers (lines) in control (left) and MSG-treated (right) rats at selected levels of the hypothalamus (A-D). Sections were matched to plates in a rat brain atlas listed in bottom right corner (Swanson, 1992). NKB-ir cells are depleted at most levels of the arcuate nucleus, with near total loss of NKB-ir fibers in the arcuate nucleus and the adjacent median eminence (C). NKB-ir fibers are also reduced in multiple hypothalamic areas (A-D). There is relative preservation of NKB-ir neurons at the level of the premammillary arcuate (D). Scale bars in B and D = 250 μm (applies to all).

Figure 4

Computer assisted drawings of NKB-ir neurons (filed circles) and fibers (lines) in control (left) and MSG-treated (right) rats at selected levels of the hypothalamus (A-D). Sections were matched to plates in a rat brain atlas listed in bottom right corner (Swanson, 1992). NKB-ir cells are depleted at most levels of the arcuate nucleus, with near total loss of NKB-ir fibers in the arcuate nucleus and the adjacent median eminence (C). NKB-ir fibers are also reduced in multiple hypothalamic areas (A-D). There is relative preservation of NKB-ir neurons at the level of the premammillary arcuate (D). Scale bars in B and D = 250 μm (applies to all).

Figure 5

Bar graphs showing quantitative analysis of NKB-immunoreactivity at multiple levels of the arcuate nucleus, median eminence and fiber tracts (A) and regions throughout the hypothalamus and adjacent areas (B) in control (gray bars) and MSG-treated (black bars) rats. Note the difference in scale between the two graphs indicative of the higher density of NKB-immunoreactivity the arcuate nucleus, median eminence and fiber tracts. Quantitative analysis revealed significantly reduced NKB-immunoreactivity in the arcuate nucleus, median eminence, fiber tracts and multiple hypothalamic areas of MSG-treated rats. n = 3 - 6 animals/group * significantly different from control, p ≤ 0.05.

Figure 6

Photomicrographs of BDA injection sites visualized by avidin-biotin-horseradish peroxidase histochemistry (A-D). The dotted lines indicate the border of the arcuate nucleus. The animal identification number is listed at the top of each photomicrograph. BDA injections were either confined to the arcuate nucleus (A, C, D) or included the arcuate nucleus and adjacent ventromedial nucleus (B). In one animal (D), the injection was midline and BDA was taken up in neurons in on both sides. Two injections (A and B) were at the mid-level of the arcuate nucleus and two (C and D) were in the posterior arcuate nucleus, corresponding to plates 28 (-2.45 from Bregma) and 31 (-3.70 from Bregma), respectively (Swanson, 1992). Scale bar in A = 200 m (applies to all).

Figure 7

A) Computer-assisted drawing showing the location of dual-labeled NKB/BDA-ir cell bodies (filled circles) and fibers (lines) near the injection site of R10 (see Fig. 6). This illustration was created by superimposing the tracings from 4 non-adjacent sections. The dotted lines indicate the borders of the arcuate nucleus. The arrow points to the location of the cell illustrated in B-D. Injection of BDA into the arcuate nucleus labeled a small number of NKB-ir neurons in the arcuate nucleus. Antereogradely labeled NKB-ir fibers were identified within the arcuate nucleus and adjacent median eminence including the lateral palisade zone (line). In addition, NKB/BDA-ir fibers were identified across the ME and within the arcuate nucleus of the contralateral side. (B-D) Demonstration of uptake of BDA in a NKB-ir neuron in the arcuate nucleus. High magnification photomicrographs of a NKB-ir cell body (B, green) in the arcuate nucleus that has taken up BDA (C, red). Color-combined image showing co-localization of NKB immunoreactivity and BDA (D, yellow). Scale bar in A = 100 μm. Scale bar in B = 10 μm (applies to B-D).

Figure 8

Confocal microscope projection image of close apposition of a NKB/BDA-ir fiber onto a NKB-ir cell in the arcuate nucleus. NKB immunoreactivity is green (A) and BDA is red (B). The arrows point to dual-labeled fibers (yellow) in close apposition to the somata and proximal dendrite of an NKB-ir neuron ipsilateral to the side of injection. The projection is a stack of seven 0.8 μm thick confocal images. Scale bar in A = 10 μm (applies to all).

Figure 9

High magnification photomicrographs of NKB (A and E, green) and BDA (B and F; red) immunofluorescence. The color-combined images (C and G) show that these NKB-immunoreactive axons are anterogradely labeled with BDA (yellow). The map in D shows the location of these dual-labeled axons. The top photomicrographs illustrate a thin, beaded anterogradely-labeled NKB-ir axon extending to the lateral ventral septal region (A, B and C, arrowheads). The bottom photomicrographs illustrate a dual-labeled axon in the medial preoptic region that is thicker and displays few varicosities (E, F, and G, arrowheads). The arrows show single-labeled fibers that are immunoreactive for either BDA (B and C) or NKB (E and G). Scale bar in A = 10 μm (applies to A-C and E-F). Scale bar in D = 200 μm.

Figure 10

Schematic diagram (horizontal plane) of arcuate NKB projections revealed by anterograde tract-tracing and NKB/dynorphin immunofluorescence (left) and MSG-ablation of arcuate NKB neurons (right). Left: The arrows indicate the location of arcuate NKB projections labeled by a BDA injection into the arcuate nucleus. The outlined areas show the where dual-labeled NKB/dynorphin-ir fibers were previously described (Burke et al., 2006). Right: The areas shaded in dark gray exhibited a significant reduction in NKB-ir fibers in MSG-treated animals. The areas shaded light gray exhibited a reduction of NKB-ir in MSG-treated animals that was not significantly different. The diverse projections of arcuate NKB neurons provide a mechanism to integrate the reproductive axis with multiple homeostatic and neuroendocrine circuits.