Definition of brainstem afferents to gonadotropin-releasing hormone neurons in the mouse using conditional viral tract tracing

Abstract

Brainstem monoamines have long been considered to play a role in regulating the activity of GnRH neurons, although their neuroanatomical relationship with these cells has remained unclear. Using a Cre-dependent pseudorabies virus (Ba2001) technique that permits retrograde tracing selectively from GnRH neurons in the mouse, we have examined the organization of brainstem inputs to rostral preoptic area (rPOA) GnRH neurons. Two days after injection of Ba2001 into the rPOA of adult female GnRH-Cre transgenic mice, five to nine GnRH neurons located immediately adjacent to the injection site were found to express green fluorescent protein (GFP), the marker of virus infection, with no GFP expression anywhere else in the brain. In mice killed 24 h later (3 d after injection), GFP-expressing cells were identified (in order of density) in the raphe nuclei, periaqueductal grey, locus coeruleus, nucleus tractus solitarius, and area postrema. This time course is compatible with these neurons representing primary afferent inputs to the GnRH neurons. Four and 6 d after Ba2001 injection, GFP-expressing cells were found in additional brain regions. Dual-label immunofluorescence experiments in 3-d postinjection mice demonstrated that 100% of GFP-expressing neurons in the raphe were positive for tryptophan hydroxylase, whereas 100% and approximately 50% of GFP neurons in the locus coeruleus and nucleus tractus solitarius, respectively, expressed tyrosine hydroxylase. These observations demonstrate that rPOA GnRH neurons receive direct projections from brainstem A2 and A6 noradrenergic neurons and that, surprisingly, the largest afferent input from the brainstem originates from raphe serotonin neurons in the mouse.

Figure 1. Locations of GnRH neurons infected with PRV 2 days after injection.

A-C. Confocal image showing a single GnRH neuron (A, red) immediately adjacent to the Ba2001 injection site (stippled line) co-labeled with GFP (B, green) as indicated by yellow pixels in overlay (C). Arrowheads indicate an uninfected GnRH fiber (red). Scale bar in C represents 10µm. D-E. Schematic representations of the location of the injection cannulae and infected GnRH neurons in a coronal 30µm-thick section from three individual mice. Red dots indicate GnRH neurons, yellow dots indicate infected GnRH neurons from where retrograde tracing will commence. AC, anterior commisure; LS, lateral septum; rPOA, rostral preoptic area.

Figure 2. Locations of brainstem afferents to GnRH neurons.

A. Table indicating locations and relative densities of GFP labeled cells in regions of the brainstem 3, 4 and 6 days following infection. –, no cells; +, >1 cell in selected region or not seen in all animals; ++, >1 cell in selected region in all animals; +++, ≥4 cells in selected region in all animals. AP, area postrema; Bar, Barrington nucleus; DRN, dorsal raphe nucleus; LC, locus coreuleus; MLF, medial longitudinal fasiculus; NTS, nucleus tractus solitarius; PAG, periaqueductial grey; VLM, ventrolateral medulla. B-D. Photomicrographs showing representative images of GFP-ir neurons in the DRN (B), LC (C) and NTS(D). Scale bar in B-D represents E-G 75μm.

Figure 3. Time-course of GFP expression in the raphe nuclei.

A-C. GFP-ir cells in the dorsal raphe nucleus (level -4.72) at 3, 4 and 6 days following Ba2002 infection. D,E. Mean (+ SEM) number of GFP-ir cells counted at 3,4 and 6 day time points in the median and paramedian raphe nucleus (D) and dorsal raphe nucleus (E) in brain sections corresponding to 0.2mm intervals between -4.04mm and -5.20mm relative to bregma. * p<0.05 compared to d3.

Figure 4. Time course of GFP expression in locus coreuleus (LC) and nucleus tractus solitarius (NTS).

A. Mean (± SEM) number of GFP-ir cells identified within the LC at 3, 4 and 6 days post infection. B. Mean (± SEM) number of GFP-ir cells identified within the NTS at 3, 4 and 6 days post infection.

Figure 5. Neurochemical phenotype of primary afferent brainstem inputs to GnRH neurons.

A,B. Low-power view of the dorsal raphe nucleus stained for tryptophan hydroxylase (TPH, red) and GFP (green). All GFP-expressing cells in this region were positive for TPH when viewed under confocal. Note that some cells express GFP so strongly that they appear green at low-power and are only found to also express TPH at high-power under the confocal. C,D. Medium-power view of two GFP expressing cells located in two different LC sections that were immunoreactive for tyrosine hydroxylase (TH). E-G, Low-power views of a single neurons located in the NTS that expressed both GFP (E) and TH (F). Scale bars represent 15μm.