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Endocrinology Vol. 148, No. 12 5884-5890
Copyright © 2007 by The Endocrine Society

Definition of Brainstem Afferents to Gonadotropin-Releasing Hormone Neurons in the Mouse Using Conditional Viral Tract Tracing

Rebecca E. Campbell and Allan E. Herbison

Centre for Neuroendocrinology and Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand 9001

Address all correspondence and requests for reprints to: Allan E. Herbison, Ph.D., Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand 9001. E-mail: allan.herbison{at}stonebow.otago.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH NEURONS ARE the final downstream neurons of the central neuronal network controlling fertility in all mammalian species. Defining the neural elements regulating GnRH neurons, the GnRH neuronal network is of fundamental importance to our understanding of how multiple internal and external cues modulate reproductive function. Taking the lead from the pioneering work of Sawyer and colleagues (1, 2), investigators over the last three decades have demonstrated that brainstem catecholamine-synthesizing neurons, and particularly those synthesizing norepinephrine (NE), are likely to be important components of the GnRH neuronal network (3, 4, 5). Neuroanatomical, lesioning, and electrical stimulation studies in several species have indicated that subpopulations of A1 and A2 NE neurons located in the ventrolateral medulla (VLM) and nucleus tractus solitarius (NTS) of the brainstem, and possibly the locus coeruleus (LC) (6), are components of the GnRH neuronal network (7, 8, 9, 10, 11, 12). Although there have been many postulated functions for NE in regulating the activity of GnRH neurons, the most consistent role across experiments and species has been that of stimulating GnRH neurons located in the rostral preoptic area (rPOA) to help evoke the LH surge (3, 5, 13).

Despite the apparent importance of NE inputs to GnRH neurons, it remains controversial whether NE neurons innervate GnRH neurons directly and from exactly which brainstem populations they arise. Conventional tract tracing studies have only limited ability to define whether specific inputs arising from a particular brain region make synapses with identified neurons. In the case of the GnRH neurons, there is abundant evidence that NE inputs from the brainstem terminate in the vicinity of the GnRH neuron cell bodies (7, 8, 9). However, to date, the only electron-microscopic evidence for NE terminals synapsing on GnRH neurons comes from a study in which rats pretreated with tritiated NE were found to have radioactively labeled boutons synapsing on GnRH neurons (14). Nevertheless, the more recent confocal visualization of dopamine-ß-hydroxylase (DBH) fibers apposed to GnRH neuron soma (15) and the presence of adrenergic receptor transcripts in mouse GnRH neurons identified through microarray profiling (16) suggest that NE inputs are direct to GnRH neurons. Where these direct inputs originate from within the various NE cell populations is unknown.

Recently, the development of novel, GnRH neuron-specific viral tract tracing methodologies have enabled the definition of primary and higher-order afferents within the GnRH neuronal network (17). One such technique harnesses the natural ability of a pseudorabies virus (PRV) to cross synapses in a strictly retrograde fashion. To trace specifically from GnRH neurons, a Cre-Lox approach has been used in which a PRV strain, engineered replication deficient in the absence of Cre recombinase (Cre), is coupled with a GnRH-Cre transgenic mouse line. With this approach, the presence of Cre within only GnRH neurons results in the excision of a stop-Flox sequence that then enables replication of the virus within GnRH neurons initiating retrograde tracing and the expression of GFP in each neuronal afferent. This provides a convenient and powerful methodology for tracing out the synaptic inputs to GnRH neurons in vivo (17). To date, two studies focusing on the olfactory (18) and estrogen-sensitive primary afferents to GnRH neurons (19) have reported on the GnRH neuronal network using this approach. Given the importance of brainstem catecholaminergic inputs in activating GnRH neurons, we aimed here to use this methodology to define the primary and higher-order brainstem afferent inputs to rPOA GnRH neurons in the mouse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Two independently generated GnRH-Cre mouse lines were used in the present study (18, 19). Female GnRH-Cre and wild-type littermates were housed under conditions of 12 h light (lights on at 0700 h) with ad libitum access to food and water. Cre-expressing transgenic mice were identified by PCR analysis of genomic DNA isolated from tail biopsies. The University of Otago Animal Welfare and Ethics Committee approved all experimentation.

Conditional PRV tract tracing
A conditional strain of PRV, Ba2001, was generated as described previously (20) and stored at –80 C until use. Our experimental protocol using this virus has been reported previously (19). After Avertin anesthesia, a single stereotaxic injection of Ba2001 (500 nl, 3.8 x 108 pfu/ml) was administered into the rPOA (coordinates: 0.5 mm bregma, 0 mm lateral, –5.1 mm dorsal-ventral) at a rate of 20 nl/min through a Hamilton syringe. The needle was left undisturbed for 5 min after injection. Control injections were made into wild-type mice and into the striatum of GnRH-Cre mice. PRV typically requires approximately 24 h to replicate and pass from a neuron into its afferents (21). To identify primary and higher-order afferent inputs, a time-course study was carried out by killing mice at various times after injection of PRV (24 and 30 h and 2, 3, 4, and 6 d). Mice were killed by pentobarbital overdose and perfusion of 4% paraformaldehyde through the heart. Brains were removed, postfixed in 4% paraformaldehyde for 1 h at room temperature, and then saturated in 30% sucrose in Tris-buffered saline overnight. Three sets of 30-µm-thick sections throughout the forebrain and brainstem were cut on a freezing sledge microtome in the coronal plane and processed for immunohistochemistry as described below.

Immunohistochemistry
Single labeling.
One set of one-in-three sections extending from the caudal aspect of the POA through to the caudal medulla was stained for green fluorescent protein (GFP) using a rabbit anti-GFP antibody (1:5000; Molecular Probes, Eugene, OR), followed by biotinylated antirabbit IgGs (1:400) and then the Vectastain ABC kit with DAB as the chromogen (Vector Laboratories, Burlingame, CA). The expression of GFP marks the location of neurons infected with PRV. Controls included the omission of the primary antibody. Sections were dehydrated through a graded series of ethanols and xylene and then coverslipped with DPX (Sigma, St. Louis, MO).

Dual-immunofluorescence labeling.
Every coronal section containing the medial septum and rPOA was dual-labeled for GnRH and GFP using a cocktail of rabbit anti-GnRH (LR1, 1:40,000, a kind gift of R. Benoit, Montreal, Canada) and chicken anti-GFP (1:2500; Chemicon, Temecula, CA) primary antibodies for 48 h (4 C) followed by biotinylated antirabbit IgGs (1:200; Vector) and antichicken fluorescein isothiocyanate (1:200; Jackson Immunolabs, West Grove, PA) for 2 h and then strepavidin-568 (1:200; Molecular Probes) for 2 h, both at room temperature. The two remaining one-in-three sets of brainstem sections were dual-labeled for GFP and tryptophan hydroxylase (TPH) or GFP and tyrosine hydroxylase (TH) using rabbit anti-GFP (1:5000; Molecular Probes) combined with sheep anti-TPH (1:2000; Chemicon) or monoclonal anti-TH (1:5000; Chemicon). This was followed by a cocktail of biotinylated antirabbit IgGs (1:200; Vector) and either antisheep rhodamine (1:200; Jackson Immunolabs) or antimouse 568 (1:200; Molecular Probes), respectively, followed by streptavidin 488 (1:200; Molecular Probes). Controls were undertaken by removing one of the primary antibodies in each of the cocktails but retaining all the secondary and tertiary reagents. Sections were coverslipped with Vectashield (Vector).

Analysis of immunocytochemistry
Single labeling.
GFP-immunoreactive (GFP-ir) neurons were initially mapped in every third section throughout the brainstem (3 d, n = 6; 4 d, n = 4; 6 d, n = 3). In brain regions where GFP-labeled cells were found, quantitative analyses were undertaken by counting GFP-ir cells in two representative sections of each brain region listed. Due to the large number of GFP-labeled cells encountered in the raphe nuclei, quantitative analysis was conducted by counting GFP-ir cells in the different divisions of the raphe at six different anteroposterior levels between plates –4.04 and –5.20 mm (22) in the one-in-three series. Because smaller numbers of GFP-ir cells were observed in the LC and NTS, quantification was undertaken by counting cells at all levels of these brain regions for the given time points. Data are given as mean + SEM and statistical comparison between 3- and 4-d measurements undertaken with ANOVA followed by post hoc Student-Newman-Keuls tests.

Dual-immunofluorescence labeling.
Every section throughout the rPOA of 24-h (n = 3), 30-h (n = 3), 2-d (n = 4) and 3-d (n = 3) post-infected brains was analyzed for GnRH/GFP double labeling using an Olympus BX51 epifluorescence microscope. Each GnRH neuron was examined at x40 magnification and scored for the presence or not of GFP-immunofluorescence. GFP/TPH double labeling was analyzed in every third section throughout the raphe nuclei, and GFP/TH double labeling was analyzed in every third section throughout the LC and NTS. To confirm colocalization, confocal microscopy was used. Stacks of confocal images were captured using x20 Plan Neofluar (numerical aperture 1.3) and x63 PlanApochromat (numerical aperture 1.4) objectives with x2 zoom function. A red helium-neon laser exciting at 633 nm was used to image the Texas Red fluorophore, and an argon laser exciting at 488 nm was used to image GFP. A series of images at 1.5- or 0.36-µm intervals throughout defined region were collected for analysis. Images are presented as projections of optical image stacks or as individual optical slices. The brightness and contrast of the images were adjusted in Photoshop (Adobe Systems, San Jose, CA) to match microscope visualization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of infection of GnRH neurons after Ba2001 injection
Two different lines of GnRH-Cre mice were used and gave identical results. At 24 h (n = 3) and 30 h (n = 3) after Ba2001 injection, no GFP-ir cells were detected in the brain. At 2 d (n = 4) after injection, GFP immunofluorescence was found exclusively in small numbers (between five and nine) of rPOA GnRH neurons (Fig. 1Go, A–C) immediately adjacent to the injection site in each animal (Fig. 1Go, D–F). No GFP-ir was identified in wild-type animals killed 2 d after receiving identical injections of Ba2001 (n = 2), and the removal of the GFP primary antibody resulted in a complete absence of staining.


Figure 1
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  		FIG. 1. Locations of GnRH neurons infected with PRV 2 d after injection. A–C, Confocal image showing a single GnRH neuron (A, red) immediately adjacent to the Ba2001 injection site (stippled line) colabeled with GFP (B, green) as indicated by yellow pixels in the overlay (C). Arrowheads indicate an uninfected GnRH fiber (red). Scale bar in C, 10 µm. D–F, Schematic representations of the location of the injection cannulae and infected GnRH neurons in a coronal 30-µm-thick section from three individual mice. Red dots indicate GnRH neurons, and yellow dots indicate infected GnRH neurons from where retrograde tracing will commence. AC, Anterior commissure; LS, lateral septum.

 
Time course of infection and distribution of labeled brainstem neurons after Ba2001 injection
At 3 d (n = 6) after Ba2001 injection, GFP-ir cells were identified for the first time outside of the GnRH neuronal population. At this time point, GFP-ir neurons were observed in the raphe nuclei, periaqueductal gray area (PAG), LC, NTS, and area postrema (AP) of the brainstem (Fig. 2Go, A–D). The numbers of GFP-ir cells identified in these areas was variable across the six mice; however, the highest density of GFP-ir cells was consistently detected in the dorsal raphe nuclei (DRN) (Fig. 2Go, A and B). There was no evidence for GFP-ir cells in the VLM of mice at 3 d. Wild-type mice (n = 2) with rPOA injections or GnRH-Cre mice (n = 2) with striatal injections of Ba2001 exhibited no GFP-ir cells in the brainstem.


Figure 2
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  		FIG. 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 d after infection. –, No cells; +, more than one cell in selected region but not seen in all animals; ++, one to three cells in selected region in all animals; +++, four or more cells in selected region in all animals. PRN, Pontine reticular nucleus; Bar, Barrington nucleus; MLF, medial longitudinal fasciculus. B–D, Photomicrographs showing representative images of GFP-ir neurons in the DRN (B), LC (C), and NTS (D). Scale bar in B–D, 75 µm.

 
At 4 d (n = 4) after injection, GFP-ir cells were found in the same brain regions observed in 3-d mice in addition to the pontine reticular nucleus and Barrington nucleus and a very few cells in the VLM. At 6 d (n = 3) after injection, the only new region identified to have GFP-ir cells was that around the medial longitudinal fasciculus. However, at 6 d, several of the brain areas previously expressing GFP-ir cells exhibited many fewer or even no labeled cells (Figs. 2AGo and 3Go, A–C).


Figure 3
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  		FIG. 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 d after Ba2002 infection. D and E, Mean (+SEM) number of GFP-ir cells counted at 3-, 4-, and 6-d time points in the median and paramedian raphe nucleus (D) and DRN (E) in brain sections corresponding to 0.2-mm intervals between –4.04 and –5.20 mm relative to bregma. *, P < 0.05 compared with d 3.

 
Within the raphe nuclei, GFP-ir cells were detected in the median and paramedian nuclei as well as the DRN, where they predominated (Fig. 3Go). For most regions, and at all anterior-posterior levels, the numbers of GFP-ir cells detected at 3 d was the same as that found at 4 d. The exceptions were the caudal aspect of the medial raphe (level –4.48) and rostral pole of the DRN (level –4.48) where significantly (P < 0.05) greater numbers of GFP-expressing cells were detected at 4 d compared with 3 d (Fig. 3Go, D and E). At 6 d, very few GFP-ir cells were detected anywhere within the raphe nuclei (Fig. 3Go).

A very similar time course of GFP expression was observed in the LC (Fig. 4AGo) and NTS (Fig. 4BGo) where low numbers of positive cells were detected at 3 and 4 d, and these were not found to be significantly different. GFP-ir cells were found scattered throughout the anterior-posterior extent of the LC and NTS. Although cells were absent from the NTS at 6 d, they remained present in the LC.


Figure 4
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  		FIG. 4. Time course of GFP expression in LC and NTS. A, Mean (+SEM) number of GFP-ir cells identified within the LC at 3, 4, and 6 d after infection. B, Mean (+SEM) number of GFP-ir cells identified within the NTS at 3, 4, and 6 d after infection.

 
Phenotypes of brainstem primary afferents to GnRH neurons
The GFP-ir cells identified at 3 d are likely to represent primary afferents to GnRH neurons. To establish the identity of these cells, dual-label immunofluorescence was undertaken in 3-d postinfection tissue from four mice. All GFP-ir cells identified throughout the median and paramedian raphe nuclei and DRN colocalized with TPH, the rate-limiting enzyme for serotonin production (Fig. 5Go, A and B). GFP-expressing cells in the PAG were not stained with TPH.


Figure 5
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  		FIG. 5. Neurochemical phenotype of primary afferent brainstem inputs to GnRH neurons. A and B, Low-power view of the DRN stained for TPH (red) and GFP (green). All GFP-expressing cells in this region were positive for TPH when viewed under confocal microscopy. Note that some cells express GFP so strongly that they appear green at low power and are found to also express TPH at high power only under the confocal microscope. C and D, Medium-power view of two GFP-expressing cells located in two different LC sections that were immunoreactive for TH. E–G, Low-power views of a single neuron located in the NTS that expressed both GFP (E) and TH (F). Scale bars, 15 µm.

 
Similar to the raphe nuclei, all GFP-ir cells identified within the LC were found to be colocalized with TH, the rate-limiting enzyme for catecholamine synthesis (Fig. 5Go, C and D). However, only approximately half of the GFP-ir cells located in the NTS were immunoreactive for TH (Fig. 5Go, E–G), whereas none of the GFP-ir cells located in the AP were positive for TH.

The positive dual-label results were confirmed using confocal microscopy to investigate colocalization of pixels in less than 0.36-µm-thick optical sections (Fig. 5Go). Controls in which primary antibodies were removed from the cocktail resulted in a complete absence of immunofluorescence.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These experiments have used the new approach of Cre-dependent PRV tracing to define the brainstem afferent inputs to rPOA GnRH neurons in the female mouse. Unexpectedly, we found that the largest brainstem input arises from serotonin neurons of the raphe nuclei; the numbers of inputs from the DRN, in particular, were at least 10-fold greater than those from any other region. We also provide evidence that NE neurons project directly to rPOA GnRH neurons and that they are, somewhat surprisingly, located in the NTS and LC but not the VLM. Although most of the brainstem primary afferents can be characterized as being catecholaminergic or serotoninergic, there remain small populations of neurochemically uncharacterized afferent neurons in the NTS and PAG.

The Cre-dependent PRV tracing approach used here can be a powerful tool for determining the afferent neuronal network of a specific neuronal phenotype (17). Greater than 95% of GnRH neurons express Cre in the two mouse lines used in this study, and within the rPOA, no other cells express Cre (18, 19). Hence, as demonstrated here, the injection of Ba2001 into the rPOA results in selective PRV replication within the GnRH neurons. An important consideration of this approach is that only those GnRH neurons immediately adjacent to the injection site express GFP, the marker of Ba2001 replication. Despite the 100-nl volume of the injection site, it is thought that a titer of virus sufficient to infect a neuronal cell body exists only immediately adjacent to the injection needle. In our hands, this results in retrograde tracing from less than 10 GnRH neurons in each animal. The advantage of this situation is that GnRH neurons in highly circumscribed areas can be targeted; in this case, we sought to trace out from the rPOA GnRH neurons because, on the basis of c-Fos staining, the great majority of these cells appear to be involved in the GnRH surge in the mouse (19). The disadvantage, however, is that it is very hard to target exactly the same GnRH neuron population in each animal, and as such, quantitative assessments are difficult. Equally, a negative finding may not hold true for the whole GnRH neuronal population because only a very few GnRH neurons have been targeted.

Studies undertaken with PRV in other neuronal networks indicate that it takes 24 h for PRV to move from one neuron to the next and that the distance traveled by the virus along the axon is not a major factor in determining this time course of infection (21, 23). At 2 d after Ba2001 injection, we observed that only GnRH neurons express GFP, whereas 24 h later at 3 d, neurons located in highly specific brainstem areas were positive for GFP. This suggests that neurons expressing GFP at 3 d are primary afferents to GnRH neurons.

The number of GFP-expressing cells was found to be dramatically reduced in most areas of the brainstem at 6 d after infection compared with earlier time points. Previous studies with PRV have found the same phenomenon, and this is thought to result from the clearance of infected cells by the immune system (24). Thus, at any time point after the initial infection of GnRH neurons with PRV, the resultant GFP expression is likely to reflect a balance between newly infected cells and those previously infected cells being phagocytosed by macrophages. Although there was a general trend throughout the brainstem for greater densities of GFP-expressing cells at 4 d compared with 3 d, this was found to be significant only for restricted serotoninergic neuron groups. Whereas it is very likely that GFP-infected brainstem cells are primary afferents, it is not possible to assess the degree to which 4-d GFP cells in the same area are new second-order neurons or existing primary afferents. Only three regions were detected for which a sudden appearance of GFP-expressing cells occurred at 4 d: the pontine reticular nuclei, Barrington nucleus, and VLM. These neurons are likely to be second-order afferents within the GnRH neuronal network, although which neurons they project to themselves cannot be determined. We speculate that the Barrington nucleus cells may be innervating LC neurons because it is well characterized to provide direct inputs to the LC, and these afferents are thought to be involved in their activation after stressful stimuli (25). It is also possible that the very few VLM neurons detected in 4-d mice are projecting to NTS primary afferents because reciprocal connections between A1 and A2 neurons are known to exist in the rat (26).

Whereas there is general agreement that NE has a role in regulating LH secretion (13), the neuroanatomical relationship between NE terminals and GnRH neurons has remained controversial. The evidence presented here indicates that specific brainstem NE neurons project directly to rPOA GnRH neurons in the mouse. This result is in accord with previous studies in the mouse showing that DBH-immunoreactive terminals are in close apposition to GnRH cell bodies (15) and that GnRH neurons express specific {alpha}- and ß-adrenergic receptor subunit mRNAs (16). Thus, it is very likely that brainstem NE inputs regulate rPOA GnRH neuron excitability directly and that the source of these inputs is the A2 and A6 NE cell populations in mice. The absence of evidence for direct inputs from the A1 was not expected but may result from our tracing from less than 10 GnRH neurons in any one animal. Tract-tracing studies in the rat have demonstrated that NE inputs to the vicinity of the rPOA GnRH neurons arise from the A1 and A2 populations (7, 8) with only a small contribution from the A6 to the hypothalamus (27). In contrast, studies in the sheep indicate that NE inputs to the vicinity of the GnRH neuron cell bodies in the rPOA arise predominantly from A1 and A6 neurons (9, 10). Thus, it seems likely that species differences exist in the neuroanatomical organization of ascending NE inputs within the GnRH neuronal network. The functional consequences of this are unknown. For example, despite the A1-A2 differences between rats and sheep, estradiol is able to impact directly upon putative NE afferents to GnRH neurons in both species (7, 10, 28). The estrogen receptor expression profile of NE neurons has yet to be established in the mouse. However, the LC and NTS are known to contain cells expressing estrogen receptor {alpha} and ß, respectively, (29), and estradiol regulates TH gene expression in the A6 in this species (30).

The involvement of the A6 NE cells of the LC in the GnRH neuronal network of the mouse is noteworthy. These neurons are proposed to have key roles in the integration of sensory information relevant to wide-ranging neuronal networks within the forebrain (31). Thus, LC neurons may play a central role in integrating and transmitting information about the environment within the GnRH neuronal network. Although nothing is known about the role of NE neurons in regulating LH secretion the mouse, studies in the rat suggest that LC inputs are involved in the surge mechanism because lesions decrease the NE content in the POA and block the preovulatory LH surge (6, 32).

The most surprising observation of this study has been the definition of a very large primary afferent input from raphe serotoninergic neurons to GnRH neurons. Previous neuroanatomical studies in rats and sheep have indicated that raphe serotonin neurons project to the POA (33, 34) and an electron-microscopic study in male rats suggested that up to 5% of synapses on GnRH dendrites were serotoninergic (35). However, pharmacological studies have provided contradictory results when investigating the physiological role of serotonin in the control of LH secretion (for review see Ref. 36). Alongside the gonadal steroid dependency of serotonin action on LH secretion, typical of many neurotransmitter systems (4), evidence has been presented for both inhibitory and facilitatory effects of serotonin on the LH surge mechanism (36). The reasons for this discrepancy are not clear, but there is recent evidence that adult and embryonic GnRH neurons express a variety of different serotonin receptor subtype mRNAs (16, 37) and that the effects of serotonin on embryonic GnRH neurons are dose dependent (37). Equally, it has been suggested that serotonin neurons located in different parts of the raphe complex may be responsible for differing actions upon the GnRH neurons (38). Furthermore, serotonin is likely to act both directly and indirectly within the network to modulate GnRH neuron activity (39). As such, the intracerebroventricular or parenteral administration of serotonin-active compounds would have little prospect of defining the role of specific serotoninergic pathways in regulating GnRH neuron excitability. Although we have found evidence for serotonin inputs from the dorsal as well as the median raphe nuclei, it is the former that predominates to a very large extent. The observation here of a major serotoninergic primary afferent input to GnRH neurons in the mouse suggests that a careful reevaluation of the physiological role of serotonin in the control of fertility is necessary.

In summary, we report here that the brainstem provides multiple monoaminergic primary afferent inputs to the GnRH neurons in the mouse. The NE inputs derive from the A2 and A6 cell groups, whereas a substantial serotonin input originates principally from the DRN. These observations, made possible through the use of a GnRH neuron-selective retrograde tracing technique, provide evidence that NE and serotonin regulate the activity of GnRH neurons in the rPOA in a direct manner. Such findings provide a neuroanatomical framework for the detailed evaluation of the physiological roles of NE and serotonin in the regulation of GnRH neuronal activity in the mouse.


   Acknowledgments
 
We thank Prof. Lynn Enquist (Princeton University) for expert advice and help with Ba2001 and Ms. Zhi-Yi Ong for technical assistance.


   Footnotes
 
This work was supported by The Wellcome Trust (United Kingdom), The Marsden Fund (New Zealand), and the Foundation for Research Science and Technology (New Zealand).

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 6, 2007

Abbreviations: AP, Area postrema; DBH, dopamine-ß-hydroxylase; DRN, dorsal raphe nuclei; GFP, green fluorescent protein; GFP-ir, GFP-immunoreactive; LC, locus coeruleus; NE, norepinephrine; NTS, nucleus tractus solitarius; PAG, periaqueductal gray; PRV, pseudorabies virus; rPOA, rostral preoptic area; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; VLM, ventrolateral medulla.

Received June 26, 2007.

Accepted for publication August 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. B. Roberts and K. J. Suter
Emerging methodologies for the study of hypothalamic gonadotropin-releasing-hormone (GnRH) neurons
Integr. Comp. Biol., November 1, 2008; 48(5): 548 - 559.
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