help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-1796
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bingham, B.
Right arrow Articles by Viau, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bingham, B.
Right arrow Articles by Viau, V.
Endocrinology Vol. 149, No. 7 3581-3591
Copyright © 2008 by The Endocrine Society

Neonatal Gonadectomy and Adult Testosterone Replacement Suggest an Involvement of Limbic Arginine Vasopressin and Androgen Receptors in the Organization of the Hypothalamic-Pituitary-Adrenal Axis

Brenda Bingham and Victor Viau

Neuroscience Program, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

Address all correspondence and requests for reprints to: Victor Viau, Department of Cellular and Physiological Sciences, Life Sciences Centre, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. E-mail: viau{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Testosterone exposure during critical periods of development exerts major organizing effects on the hypothalamic-pituitary-adrenal (HPA) axis. Here we examined how neonatal gonadectomy (GDX) with or without testosterone treatment during the first week of life alters the HPA response to adult testosterone replacement in 65-d-old male rats. As adults, neonatal GDX rats showed higher levels of plasma corticosterone and Fos activation in medial parvocellular part of the paraventricular nucleus of the hypothalamus under basal conditions and during 30 min of restraint exposure. These responses were normalized with testosterone treatment on postnatal d 1–5 but were not restored with adult testosterone replacement. As adults, neonatal GDX rats also showed a decrease in the number of androgen receptor and arginine vasopressin-positive cells in the bed nucleus of the stria terminalis and in the medial nucleus of the amygdala, and both of these responses were reversed with postnatal testosterone treatment. In stressed and unstressed animals, the number of androgen receptors and arginine vasopressin-expressing neurons in both of these nuclei correlated negatively with corticosterone concentrations in plasma and Fos levels in the paraventricular nucleus. Taken together, our findings suggest that testosterone exposure during the neonatal period primes the adult HPA response to testosterone by altering androgen receptor levels and function within afferent mediators of basal and stress-related input to the HPA axis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL PROCESSES influence the maturation and the response of the hypothalamic-pituitary-adrenal (HPA) axis. Manipulations that alter environmental variables, maternal-infant relationships, and sex steroid hormone levels during critical periods of development permanently modify the HPA axis. Alterations in androgen exposure and its active metabolites over the perinatal period in the male rat have been shown to exert remarkable changes in brain morphology and behavior as well as pituitary-adrenal function during adulthood (reviewed in Refs. 1, 2, 3, 4).

Levine and Mullins (5) were one of the first to report effects of neonatal testosterone and estrogen treatment on the corticosterone response to stress in the adult rat. Since then, several studies have demonstrated that testosterone and its conversion to estrogen act during different periods of development to regulate multiple components of the HPA system. For example, male rats that are gonadectomized within 16 h of birth show increased corticosterone release as adults in response to restraint (4). This elevated adrenal response is attenuated with neonatal but not with adult testosterone replacement, indicating that neonatal androgens determine how the HPA axis responds to testosterone later in life. Furthermore, adult male rats show higher levels of CRH and arginine vasopressin (AVP) mRNA in the paraventricular nucleus of the hypothalamus (PVN) when treated with the androgen receptor (AR) antagonist flutamide or the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) between d 13 of gestation and postnatal day (6). These treatments also increase the mean level and amplitude of corticosterone release under basal conditions and the HPA response to auditory stress and lipopolysaccharide administration.

Despite the wealth of studies showing that the neonatal sex steroid hormone environment plays an important role in organizing the HPA axis, where this occurs in the brain remains poorly understood. Our connectional studies indicate that the AR and the estrogen receptor β-subtype are not distributed within PVN cells directed at the median eminence (7). This raises the possibility that the central organizing influences of neonatal androgens are mediated outside the PVN. Notably, androgen and estrogen receptors are concentrated within several regions of the brain identified as regulating the HPA axis or projecting to the PVN region (8, 9, 10), including within the bed nuclei of the stria terminalis (BST) and the medial nucleus of the amygdala (MeA). Compared with their neuroendocrine counterparts in the PVN, AVP projections of the BST and MeA are extremely and uniquely sensitive to adult testosterone levels (11, 12) and appear to exert an inhibitory influence on the neuroendocrine hypothalamus, including the PVN (reviewed in Ref. 9). Thus, the central inhibitory effects of testosterone on the HPA axis could involve some components of the limbic-AVP system. All the more intriguing is that AVP expression in the posterior BST and MeA as well as its regulation by testosterone in adults depend on androgen exposure during the first week of life (reviewed in Refs. 11 , 13 , and 14).

Based on these findings, we hypothesized that the organizing effects of neonatal testosterone exposure could involve a shift in the capacity of neurons to express ARs and/or respond to adult levels of testosterone, particularly within brain nuclei projecting to or regulating the HPA axis. To examine this possibility, we tested the effects of neonatal gonadectomy (GDX) with or without testosterone treatment during the first week of life on HPA function in adult animals bearing similar levels of testosterone replacement. Central AVP and AR expression levels were also assessed in the same animals to determine the impact of neonatal GDX on AR function as well as to reveal potential sites of testosterone’s upstream and organizational influences on the HPA axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Postnatal gonadectomy and testosterone treatment
Sprague Dawley rats were mated at the South Campus Animal Care Facility at the University of British Columbia, where they were housed in a controlled environment with a 12-h light, 12-h dark cycle, lights on at 0600 h, with food and water available ad libitum. Dams were preconditioned to pup removal by exposing them to a piece of latex glove laced with alcohol and Vetbond every day of gestation. On the day of birth, rat pups were subjected to sham GDX (neo-sham-GDX), GDX (neo-GDX), or GDX with testosterone treatment (neo-GDX + T). Two experimental male pups were used from each litter, one kept on a heating pad in a cage until surgery and the other pup anesthetized with isoflurane without added heat supplementation. Incisions were made between the umbilicus and prepuce, and each testicle was brought out and clamped with a hemostat. Heated forceps were then used to cauterize the blood supply to the testicle, and the muscle layer was closed with 5-0 absorbable sutures, closing the skin with a drop of Vetbond tissue glue. Sham surgeries involved all the same procedures as the GDX surgeries, excluding testes removal. On postnatal d 1, 3, and 5, sham-operated pups received daily injections of 0.05 ml sesame oil (neo-sham-GDX), GDX rats received an injection of sesame oil either alone (neo-GDX) or with 500 µg testosterone propionate (neo-GDX + T), as previously described (15). Animals were weaned at 21 d of age and delivered to our animal housing facility, where they were housed two per cage under controlled temperature and lighting conditions (12-h light, 12-h dark cycle; lights on at 0600 h), with food and water available ad libitum. Rats were weighed every other day for 30 d before testing to monitor their health and to habituate the animals to the experimental conditions.

We are aware that pup litter environment plays an instrumental role in brain development. Unlike the majority of previous studies employing neonatal GDX ± T procedures, we adopted a more conservative approach to minimize litter disruption and to control for between-litter effects in maternal care (16) and nutritional load (17). First, only two male rats per litter were experimentally manipulated. Second, total litter size in all cases was maintained at 12 pups per litter (two experimental, five male, and five female littermates). Third, litters remained intact and undisturbed until weaning at 21 d of age, and control, sham, and GDX ± T males were then pair housed for the remainder of the experiments. Finally, each study used a minimum of six litters per treatment and employed one experimental animal per litter in any given test as a single measure, empirically controlling for litter effects (18). To test the potential impact of handling and surgery on our results, we included in some of our studies animals from a subset of litters that were never handled. We found no significant differences in CRH mRNA expression levels in the PVN and no significant differences in restraint-induced plasma corticosterone levels between neo-sham-GDX and unhandled control animals. Pilot studies on maternal behavior, including temporal assessments of passive, arch-back and blanket nursing, licking and grooming, and maternal separation (time away from pups), indicated no obvious between-litter differences in maternal care.

Adult testosterone replacement
To unmask effects of neonatal androgen exposure on adult responses to testosterone, all rats received equivalent levels of testosterone replacement at 45 d of age. Testes from adult animals that were sham-GDX as neonates were removed under ketamine-xylazine-acepromazine anesthesia (administered sc, 77:1.5:1.5 mg/ml, respectively, 1 ml/kg). Each testis was delivered through the scrotal incision and exteriorized by severing the vas deferens and spermatic artery and ligated to maintain hemostasis. GDX was completed by closing the scrotal incision with 4-0 nonabsorbable sutures. As adults, neo-GDX and neo-GDX + T-treated animals were likewise subjected to ketamine-xylazine-acepromazine anesthesia but with sham GDX surgery. All animals received two sc SILASTIC brand (Dow Corning, Midland, MI) capsule implants (inner diameter 1.57 mm, outer diameter 3.18 mm, length 3.5 cm) packed with crystalline testosterone designed to deliver stable, adult levels of plasma testosterone (19).

Tissue and blood collection
On d 20 of adult testosterone replacement, 65-d-old rats were anesthetized with a lethal dose of chloral hydrate (35% wt/vol, 1 ml per 100 g body weight, ip) for perfusion, either immediately after removal from the home cage or at the end of a single 30-min exposure to restraint. As verified by corneal, pedal, and tail-pinch reflexes, deep anesthesia was reliably achieved within 45–60 sec of chloral hydrate administration. Blood samples were obtained by tail nick immediately after the rat was placed in a restrainer (6.3 x 15 cm Plexiglas restrainer; Kent Scientific, Litchfield, CT), at 30 min of restraint exposure, and/or via the right auricle just before perfusate delivery. Blood samples collected in ice-chilled EDTA-treated Eppendorf tubes (3.75 mg EDTA/100 µl blood) were centrifuged at 3000 x g for 20 min and stored at –80 C until assayed.

Animals were perfused via the ascending aorta with ice-cold 0.9% saline (125 ml), followed by 500 ml ice-cold 4% paraformaldehyde (pH 9.5). The brains were postfixed for 4 h in a solution of the same fixative and cryoprotected in 15% sucrose in 0.1 M potassium PBS (KPBS, pH 7.4) overnight at 4 C. Five adjacent 1-in-5 series of 30-µm-thick frozen sections were collected and stored in cryoprotectant (30% ethylene glycol and 20% glycerol in 0.05 M KPBS buffer) at –20 C until processing. Adjacent series of tissues from each animal were used for in situ hybridization and immunohistochemical analyses. In all cases, one series was counterstained with thionin and alternately compared with dark- and bright-field illuminations for morphological and anatomical reference.

Plasma hormones
Plasma testosterone (25 µl) and corticosterone (5 µl) were measured using commercial RIA kits (MP Biomedicals, Costa Mesa, CA). For corticosterone, the plasma samples were diluted 1:100 and 1:200 for prestress and poststress time intervals, respectively, to render hormone detection within the linear part of the standard curve. The intra- and interassay coefficients of variation for these assays ranged from 4–6 and 10–12%, respectively. 125I-labeled ligands were used as tracer in both cases. The testosterone antibody cross-reacts 100% with testosterone and slightly with 5{alpha}-dihydrotestosterone (3.40%), 5{alpha}-androstane-3β,17β-diol (2.2%), and 11-oxotestosterone (2%). The standard curve ED50 for the testosterone RIA was 1.2 ng/ml, with a detection limit of 0.1 ng/ml. The corticosterone antibody cross-reacts 100% with corticosterone and slightly with desoxycorticosterone (0.34%), and testosterone and cortisol (0.10%). The standard curve ED50 for the corticosterone RIA was 15 µg/dl, with a detection limit of 0.2 µg/dl. A freezer breakdown after these steroids were assayed prevented us from measuring plasma ACTH.

Fos immunohistochemistry
Cellular activation of the PVN under basal conditions and during restraint was determined by Fos-ir detection within the medial parvocellular part of the nucleus. Based on our previous time course studies, the 30 min poststress interval is optimal for detecting, within individual animals, both relative differences in stress-induced indices of HPA function and intervening levels of Fos in PVN attributable to differences in gonadal status (20). Fos-ir was detected using a standard avidin-biotin-immunoperoxidase procedure (Vectastain Elite ABC kit; Vector Laboratories, Burlington, CA) to localize a primary antiserum (1:40000) raised against amino acids 4–17 of human Fos protein (Oncogene Research Products, Boston, MA). As previously described (21), specific staining was abolished by preabsorbing primary antiserum with 50 µM synthetic Fos (Oncogene Research Products). An observer blind to treatment status took Fos-ir cell counts in regularly spaced (150 µm) intervals through the extent of the PVN. Positive cells were identified as those expressing a black nuclear reaction product. Analyses of the number of cells recruited to express Fos protein in the medial parvocellular, dorsal part of the PVN was assisted by redirected sampling of an adjacent series of thionin stained tissue for anatomical reference. Cell number estimates were generated by counting bilaterally the number of Fos-positive cells through three levels of the medial parvocellular cell population located immediately adjacent to the laterally displaced posterior magnocellular cell group, averaged by dividing the total number of cell counts by slice number, corrected for double counting errors (22, 23), and multiplying this product by a factor of five to account for slice frequency (one in five sections). Results represent estimates of the total number of Fos-positive cells through the medial parvocellular, dorsal part of the PVN.

AR immunohistochemistry
AR-immunoreactivity (AR-ir) was localized through several HPA-regulating brain regions, including the medial preoptic nucleus (MPN) and the CA1 region of the hippocampus, in addition to the posterior division of the BST and the posterodorsal part of the MeA. The CA1 region of the hippocampus was chosen as a negative control, because AR expression in this region appears to vary as a function of adult but not neonatal levels of testosterone (24).

Regions of interest were analyzed in tissues obtained under basal conditions and after acute restraint exposure so that correlations could be made between AR-ir cell number and HPA function. Because all of the animals were replaced with static levels of testosterone replacement as adults, we expected AR (as well as AVP in the BST and MeA) to change as a function of neonatal testosterone but not as a consequence of acute restraint exposure.

AR-ir was detected using a standard avidin-biotin-immunoperoxidase procedure to localize a primary antiserum (1:8000) raised against amino acids 1–20 of the rat AR (Santa Cruz Biotechnology, Santa Cruz, CA). Control experiments, in which the primary antiserum to AR was preadsorbed for 24 h at 4 C with 6.7 µM (10-fold excess) synthetic peptide immunogen, corresponding to N-terminal amino acids 1–21 or N-terminal amino acids 2–21 of the rat AR, failed to yield any evidence of specific AR staining (7). Additional control experiments for antisera cross-reactivity, involving the omission of either primary or secondary antibody, yielded no specific labeling (10).

Light-level images of AR-positive neurons were counted bilaterally on an equal number of sections per region of interest per animal. The planes of the sections were standardized according to the atlas of Swanson (25) and assisted by the morphological features provided by thionin staining of adjacent series of tissue. Areas to be measured were outlined using a standard frame for each of the following regions (26): posterior division of the BST (0.67 mm2), posterodorsal part of the MeA (0.42 mm2), MPN (0.26 mm2), and CA1 region of the hippocampus (0.10 mm2). Final estimates of the total number of AR-positive cells were generated as described for Fos detection.

Hybridization histochemistry
cRNA probes were used to determine the relative expression levels of CRH and AVP mRNA in the PVN, AVP mRNA in the posterior BST and medial nucleus of the amygdala, and AR mRNA in the posterior BST, medial nucleus of the medial amygdala, and MPN. Hybridization histochemistry was carried out using [33P]UTP-labeled (GE Healthcare Bio-Sciences, Baie d’Urfe, Canada) antisense cRNA exon-specific probes transcribed from a 680-bp fragment of the CRH gene, a 229-bp fragment of the rat vasopressin gene, and a 572-bp fragment of the AR.

Techniques for riboprobe synthesis, hybridization (27), and the patterns of hybridization for these probes are described in greater detail elsewhere (8, 28, 29, 30). Briefly, free-floating sections were first rinsed in KPBS to remove cryoprotectant and then mounted and vacuum dried on glass slides overnight. After postfixation with 10% formaldehyde for 30 min at room temperature, sections were digested in proteinase K (10 mg/ml, 37 C) for 30 min, acetylated for 10 min (2.5 mM acetic anhydride and 0.1 M triethanolamine, pH 8.0), rapidly dehydrated in ascending ethanol concentrations (50–100%), and then vacuum dried. Radionucleotide antisense cRNA probes were used at concentrations approximating 3 x 107 cpm/ml in a solution of 50% formamide, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 0.05% tRNA, and 10 mM dithiothreitol, 1x Denhardt’s solution, and 10% dextran sulfate and applied to individual slides. Slides were coverslipped and then incubated overnight at 57.5 C, after which the coverslips were removed and the sections washed three times in 4x standard saline citrate (SSC; 0.15 M NaCl and 15 mM citric acid, pH 7.0) at room temperature, treated with ribonuclease A (20 µg/ml) for 30 min at 37 C, desalted in descending SSC concentrations (2–0.1x SSC), washed in 0.1x SSC for 30 min at 60 C, and dehydrated in ascending ethanol concentrations. Hybridized sections were then exposed to x-ray film (β-max; GE Healthcare, Piscataway, NJ), defatted in xylene, coated with Kodak NTB2 liquid autoradiographic emulsion, and exposed at 4 C in the dark with desiccant. The duration on emulsion was determined by the strength of signal on x-ray film (6 d for AVP mRNA and 9 d for CRH mRNA in the PVN; 15 and 18 d for AVP mRNA in the posterior BST and MeA, respectively; and 21 d for AR mRNA in the MPN, BST, and MeA). Slides were developed at 14 C with Kodak D-19 for 3.5 min, briefly rinsed in distilled water for 15 sec, fixed in Kodak fixer for 6.5 min, and then washed in running water for 45 min at room temperature.

Based on the strength of the autoradiographic signal, exposure time to emulsion was optimized to ensure that mRNA levels detected were within the linear range of the assay and could be quantified by making relative comparisons in OD levels. OD readings, corrected by background subtraction, were taken at regularly spaced (150-µm) intervals through each region of interest. In the MPN, BST, and MeA, the planes of sections for in situ hybridization were analyzed using the same frame dimensions described above. Hybridized tissue series between animals were aligned along the rostrocaudal plane using white matter morphology as anatomical reference points in addition to the cytoarchitectonic features provided by an adjacent thionin stained tissue series. Analysis of the relative levels of CRH and AVP mRNA in the PVN was restricted to the medial parvocellular dorsal (mpd) region of the nucleus, likewise assisted by redirected sampling of adjacent thionin-stained material, as previously described (30).

The dispersed nature by which AVP mRNA is expressed by neurons in the posterior BST and MeA allowed us to quantify the number of AVP-expressing cells in these nuclei. As previously described (29), any individual cell showing a developed silver grain density greater than five times background was deemed positive and counted. As discussed above with respect to AR, we expected changes in AVP mRNA levels in the BST and AVP to occur, in largest part, as a function of the organizing effects of neonatal testosterone but not as a consequence of acute restraint exposure. As shown in Results, the stability of AVP mRNA (and AR cell numbers) across basal and stress conditions within each treatment group allowed us to correlate indices of HPA activity as a function of central AVP.

Assessing the number of grains per cell within individual neurons directly was impractical given the enormity of samples and numbers of AVP cells detected. Thus, to provide an indirect estimate of the capacity of individual neurons to express AVP, we divided the AVP OD values by the total number of AVP cells encountered through each region of interest.

Parceling of the rat brain followed the mapping of AVP expression and AR staining as defined by the morphological features provided by thionin staining of adjacent series of tissue, based on the terminology of Swanson (25) to describe the posterior BST and Canteras et al. (31) to describe the MeA. Light- and dark-level images were captured using a Retiga 1300 CCD digital camera (Q-imaging, Burnaby, British Columbia, Canada), analyzed using Macintosh OS X-driven, Open Lab Image Improvision version 3.0.9 (Quorum Technologies, Guelph, Ontario, Canada) and Image J version 1.3.5 software (National Institutes of Health, Bethesda, MD), and exported to Adobe Photoshop (version 7.0; San Jose, CA), where standard methods were used to adjust contrast and brightness and final assembly at a resolution of 300 dpi.

Statistics
Data are expressed as the mean ± SEM and were analyzed by using one- and two-way ANOVA to detect neonatal treatment and neonatal treatment x stress effects, respectively. Post hoc analyses were performed when appropriate using Newman-Keuls test for multiple pairwise comparisons. Correlational analyses were performed using simple regressions to determine relations between plasma corticosterone and Fos-ir cell counts in the mpd PVN. Likewise, linear regressions were also performed between basal and stress-induced parameters of HPA function and the number of cells expressing AVP mRNA and AR-ir. Immunohistochemical, in situ hybridization-histochemical, and statistical comparisons were made observer-blind by assigning coded designations to the tissue and data sets in advance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adult testosterone replacement and corticosterone levels
Testosterone replacement levels were comparable between neonatal treatment groups. Plasma testosterone concentrations = 3.4 ± 0.4, 3.8 ± 0.5, and 3.6 ± 0.5 ng/ml in neo-sham-GDX, neo-GDX, and neo-GDX + T-treated animals, respectively. Analysis of plasma corticosterone concentrations revealed significant main effects of neonatal treatment [F(2,30) = 6.70; P = 0.003] and stress [F(1, 30) = 338.00; P < 0.0001], and a significant neonatal treatment x stress interaction [F(2, 30) = 3.59; P = 0.04]. This interaction was attributed to significantly higher levels of corticosterone in adult testosterone-replaced, neo-GDX rats under basal conditions (Fig. 1Go) and in response to restraint stress exposure (Fig. 2Go).


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		FIG. 1. A and B, Mean ± SEM plasma corticosterone concentrations (A) and Fos-ir cell numbers in the mpd region of the PVN (B) under basal conditions in adult male rats bearing similar levels of testosterone replacement (see Results, testosterone replacement levels). As neonates, these animals were either neo-sham), neo-GDX, or neo-GDX + T during the first week of life. *, P < 0.05 vs. neo-sham and neo-GDX + T animals (n = 6 per group). C, Scattergram shows a strong positive correlation between the numbers of Fos cells in the mpd region of the PVN and plasma corticosterone concentrations in unstressed animals (n = 18).

 

Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		FIG. 2. A and B, Mean ± SEM plasma corticosterone concentrations (A) and Fos-ir cell numbers in the mpd region of the PVN (B) under stress conditions in adult male rats bearing similar levels of testosterone replacement. *, P < 0.05 vs. neo-sham and neo-GDX + T animals (n = 6 per group). C, Scattergram shows a strong positive correlation between the numbers of mpd PVN cells recruited to express Fos protein and plasma corticosterone concentrations in stressed animals (n = 18).

 
Fos-ir, CRH, and AVP mRNA in mpd PVN
Quantitative assessment of the number of Fos-responding neurons in the HPA-regulating, mpd part of the PVN revealed significant main effects of neonatal treatment [F(2,30) = 8.61; P = 0.001] and stress [F(1,30) = 413.37; P < 0.0001] but no significant neonatal treatment x stress interaction [F(2,30) = 2.01; P = 0.15]. Compared with neo-sham-GDX and neo-GDX + T-treated rats, neo-GDX rats showed higher numbers of Fos-ir cells under basal conditions (Fig. 1Go) as well as higher numbers of mpd cells recruited to express Fos-ir during restraint (Fig. 2Go). Correlational analyses revealed reliable positive relationships between plasma corticosterone concentrations and Fos cell counts under basal [F(1,16) = 7.65; P = 0.014] and stress [F(1,16) = 11.4; P = 0.001] conditions (Figs. 1Go and 2Go, respectively).

Densitometric measurements within the mpd region of the PVN revealed no main effect of neonatal treatment on CRH mRNA under basal conditions [F(2,15) = 0.77; P = 0.92] or at 30 min of restraint exposure [F(2,15) = 1.52; P = 0.25]. Likewise, there was no main effect of neonatal treatment on AVP mRNA levels under basal conditions [F(2,15) = 1.66; P = 0.22] or after restraint [F(2,15) = 1.12; P = 0.35].

AVP mRNA responses to neonatal GDX and adult testosterone replacement
ANOVA indicated no significant effect of stress or a significant stress x neonatal interaction on AVP mRNA levels in the BST and MeA (P ≥ 0.52 in all cases). Thus, the relative levels of the transcript through these regions were assessed in basal and stress tissue combined and analyzed, a priori, as a function of neonatal treatment. As revealed by densitometric analyses, there was a significant main effect of neonatal treatment on AVP mRNA in the BST [F(2,33) = 12.95; P < 0.0001], credited to significantly lower levels of AVP mRNA in neo-GDX animals compared with neo-sham-GDX and neo-GDX + T-treated animals (Fig. 3Go, top panel). There was a main effect of neonatal treatment on AVP cell numbers in the BST [F(2,33) = 43.06; P < 0.0001]. This main effect was likewise credited to the neo-GDX group, because they showed significantly lower numbers of AVP-expressing cells compared with neo-sham-GDX and neo-GDX + T-treated animals (Fig. 3Go, middle panel). Taken as an index of the capacity of individual cells to express AVP (see Fig. 3Go, bottom panel), there was no neonatal treatment effect on the AVP OD to cell number ratio [F(2,33) = 0.27; P = 0.76]. As illustrated in Fig. 4Go, the bulk of AVP mRNA in the posterior BST was found to be most concentrated within the vicinity of the transverse and intrafascicular nuclei, although scattered and far less intense clusters of hybridized cells were routinely found within the principal nucleus. Furthermore, qualitative assessment of the spatial pattern of cells hybridized for AVP indicated that the effects of neo-GDX ± T were most apparent within the transverse and intrafascicular nuclei but far less reliable within the principal nucleus (Figs. 4Go). As we optimized the time on emulsion to capture the majority of AVP mRNA-expressing cells in the posterior division of the BST, our inability to detect reliable differences in the principal nucleus may be a matter of detection given the scarcity and relatively weaker labeling of this AVP population.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 3. The AVP response to adult testosterone replacement in the posterior division of the BST is reduced by neonatal GDX and restored with neonatal testosterone treatment. Mean ± SEM relative levels of AVP mRNA (A), AVP mRNA-expressing cell numbers (B), and AVP mRNA to AVP cell number ratios (C) in neo-sham and neo-GDX ± T rats bearing similar levels of adult testosterone replacement. *, P < 0.05 vs. neo-sham and neo-GDX + T animals (n = 12 per group).

 

Figure 4
View larger version (40K):
[in this window]
[in a new window]

  		FIG. 4. A–D, Dark-field photomicrographs at 150-µm intervals through the rostrocaudal extent of the posterior division of the BST to show the relative distribution and strength of hybridization signal for AVP mRNA in control animals. Scale bar, 500 µm (A–D). E, Mean ± SEM relative levels of AVP mRNA in neo-sham and neo-GDX ± T-treated rats through the rostrocaudal extent of the posterior division of the BST. Intervals A–D shown on the x-axis correspond to the slice intervals illustrated in the photomicrographs. Group differences in AVP mRNA were detected through the rostral to mid-extent of the posterior BST (slice levels B and C), most apparent within cells hybridized for AVP in the vicinity of the intrafascicular and transverse nuclei. *, P < 0.05 vs. neo-sham and neo-GDX + T animals (n = 12 per group).

 
In the MeA, there was a significant main effect of neonatal treatment on the density of AVP mRNA [F(2,33) = 8.33; P = 0.0012] within the anterodorsal region of the nucleus (Fig. 5Go). The neonatal treatment effect was credited to both the neo-GDX and the neo-GDX + T treatment group, showing significantly lower levels of AVP mRNA compared with neo-sham-GDX animals (see Fig. 5Go, top panel). There was a main effect of neonatal treatment on AVP cell numbers in the MeA [F(2,33) = 5.71; P < 0.0074] attributed to neo-GDX but not to neo-GDX + T-treated rats. Thus, neo-GDX animals showed significantly lower numbers of AVP mRNA-expressing cells compared with neo-sham-GDX and neo-GDX + T-treated rats (Fig. 5Go, middle panel). Finally, there was a main effect of neonatal treatment on the capacity of individual cells to express AVP [F(2,33) = 5.64; P = 0.0078]. This main effect was credited to significantly lower AVP OD: cell number ratios in neo-GDX and neo-GDX + T-treated animals compared with neo-sham-GDX rats (see Fig. 5Go, bottom panel). AVP mRNA cells were detected through the longitudinal extent of the MeA, represented by concentrated clusters in the anterodorsal part and by a vastly smaller and weakly hybridized contingent in the posterodorsal part. As illustrated in Fig. 6Go, neonatal GDX ± T treatment effects on AVP were clearly pronounced within the anterodorsal part.


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 5. Neonatal testosterone treatment restores the number of AVP cells in the anterodorsal part of the medial amygdala but not the capacity of individual cells to express AVP mRNA. A–C, Mean ± SEM relative levels of AVP mRNA (A), AVP mRNA-expressing cell numbers (B), and AVP mRNA to AVP cell number ratios (C) in neo-sham and neo-GDX ± T rats bearing similar levels of adult testosterone replacement. *, P < 0.05 vs. neo-sham (A and C); *, P < 0.05 vs. neo-sham and neo-GDX + T (C) (n = 12 per group).

 

Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 6. Hybridization histochemical localization of AVP mRNA in the anterodorsal part of the MeA. A–C, Dark-field photomicrographs of coronal sections through a comparable level of the BST to show the distribution and relative strength of hybridization signal in neo-sham (A), neo-GDX (B), and neo-GDX + T-treated rats (C) bearing similar adult testosterone replacement levels. Note, as described in Fig. 5Go, that although neo-GDX + T restored the number of AVP-expressing cells in the MeA, this treatment did not reverse the inhibitory effect of Neo-GDX on the AVP signal within individual cells (compare B and C). For anatomical reference, the optic tract is illuminated in the upper left corners. Scale bar, 100 µm (A–C).

 
AR-ir responses to neonatal GDX and adult testosterone replacement
ANOVA indicated main effects of neonatal treatment [F(2,30) = 22.78; P < 0.0001] and region [F(3,90) = 167.97; P < 0.0001] but no significant effect of stress [[F(1,30) = 0.16; P = 0.69]. Thus, the relative levels of AR staining were assessed in basal and stress tissue combined. There was a significant main effect of neonatal treatment on AR cell numbers in the posterior BST [F(2,33) = 16.11; P < 0.0001] and in the posterodorsal part of the MeA [F(2,33) = 7.62; P = 0.0019] but not in the CA-1 region of the hippocampus [F(2,33) = 0.44; P = 0.90], although a trend toward significance was found in the MPN [F(2,33) = 3.39; P = 0.084]. Treatment effects in both the posterior BST and MeA were attributed to the neo-GDX group, showing significantly lower numbers of AR-positive cells than neo-sham-GDX and neo-GDX + T-treated rats (Figs. 7Go and 8Go, respectively). No significant effect of neonatal treatment was found on the relative levels of AR mRNA in the MPN, BST, and MeA (P = 0.65). Relative to neo-sham-GDX and neo-GDX + T-treated animals, neo-GDX rats showed a substantial reduction in the number of AR-ir cell numbers throughout the posterior BST but without any apparent bias between the principal, transverse, and intrafascicular nuclei (Fig. 7Go). Group differences in AR staining (lowest in neo-GDX animals) were noted throughout the region of the MeA, most conspicuously in the posterodorsal part of the nucleus (Fig. 8Go).


Figure 7
View larger version (122K):
[in this window]
[in a new window]

  		FIG. 7. Histochemical localization of AR-ir in the posterior division of the BST. A–C, Bright-field photomicrographs of coronal sections through a comparable level of the BST to show the distribution and relative strength in AR staining in neo-sham (A), neo-GDX (B), and neo-GDX + T-treated rats (C) bearing similar adult testosterone replacement levels. Scale bar, 250 µm (A–C). At the level depicted, neo-GDX resulted in a decrease in AR staining within the vicinity of the principal nucleus, located just lateral to the stria medullaris (sm) and fornix (fx). A decrease in staining was also apparent within the vicinity of the intrafascicular and transverse nuclei, located medial to the ventral tips of the stria terminalis (st) and the internal capsule (int), respectively. Neo-GDX + T restored AR staining in both regions (compare B and C). D, Mean ± SEM total estimates of AR-ir cell numbers in neo-sham and neo-GDX ± T-treated rats bearing similar adult testosterone replacement levels. *, P < 0.05 vs. neo-sham and neo-GDX + T animals (n = 12 per group).

 

Figure 8
View larger version (121K):
[in this window]
[in a new window]

  		FIG. 8. Histochemical localization of AR-ir in the posterodorsal part of the MeA. A–C, Bright-field photomicrographs of coronal sections through a comparable level of the MeA to show the distribution and relative strength in AR staining in neo-sham (A), neo-GDX (B), and neo-GDX + T-treated rats (C) bearing similar adult testosterone replacement levels. Scale bar, 250 µm (A–C). Neo-GDX resulted in a decrease in AR staining throughout the entire posterodorsal part, reversed with neonatal testosterone treatment. Structures are labeled for reference: ot, optic tract; st, stria terminalis. D, Mean ± SEM total estimates of AR-ir cell numbers in neo-sham and neo-GDX ± T-treated rats bearing similar adult testosterone replacement levels. *, P < 0.05 vs. neo-sham and neo-GDX + T animals (n = 12 per group).

 
Correlations between AVP and AR-ir cells and HPA function
Several of our findings implied relations between the capacity of animals to express AVP and AR-ir and to respond to adult testosterone replacement. The stability of AR-ir and AVP mRNA across basal and stress conditions within each treatment group allowed us to perform linear regressions between these variables and basal and stress indices of HPA across animals (see Table 1Go).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Correlations between AR and AVP cell numbers and basal and stress-HPA function

 
Thus, under nonstressed conditions, a strong negative correlation was found between the number of AR-ir cells in the MeA and resting-state levels of Fos in the mpd part of the PVN. A strong negative correlation was also found between AVP cells in the posterior BST and basal levels of Fos-ir in the PVN. Basal corticosterone levels tended to vary as a function of AVP in the BST and AR cell numbers in the MeA (P = 0.07 for both). In animals exposed to 30 min of restraint, stress-induced levels of Fos in the PVN and corticosterone in plasma varied strongly and negatively with AR and AVP in the posterior BST.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is becoming increasingly clear that the sex steroid hormone environment during the perinatal period plays an important role in organizing the HPA axis, where this occurs in the brain remains poorly understood. The present findings suggest that the organizing effects of testosterone might be mediated upstream from the PVN, particularly within brain regions previously acknowledged as regulating the HPA axis and concentrating ARs. Thus, we now have a testable framework for examining in future studies whether the critical effects of neonatal androgens occur within AR- and/or AVP-expressing cells that communicate with the HPA axis.

Our findings support previous reports showing a stimulatory effect of neonatal gonadectomy on both basal and stress-induced corticosterone release in adult male rats (4). The effects of neo-GDX on HPA function that we observed were restored with neonatal testosterone treatment but not with adult testosterone replacement. Based on our previous GDX and dose-replacement experiments (19), the adult testosterone replacement levels achieved in the present study were more than sufficient to reverse the stimulatory effects of GDX on HPA function in adults. Importantly, it has been previously demonstrated that neo-GDX animals show similar elevations in HPA responses to stress regardless of testosterone (4, 32). Thus, because we did not study adult animals without testosterone replacement, we probably underestimated the capacity of neo-GDX animals to show reduced HPA responses to testosterone. Nonetheless, relative to neo-sham and neo-GDX + T-treated animals, HPA activity was significantly higher in neo-GDX animals despite similar levels of adult testosterone replacement.

CRH and AVP expression in the PVN did not vary as a function of neonatal treatment. However, basal and stress-induced levels of corticosterone varied strongly and positively with the number of Fos-positive cells in the mpd part of the PVN. These findings provide a strong indication that neonatal testosterone can organize the cellular activation of PVN motor neurons governing ACTH release under basal and stress conditions. Seale et al. (6) recently demonstrated elevated CRH and AVP mRNA levels in animals that received either the AR antagonist flutamide or the aromatase inhibitor ATD during the perinatal period. This discrepancy with our findings may be explained by normal androgenic and estrogenic influences exerted before our postnatal manipulation (33, 34). Thus, there are likely several critical periods over which testosterone and its metabolites are capable of organizing and operating on different aspects of the HPA system, including ACTH secretagogue synthesis and release and, as our current findings suggest, afferents supplying androgen-sensitive input to the PVN motor neurons.

Mechanisms to explain changes in AR and AVP
Neuronal cell numbers in the posterior BST and medial amygdala are greater in adult male rodents than in females, and testosterone contributes to this sex difference. Neonatal GDX results in a decrease in neuronal soma size and cell numbers in the posterior BST of adult males, and this testosterone-reversible effect is most conspicuous in the vicinity of the principal nucleus (reviewed in Ref. 11 , and see Ref. 35). The sex difference in neuronal cell numbers in the MeA is most apparent with the posterodorsal part of the nucleus and likewise appears to be organized by testosterone, at least before adulthood (36). As we observed, neonatal GDX ± T altered AVP expression within the vicinity of the transverse and intrafascicular nuclei of the posterior BST (Figs. 4Go and 5Go) and in the anterodorsal part of the MeA (Fig. 6Go), regions least associated with testosterone- and gender-dependent differences in neuronal volume and cell number. Effects on AR-ir cell numbers were also localized, but not limited to, these same regions of the BST and MeA (Figs. 7Go and 8Go). Taken together, the neurotrophic effects of testosterone that occur during the neonatal period remain an important consideration; however, the group differences in AVP expression and AR staining are unlikely to be explained solely by developmental influences on neuronal cell number.

Our findings could also reflect a change in the ability of existing neurons to express the AR. A shift in the capacity of these neurons to register and respond to circulating levels of testosterone is also possible, considering that androgens often regulate the synthesis of their own receptors (24). Because we detected no differences in AR mRNA, regional differences in AR-ir detection could be explained by site- and cell-specific differences in testosterone metabolism and, ultimately, AR and mRNA stability (see Ref. 37). In addition, several AR coregulators have been identified as candidate molecules capable of influencing the stability of the AR ligand complex and nuclear translocation (reviewed in Ref. 38). Thus, local mechanisms regulating the capacity of cells to register circulating levels of testosterone might explain why neo-GDX animals displayed less AR staining in the cell nucleus and functionally lower HPA and central AVP responses to adult testosterone replacement.

Mechanisms to explain changes in the adult HPA response to testosterone
The strong associations observed between AR-ir cell numbers, plasma corticosterone, and intervening levels of Fos in the PVN (see Table 1Go) suggest that the posterior BST and MeA are important candidate mediators of the developmental effects of testosterone on HPA function in adulthood. The posterodorsal part of the MeA and the posterior division of the BST, including the principle, intrafascicular, and transverse subnuclei, are among several forebrain regions with the highest densities of neurons that express androgen and estrogen receptors (8, 39). The posterior BST send robust projections to the PVN region, toward the medial parvocellular part of the nucleus (40), and to the PVN surround, targeting several cell groups that are in a position to integrate, albeit indirectly, input from the limbic forebrain, including the prefrontal cortex, lateral septum, MeA, and ventral subiculum (reviewed in Ref. 10). Lesions in the vicinity of the posterior division of the BST and the dorsal aspect of the MeA indicate that both of these structures are capable of providing inhibitory inputs to the PVN (41, 42). Superimposing our current findings onto this functional and connectional data, we propose that neonatal testosterone primes the adult HPA response to testosterone by defining the number of ARs within the posterior BST and MeA. Although our previous connectional studies show a high containment of ARs within PVN projecting nuclei in the posterior BST, and a small contingent in the MeA that project to the perinuclear region of the PVN (10), the extent to which neonatal testosterone alters ARs within these PVN afferents remains to be confirmed.

The group differences in the number of AVP-expressing cells in the BST and MeA confirm several previous reports showing that testosterone stimulation of AVP expression within these regions depends on the level of testosterone exposure during the neonatal period (reviewed in Ref. 11). AVP mRNA expression and synthesis in the BST and MeA, as well as their projections, are extremely dependent on testosterone levels in adult males (reviewed in Refs. 11 and 12). Despite similar adult testosterone replacement levels, however, neo-GDX rats expressed lower levels of AVP mRNA in both the BST and MeA (Figs. 3Go and 6Go, respectively). This could reflect a loss in testosterone responsiveness within existing AVP neurons, at least in the MeA. In addition to the differences in the number of cells that express AVP, neo-GDX animals showed less labeling per cell in the MeA (Figs. 6Go and 7Go). In the BST, our analyses suggest that alterations in AVP mRNA expression are produced by changes in the number of neurons expressing the transcript and not by changes in the amount of AVP mRNA expressed per cell. The dense packing of AVP-expressing neurons in the posterior BST, particularly within the vicinity of the intrafascicular and transverse nuclei, could have impacted our ability to detect neonatal-dependent changes in the capacity of individual neurons to express AVP. Otherwise, the testosterone-dependent organization of AVP neurons in the BST and MeA may be mediated by distinct mechanisms.

In line with this possibility, neo-GDX + T-treated animals showed region-specific differences in their cellular responsiveness to adult testosterone replacement. In the BST, neonatal testosterone treatment restored the number of neurons expressing the AVP transcript. In the MeA, neonatal testosterone treatment likewise restored the number of AVP cells, but not the capacity of individual cells to express AVP mRNA (Fig. 6Go). Previous labeling studies in gonadal-intact, adult male rats indicate that virtually all AVP neurons in the BST and MeA colocalize with androgen and estrogen receptors in the BST and MeA (reviewed in Ref. 11). Based on our current findings, however, we are compelled to conclude that in contrast to the MeA, the AVP response to adult testosterone in the BST may be more evenly coupled to changes in ARs and/or similar periodic influences of testosterone. What should be emphasized at this point is that the animals used in the current study were robbed of testosterone exposure up to the age of 45 d. During this time in male rodents, major age-related increases in AVP mRNA, AVP-ir, and AR cell numbers in the BST and MeA occur, attributed to an increase in plasma testosterone (43, 44). Furthermore, a recent report suggests that puberty is also met by a testosterone-dependent increase in neurogenesis in the MeA (45). Thus, the differential AVP cellular responses in the MeA may have been shaped by the absence of testosterone exposure over the course of pubertal development.

Notwithstanding these caveats, we found several correlations in support of our hypothesis that the limbic system may be responsible for organizing the adult HPA response to testosterone and that AVP may be a key contributor to this response, at least within the posterior BST (see Table 1Go). We are not the first to make this association between central AVP and HPA function. Gomez et al. (44) identified strong negative relationships between the number of AVP-positive cells in the BST and the capacity of mpd neurons in the PVN to express CRH- or AVP-ir. Although these studies were performed in the context of puberty, steady-state and stress-induced levels of AVP in the BST were also shown to vary as a function of resting and stress-induced levels of testosterone between pre- and postpubertal animals. Taken together with our findings, changes in AVP expression and its regulation by testosterone in the BST could signify a means by which the central and organizing influences of testosterone are transmitted to the PVN. Compared with the BST, the MeA is functionally at least one or several steps removed from the PVN, which might explain why we were unable to detect significant correlations between AVP cell numbers in this region and basal and stress-induced indices of HPA function.

Conclusion and future considerations
We have shown that neonatal testosterone exerts profound effects on the HPA response to testosterone during adulthood. The extent to which this is executed by changes in AVP and AR within the same cells and/or represented within unique projections to the HPA axis requires further clarification. As discussed earlier, neonatal gonadectomy exerts remarkable changes in adult brain morphology, including effects on neuronal soma size and cell numbers within subregions of the posterior BST and the MeA. This could have an important bearing on the extended circuitries of the PVN, including its functional connectivity with the limbic system. Using an anterograde tract-tracing tracer approach, Gu et al. (46) showed that neonatal gonadectomy can decrease the projection densities of the principal nucleus of the posterior BST, at least within the anteroventral periventricular nucleus and the ventral premammillary nucleus of the hypothalamus. Thus, future studies employing a retrograde tracer injection approach in the PVN could answer whether neonatal androgens are capable of altering the number of neurons targeting the PVN directly as well as their containment of ARs and AVP.

Because the group differences in the number of AR cells were several orders of magnitude higher than the number of AVP cells, we anticipate that there are likely several androgen-sensitive, candidate neurotransmitters driving the organizational actions of testosterone. Nonetheless, the group difference in AVP responsiveness to adult testosterone replacement provides some indication that AR function may be altered by neonatal testosterone exposure.

A clear majority of studies have shown that the organizing effects of testosterone are mediated, in large part, by its conversion to estrogen (reviewed in Ref. 47). Furthermore, testosterone and estrogen interact on the central regulation of AVP during adulthood (48, 49) as well as during the neonatal period (50). Thus, in addition to ARs, the organization of the adult HPA response to testosterone probably involves important changes in estrogen receptor expression and function as well as cellular and region-specific differences in aromatase activity (51). Based on the strength of our current findings, we now have an anatomical and functional framework for exploring these possibilities.


   Acknowledgments
 
We thank the staff of the Animal Care Centre, Dr. Tamara Godbey, and Dr. Bev Chua for technical assistance. This study is dedicated to the memory of Dr. Seymour "Gig" Levine for his kind suggestions and inspiration to our lab.


   Footnotes
 
This study was supported by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada (to V.V.). B.B. is supported by a Canadian Graduate Student Scholarships Doctoral Award from the Canadian Institutes of Health Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 10, 2008

Abbreviations: AR, Androgen receptor; ATD, ,4,6-androstatriene-3,17-dione; AVP, arginine vasopressin; BST, bed nuclei of the stria terminalis; GDX, gonadectomy; HPA, hypothalamic-pituitary-adrenal; ir, immunoreactivity; MeA, medial nucleus of the amygdala; mpd, medial parvocellular dorsal; MPN, medial preoptic nucleus; neo-GDX, neonatally gonadectomized; neo-sham-GDX, neonatally sham gonadectomized; neo-GDX+T, neonatally gonadectomized with testosterone treatment; PVN, paraventricular nucleus of the hypothalamus; SSC, standard saline citrate.

Received December 26, 2007.

Accepted for publication March 31, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Negri-Cesi P, Colciago A, Celotti F, Motta M 2004 Sexual differentiation of the brain: role of testosterone and its active metabolites. J Endocrinol Invest 27:120–127[Medline]
  2. Wallen K 2005 Hormonal influences on sexually differentiated behavior in nonhuman primates. Front Neuroendocrinol 26:7–26[CrossRef][Medline]
  3. Wilson CA, Davies DC 2007 The control of sexual differentiation of the reproductive system and brain. Reproduction 133:331–359[Abstract/Free Full Text]
  4. McCormick CM, Furey BF, Child M, Sawyer M, Donohue SM 1998 Neonatal sex hormones have ‘organizational’ effects on the hypothalamic-pituitary-adrenal axis of male rats. Brain Res Dev Brain Res 105:295–307[Medline]
  5. Levine S, Mullins R 1967 Neonatal androgen or estrogen treatment and the adrenal cortica response to stress in adult rats. Endocrinology 80:1177–1179[Abstract/Free Full Text]
  6. Seale JV, Wood SA, Atkinson HC, Lightman SL, Harbuz MS 2005 Organizational role for testosterone and estrogen on adult hypothalamic-pituitary-adrenal axis activity in the male rat. Endocrinology 146:1973–1982[Abstract/Free Full Text]
  7. Bingham B, Williamson M, Viau V 2006 Androgen and estrogen receptor-β distribution within spinal-projecting and neurosecretory neurons in the paraventricular nucleus of the male rat. J Comp Neurol 499:911–923[CrossRef][Medline]
  8. Simerly RB, Chang C, Muramatsu M, Swanson LW 1990 Distribution of androgen and estrogen mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294:76–95[CrossRef][Medline]
  9. Williamson M, Bingham B, Viau V 2005 Central organization of androgen-sensitive pathways to the hypothalamic-pituitary-adrenal axis: implications for individual differences in responses to homeostatic threat and predisposition to disease. Prog Neuropsychopharmacol Biol Psychiatry 29:1239–1248[CrossRef][Medline]
  10. Williamson M, Viau V 2007 Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus in the male rat. J Comp Neurol 503:717–740[CrossRef][Medline]
  11. De Vries GJ 2006 Sex differences in neurotransmitters systems: vasopressin as an example. In: Blaustein J, ed. Handbook of neurochemistry and molecular neurobiology. New York: Springer-Verlag; 487–512
  12. Kalsbeek A, Palm IF, Buijs RM 2002 Central vasopressin systems and steroid hormones. Prog Brain Res 139:57–73[Medline]
  13. Del Abril A, Segovia S, Guillamon A 1987 The bed nucleus of the stria terminalis in the rat: regional sex differences controlled by gonadal steroids early after birth. Dev Brain Res 32:295–300[CrossRef]
  14. Wang Z 1994 Testosterone effects on development of vasopressin messenger RNA expression in the bed nucleus of the stria terminalis and medial amygdaloid nucleus in male rats. Brain Res Dev Brain Res 79:147–150[CrossRef][Medline]
  15. Han TM, De Vries GJ 2003 Organizational effects of testosterone, estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis. J Neurobiol 54:502–510[CrossRef][Medline]
  16. Liu D, Diorio J, Tannenbaum B, Caldji C, Francis D, Freedman A, Sharma S, Pearson D, Plotsky PM, Meaney MJ 1997 Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277:1659–1662[Abstract/Free Full Text]
  17. Lesage J, Dufourny L, Laborie C, Bernet F, Blondeau B, Avril I, Breant B, Dupouy JP 2002 Perinatal malnutrition programs sypathoadrenal and hypothalamic-pituitary-adrenal axis responsiveness to restraint stress in adult male rats. J Neuroendocrinol 14:135–143[CrossRef][Medline]
  18. Abbey H, Howard E 1972 Statistical procedure in developmental studies on species with multiple offspring. Dev Psychobiol 6:329–335[CrossRef]
  19. Viau V, Meaney MJ 1996 The inhibitory effect of testosterone on hypothalamic-pituitary-adrenal responses to stress is mediated by the medial preoptic area. J Neurosci 16:1866–1876[Abstract/Free Full Text]
  20. Viau V, Lee P, Sampson J, Wu J 2003 A testicular influence on restraint-induced activation of medial parvocellular neurons in the paraventricular nucleus in the male rat. Endocrinology 144:3067–3075[Abstract/Free Full Text]
  21. Viau V, Bingham B, Davis J, Lee P, Wong M 2005 Gender and puberty interact on the stress-induced activation of parvocellular neurosecretory neurons and corticotropin-releasing hormone messenger ribonucleic acid expression in the rat. Endocrinology 146:137–146[Abstract/Free Full Text]
  22. Abercrombie M 1946 Estimation of nuclear population of microtome sections. Anat Rec 94:239–247[CrossRef]
  23. Guillery RW 2002 On counting and counting errors. J Comp Neurol 447:1–7[CrossRef][Medline]
  24. Xiao L, Jordan CL 2002 Sex differences, laterality, and hormonal regulation of androgen receptor immunoreactivity in rat hippocampus. Horm Behav 2002 42:327–336[CrossRef][Medline]
  25. Swanson LW 1998 Brain maps: structure of the rat brain. 2nd ed. Amsterdam: Elsevier
  26. Portillo W, Díaz NF, Cabrera EA, Fernández-Guasti A, Paredes RG 2006 Comparative analysis of immunoreactive cells for androgen receptors and oestrogen receptor {alpha} in copulating and non-copulating male rats. J Neuroendocrinol 18:168–176[CrossRef][Medline]
  27. Simmons DM, Arriza JL, Swanson LW 1989 A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J Histotechnol 12:169–181
  28. Herman JP, Schafer MKH, Thompson R, Watson SJ 1992 Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mol Endocrinol 6:1061–1069[Abstract/Free Full Text]
  29. Viau V, Soriano L, Dallman M 2001 Androgens alter corticotropin releasing hormone and arginine vasopressin mRNA within forebrain sites known to regulate activity in the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 13:442–452[CrossRef][Medline]
  30. Viau V, Sawchenko PE 2002 Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress. J Comp Neurol 445:293–307[CrossRef][Medline]
  31. Canteras NS, Simerly RB, Swanson LW 1995 Organization of projections from the medial nucleus of the amygdala: a PHAL study in the rat. J Comp Neurol 360:213–245[CrossRef][Medline]
  32. Libertun C, Lau C 1972 Adrenocortical function in prepubertal rats: neonatal effects of testosterone. J Endocrinol 55:221–222[Abstract/Free Full Text]
  33. Weisz J, Ward IL 1980 Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106:306–316[Abstract/Free Full Text]
  34. Baum MJ, Brand T, Oomss M, Vreeburg JT, Slob AK 1988 Immediate postnatal rise in whole body androgen content in male rats: correlation with increased testicular content and reduced body clearance of testosterone. Biol Reprod 38:980–986[Abstract]
  35. Hines M, Allen LS, Gorski RA 1992 Sex differences in subregions of the medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain Res 579:321–326[CrossRef][Medline]
  36. Morris JA, Jordan CL, Breedlove SM 2008 Sexual dimorphism in neuronal number of the posterodorsal medial amygdala is independent of circulating androgens and regional volume in adult rats. J Comp Neurol 506:851–859[CrossRef][Medline]
  37. Lu S, Simon NG, Wang Y, Hu S 1999 Neural androgen receptor regulation: effects of androgen and antiandrogen. J Neurobiol 41:505–512[CrossRef][Medline]
  38. Heinlein CA, Chang C 2002 Androgen receptor (AR) coregulators: an overview. Endocr Rev 23:175–200[Abstract/Free Full Text]
  39. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen receptor-{alpha} and -β mRNA in the rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
  40. Dong HW, Swanson LW 2004 Projections from bed nuclei of the stria terminalis, posterior division: implications for cerebral hemisphere regulation of defensive and reproductive behaviors. J Comp Neurol 471:396–433[CrossRef][Medline]
  41. Dayas CV, Buller KM, Day TA 1999 Neuroendocrine responses to an emotional stressor: evidence for involvement of the medial but not the central amygdala. Eur J Neurosci 11:2312–2322[CrossRef][Medline]
  42. Choi DC, Furay AR, Evanson NK, Ostrander MM, Ulrich-Lai YM, Herman JP 2007 Bed nucleus of the stria terminalis subregions differentially regulate hypothalamic-pituitary-adrenal axis activity: implications for the integration of limbic inputs. J Neurosci 27:2025–2034[Abstract/Free Full Text]
  43. Romeo RD, Diedrich SL, Sisk CL 2000 Effects of gonadal steroids during pubertal development on androgen and estrogen receptor-{alpha} immunoreactivity in the hypothalamus and amygdala. J Neurobiol 44:361–368[CrossRef][Medline]
  44. Gomez F, Manalo S, Dallman MF 2004 Androgen-sensitive changes in regulation of restraint-induced adrenocorticotropin secretion between early and late puberty in male rats. Endocrinology 145:59–70[Abstract/Free Full Text]
  45. Zehr JL, Ahmed EI, Schulz KM, Sisk CL, Sex differences in addition of new cells in the rat medial amygdala during puberty. Proc Annual Meeting of the Society for Neuroscience, Atlanta, GA, 2006 (Abstract 152.2)
  46. Gu G, Cornea A, Simerly RB 2003 Sexual differentiation of projections from the principal nucleus of the bed nuclei of the stria terminalis. J Comp Neurol 460:542–562[CrossRef][Medline]
  47. Baum MJ 2003 Activational and organizational effects of estradiol on male behavioral neuroendocrine function. Scand J Psychol 44:213–220[CrossRef][Medline]
  48. Brot MD, De Vries GJ, Dorsa DM 1993 Local implants of testosterone metabolites regulate vasopressin mRNA in sexually dimorphic nuclei of the rat brain. Peptides 14:933–940[CrossRef][Medline]
  49. De Vries GJ, Wang Z, Bullock NA, Numan S 1994 Sex differences in the effects of testosterone and its metabolites on vasopressin messenger RNA levels in the bed nucleus of the stria terminalis of rats. J Neurosci 14:1789–1794[Abstract]
  50. Kühnemann S, Brown TJ, Hochberg RB, MacLusky NJ 1995 Sexual differentiation of estrogen receptor concentrations in the rat brain: effects of neonatal testosterone exposure. Brain Res 691:229–234[CrossRef][Medline]
  51. Balthazart J 1997 Steroid control and sexual differentiation of brain aromatase. J Steroid Biochem Mol Biol 61:323–339[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bingham, B.
Right arrow Articles by Viau, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bingham, B.
Right arrow Articles by Viau, V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals