Androgen receptor expressing neurons that project to the paraventricular nucleus of the hypothalamus...

Androgen Receptor Expressing NeuronsThat Project to the ParaventricularNucleus of the Hypothalamus in the

Male Rat

MARTIN WILLIAMSON AND VICTOR VIAU*

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

ABSTRACTAndrogen receptors are distributed throughout the central nervous system and are

contained by a variety of nuclei that are known to project to or regulate the paraventricularnucleus (PVN) of the hypothalamus, the final common pathway by which the brain regulatesthe hypothalamic–pituitary–adrenal (HPA) response to homeostatic threat. Here we charac-terized androgen receptor staining within cells identified as projecting to the PVN in malerats bearing iontophoretic or crystalline injections of the retrograde tracer FluoroGold aimedat the caudal two-thirds of the nucleus, where corticotropin-releasing hormone-expressingneurons are amassed. Androgen receptor (AR) and FluoroGold (FG) double labeling wasrevealed throughout the limbic forebrain, including scattered numbers of cells within theanterior and posterior subdivisions of the bed nuclei of the stria terminalis; the medial zoneof the hypothalamus, including large numbers of AR-FG-positive cells within the anteroven-tral periventricular and medial preoptic cell groups. Strong and consistent colabeling wasalso revealed throughout the hindbrain, predominantly within the periaqueductal gray andthe lateral parabrachial nucleus, and within various medullary cell groups identified ascatecholaminergic, predominantly C1 and A1 neurons of the ventral medulla. These connec-tional data predict that androgens can act on a large assortment of multimodal inputs to thePVN, including those involved with the processing of various types of sensory and limbicinformation, and provide an anatomical framework for understanding how gonadal statuscould contribute to individual differences in HPA function. J. Comp. Neurol. 503:717–740,2007. © 2007 Wiley-Liss, Inc.

Indexing terms: androgen receptor; FluoroGold; paraventricular nucleus of the hypothalamus,

hypothalamic-pituitary-adrenal axis, catecholamine neurons

In the vertebrate central nervous system, adaptive re-sponses to homeostatic threat (i.e., stress) rely on theintegrative capacity of several major visceromotor sys-tems. Depending on the nature of the stressor (e.g., inten-sity, duration, physiology), at least four major responsessystems are recruited to various degrees and overlap, in-cluding those mediating behavior, autonomic, neuroendo-crine, and immune output responses (Dallman, 2003;McEwen and Wingfield, 2003). Proper maintenance of nor-mal homeostasis and successful adaptation to any chal-lenge rests not only within the accuracy by which any oneof these systems act, but also within the extent to whichthe end products or outcomes of these systems interact. Awell-documented fact is that stress can influence the out-put of the hypothalamic–pituitary–gonadal axis, includ-

ing effects on the synthesis and release of the sex steroidsestrogen, progesterone, and testosterone (Vreeburg et al.,1984; Rivier et al., 1986; Rivier and Rivest, 1991; Henry,1992; Van den Berghe, 2002; Wingfield and Sapolsky,

Grant sponsor: Canadian Institutes of Health Research; Grant sponsor:National Alliance for Research On Schizophrenia and Depression.

*Correspondence to: Victor Viau, Department of Cellular and Physiolog-ical Sciences, Life Sciences Centre, University of British Columbia, 2350Health Sciences Mall, Vancouver BC V6T 1Z3, Canada.

E-mail: [email protected] 15 December 2006; Revised 3 April 2007; Accepted 24 April

2007DOI 10.1002/cne.21411Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 503:717–740 (2007)

© 2007 WILEY-LISS, INC.

2003). Conversely, the magnitude of the hypothalamic–pituitary–adrenal (HPA) response to stress also varies asa function of gonadal status in males and females (Viauand Meaney, 1991; Handa et al., 1994; Patchev andAlmeida, 1998; Young, 1998; Rhodes and Rubin, 1999;Figueiredo et al., 2002; Isgor et al., 2003; Viau et al., 2003;Lund et al., 2004; Young and Altemus, 2004; Williamsonet al., 2005). Of interest here, testosterone can act andinteract with corticosterone on almost every conceivableaspect of the HPA axis, including inhibiting the recruit-ment of hypophysiotropic neurons that are activated dur-ing stress, the regulation of the principle adrenocortico-tropin cosecretagogues, corticotropin-releasing hormoneand arginine vasopressin, and cooperatively on the pro-cess of glucocorticoid-mediated negative feedback (Viau etal., 1999). More detailed descriptions of how testosteroneand corticosterone interact on HPA function are reviewedelsewhere (Viau, 2002; Williamson et al., 2005).

The shared inhibitory characteristics by which testos-terone and corticosterone operate on the HPA axis wouldplace such influences within the central nervous systemand the PVN. Studies examining the distribution of sexsteroid hormone receptors within the PVN on spatial(Rhodes et al., 1982; Simerly et al., 1990; Zhou et al., 1994)and connectional grounds (Stern and Zhang, 2003; Bing-ham et al., 2006) indicate that the androgen receptor andthe estrogen receptor-beta isoform are not expressed bycells occupying the medial parvocellular part of the PVNdirected at the median eminence, but by cells projecting tothe spinal cord and medulla. While this signifies a meansby which sex steroids can influence autonomic and sensoryfunction within the PVN directly, these findings alsolikely place androgenic (and estrogenic) influences onHPA function upstream from the PVN (but see Lund et al.,2006). Androgen receptors are distributed throughout thebrain, including within several cortical, hypothalamic,limbic, and brainstem cell groups that regulate HPA func-tion and/or project to the PVN region (Sar and Stumpf,1975; Simerly et al., 1990; Bingaman et al., 1994; Lisciottoand Morrell, 1994; Kerr et al., 1995; Romeo et al., 2000;Murphy and Hoffman, 2001; Hamson et al., 2004). Be-cause circulating levels of testosterone vary as a functionof age, sexual experience, social status, and in response tostress (Bartke et al., 1973; Sencar-Cupovic and Milkovic,1976; De Goeij et al., 1992; Blanchard et al., 1993; Romeroet al., 1995; Gomez and Dallman, 2001; Seeman et al.,2001; Dallman et al., 2002; Gomez et al., 2004; Romeo etal., 2004; Tamashiro et al., 2004), situation-specific andstate-dependent changes in gonadal status may, there-fore, act to harmonize several independent, yet converginginfluences on PVN function. This could hold the key forsuccessful adaptation to complex homeostatic challengesdemanding multiple types of system responses.

Tract-tracing studies used in combination withimmediate-early-gene and phenotypic approaches haveproven incredibly instructive in determining the specific-ity by which distinct classes of homeostatic threat (neuro-genic, systemic) influence different central pathways andeffector motor neurons in the PVN (reviewed in Cullinanet al., 1995; Sawchenko et al., 2000; Dayas et al., 2001a;Herman et al., 2003; Day, 2005). Further, changes ingonadal and reproductive status in males and females caninfluence the pattern and the magnitude of stress-inducedexpression of the c-fos gene and its protein counterpart,Fos, within the PVN and several putative central regula-

tors of the HPA axis (Da Costa et al., 1996; Kerr et al.,1996; Cheung et al., 1997; Nappi et al., 1997; Rachman etal., 1998; Figueiredo et al., 2002; Ceccarelli et al., 2003,2006; Viau et al., 2003, 2005). However, the extent towhich regional differences in androgen receptors contrib-ute to this influence, and their containment within PVN-projecting nuclei, has not been determined. In the currentstudy we sought to build on the latter by employing acombined retrograde transport and immunohistochemicalprocedure to 1) characterize the distribution of androgenreceptors within PVN-projecting cell groups directly, and2) assess the relative densities of androgen receptor stain-ing within these candidate afferent mediators of gonadalstatus. Superimposing the results of these two aims ontothe results of previous anterograde experiments allowedus to examine the potential by which testosterone couldinfluence different classes of effector motor neurons in thePVN. Portions of these results have been presented pre-viously in abstract form (Williamson et al., 2004).

MATERIALS AND METHODS

Animals

Sixty adult male Sprague-Dawley rats (Charles River,St. Constant, Canada) were used, weighing 250–260 g onarrival (40 days old) and 345–365 g when sampled (�60days old). Animals were pair-housed under controlledtemperature and lighting conditions (12:12-hour light:dark cycle, lights on at 0600 hours) with food and wateravailable ad libitum. All experimental protocols were ap-proved by the University of British Columbia Animal CareCommittee.

Mapping candidate androgen-responsiveafferents to the PVN

To identify AR-immunoreactive (ir) neurons in brain re-gions that project to the region of the PVN, a combinedretrograde transport and immunohistochemical procedurewas used in all animals bearing appropriate tracer injectionsinto the PVN as previously described (Li and Sawchenko,1998). Under ketamine-xylazine-acepromazine anesthesia-analgesia (25, 5, and 1 mg/mL, respectively, 1 mL/kg subcu-taneously), animals received stereotaxically guided ionto-phoretic injections or crystalline implants of the retrogradelytransported tracer, FluoroGold (FG; Fluorochrome, Denver,CO), directed at the PVN. Stereotaxic coordinates wereadapted from Bregma according to Swanson (1998), AP:�1.45 mm; ML: 0.25 mm; DV: �7.4 mm; bite bar set at 3below interaural zero. Iontophoretic injections of FG (2% w/vin 0.9% saline) were performed using a glass micropipette(20–25 �m outer diameter; Sutter Instruments, Novato, CA)backfilled with a 2% FG solution. The pipette was positionedto the desired coordinates and left in place for 5 minutesprior to iontophoresis (5 mA alternating current, 7 secondson/off, 5 minutes), then left in place for an additional 5minutes before removal. Crystalline implants were per-formed using a glass micropipette (100–175 �m outer diam-eter; Sutter Instruments) filled with a suspension of FG(100% w/v in MilliQ) that was dried at 37°C for 48 hours toproduce a tracer plug. This assembly was aimed 0.5 mmdorsal to the PVN and left in place for a period of 1 minute.The FG plug was then ejected with a wire plunger (76 mmdiameter; Hamilton, Reno, NV) extending 0.5 mm beyondthe pipette tip. The pipette and plunger were left in place for

The Journal of Comparative Neurology. DOI 10.1002/cne

718 M. WILLIAMSON AND V. VIAU

an additional 10 minutes before removal to minimize thepotential uptake and transport of tracer along the pipetteand plunger track.

Both of these methods proved effective in delivering FGto the region of the PVN in terms of providing a concen-trated amount of tracer directed at the caudal two-thirdsof the nucleus (Swanson et al., 1981). Although traceraccumulation was noticeably more intense or completewithin individual cells using the crystal implant tech-nique, the total number of FG cells encountered withinmost forebrain cell groups was, nonetheless, relativelystable using both delivery methods. In the brainstem, bothmethods produced similar patterns of FG distribution.However, relatively greater disparities in cell stainingintensity and density were encountered among animalsbearing iontophoretic injections. In contrast, retrogradelabeling within individual brainstem neurons was muchmore intense and uniform using the crystal implant tech-nique. This discrepancy between the two injection tech-niques likely reflects a difference in the amount of tracerdeposited, detectable only over long transport distances.The brainstem results described, therefore, were takenonly from animals bearing the most appropriate PVNcrystal deposits.

Tracer deposits and resulting patterns of retrogradeaccumulation were further controlled by including theinitial assessment of the extent to which any deposit (ei-ther by injection or implant) was centered and concen-trated within the caudal two-thirds of the PVN. Most ofthe major functional groups of the PVN are found at(although not entirely restricted to) this level, includingneurosecretory cell groups that project to the median em-inence and posterior pituitary, and preautonomic cellgroups giving rise to preganglionic brainstem and spinalcord projections. Assisted by redirected sampling of adja-cent thionin-stained sections and alternately viewing thesections under darkfield illumination, material showingtracer delivery that was not centered within the posteriorregion of the PVN, or not fully encompassing its bound-aries, served as controls. Accumulation beyond the nu-clear boundaries of the nucleus was expected (seeSawchenko and Swanson, 1983). Cases in which this la-beling was extended to the contralateral PVN were notincluded. Controlling FG delivery using these criteria ren-dered stable patterns and densities of retrograde accumu-lation between animals. Guided by previous anatomicalretrograde and anterograde transport studies (see Table1), 10 animals were ultimately used to describe forebrainafferents (five iontophoretic and five crystal injections),and five animals (crystal only) were used to describe hind-brain afferents to the PVN. Illustrations representing ap-propriate iontophoretic and crystal FG injections into thePVN are shown in Figure 1. We also relied on animalsshowing FG deposits that missed the PVN (Fig. 1D) tohelp describe projections to the PVN surround. A total of30 cases showed independent and overlapping profiles ofFG that were concentrated dorsal, ventral, and lateral tothe caudal part of the PVN.

Tissue preparation

Two weeks following retrograde tracer injections intothe region of the PVN, optimal for FG transport (Schmuedand Fallon, 1986; Moga and Saper, 1994; Tillet et al.,2000), rats were deeply anesthetized with chloral hydrate(200 mg/kg) and perfused via the ascending aorta with

ice-cold 0.9% saline (125 mL), followed by 500 mL of ice-cold 4% paraformaldehyde (pH 9.5). The brains were post-fixed for 4 hours in a solution of the same fixative andcryoprotected in 15% sucrose in 0.1 M potassiumphosphate-buffered saline (KPBS, pH 7.4) overnight at4°C. Five adjacent 1-in-5 series of 30-�m-thick frozen sec-tions were collected and stored in cryoprotectant (30%ethylene glycol and 20% glycerol in 0.05 M KPBS buffer)at –20°C until histochemical processing. One series wasused to examine the injection site and the distribution ofretrogradely labeled cells. One series was counterstainedwith thionin and alternately compared with darkfield il-lumination to morphologically mark the location of retro-grade labeling. Based on the quality and confinement ofthe tracer deposit (see above), the remaining adjacentseries were processed to determine the relative extent towhich cell groups innervating the PVN region localizeAR-ir.

Localization and connectivity of androgen-sensitive neurons

To detect cells displaying nuclear AR-ir and cytoplasmictracer accumulation, we employed a sequential nickel- andnon-nickel-intensified avidin-biotin-immunoperoxidase pro-cedure, respectively. This was performed by first localizingAR-ir using antiserum directed at the N-terminal fragmentof the AR, followed by incubation in rabbit anti-FG serum.AR-ir was initially localized using the PG-21 antiserum,raised against N-terminal amino acids 1–21 (Chemicon,Temecula, CA, 06-680, lot 26042; 0.67 �g/mL). However, aportion of the material was processed using the N-20rabbit anti-AR antibody raised against N-terminal aminoacids 2–21 (Santa Cruz Biotechnology, Santa Cruz, CA,sc-816, lot E1004; 0.025 �g/mL; 1:8,000) due to a limitedsupply of PG-21. Both antibodies are specific to theN-terminal of AR (Prins et al., 1991; Kritzer, 1997), andconsistent with the findings of Kritzer (2004) our controlstudies showed that both of these antibodies produce sim-ilar distribution and density profiles for AR in adjacenttissue series taken from the same animal. Free-floatingsections were first rinsed in KPBS buffer to remove cryo-protectant, then pretreated with 0.3% hydrogen peroxidefor 10 minutes to quench endogenous peroxidase activity.This was followed by four rinses in KPBS, and then insodium borohydride (1% w/v in KPBS) for 5 minutes toreduce free aldehydes. Sections were then incubated for 48hours at 4°C in a KPBS-Triton (0.3% Triton-X; Sigma-Aldrich, Oakville, ON) solution containing 2% normal goatserum and the primary antiserum to detect AR. AR pri-mary antiserum was detected using a conventional nickel-intensified, avidin-biotin-immunoperoxidase (VectastainElite ABC kit; Vector Laboratories, Burlington, CA) pro-cedure (Li and Sawchenko, 1998). This procedure wasthen followed by a non-nickel variant of the proceduredescribed above using primary antisera against FG(Chemicon AB153, lot 24010565; 1:15,000).

Concurrent immunofluorescence detection of AR andFG involved the N-20 anti-AR antibody and tissue pre-treatment as described above, except that the hydrogenperoxide step was eliminated. Primary antiserum againstAR, in this case, was detected with a conjugated antirabbitIgG fluorescent secondary antibody (Alexa 594, Invitro-gen, Burlington, ON; 1:500). Cells displaying nuclearAR-ir and cytoplasmic FG were identified under fluores-cence wavelength and ultraviolet excitation, respectively.


719ANDROGEN-SENSITIVE PATHWAYS TO THE PVN

The pattern of AR staining in the brainstem (Sar andStumpf, 1975; Simerly et al., 1990; Hamson et al., 2004),either alone or in combination with FG labeling, suggesteda distribution within several catecholamine-producing,PVN-projecting cell groups (Chan and Sawchenko, 1994;Palkovits et al., 1997; Li and Sawchenko, 1998; Buller etal., 2001; Dayas et al., 2001b). Histochemical character-ization of AR-ir neurons in this population was performed

by using a retrograde transport-double immunohisto-chemical labeling technique including a mouse-derivedmonoclonal antibody against tyrosine hydroxylase (TH)purified from rat phenochromocytoma (Pel-Freeze, Rog-ers, AK, P80101-0, lot 15827; 1:2,000; see Liao et al.[1996]) and sheep polyclonal antibody against phenylethanolamine-N-methyltransferase purified from bovineadrenals (PNMT; Chemicon AB146, lot 0602021851;

TABLE 1. Relative Density of AR Staining within Candidate Afferent Mediators of Gonadal Status on the PVN Proper and Surround1,2,3

Cell Group

PVN Compartment

PVN Surround Referencesmpd pm dp/lp/mpv4

Forebrain LimbicPrefrontal cortex – – – � 14, 28Lateral septum – – – �� 22, 23Medial amygdala – – – � 5, 21Ventral subiculum – – – � 6Ventral premammillary n. – – – �� 3, 26

Bed n. Stria terminalisAnteromedial area �� 10, 20Dorsomedial n. �� 8Dorsolateral n. – – – �� 8Fusiform n. � � – – 8Principle n. �� – �� 9Interfascicular n. – – – �� 9Transverse n. – – – �� 9

Preoptic areaAnteroventral periventricular n. �� 29, 32Preoptic periventricular n. �� – �� 26, 30Median preoptic n. � � � � 26, 32Medial preoptic area �� – �� 26Medial preoptic n. �� – �� 29Lateral preoptic area � � – � 26, 30, 17

HypothalamusParastrial n. � � � � 29, 32Anterior hypo. n. �� – 22, 24, 26Lateral hypo. area �� – �� 12, 17, 26Arcuate n. � � � � 33Ventromedial n. �� – �� 4, 26Posterior hypo. area �� – – 23

ThalamusPosterior complex �� – 2

Hindbrain-SensorySolitary tract n., medial � � � – 7Parabrachial n., lateral �� 1, 15, 16, 19Parabrachial n., medial � � – � 19, 25

Hindbrain-ReticularPeriaqueductal gray, ventral lateral �� 11Periaqueductal gray, dorsal lateral – – – �� 23Locus coeruleus � � � � 7Dorsal raphe n. � � – � 18, 27Median raphe n. � � – � 18Raphe magnus n. � � � � 18Dorsal tegmentum � � � – 13, 23Pedunculopontine n. �� – 23Ventrolateral medulla �� – 7

AR, androgen receptor; PVN, paraventricular nucleus of the hypothalamus.1. Density of AR-immunoreactivity (AR-ir) in select cell groups showing the highest and most reliable incidence of AR�FG double labeling and projecting to the PVN region, asbased on previous anterograde labeling studies. Nomenclature based on the atlas of Swanson [31].2. A three point rating scale was used for comparison in which (��) represents AR staining in a substantial majority (�40%) of FG-labeled neurons in a given cell group, (��)represents a moderate density (20-39%), and (�) represents a low, but consistent density (5-19%) of AR staining within scattered populations of retrogradely labeled cells. Cellgroups showing very low and unreliable (0-5%) detection are not included (see Results).3. Based on previous anterograde tracing experiments, (–) indicates a lack of identified projections to the PVN region.4. Projections to one or more of the autonomic-related compartments of the PVN, described in detail elsewhere (see appropriate reference).

1. Bester et al., 1997.2. Campeau and Watson, 2000.3. Canteras et al., 1992.4. Canteras et al., 1994.5. Canteras et al., 1995.6. Cullinan et al., 1993.7. Cunningham and Sawchenko, 1988.8. Dong et al., 2001.9. Dong and Swanson, 2004.10. Dong and Swanson, 2006a.11. Floyd et al., 1996.

12. Goto et al., 2005.13. Groenewegen and Van Dijk, 1984.14. Hurley et al., 1991.15. Jhamandas et al., 1992.16. Krukoff et al., 1993.17. Larsen et al., 1994.18. Larsen et al., 1996.19. McKellar and Loewy, 1981.20. Mulders et al., 1997.21. Prewitt and Herman, 1998.22. Risold et al., 1994.

23. Risold et al., 1997.24. Roland and Sawchenko, 1993.25. Saper and Loewy, 1980.26. Sawchenko and Swanson, 1983.27. Sawchenko et al., 1983.28. Sesack et al., 1989.29. Simerly and Swanson. 1988.30. Swanson, 1976.31. Swanson, 1998.32. Thompson and Swanson, 2003.33. Watts et al., 1987.



1:5,000; see Legradi and Lechan [1998]). Free-floating tissueencompassing the brainstem from animals bearing PVNtracer implants were prepared as described above withslight modifications of these methods to optimize doublelabeling for AR-ir, and TH-ir, or PNMT-ir, including 1) theelimination of hydrogen peroxide pretreatment, and 2) usingbovine serum albumin as a blocking agent. Primary antiseraagainst AR and TH or PNMT were detected using conju-gated antirabbit (Alexa 594, Invitrogen; 1:500) and anti-mouse IgG (Alexa 488, Invitrogen; 1:500) or antisheep IgG(Alexa 488, Invitrogen; 1:500) fluorescent secondary antibod-ies, respectively. Concurrent immunofluorescence detectionof AR-ir, TH-ir, or PNMT-ir and FG-accumulating neuronswas achieved under appropriate fluorescence wavelengthand ultraviolet excitation. Counts using antisera against TH

and PNMT were obtained from adjacent series of sectionsfrom the same animal (n � 4).

Control experiments, in which the primary antiserum toAR was preadsorbed for 24 hours at 4°C with 6.7 �M(10-fold excess) synthetic peptide immunogen, correspond-ing to N-terminal amino acids 1–21 (MEVQLGL-GRVYPRPPSKTYRG; SynPep, Dublin, CA) or N-terminalamino acids 2–21 (sc-816 P, Santa Cruz Biotechnology) ofthe rat androgen receptor, failed to yield any evidence ofspecific AR staining (Bingham et al., 2006). Additionalcontrol experiments for antisera crossreactivity, involvingthe omission of either primary or secondary antibody,yielded no specific labeling. Finally, the staining patternfor either AR or FG was similar whether detected alone orprocessed in combination.

Fig. 1. Photomicrographs showing the appearance of iontophoretic(A) and crystalline (B) FluoroGold injection sites. Injection placementwas precisely determined by locating the core and spread of thedeposit under UV lighting and alternately viewing the section underdarkfield illumination (A and C, respectively). The schematic repre-sentation (D) describes the injection core (dark blue) and local uptakeand transport (light blue) in animals showing appropriate ionto-phoretic (left) and crystalline (right) tracer injections (n � 5 each).

Animals showing tracer injections that were not centered within theposterior region of the PVN (green), or not fully delimited to itsboundaries (red) served as controls (n � 30). Structures labeled forreference: AHN, anterior hypothalamic nucleus; fx, fornix; mpd, me-dial parvicellular, dorsal part (PVN); pm, posterior magnocellularpart (PVN); RE, nucleus reunions; V3, 3rd ventricle; ZI, zona incerta.Scale bar � 250 �m (applies to A–C).



Analysis and imaging

To provide an estimate of the relative number of AR-expressing neurons contained within PVN-projecting cellgroups, cell counts taken from immunoperoxidase mate-rial in animals bearing appropriate FG deposits were de-termined under brightfield conditions using a 40� objec-tive (NA 0.8). Doubly labeled (AR�FG) cells within eachpopulation of interest were defined as those showing ablack and brown reaction product in the nucleus and cy-toplasm, respectively. Counts were taken in complete, reg-ularly spaced (150-�m intervals) series of sectionsthrough the rostrocaudal extent of the cell groups in ques-tion and corrected for double-counting error using Aber-crombie’s formula (Abercrombie, 1946; and see Guillery,2002), factoring in regional differences in cell diameterwhere appropriate. Data describing the distribution ofAR-ir profiles within cells concurrently displaying cytoso-lic tracer are extrapolated estimates derived from the totalcorrected number of profiles encountered in each series ofsections multiplied by the sectioning interval of five. Par-celing of the rat brain followed the mapping of FG accu-mulation and AR staining as defined by the morphologicalfeatures provided by thionin staining of adjacent series oftissue, based on the terminology of Swanson (1998), and ofDong and Swanson (2004, 2006c) to describe the major bednuclei of the stria terminalis. Light-, dark-, andfluorescence-level images were captured using a Retiga1300 CCD digital camera (Q-imaging, Burnaby, BC), an-alyzed using Macintosh OS X-driven, Open Lab ImageImprovision software v. 3.0.9 (Quorum Technologies,Guelph, ON), and exported to Adobe Photoshop (v. 7.0,San Jose, CA), where standard methods were used toadjust contrast and brightness, and final assembly at aresolution of 300 dpi.

RESULTS

FG injection and retrograde labeling

As detailed above, several criteria were imposed to en-sure that FG injections were centered at the caudal two-thirds of the PVN region, specifically. Discrete and con-centrated injections were produced when deliverediontophoretically; and intense, but less restricted, depositswhen delivered by crystal implantation. The crystallinedeposit also produced minimal spread along the pipettetract, which typically occurs with volume or iontophoreticinjections (see also Lind, 1986; Li and Sawchenko, 1998).In most experiments the tracer injections were concen-trated in, but not completely restricted to, the morpholog-ical confines of the PVN (Fig. 1). The largest depositsspread dorsally to the nucleus reunions of the thalamusand the zona incerta or ventrally to involve aspects of theanterior hypothalamic area. The injections never ex-tended laterally into or beyond the fornix. In several in-stances, injections made outside the PVN proper wereuseful in describing potential indirect sources ofandrogen-sensitive, limbic forebrain-related input to thePVN (Roland and Sawchenko, 1993; Herman et al., 2003,2005). As described below, and in agreement with Rina-man et al. (1995), the pattern and density of FG labelingin the brainstem (e.g., nucleus of the solitary tract, ven-trolateral medulla) in animals bearing the smallest andmost concentrated injections in the PVN was comparableto those showing larger tracer deposits encompassing the

anterior one-third of the PVN, the ventral tip of the nu-cleus reunions, and/or deposits centered toward the dorsalaspect of the PVN nucleus. As previously described, theseobservations indicate that neurons adjacent to, or in theimmediate vicinity of, the PVN do not substantially con-tribute to retrograde labeling in the brainstem. Thesefindings are consistent with previous phenotypic studiesdescribing only meager adrenergic input to neighboringcell groups such as the thalamic nucleus reunions andanterior hypothalamic area (Swanson et al., 1981), and inagreement with previous anterograde studies confirmingthe existence of brainstem afferents to discrete aspects ofthe parvicellular and magnocellular divisions of the PVN(Cunningham and Sawchenko, 1988; Cunningham et al.,1990).

The pattern of retrograde labeling in the hypothalamuswas predominately ipsilateral to the side of the injection,although bilateral labeling was most evident in those an-imals showing tracer accumulation or spread to the con-tralateral PVN. This was most obvious when injectionswere biased toward the dorsal part of the PVN and/orencroached upon the ventromedial tip of the ipsilateralnucleus reuniens. Bilateral retrograde labeling was evi-dent in the lower brainstem, even when the smallest in-jections were entirely confined within the nuclear bound-aries of the PVN. Although the clear majority of brainstemstructures labeled ipsilaterally, in some instances bilat-eral (albeit sparse) retrograde labeling was observedwithin the lateral tegmental nucleus, the ventrolateraldivisions of the periaqueductal gray, and the caudal as-pect of the ventrolateral medulla.

To minimize potential differences in the amount oftracer delivered to the PVN, so that numerical assess-ments in FG accumulation between regions of interestcould be made with confidence, we limited quantificationto those animals showing comparable levels of tracerspread and accumulation within the immediate vicinity ofthe nucleus. This was initially determined in unreactedmaterial, in which FG transport and accumulation wasvisualized under ultraviolet illumination, using size,shape, and contralateral diffusion as an index of place-ment and accumulation. Some discrepancy between thetwo methods of tracer delivery was expected at the cellularand regional levels, depending on the area of interest anddistance required for tracer transport (described in Mate-rials and Methods). Subsequent light-level analysis ofimmunoperoxidase-reacted tissue indicated comparablenumbers and distribution of detectable FG-labeled cells inthe forebrain using either tracer delivery method, in gen-eral, although in some cases the circumscribed injectionsproduced by iontophoresis were more effective in identify-ing the most local of afferent sources to the PVN (e.g.,projections from the anterior hypothalamic nucleus). Onthe other hand, we observed far less variance in the num-ber of detectable neurons in the brainstem of animalsbearing discrete crystalline implants (e.g., pontine andmedullary neurons). Thus, the forebrain data presentedhere were derived from both injection methods, while thebrainstem data presented is from animals bearing crystalinjections only. Controlling FG injections as such provideda reliable means with which to gauge the relative contri-butions of AR-expressing afferents to the PVN.



Forebrain retrograde- and androgenreceptor-labeled neurons

Limbic-related; PFC, septum, amygdala, and hip-

pocampus. In agreement with previous anatomicalstudies, no area of the prefrontal cortex (Sesack et al.,1989; Hurley et al., 1991; Radley et al., 2006), septum(Risold et al., 1994; Risold and Swanson, 1997), amygdala(Canteras et al., 1995; Prewitt and Herman, 1998), orhippocampus (Canteras and Swanson, 1992) showed reli-able retrograde labeling following the most discrete tracerinjections. In animals bearing FG deposits centered orextending beyond the nuclear boundaries of the PVN,retrograde labeling was occasionally observed in the pre-frontal cortex (0–25 labeled neurons per section), lateralseptum (15–35), medial amygdala (15–35), and hippocam-pal ventral subiculum (0–25). Injections placed along thedorsal borders of the PVN, impinging upon the ventralaspects of the nucleus reunions and zona incerta, yieldeda cluster of FG-labeled cells in the infralimbic and cingu-late cortex (areas 25 and 24/29, respectively) without anyapparent dorsal-ventral bias. On the other hand, tracerdeposits extending ventrally into the surrounding sub-paraventricular zone and anterior hypothalamic area con-sistently yielded tracer uptake in the ventrolateral part ofthe septal complex. Within the amygdala, tracer depositsshowing a ventral bias in the PVN often labeled thepostero-dorsal and -ventral parts of the medial amygdala(Fig. 2), while the central and basolateral amygdaloidnuclei were consistently devoid of retrograde labeling (Sil-verman et al., 1981; Ono et al., 1985; Canteras et al., 1992,1995; Prewitt and Herman, 1998; Campeau and Watson,2000). Similarly, FG deposits that invaded the subpara-ventricular zone resulted in reliable retrograde accumu-lation within the temporal aspect of the ventral subiculum(Canteras and Swanson, 1992; Cullinan et al., 1993). ARsare abundantly expressed in the lateral septum, medial

amygdala, and subiculum, and to a much lesser extent inthe prefrontal cortex (Sar and Stumpf, 1975; Simerly etal., 1990; Clancy et al., 1992). In animals bearing PVNinjections that were off-centered, only modest numbers ofdoubly labeled (AR�FG) cells were detected in these re-gions, never exceeding �30% of the total number of FG-labeled cells encountered.

Bed nuclei of the stria terminalis. Parceling and ter-minology used for describing FG accumulation followedDong and Swanson (2004, 2006c), in which we comparedthe pattern of FG labeling and AR staining against themorphological features of adjacent thionin-stained sec-tions. Within the anterior division of the bed nuclei of thestria terminalis (BST), FG labeling was detected withinthe anteromedial group, including the anterodorsal andanteroventral nuclei, in agreement with several previousretrograde and anterograde mapping studies (Sawchenkoand Swanson, 1983; Weiss and Hatton, 1990; Cullinan etal., 1993, 1996; Moga and Saper, 1994; Prewitt and Her-man, 1998; Spencer et al., 2005). The incidence of AR�FGdouble labeling was very consistent in both the anterodor-sal (31%) and anteroventral (24%) nuclei. While very highnumbers of AR-ir cells were encountered within the adja-cent lateral, juxtacapsular, and oval nuclei, doubly labeledcells in these nuclei were rarely encountered. Within themedial part of the rostrocaudal extent of the BST, FGinjections appropriately centered in the PVN routinelylabeled a dense packing of cells in the dorsomedial, ven-tral magnocellular, and fusiform bed nuclei (Fig. 3), con-sistent with recent anterograde surveys (Dong and Swan-son, 2006a–c). Moderate numbers of doubly labeled cellswere reliably encountered within all of these nuclei, con-sistently highest within the dorsomedial nucleus.

The posterior division of the BST constitutes a majorsource of input to the PVN (Ju and Swanson, 1989; Culli-nan et al., 1996; Gu et al., 2003; Dong and Swanson, 2004),

Fig. 2. Photomicrographs showing the appearance of FluoroGold(FG)-labeled cells in the medial amygdala, typically encounteredwhen retrograde tracer injections diffused beyond the borders of thePVN or were centered in the subparaventricular zone. Darkfield viewof retrograde labeling within the posterodorsal and posteroventralparts of the medial amygdala (A). Enlarged brightfield view (B) of the

boxed region showing dual immunoperoxidase labeling for nuclearAR-ir and cytoplasmic FG-ir. Solid arrows show doubly labeled neu-rons and open arrowhead marks an AR-positive, FG-negative cell.Structure labeled for reference: ot, optic tract; MeApd and MeApv,posterodorsal and posteroventral medial amygdala. Scale bar � 50�m in B; 250 �m for A.



and is concentrated by both estrogen and ARs (Simerly etal., 1990; Simerly, 1993, 2002; Shughrue et al., 1997;Laflamme et al., 1998; Greco et al., 2001; Auger and DeVries, 2002). Not surprisingly, this aspect of the BSTshowed a very high incidence of double labeling (Fig. 3),predominately within the principle nucleus (Fig. 4), whichpreferentially innervates the medial parvicellular part ofthe PVN (Dong and Swanson, 2004). Relatively moderatenumbers of double-labeled cells were encountered withinthe interfascicular and transverse nuclei. Anterogradeanalysis by Dong and Swanson (2004) indicated that thetransverse BST nucleus innervates, at best, the anteriorparvicellular part of the PVN. Clearly distinct, the inter-fascicular BST nucleus innervates regions located outsideor immediately adjacent to the PVN proper, includingprojections to the ventral aspect of the nucleus reuniens,the dorsomedial portion of the anterior hypothalamicarea, and the subparaventricular zone of the hypothala-mus (Dong and Swanson, 2004). Perhaps consistent withthis unique projection profile, our initial assessment ofinjection placement in the PVN suggested a passing influ-ence of ventral FG deposits on retrograde labeling in theregion occupied by the interfascicular nucleus.

Circumventricular organs. A moderate number ofFG-labeled cells were consistently found within the lateralparts of the subfornical organ, to a lesser extent within theregion of the organ of the lamina terminalis, and not at allwithin the area postrema (Berk and Finkelstein, 1981;Sawchenko and Swanson, 1983; Plotsky et al., 1988; Weissand Hatton, 1990; Thellier et al., 1994; Pan et al., 1999).AR detection was low to moderate in the subfornical organand lamina terminalis, and most concentrated in the areapostrema (Sar and Stumpf, 1975; Simerly et al., 1990;Hamson et al., 2004), and doubly labeled cells werescarcely, if at all, detected in these regions.

Hypothalamus. Almost all major areas of the hypo-thalamus showed retrograde accumulation and showeddouble labeling to various degrees (Figs. 5, 6). While theuptake of FG in most of the forebrain proved comparablebetween the most circumscribed crystal deposits and dis-crete iontophoretic injections into the PVN, we relied moreheavily on the iontophoretic method when gauging traceraccumulation in the immediate vicinity of the PVN, in-cluding within the anterior hypothalamic area. Other-wise, comparable numbers of FG accumulating cells in theremainder of the hypothalamus were achieved in animalsbearing the smallest crystal injections and the most cir-cumscribed iontophoretic injections of tracer in the PVN.Within the diagonal band complex, small numbers of FG-positive cells were occasionally found. The density of FG-labeled cells in this region appeared to vary with tracerinjection size, likely delivered by terminals occupying theperimeter of the PVN (see Larsen et al., 1994). Small tomoderate numbers of AR-ir neurons were scatteredamong, but seldom colocalized to, FG-labeled cells in thediagonal band complex.

The medial preoptic area represents a major source ofinput to the PVN (Tribollet and Dreifuss, 1981;Sawchenko and Swanson, 1983; Simerly and Swanson,1988; Weiss and Hatton, 1990; Larsen et al., 1994; Culli-nan et al., 1996; Champagne et al., 1998; Tribollet et al.,1999; Campeau and Watson, 2000). Consistent with thisprofile, a very high density of FG-labeled cells was reliablyfound throughout this region (Fig. 7). FG-accumulatingneurons were also encountered in the lateral part of thepreoptic area, but to a much lesser extent (�350 cells).This number increased when tracer injections were biasedtoward, or extended beyond, the lateral border of the PVN.Most striking was the amount of FG-labeling in the me-dial preoptic nucleus (Fig. 7). Notably, the medial preopticnucleus projects strongly and preferentially to the dorsalmedial parvocellular part of PVN (Simerly and Swanson,1988). Of all the forebrain regions surveyed, the medialpreoptic nucleus showed the highest number of FG-accumulating neurons in total (Campeau and Watson,2000), and almost 70% of these stained for AR (Fig. 5). Asdiscerned along morphological grounds, by redirectedsampling of adjacent thionin-stained sections, a relativelysmall to moderate population of doubly labeled cells wasfound within the medial (27%) and lateral (16%) aspects ofthe preoptic area and within the preoptic periventricularnucleus (see Fig. 7).

Retrograde labeling was moderate, but consistently de-tected through the rostrocaudal extent of median preopticnucleus, and with an apparent greater density toward theposterior aspect of the nucleus adjoining the medial pre-optic area. Low levels of AR staining was detectedthroughout the median preoptic nucleus; however, theincidence of double labeling was scarce at best (5–10%).The PVN is strongly innervated by the parastrial nucleus(Sawchenko and Swanson, 1983; Swanson, 1987; Simerlyand Swanson, 1988; Cullinan et al., 1996; Thompson andSwanson, 2003). While moderate levels of AR stainingwere most readily detected along the dorsal and ventralborders of the parastrial nucleus (Fig. 7), the number ofdouble-labeled cells encountered varied considerably be-tween animals (Figs. 6, 7).

Consistent with previous experiments (Sawchenko andSwanson, 1983; Watts and Swanson, 1987), scatterednumbers of FG-labeled cells (10–15 cells per section) were

Fig. 3. Estimated cell counts of FG-labeled neurons and AR con-tainment within bed nuclei of the stria terminalis. Mean � SEM totalnumber of FG neurons (stacked open bars) and the number of doublylabeled (AR�FG) neurons (solid bars) detected per BST region. Thenumber above each solid bar indicates the percent of double labeling,providing a relative index of AR containment between each region(n � 10). AM, anteromedial area; DM, dorsomedial nucleus; FU,fusiform nucleus; PR, principal nucleus; IF, interfascicular nucleus;TR, transverse nucleus.



consistently found within the medioventral part of thesuprachiasmatic nucleus in animals bearing the most cir-cumscribed FG injections. In contrast, considerablygreater numbers were encountered in animals bearinginjections that were centered in or invaded the subpara-ventricular zone of the PVN. This finding agrees with theanterograde experiment of Watts et al. (1987) suggesting

that the suprachiasmatic nucleus provides minor input tothe PVN proper. AR staining was not detected in thesuprachiasmatic nucleus, consistent with the absence ofthe AR transcript in this structure (Simerly et al., 1990).

A very large assortment of FG-labeled cells were scat-tered throughout the rostrocaudal extent of the anteriorhypothalamic area. While comparable numbers of FG cells

Fig. 4. Photomicrographs showing the accumulation of FG withinthe posterior division of the bed nucleus of the stria terminalis at thelevel of the rostral extent of the PVN. The darkfield view (A) illus-trates a high density of FG labeling within anterior parvicellular PVNneurons and a scattered profile of retrograde labeling in the immedi-ate (lateral) vicinity of this PVN population, including within theprinciple nucleus of the BST (boxed region). AR staining was detected

throughout the bed nucleus, but not within anterior parvicellularPVN neurons. Enlarged brightfield view (B) of the boxed region show-ing dual immunoperoxidase labeling for AR-ir and FG-ir. Solid arrowsshow doubly labeled neurons and open arrowhead marks AR-positive,FG-negative cell. ap, anteroparvicellular PVN; fx, fornix; V3, thirdventricle. Scale bar � 50 �m in B; 500 �m for A.

Fig. 5. Estimated cell counts of FG-labeled neurons and AR con-tainment through the rostrocaudal extent of the medial hypothala-mus. Mean � SEM total number of FG neurons (stacked open bars)and the number of doubly labeled (AR�FG) neurons (solid bars)detected per hypothalamic region (n � 5). MPA, medial preoptic area;MPN, medial preoptic nucleus, AHNc, central part of the anteriorhypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus;VMHd and VMHd, ventromedial hypothalamic nucleus, dorsal andventral part.

Fig. 6. Estimated cell counts of FG-labeled neurons and AR con-tainment through the rostrocaudal extent of the periventricular andlateral hypothalamus. Mean � SEM total number of FG neurons(stacked open bars) and the number of doubly labeled (AR�FG) neu-rons (solid bars) detected per hypothalamic region (n � 5). PS, paras-trial nucleus; AVPV, anteroventral periventricular nucleus; PVpo,preoptic periventricular nucleus; PVp, posterior periventricular nu-cleus; LHA, lateral hypothalamic area; PMv, premammillary nucleus,ventral part.



were found in the anterior and central parts of the ante-rior hypothalamic area (�500 and 750 cells, respectively),the incidence of double labeling was consistently higher inthe central part (34%, see Fig. 5) compared to the anteriorpart (14%). In comparison, relatively few FG-labeled cellswere found in the posterior part of the anterior hypotha-lamic area (�50 cells), but with an incidence of doublelabeling equivalent to that of the central part (34%). Atthe level of the posterior part of the PVN, noticeableclusters of AR-ir cells were reliably detected within theperifornical part of the anterior hypothalamic area (seeRoland and Sawchenko, 1993; Cullinan et al., 1996;Champagne et al., 1998; Campeau and Watson, 2000).Small, but generally consistent proportions of FG-accumulating cells in this region stained positively for AR(�15%).

A substantial number of FG-labeled cells were consis-tently found within the ventromedial and dorsomedialhypothalamic nuclei (Fig. 5), in agreement with previousfindings (Canteras et al., 1994; Cullinan et al., 1996;Thompson and Swanson, 1998, 2003). The distributionand concentration of tracer accumulating neurons wasquite uniform across all parts of the dorsomedial nucleusin animals showing the most circumscribed injections ofFG. In contrast, fewer, but no less uniform, numbers ofretrograde-labeled cells were observed in animals bearingFG injections that occurred either above of below the PVNproper. These results are in agreement with several pre-vious anterograde (Ter Horst et al., 1984; Ter Horst andLuiten, 1986, 1987; Thompson et al., 1996) and retrogradetracing experiments (Sawchenko and Swanson, 1983;Levin et al., 1987; Fodor et al., 1994; Champagne et al.,1998). While the dorsomedial nucleus reliably stained forAR, the incidence of double labeling was scarce, if at alldetectable.

Within the small circular anterior part of the ventrome-dial nucleus, at the level of the posterior part of the ante-

rior hypothalamic area, few and variable numbers of scat-tered retrograde labeled cells were detected. FG labelingwas most evident toward the posterior aspect of the nu-cleus, concentrated within the larger cell-dense dorsome-dial and ventrolateral parts of the ventromedial nucleus(Silverman et al., 1981; Tribollet and Dreifuss, 1981;Sawchenko and Swanson, 1983; Campeau and Watson,2000). FG labeling was relatively sparse within the diag-onally oriented central part separating the dorsal andventral parts of the ventromedial nucleus. Consistent withprevious anterograde tracing studies (Saper et al., 1976;Canteras et al., 1994), the number of FG-labeled cellswithin the ventromedial nucleus increased dramaticallyin animals bearing FG injections that were biased towardthe subparaventricular zone of the PVN. All parts of theventromedial nucleus, with the exception of the cell-poorcentral part, stained strongly for AR. While the dorsome-dial and ventrolateral parts contained similar numbers oftracer accumulating and AR-expressing cells in total, theincidence of double labeling was consistently higherwithin the dorsomedial part (Fig. 5).

Tracer accumulating cells were found scatteredthroughout the extent of the lateral hypothalamic area,and distributed within each of its subregions (Saper et al.,1979; Silverman et al., 1981; Larsen et al., 1994; Cullinanet al., 1996). Specifically, FG-labeled cells were concen-trated within the ventral zone of the nucleus, located justdorsal to the optic tract, in agreement with the profiledescribed by Larsen et al. (1994). AR staining was scat-tered throughout the lateral hypothalamic region withoutany obvious topographical bias. Double-labeled cells wereencountered in all parts of the lateral hypothalamic area(26%), and a clear majority of these were located withinthe ventral zone of the nucleus.

A moderate number of tracer-accumulating neuronswere detected throughout the rostrocaudal extent of thearcuate nucleus. The highest density of FG labeling was

Fig. 7. Photomicrographs showing the accumulation of FG at thelevel of the medial preoptic area. The darkfield view illustrates aunilateral distribution of FG uptake (A), prominent within theperiventricular and medial zones of the hypothalamus, including adense collection of cells within the medial preoptic nucleus (boxedregion). Note also, dense clusters of FG-labeled cells within fusiform

(Fu) and parastrial (PS) nuclei. Enlarged view (B) of the boxed regionshowing concurrent fluorescent retrograde tracer uptake (blue cyto-plasm) and AR staining (red nucleus) in the medial preoptic nucleus.Solid arrows show doubly labeled neurons and open arrowhead marksan AR-positive, FG-negative cell. ac, anterior commissure; och, opticchiasm; V3, third ventricle. Scale bar � 50 �m in B; 1,000 �m for A.



reliably encountered along the medioventral aspect of thenucleus, at the level of the dorsal extent of the dorsome-dial hypothalamic nucleus (see Silverman et al., 1981;Sawchenko et al., 1982; Baker and Herkenham, 1995). ARstaining was scattered through the rostrocaudal extent ofthe arcuate nucleus, and the incidence of double labelingwas likewise represented by a scattered complement ofcells (12%).

Within the most caudal reaches of the hypothalamus,low to moderate numbers of FG-labeled cells were rou-tinely detected within the dorsal premammillary nucleus.Most conspicuous was the distribution of retrograde accu-mulation within the ventral premammillary nucleus, par-ticularly its anterior part (Fig. 6), in addition to the pos-terior periventricular nucleus (Silverman et al., 1981;Sawchenko and Swanson, 1983; Campeau and Watson,2000). Approximately 44% of FG-labeled cells in the ven-tral premammillary nucleus stained positively for AR.Higher numbers of cells were detected within the ventralpremammillary nucleus in animals bearing injections thatwere biased toward the anterior part of the PVN. Takentogether, the profile of FG accumulation and sensitivity toinjection placement are in line with the anterograde studyof Canteras et al. (1992), showing that the premammillarynucleus projects primarily to the anterior and periven-tricular regions of the PVN. The medial and lateral mam-millary nuclei were consistently devoid of FG labeling. Onthe whole, a relatively dense number of FG-labeled cells(�250 cells) were detected throughout the posterior hypo-thalamic area (see Simerly et al., 1990; Yokosuka andHayashi, 1996), and a reliable number of these stained forAR (24%).

Periventricular zone. Consistent with previous find-ings (Tribollet and Dreifuss, 1981; Sawchenko and Swan-son, 1983), a very high density of tracer-accumulatingcells was found through the median preoptic, anteroven-tral, preoptic, and posterior periventricular nuclei, andwithin the ventrally displaced suprachiasmatic preopticnucleus. FG labeling was occasionally detected in the vi-cinity of the posterior part of the vascular organ of thelamina terminalis, but clearly biased toward the caudalextent of the anteroventral periventricular nucleus. ARstaining was prevalent throughout the periventricularzone of the hypothalamus; however, the incidence of dou-ble labeling was evidently highest in the anteroventraland posterior periventricular nuclei (Fig. 6).

Thalamus. Small contingents of tracer-accumulatingneurons were routinely identified within the paraven-tricular nucleus of the thalamus. In agreement with pre-vious retrograde experiments (Sawchenko and Swanson,1983; Campeau and Watson, 2000), FG-labeled cells werescattered through the rostrocaudal extent of the nucleus,and always distributed ipsilateral to the side of the injec-tion. The density of retrograde labeling in this cell groupwas typically greater when the injections were placedmore dorsally, invading the zona incerta. AR-ir neuronswere not detected in the paraventricular nucleus of thethalamus. The highest density of FG-labeling was ob-served within the posterior thalamic nuclei, including theparvicellular part of the subparafascicular nucleus, in ad-dition to the peripeduncular and posterior intralaminarthalamic nuclei (Campeau and Watson, 2000). Within theparvicellular part of the subparafascicular nucleus, spe-cifically, retrograde labeling was reliably detected in twodiscrete groups of cells, including a small cluster of hori-

zontally oriented cells near the lateral borders of the ven-tral posteromedial thalamic nucleus and beneath the fas-ciculus retroflexus (LeDoux et al., 1985; Price, 1995). Thispattern of FG labeling extended caudally into the peripe-duncular and posterior intralaminar thalamic nuclei.Within the posterior intralaminar nucleus retrograde la-beling was most apparent along the dorsal borders of thesubstantia nigra, pars compacta, and a significant comple-ment of FG-labeled cells along this continuum stained forAR (Fig. 8).

Fig. 8. Estimated cell counts of FG-labeled neurons and AR con-tainment through the hindbrain reticular formation and sensory as-sociated nuclei. Mean � SEM total number of FG neurons (stackedopen bars) and the number of doubly labeled (AR�FG) neurons (solidbars) detected per region (n � 5). PoT, posterior thalamic complex;PBL, lateral parabrachial nucleus; PBm, medial parabrachial nu-cleus; NTSm, nucleus of the solitary tract, medial division; VLM,ventrolateral medulla; PPN, pedunculopontine nucleus.

Fig. 9. Estimated cell counts of FG-labeled neurons and AR con-tainment through the hindbrain reticular core nuclei. Mean � SEMtotal number of FG neurons (stacked open bars) and the number ofdoubly labeled (AR�FG) neurons (solid bars) detected per region (n �5). PAGvl and PAGdl, periaqueductal gray, ventrolateral and dorso-lateral column; DT, dorsal tegmentum; LDT, laterodorsal tegmentalnucleus; LC, locus coeruleus; RMg, raphe magnus nucleus.



Hindbrain retrograde- and androgenreceptor-labeled neurons

Reticular core. The pattern of FG labeling (Figs. 8, 9)is in agreement with several previous studies showingsparse to moderate labeling throughout the hindbrain(Berk and Finkelstein, 1981; Tribollet and Dreifuss, 1981;Sawchenko and Swanson, 1982; Campeau and Watson,2000). Retrograde labeling was substantial within theperiaqueductal gray and its pontine continuation, includ-ing the lateral, ventrolateral, and dorsolateral columns(Bandler and Keay, 1996; Bandler et al., 2000). In thedorsolateral column of the periaqueductal gray, FG label-ing was most pronounced in animals bearing tracer injec-tions biased toward the dorsal border of the PVN, in whichthe rostral division of the nucleus reunions was invaded(Risold et al., 1997). FG labeling was highest and mostreliable within the ventrolateral column (Figs. 9, 10), con-sistent with previous retrograde (Tribollet and Dreifuss,1981) and anterograde tracing experiments (Cameron etal., 1995; Floyd et al., 1996) showing that this aspect of theperiaqueductal gray provides a rich and unique source ofinput to the PVN. AR staining was prominent throughoutthe rostrocaudal extent of the PAG (see Sar and Stumpf,1975; Simerly et al., 1990; Hamson et al., 2004), anddouble labeling was most pronounced within the ventro-lateral part and to a lesser extent within the dorsolateralpart of the periaqueductal gray (Figs. 9, 10). Small clus-ters of FG-labeled cells were reliably detected within thecuneiform, central tegmental, and mesencephalic reticu-lar retrorubral fields, and within the superior colliculusand the rostral linear nucleus (Silverman et al., 1981;Sawchenko and Swanson, 1983). ARs were detectedwithin all of these nuclei, although the incidence of doublelabeling was scarce, if at all indicated (0–5%).

Substantial numbers of FG-labeled neurons were con-sistently detected within and in the vicinity of the dorsaltegmental nucleus (Berk and Finkelstein, 1981; McKellarand Loewy, 1981; Tribollet and Dreifuss, 1981; Rye et al.,1987; Bittencourt et al., 1991; Champagne et al., 1998).FG labeling was sparse to undetectable within the largestexpanse of the dorsal tegmental nucleus, and this numberincreased significantly toward the region occupying thecaudal extent of the nucleus. FG distribution at this levelwas most noticeable within the dorsolateral part of thepontine central gray, in the vicinity of the rostral part ofthe locus coeruleus and Barrington’s nucleus (Fig. 11). Asmall cluster of FG-labeled cells was also detected withinthe diffuse part of the nucleus incertus, spanning themidline below the floor of the cerebral aqueduct at thelevel of the caudal part of the locus coeruleus (Berk andFinkelstein, 1981; Sawchenko and Swanson, 1982, 1983).AR staining was most conspicuous within the nucleusincertus, and a small but reliable number of FG-labeledcells in this nucleus stained for AR (18%).

FG injections in the PVN gave rise to retrograde label-ing within distinct mesencephalic 5-hydroxytryptaminecell groups, most prominent at the level of the midbraindorsal (B7), median (B8), and pontine raphe magnus (B3cell group) nuclei (see Silverman et al., 1981; Sawchenkoand Swanson, 1983; Sawchenko et al., 1983; Petrov et al.,1992; Larsen et al., 1996; Champagne et al., 1998). Withinthe nucleus raphe magnus, FG accumulation was highesttoward the rostral extent of the nucleus, situated justdorsal to the medial lemniscus, and the number of FG cells

detected showed little variation between animals bearingappropriate tracer injections or implants. In contrast, ret-rograde labeling in the dorsal and median raphe variedconsiderably as a function of injection size and type. Ani-mals bearing the smallest and most circumscribed tracerinjections showed considerably fewer numbers of FG cellsin the dorsal (350 cells) and median (175 cells) raphenuclei compared to animals bearing large injections thatinvaded the PVN surround. These findings are in agree-ment with the anterograde tracing study by Larsen et al.(1996), indicating that the raphe magnus provides thelargest amount of input directed at the PVN proper, incontrast to the dorsal and median raphe nuclei that pref-erentially innervate the PVN surround. AR staining wasscattered throughout the midline of the midbrain, pons,and medulla, and all three raphe nuclei showed low tomoderate levels of AR staining, in agreement with Simerlyet al. (1990) and Hamson et al. (2004). A low, but consis-tent degree of double labeling was observed within allthree raphe nuclei, highest within the median raphe nu-cleus (9%, see Fig. 9) and to a lesser extent in the dorsal(5%) and median raphe (5%) nuclei.

Pons-medulla. A substantial number of FG-labeledcells were detected in the pontine parabrachial nucleus.Consistent with previous retrograde surveys (Larsen andMikkelsen, 1995; Pan et al., 1999) and in agreement withanterograde tracing studies (Saper and Loewy, 1980;McKellar and Loewy, 1981; Fulwiler and Saper, 1984;Jhamandas et al., 1992; Bester et al., 1997), the vastmajority of retrograde labeling was found within the lat-eral subdivision of the nucleus and only very few FG-labeled cells were detected in the medial subdivision of theparabrachial nucleus. Based on cytoarchitectonic criteriaprovided by Fulwiler and Saper (1984), FG labeling wasmost concentrated within the dorsal and central parts ofthe lateral parabrachial subdivision, and to a much lesserextent within the external lateral part, consistent withprevious retrograde experiments specifically involving thecaudal half of the PVN (Larsen and Mikkelsen, 1995; Panet al., 1999). Anterograde studies indicate that each of thelateral parabrachial nuclei are preferentially directed atthe parvicellular division of the PVN (Saper and Loewy,1980; Jhamandas et al., 1992), including its dorsal parvo-cellular part (Bester et al., 1997). AR staining was concen-trated throughout the lateral parabrachial subdivision,although to a much lesser extent within the medial sub-division of the nucleus. A high degree of double labelingwas reliably observed within the dorsal and central partsof the lateral parabrachial division nucleus, and to a mi-nor extent within the medial division (Fig. 8).

FG injections appropriately centered in the PVN consis-tently labeled significant numbers of cells in the ventralmedulla (the C1 and A1 cell groups) and moderate num-bers in the dorsal medulla, primarily within the nucleus ofthe solitary tract, representing the C2 and A2 cell groups(McKellar and Loewy, 1981; Sawchenko and Swanson,1982; Sawchenko et al., 1985; Cunningham andSawchenko, 1988; Cunningham et al., 1990; Rinaman etal., 1995; Pan et al., 1999; Card et al., 2006). Reliable, butfar less retrograde labeling (�50 cells total), was observedin the locus coeruleus (A6 cell group). Double labeling(FG�AR) was highest in the ventral medulla (Fig. 8),observed as a conspicuous cluster of cells situated betweenthe principle and subtrigeminal parts of the lateral retic-ular nucleus (Sawchenko et al., 1985; Rinaman et al.,



1995). In the nucleus of the solitary tract the majority ofFG-labeled cells were detected within the medial part ofthe nucleus, and relatively less retrograde labeling wasencountered within the lateral part of the nucleus(Sawchenko and Swanson, 1982; Sawchenko et al., 1985).AR staining was uniformly scattered throughout the soli-tary tract nucleus and locus coeruleus (Simerly et al.,1990; Hamson et al., 2004). Double labeling was reliablyencountered within the medial part of the solitary tractnucleus (Fig. 8) and only a very small contingent of FG-labeled cells in the locus coeruleus stained for AR (Fig. 9).

The spatial profile of AR staining within FG-accumulating cells in the locus coeruleus, medulla, anddorsal vagal complex suggested a possible distributionwithin the catecholaminergic cell groups (Sawchenko andSwanson, 1982; Sawchenko et al., 1985; Rinaman et al.,1995). Thus, we used a combined retrograde transport-double immunohistochemical-labeling technique to gaugethe extent to which AR-expressing, PVN afferents withinthese cell groups are represented by noradrenergic andadrenergic neurons (Figs. 11–13). In agreement with pre-vious findings (Sawchenko et al., 1985), a clear majority ofFG-labeled cells spanning the rostrocaudal extent of theventrolateral medulla, the nucleus of the solitary tract,and in the locus coeruleus (70–85%) stained positively forTH (FG�TH, Figs. 11–13D). As expected, PNMT-ir wasnot detected in the locus coeruleus (A6 cell group; seeSawchenko and Swanson, 1982; Sawchenko et al., 1985).

Independent assessment of AR and PNMT double label-ing suggested an almost even distribution of AR stainingamong the noradrenergic (60%) and adrenergic (40%) neu-rons of the ventrolateral medulla, and a reliable propor-tion (20–45%) of the retrogradely labeled, TH-positivecells through the extent of the ventrolateral medulla alsostained for AR (FG�TH�AR, Fig. 12D). Similar to theidentity of AR cells in the ventral medulla, roughly halfthe total of AR cells spanning the rostrocaudal extent of

the solitary tract nuclei stained for PNMT. A significantproportion (88%) of TH cells in the nucleus of the solitarytract stained positively for AR (TH�AR, Fig. 13D); how-ever, only rarely and unreliably (0–40%) were triple-labeled (FG�TH�AR) cells encountered (Fig. 13D). In thelocus coeruleus, TH and AR double labeling was consider-able (66%); however, triple labeling was inconsistent (0–25%; Fig. 11D). In sum, of the PVN projecting cat-echolaminergic cell groups, AR was most largelycontained within C1 and A1 neurons of the ventral me-dulla.

Based on the inherent limitations and difficulties inconcurrently visualizing and superimposing fluorescentmaterial, the results of the triple labeling experiments,at best, provide a qualitative estimate of AR contain-ment between catecholaminergic afferents. However,noninclusive assessment of AR and TH staining withinFG cells from separate experiments provided some in-dication that our triple labeling estimates were valid.Thus, the amount of triple labeling predicted by thearithmetic product of (FG�AR) � (FG�TH) percentvalues in the ventrolateral medulla, solitary tract nu-cleus, and in the locus coeruleus yielded values of 19.8 �2.0%, 11.1 � 1.2%, and 6.1 � 0.6%, respectively, over-lapping considerably with the mean percentage andrange of FG-positive cells displaying both TH and AR inthese regions, 30 � 6.1%, 11.3 � 7.9%, and 8.8 � 5.5%,respectively (FG�TH�AR, Figs. 11–13D).

DISCUSSION

In the present study we characterized the anatomicalnature by which androgen-sensitive targets in the braincommunicate directly with the PVN of the hypothalamus.Several criteria were imposed with respect to retrogradetracer placement in the PVN, allowing us to quantify therelative densities of AR containment within candidate

Fig. 10. Photomicrographs showing the accumulation of FG in theperiaqueductal gray. The darkfield view (A) illustrates a very highdensity of FG labeling particularly within the ventrolateral column(PAGvl). Enlarged brightfield view (B) of the boxed region showingdual immunoperoxidase labeling for AR-ir and FG-ir (see Fig. 9). Solid

arrows show doubly labeled neurons and open arrowheads markAR-positive, FG-negative cells. Aq, aqueduct of Sylvius; IC, inferiorcolliculus; mlf, medial longitudinal fasciculus; scp, superior cerebellarpeduncle. Scale bar � 50 �m in B; 500 �m for A.



afferent cell populations. Taking previous anterogradesurveys into account (see Table 1), our current findingsprovide an index of testosterone’s potential influence onvarious visceromotor systems of the PVN.

To determine potential sites of androgen influence, weemployed a dual immunohistochemical procedure to con-currently label cells displaying nuclear AR staining andcytoplasmic FG accumulation in the PVN. Delivery of FGby iontophoresis produced discrete injection sites, al-though occasionally this yielded suboptimal neuronaltracer uptake. Crystalline tracer deposits, on the otherhand, produced denser injections and more intense label-ing, although tracer uptake and transport typically ex-

tended beyond the border of the PVN proper. Both deliv-ery methods were controlled by examining patterns oftracer diffusion and accumulation within individual ani-mals (see Materials and Methods). As such, we restrictedour analysis only to those animals displaying the mostcircumscribed FG injections, centered within the caudalportion of the PVN. Analysis was further restricted toanimals showing comparable numbers in FG cell countsand patterns of accumulation similar to previous retro-grade transport experiments (see Results). Stable num-bers in retrograde labeling were reliably observed in theparastrial, medial preoptic, dorsomedial, and in ventrome-dial hypothalamic nuclei, for example, and FG cells were

Fig. 11. Anatomical and immunohistochemical characterization ofFG-labeled and AR-ir cells in the vicinity of locus coeruleus (LC).A: Photomicrograph of separate avidin-biotin immunoperoxidase and-fluorescent preparations combined to show a weak superimposition ofFG accumulating cells (brown) over tyrosine hydroxylase-immunoreactive (TH-ir) positive cells (green) in the locus coeruleus.Enlarged view (B) of the boxed region to show concurrent fluorescentretrograde tracer uptake (blue cytoplasm) and AR staining (red nu-cleus). Note that the bulk of doubly labeled cells locate within thecentral gray and a tendency for this distribution to occur along themedial border of the locus coeruleus. Enlarged view (C) of the boxed

region to show concurrent double immunofluorescent detection of TH(green) and AR (red) stained cells within the locus coeruleus. Esti-mated percent (mean � SEM) of locus coeruleus TH-positive neuronsdisplaying AR staining, and of locus coeruleus FG-positive neuronsdisplaying TH and/or both TH and AR (D). This was determined inseparate preparations, obtained from adjacent series of sections fromindividual animals (n � 4). Solid arrows show doubly labeled neuronsand open arrowheads mark AR-positive, FG-negative cells. CG, cen-tral gray; DT, dorsal tegmental nucleus; V4, fourth ventricle. Scalebar � 100 �m for B,C; 400 �m for A.



rarely encountered within the lateral septal and amygda-loid complex (Cullinan et al., 1996). Achieving reliable FGcell counts between animals provided a stable frameworkfor indexing regional differences in AR containmentwithin PVN projecting cell populations.

FG injections centered within the PVN proper yielded apattern of staining that complemented several previousretrograde transport-based characterizations (see Re-sults), and will not be reviewed in great detail here.Briefly, PVN-projecting cells were located throughout therostrocaudal extent of the brain, extending from the ante-rior division of the stria terminalis to the caudal aspects ofthe ventral lateral medulla and nucleus of the solitarytract. Within the forebrain, significant uptake was consis-tently observed within the periventricular and medial

zone nuclei, with the strongest projections arising fromthe preoptic and hypothalamic cell groups. In the hind-brain, extensive FG labeling was detected within severalmidline structures, including the periaqueductal gray,and within several laterally displaced regions, includingthe posterior thalamus, lateral parabrachial nucleus,and throughout the ventrolateral catecholaminergic cellgroups of the medulla.

Within the forebrain, the highest densities of AR-expressing, PVN afferents encountered were found withinthe periventricular and medial zones of the preoptic areaand hypothalamus, and within the bed nuclei of the striaterminalis. Doubly labeled cells were generally detected inlower densities throughout the brainstem, although reli-able complements of AR�FG cells were found within the

Fig. 12. Anatomical and immunohistochemical characterization ofFG-labeled and AR-ir cells within the ventral medulla. Darkfieldphotomicrograph of an immunofluorescent preparation to show aTH-ir cell group (A). Higher magnification view (B) of the ventrolat-eral medulla to show concurrent fluorescent retrograde tracer uptake(blue cytoplasm) and AR staining (red nucleus). Enlarged view (C) ofthe same preparation in panel A to show concurrent double immuno-

fluorescent detection of TH (green) and AR (red) stained cells. Esti-mated percent (mean � SEM) of TH-positive neurons displaying ARstaining, and of FG-positive neurons displaying TH and/or both THand AR in the ventrolateral medulla (D). Solid arrow in each panelidentifies the same cell in the ventral medulla showing TH (A), FGand AR (B), and TH and AR staining (C). Scale bar � 100 �m for B,C;200 �m for A.



posterior thalamic complex, in the lateral part of the para-brachial nucleus, and in particular, the ventral lateralpart of the periaqueductal gray. The pattern of AR stain-ing within FG-accumulating neurons in the medulla andbrainstem suggested a possible distribution within cat-echolaminergic cell groups. Using a combined retrogradetransport-double immunohistochemical-labeling tech-nique, our findings indicated that the most reliable con-tingent of AR staining was found within both noradrener-gic and adrenergic components of the ventral medulla. Incontrast, AR staining within cells identified as projectingto the PVN was rare to inconsistent within the locuscoeruleus and solitary tract nuclei. Taken together, theextensive distribution of AR-expressing, PVN afferentsindicate that a vast set of forebrain and hindbrain nuclei

are positioned to mediate androgen-sensitive input to thePVN.

In drawing conclusions as to the potential targets ofcandidate populations described, it is important to em-phasize that the borders between the classes of majoreffector neurons in the PVN are neither clear nor dis-tinct, that neurons of one output class can be found incompartments dominated by other cell types, and thatdendrites of neurons of any subdivision seldom respectnuclear boundaries (Swanson and Sawchenko, 1983;Swanson et al., 1987; Rho and Swanson, 1989). None-theless, the findings of Rho and Swanson (1989) indicatethat the bulk of the dendritic mass of any functionallyrelated cell group in the PVN is confined to the samePVN compartment. Therefore, based on the results of

Fig. 13. Anatomical and immunohistochemical characterization ofFG-labeled and AR-ir cells within the dorsomedial medulla. Darkfieldphotomicrograph of an immunofluorescent preparation to show TH-ircell groups in the nucleus of the solitary tract (A). Higher-magnification view (B) of the boxed region to show concurrent fluo-rescent retrograde tracer uptake (blue cytoplasm) and AR staining(red nucleus) in the NTS. Enlarged view (C) of the boxed region of thesame preparation in panel A (left A2 group) to show concurrent double

immunofluorescent detection of TH (green) and AR (red) stained cells.Estimated percent (mean � SEM) of TH-positive neurons displayingAR staining, and of FG-positive neurons displaying TH and/or bothTH and AR in the NTS (D). Solid arrow in each panel identifies thesame TH-positive cell (boxed in panel A), showing FG and AR (B), andTH and AR staining (C). ap, area postrema; Aq, aqueduct of Sylvius;NTSm, nucleus of the solitary tract, medial division; V4, fourth ven-tricle. Scale bar � 50 �m for B,C; 375 �m for A.



several previous anterograde transport studies, takentogether with our current findings, inferences can bemade with some degree of confidence as to which func-tional cell types in the PVN are preferentially targetedby candidate AR-expressing, PVN projecting cell popu-lations, as summarized in Table 1. Importantly, thetopographic distribution of double-labeled (AR�FG)cells throughout the forebrain and brainstem suggests apotential modulation of neurosecretory and non-neurosecretory (autonomic) cells in the PVN, and somecapacity for affecting certain cell types uniquely.

Another important consideration is that many brainregions regulating the PVN and HPA axis do not directlyinnervate cells occupying the PVN proper, but instead actvia polysynaptic relays. Thus, in contrast to direct PVNprojections arising from the fusiform nucleus of the striaterminalis, anterior hypothalamic, dorsal tegmental, orpedunculopontine nuclei, for instance, a number of projec-tions described here ramify at least as prominently andquite often preferentially to regions immediately adjoin-ing the boundaries of the PVN (see Table 1). A collection ofcell groups surrounding the caudal PVN, including themedial aspects of the zona incerta, the perifornical region,the dorsal aspects of the anterior part of the anteriorhypothalamic area, and the rostral (anterior parvicellular)aspects of the PVH contain ARs and provide substantialinput to the hypophysiotropic zone of the PVN (Rolandand Sawchenko, 1993; Boudaba et al., 1996; Bowers et al.,1998; Herman et al., 2002a; Bingham et al., 2006). These cellgroups closely positioned around the PVN are thought toconstitute an organized perinuclear zone which may serve tointegrate information provided by afferents that distributepoorly, if at all, within the borders of the PVN proper, or toactivate local inhibitory neurons. As reviewed by Herman etal. (2002a,b, 2003, 2005), this -aminobutyric acid (GABA)-rich PVN surround is ideally positioned to integrate inputfrom the limbic forebrain, including the prefrontal cortex,lateral septum, ventral subiculum, and medial amygdaloidnuclei (and see Results). The peri-PVN region also receivessignificant cholinergic and serotonergic input, which alsogreatly exceeds that of the PVN proper (Sawchenko et al.,1983; Ruggiero et al., 1990), and it is clear that this peri-PVNcomplex represents a principle target of both ascending anddescending regulatory systems. Of note, several structurescomprising the PVN surround, including the dorsal anteriorhypothalamic area and the perifornical region, contain sig-nificant complements of GABA-producing, AR-expressingcells (see Bingham et al., 2006).

Several additional forebrain limbic-related and hind-brain cell groups target the PVN only indirectly, commu-nicating with such upstream relay centers as the medialpreoptic area, the bed nuclei of the stria terminalis, thedorsomedial hypothalamic nucleus, the lateral hypotha-lamic area, and the solitary tract nucleus (Herman et al.,2003). The medial preoptic area stands out as an impor-tant focal point in this regard. A large contingency ofneurons within the medial preoptic area receive and re-ciprocate input to other AR-rich groups that, in turn,impinge upon the PVN region, including the bed nuclei ofthe stria terminalis, the dorsomedial hypothalamus, andthe solitary tract nucleus (Simerly and Swanson, 1986,1988). Varying degrees of AR-ir were detected within eachof these cell groups, thus imparting several additionallevels of gonadal influence on converging homeostatic in-formation to the PVN.

The present findings would suggest a very close if notoverlapping relationship between systems governing re-production and HPA control. Indeed, many forebrain andhindbrain structures that contained extensive distribu-tions of AR-expressing, PVN afferents are also known tosubserve reproductive function (Coolen et al., 1998; Grecoet al., 1998b). Thus, regions of the medial basal forebrainknown to mediate copulatory behavior, such as the medialpreoptic and ventromedial hypothalamic nuclei (McGinniset al., 1996; Hull et al., 1997), contained high densities ofFG�AR cells. Similarly, structures thought to control theproduction of gonadotropic hormones, including theperiventricular preoptic and anteroventral periventricularnuclei (Wiegand and Terasawa, 1982) also showed signifi-cant complements of double-labeled cells. This distributionsuggests that androgenic regulation of gonadotropin-releasing hormone release and reproductive behavior is notentirely restricted to components of the gonadal axis, butmay also rely on concurrent regulation of both the neuroen-docrine and autonomic-related branches of the PVN (Selvageand Rivier, 2003; Selvage et al., 2004, Ulrich-Lai et al.,2006). This is consistent with the disruptive effects of adre-nalectomy and sympathetic blockade on testosterone secre-tion, androgenic regulation of gonadotropin-releasing hor-mone and luteinizing hormone release, and sexual behavior(reviewed in Kalra and Kalra, 1983; Levine et al., 1991;Herbison, 2006; and see Poggioli et al., 1984).

Beyond the realm of reproduction, the prevalence of ARswithin thalamic and brainstem nuclei suggest that multi-ple sensory systems may be under the influence of testos-terone (Gandelman, 1983; Simerly et al., 1990; Hamson etal., 2004). Recent work speaks to several possible routesthrough which this could be imposed. Ceccarelli et al.(2003) revealed that gonadectomized male rats show aheightened induction of Fos in the arcuate hypothalamicnucleus compared to their gonadal-intact counterparts inresponse to noxious formalin challenge. Indirect influ-ences mediated via the forebrain and hindbrain centersthat receive collaterals from the principle spinothalamicpathway have also been examined. Thus, substantial pro-jections from the spinal dorsal horn to the medial andlateral preoptic and hypothalamic nuclei, the posteriorthalamic complex, the ventrolateral periaqueductal gray,and parabrachial nuclei, with minor inputs to the perifor-nical and anteroventral periventricular nuclei, have beenidentified (Giesler et al., 1994). As characterized here, allof these contain a significant assortment of ARs.

The somato-visceral and -sensory systems could alsoserve as a viable target for testosterone’s inhibitory influ-ences on HPA output, consistent with the detection ofmoderate to large densities of AR�TH double labelingthroughout the catecholaminergic cell groups of the cau-dal brainstem and the reliable incidence of AR stainingwithin identified PVN afferents spanning the A1 and C1cell groups of the medulla (reviewed in Sawchenko andSwanson, 1982). Previous studies have indicated a pro-found influence of gonadectomy and hormone replacementin the rat on several brain monoaminergic systems, in-cluding TH activity, although this has been primarilystudied with respect to dopamine regulation (see Engel etal., 1979; Simerly, 1989; Kritzer, 2003). The results of ourdouble and triple labeling experiments, directed at deter-mining the percentage of FG-positive neurons displayingboth TH and AR directly (see Results) indicate that thebulk of any potential androgenic influence on cat-



echolaminergic afferents would have to be placed withinthe ventral medulla.

The remarkable display of AR staining by TH-positiveneurons in the locus coeruleus remains worthy of discus-sion, considering that this number exceeded the capacityof FG�AR-labeled cells detected within the locus coer-uleus and the number of TH-positive, AR-stained cells inthe dorsal and ventral medulla. On one hand, these find-ings speak to and confirm the small contribution of the A6cell group relative to the mass of catecholaminergic affer-ents provided by the dorsal and ventral medulla. On theother hand, the locus coeruleus is known to heavily projectto several PVN and HPA regulating structures (Wyss etal., 1979; Swanson, 1982; Simerly and Swanson, 1986;Carstens et al., 1990; Risold and Swanson, 1997; Thomp-son and Swanson, 1998), including major homeostatic-(dorsomedial hypothalamic nucleus, medial preoptic nu-cleus, ventral tegmental area), sensory- (medial thalamus,substantia nigra), and limbic-related relays (hippocam-pus, lateral septum). Taken together with the impressivedistribution of AR within the locus coeruleus, the possi-bility that testosterone could alter the activity of any ofthese pathways should not be underestimated.

Modest populations (20–25%) of cells in the nucleus ofthe solitary tract and ventral medulla identified as PVNprojecting are not catecholaminergic (see Rinaman et al.,1995), and these may be occupied by -inhibin and soma-tostatin neurons (Sawchenko et al., 1988, 1990; Verbaliset al., 1995). At present, the connectivity of these cellphenotypes and identity with respect to AR stainingwithin these areas has yet to be established. While-inhibin secretion (Roberts et al., 1989; Dalkin et al.,1994; Halvorson et al., 1994) and somatostatin expressionin the forebrain (Baldino et al., 1988; Argente et al., 1990)respond to changes in sex steroid hormone levels, theimpact of gonadal status on these phenotypes in the me-dulla, within PVN projecting afferents specifically (seeChibbar et al., 1990), has not been examined.

Based on the extensive containment of ARs within path-ways conveying somatosensory and nociceptive informa-tion to the PVN, it seems reasonable to assume thatchanges in gonadal status could play an important role inaltering or sustaining adaptive visceromotor responses toseveral types of homeostatic challenges. As mentionedabove, our findings would also place several aspects of theforebrain into this design, including the medial preopticand bed nuclei of the stria terminalis, which were consis-tently found to contain neurons identified as both PVNprojecting and containing ARs. In addition to receivingsubstantial input from putative ascending sensory sys-tems, the medial preoptic and bed nuclei also distributeinformation flow between several limbic regions, includingthe prefrontal cortex, lateral septum, and amygdala. Be-yond implicating the medial preoptic and bed nuclei asandrogen-sensitive mediators of sensory information,these nuclei would also appear well suited for bridgingemotional and neuroendocrine responses to changes ingonadal status (Rubinow and Schmidt, 1996; Toufexis etal., 2006). While several indications of this potential havebeen shown in separate studies (Gu et al., 2003; Polstonand Simerly, 2003), concurrent effects on both behavioraland PVN visceromotor responses have yet to be examinedexperimentally.

The histochemical identification of neurotransmitterand neuropeptide receptors on or surrounding the para-

ventricular neurons has provided some essential insightinto the chemical nature of PVN control (Sawchenko andSwanson, 1982; Cunningham and Sawchenko, 1988; Coleand Sawchenko, 2002; Herman et al., 2002b). The activa-tion of paraventricular motor neurons ultimately dependson the integration of excitatory and inhibitory signalsarising from first-order forebrain and hindbrain relays(Sawchenko and Swanson, 1982, 1983). Accordingly, theprime candidate neurotransmitters implicated in PVNcontrol include GABA, norepinephrine, and glutamate (Cun-ningham and Sawchenko, 1988; Van den Pol et al., 1990;Brann, 1995; Boudaba et al., 1996), and a variety of neu-ropeptides, including arginine vasopressin, corticotrophin-releasing hormone, galanin, and enkephalin (Simerly et al.,1986; Bittencourt et al., 1991; Moga and Saper, 1994; Cham-pagne et al., 1998).

Testosterone has been shown to interact with several ofthese neurotransmitter and peptidergic systems andwithin numerous cell groups identified as projecting to thePVN or regulating the HPA axis (De Vries et al., 1986;Diano et al., 1997; Herbison, 1997; Bloch et al., 1998;Ronnekleiv and Kelly, 2005). GABAergic projections em-anating from the MPN (and its afferent relays) stand outas a potential mediator of testosterone’s upstream inhib-itory influences on PVN visceromotor function. While awide variety of neurotransmitters are expressed by neu-rons of the MPN, GABA is the most prolific and highlysensitive to gonadal status (Simerly et al., 1986; Bowers etal., 1998; Herman et al., 2004). A clear majority ofGABAergic neurons in the MPN express androgen recep-tors, gonadectomy and local androgen receptor blockadedecrease GABAergic activity within this structure (Grat-tan and Selmanoff, 1993, 1994; Grattan et al., 1996), andflutamide and testosterone implants in the vicinity of theMPN increase and decrease, respectively, the plasma ad-renocorticotropin hormone (ACTH) response to restraintstress (Viau and Meaney, 1996; Williamson and Viau,2006). As reflected by the expression of glutamic aciddecarboxylase (GAD) mRNA, acute and repeated restraintexposure activate multiple GABAergic circuits in thebrain, particularly within numerous cell groups that wehave identified as both PVN projecting and containingARs, including the MPOA, the peri-PVN region, and theperifornical nucleus (Bowers et al., 1998). These GABAer-gic pathways warrant further examination as afferentmediators of testosterone’s control of the PVN, consideringthat normal HPA adaptation to repeated restraint expo-sure is reduced by gonadectomy and AR blockade (Bing-ham et al., 2005).

We have also previously observed a strong associationbetween the inhibitory effects of testosterone on stress-induced ACTH release and the expression of AVP mRNAwithin the medial amygdala and the posterior subnuclei ofthe stria terminalis (Viau et al., 2001; Bingham et al.,2005). Moreover, opposite to the effects of AVP originatingfrom neurosecretory neurons of the PVN, central AVPpathways appear to exert an inhibitory influence on HPAoutput (Makara et al., 1996; Wotjak et al., 1996). Similarto GABA regulation, AVP expression within the medialamygdala and posterior bed nuclei depend heavily on go-nadal status, as AVP mRNA levels and immunoreactivitywithin these regions and their afferent connections arepractically abolished by gonadectomy, effects readily re-versed with testosterone replacement (De Vries et al.,1986; De Vries and Miller, 1998). Although several inde-



pendent lines of research support a role for AVP in coor-dinating the central effects of testosterone on behavioraland visceromotor adaptation (reviewed in Williamson etal., 2005), the anatomical basis for this has yet to beresolved. Thus, while the posterior aspect of the bed nu-cleus of the stria terminalis is known to project stronglyand preferentially to the hypophysiotropic zone of thePVN (Dong and Swanson, 2004), for example, the extentto which this projection contains AR- and AVP-expressingneurons and responds to changes in testosterone secretionremain worthy of pursuit (see Koolhaas et al., 1998; Kals-beek et al., 2002).

Given the potency by which variations in circulatingtestosterone levels alter HPA output, we would like tobelieve that the nuclei identified in the present study forman important network that registers fluctuations in tes-tosterone release associated with changes in homeostaticdemand. However, the structures identified probably un-derestimate the substrate and the mechanisms of sex ste-roid hormone regulation involved. ARs are distributedthroughout the limbic system, including within their re-spective relays to the PVN, as discussed above. Several ofthese HPA-regulating nuclei, in addition to those we haveidentified as PVN-projecting, show various degrees ofoverlap with respect to estrogen receptor (alpha and beta)distribution (Stumpf, 1970; Pfaff and Keiner, 1973;Stumpf et al., 1975; Simerly et al., 1990; Shughrue et al.,1997; Greco et al., 1998a, 2001; Laflamme et al., 1998) andaromatase activity (Roselli and Resko, 1993; Wagner andMorrell, 1997; Zhao et al., 2007). Thus, situation-dependent and region-specific changes in aromatase activ-ity could very well play a critical role in determining howfluctuations in circulating testosterone levels alter thetype and flow of information to the PVN, beyond whatcould be attributed to AR-mediated events. Androgen re-ceptors can regulate gene expression directly by binding topotential androgen-responsive elements located withinthe promoter region of target genes or indirectly, by inter-acting with other steroid hormone receptors (Chen et al.,1997), transcription factors, and second-messenger sys-tems (Chang et al., 1995; Heinlein and Chang, 2002).While we have described several neurotransmitter andneuropeptide systems that reliably respond to changes ingonadal status, the cellular mechanisms governing theseresponses remain unclear (but see Bao et al., 2006). Nev-ertheless, the impressive distribution of ARs within sev-eral extended circuitries of the PVN provides a tenablebasis for assuming that testosterone could act to bridgeseveral independent, yet converging influences to the PVNand HPA axis.

As discussed above, many advances have been made inclarifying the anatomical and functional organization ofstress-related circuits in the brain, which have been crit-ical in identifying putative regulators of PVN and HPAfunction. It is clear that stimuli that elicit endocrine,autonomic, and behavioral responses rely on complex andcentrally coordinated patterns of neuronal activation(Sawchenko et al., 2000; Herman et al., 2003). Further, acommon consensus is that different stimuli recruit inde-pendent and overlapping neural systems, which in turnproject to the PVN, either directly or indirectly (Li andSawchenko, 1998; Dayas et al., 2001a; Day, 2005; but seePacak and Palkovits, 2001). Based on the present results(Fig. 14), and on those of previous retrograde and antero-grade studies (see Table 1), the PVN is innervated by a

substantial number of forebrain and hindbrain structuresthat receive first- or second-order somatosensory, visceral,and humoral information. Because ARs are contained by aclear majority of these, this clearly places the gonadal axisamong the principle response systems guarding the organ-ism against homeostatic threat. The challenge now is todetermine how changes in circulating testosterone levelsare registered within the circuits described, and how thisimpacts the stress-induced activation of these projectionsto the PVN. Our findings provide an important startingpoint for this pursuit.

ACKNOWLEDGMENT

We thank Dr. Richard H. Thompson (University ofSouthern California) for helpful comments and discussion.

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