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Department of Anatomy and Cell Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Address all correspondence and requests for reprints to: Dr. Victor Viau, Department of Anatomy and Cell Biology, University of British Columbia, 2177 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 1Z3. E-mail: viau{at}interchange.ubc.ca.
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Introduction |
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Although these findings indicate that sex steroids intersect on HPA function at the level of the PVN, this is not readily explainable by effects on CRH and AVP synthesis and release directly (reviewed in Ref.7). Estrogen and androgen receptors are sparsely, if at all, distributed within CRH-expressing parvocellular neurons, but are concentrated within several brain nuclei communicating with the hypophysiotropic zone of the PVN. Consistent with an upstream action of testosterone on PVN function, plasma ACTH responses to restraint are decreased by testosterone implants into the medial preoptic area (10, 11). Moreover, androgenic influences on HPA function and medial parvocellular AVP expression are also associated with altered CRH and AVP mRNA expression levels within PVN-projecting bed nuclei of the stria terminalis (12).
More dynamic approaches, using stimulus-induced patterns in Fos expression as a marker of synaptic activation, remain in keeping with the idea that sex steroid effects on HPA output are mediated centrally and distal to the PVN. This is often indicated in the parallel nature by which sex steroids regulate Fos activation within parvocellular and PVN-regulating nuclei and the magnitude of the ACTH response to stress (1, 13, 14, 15 , but see16). These findings suggest a gonadal influence on stress-related input to the PVN that extends beyond the simple activation of parvocellular motor neurons, including effects coupled to CRH and AVP release.
To begin to clarify how testosterone can operate on stress-related pathways in the brain, we compared the extent to which restraint exposure induces Fos immunoreactivity in the PVN in gonad-intact and gonadectomized male rats. Stress- and neurotransmitter-induced activation of parvocellular neurons are accompanied by marked increases in CRH and AVP transcription, which are alleged to initiate the replenishment of peptide stores consumed by release (17, 18). Thus, we also examined how restraint-induced elevations in heteronuclear CRH and AVP RNA respond to gonadectomy and as a function of individual plasma testosterone levels, toward establishing a functional link between the recruitment of parvocellular neurons, HPA output, and intervening changes in CRH and AVP transcriptional activation.
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Materials and Methods |
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To reveal a testicular influence on stress-HPA activity, male rats were divided into two weight-matched groups representing sham-gonadectomized (intact) and bilateral gonadectomized (GDX) rats. Testes were removed via a scrotal incision under ketamine-xylazine-acepromazine anesthesia (77:1.5:1.5 mg/ml, respectively; 1 ml/kg sc). Each testis was delivered separately through the scrotal incision and exteriorized by severing the vas deferens and spermatic artery, which was ligated to maintain hemostasis. Gonadectomy was completed by closing the scrotal incision with 4-0 nonabsorbable suture. Sham gonadectomy was achieved by clipping and suturing the scrotum under ketamine-xylazine-acepromazine anesthesia.
Rats were handled and weighed daily before stress testing, which occurred 11–15 d after surgery. Gonadectomy had a transient negative effect on body weight gain; final group mean body weights in sham and GDX rats were 370.6 ± 0.4 and 334.3 ± 54.2 g, respectively. However, by the 10th day postsurgery, the growth rate in GDX rats approached that in animals receiving sham surgeries (6.5 ± 0.4 vs. 7.0 ± 0.3 g weight gain/day, respectively).
Stress testing involved placing rats into Plexiglass restrainers (8.5 x 21.5 cm; Kent Scientific, Litchfield, CT) for a 30-min period. Rats were anesthetized for perfusion at various intervals, 0.5, 1, 2, 4, or 6 h after the beginning of restraint. Basal control rats were anesthetized and perfused immediately after removal from their home cage, in parallel with those perfused during stress testing. As verified by corneal, pedal, and tail pinch reflexes, deep anesthesia was reliably achieved within 45–60 sec of chloral hydrate administration. Blood samples obtained from the right atrium were collected into ice-chilled EDTA-treated tubes, centrifuged at 3000 x g for 20 min, and stored at -20 C until assayed for testosterone and corticosterone to validate gonadectomy and to measure plasma hormone responses to restraint. All testing was performed during the light phase of the cycle, beginning at 0800 h and ending at 1200 or 1400 h in some cases (4- or 6-h poststress sampling interval, respectively). All protocols were approved by the University of British Columbia animal care committee.
RIAs
Plasma testosterone was measured using an RIA kit from ICN Biomedicals, Inc. (Costa Mesa, CA), with [125I]testosterone as tracer and 25 µl sample. The testosterone antibody (liquid phase) cross-reacts 100% with testosterone and slightly with 5
-dihydrotestosterone (3.40%), 5-androstane-3ß,17ß-diol (2.2%), and 11-oxotestosterone (2%) but does not cross-react with progesterone, estrogen, or the glucocorticoids (all <0.01%). The intra- and interassay coefficients of variation were approximately 4% and 10%, respectively. The standard curve 50% effective concentration was 1.75 ng/ml, and the detection limit of the assay was 0.25 ng/ml.
Plasma corticosterone was measured using an RIA kit from ICN Biomedicals, Inc., with [125I]corticosterone as tracer and 5 µl sample diluted 1:200. The corticosterone antibody cross-reacts 100% with corticosterone and slightly with deoxycorticosterone (0.34%), testosterone, and cortisol (0.10%) but does not cross-react with the progestins or estrogens (<0.01%). The intra- and interassay coefficients of variation were approximately 4% and 14%, respectively. The standard curve 50% effective concentration was 16 µg/dl, and the detection limit of the assay was 0.63 µg/dl. Based on our previous findings (10, 19), blood corticosterone levels were expected to return to control concentrations by 4 h after the onset of restraint and were not assayed for corticosterone beyond this time point.
Fos immunohistochemistry
Restraint-responsive paraventricular neurons were localized using Fos immunoreactivity (Fos-ir) as a marker of synaptic activation. Fos-ir was detected using a conventional avidin-biotin-immunoperoxidase procedure (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) to localize a primary antiserum (1:23,000) raised against amino acids 4–17 of human Fos protein (Oncogene Research Products, Boston, MA). Specific staining was abolished by absorbing the primary antiserum with 50 µM synthetic Fos (Oncogene Research Products). Light level images were captured using a Hamamatsu optical system coupled to a Macintosh computer running Open Lab imaging and measuring software (Quorum Technologies, Guelph, Ontario, Canada).
Fos-ir cell counts were made by an observer blind to animal status and treatment in regularly spaced (150-µm) intervals through the rostrocaudal extent of the paraventricular cell group and were corrected for double counting error. Positive cells were identified as those expressing a black nuclear reaction product. Discrete localization of Fos-ir profiles to the medial parvocellular portion of the paraventricular neurons and adjoining compartments (see Fig. 3
, top) was assisted by sampling the Nissl pattern of an adjacent series of sections (19) through the PVN. Cell number estimates were generated by counting bilaterally the number of Fos-positive cells through each region of interest, averaged by dividing cell counts by a factor of 2, and were corrected for sampling frequency (one in five sections, 150-µm intervals) by multiplying this product by a factor of 5.
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Hybridization histochemistry was carried out using a [33P]UTP (Amersham Pharmacia Biotech, Arlington Heights, IL)-labeled antisense cRNA probe transcribed from a 700-bp fragment of intron I of the rat vasopressin (18, 20) and a [33P]UTP-labeled antisense cRNA probe transcribed from a 500-bp fragment of the single intron in the CRH (21). Techniques for riboprobe synthesis and hybridization, and the patterns of hybridization for these probes in the PVN were described in greater detail previously (21, 22, 23). Briefly, free-floating sections were first rinsed in KPBS to remove cryoprotectant, then mounted and vacuum-dried on glass slides overnight. After postfixation with 10% formaldehyde for 30 min at room temperature, sections were digested in proteinase K (10 mg/ml; 37 C) for 30 min, acetylated for 10 min (2.5 mM acetic anhydride and 0.1 M triethanolamine, pH 8.0), rapidly dehydrated in ascending ethanol concentrations (50–100%), and vacuum-dried. Radionucleotide antisense cRNA probes were used at concentrations approximating 3 x 107 cpm/ml in a solution of 50% formamide, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 0.05% tRNA, 10 mM dithiothreitol, 1x Denhardt’s solution, and 10% dextran sulfate and applied to individual slides containing sections through the rostrocaudal extent of the PVN with sampling intervals of 150 µm. Slides were coverslipped, and then incubated overnight at 57.5 C, after which the coverslips were removed, and the sections were washed three times in 4x SSC (SSC = 0.15 M NaCl and 15 mM citric acid, pH 7.0) at room temperature, treated with ribonuclease A (20 µg/ml) for 30 min at 37 C, desalted in descending SSC concentrations (2 to 0.1x SSC), washed in 0.1x SSC for 30 min at 60 C, and dehydrated in ascending ethanol concentrations. Hybridized sections were then exposed to x-ray film (ß-max, Amersham Pharmacia Biotech), defatted in xylenes, and subsequently coated with Kodak NTB2 liquid autoradiographic emulsion (Eastman Kodak Co., Rochester, NY), and exposed at 4 C in the dark with desiccant; the duration was determined by the strength of signal on x-ray film [21 and 28 d for heteronuclear AVP (hnAVP) and hnCRH, respectively]. Slides were developed with Kodak D-19 for 3.5 min at 14 C, briefly rinsed in distilled water for 15 sec, fixed in Kodak fixer for 6.5 min, and then washed in running water for 45 min at room temperature. The hybridization pattern of the antisense AVP and CRH probes are in agreement with previous hybridization localization studies in the PVN (18, 21), whereas labeled sense probes did not yield any positive hybridization signal (data not shown). Based on the strength of the autoradiographic signal, exposure time to emulsion was optimized to ensure that CRH and AVP hnRNA were within the linear range of the assay and could be quantified by making relative comparisons of OD levels.
Analysis of the relative levels of CRH and AVP hnRNA in the PVN and in the paraventricular and supraoptic nuclei, respectively, was performed on silver grain-developed, emulsion-coated slides using Macintosh-driven Open Lab image Improvision software version 3.0.9 (Quorom Technologies). This was assisted by redirected sampling of darkfield images aligned to corresponding Nissl-stained sections. OD readings, corrected by background subtraction, were taken at regularly spaced (150-µm) intervals. Average OD values for CRH and AVP hnRNA were determined bilaterally from three sections taken through the rostrocaudal extent of the medial parvocellular portion of the PVN. Given the presence of large, presumably ectopic, AVP-expressing magnocellular neurons within the hypophysiotropic parvocellular zone of the PVN (18, 24), analysis of relative AVP hnRNA levels was also approached by counting the number of individual AVP-expressing medial parvocellular nuclei, identified as showing silver grain clusters of 5 times background or greater.
To confirm the selectivity by which gonadal status and restraint operate on the parvocellular cell type, AVP hnRNA expression levels were also surveyed within two magnocellular populations, including the adjoining posterior magnocellular subdivision of the PVN and the supraoptic nucleus. Average OD values for AVP hnRNA in the posterior magnocellular division were determined bilaterally in the same sections used to survey hypophysiotropic parvocellular cells and along the same rostrocaudal plane for the supraoptic nucleus (three sections each).
Fos immunochemistry and CRH hybridization histochemistry
A combined immunoperoxidase-hybridization approach was used to qualitatively assess under restraint conditions medial parvocellular dorsal cells concurrently displaying Fos-ir and the CRH transcript using a cRNA probe encoding for CRH (Dr. K. Mayo). Fos-ir was carried out first, followed by hybridization localization of CRH mRNA using a 33P-labeled antisense cRNA probe transcribed from a full-length (1.2-kb) cDNA encoding CRH mRNA as previously described (19). To optimize dual localization of Fos-ir and the CRH transcript, H2O2 and NaBH4 pretreatments were eliminated, blocking serum (normal goat serum) was replaced with 2% BSA and 2% heparin sulfate, and nickel enhancement from the avidin-biotin-peroxidase procedure was eliminated. Finally, the reaction product was developed using reagents chilled to 0–4 C to prevent RNA degradation attributable to the exothermic nature of the reaction. Double-labeled cells were identified as those displaying Fos-ir nuclear profiles overlaid by clusters of reduced silver grains whose density was more than 5 times the background.
Statistical analysis
Data are expressed as the mean ± SEM, and were analyzed by using one- and two-way ANOVA. Post hoc analysis was performed, when appropriate, using Newman-Keuls test for multiple pairwise comparisons. Portions of this work have been presented in abstract form (25).
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, top).
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(bottom), the peak corticosterone level was similar between intact and GDX rats, evident at the termination of restraint exposure (peak corticosterone, 39.4 ± 1.0 and 45.3 ± 2.1 µg/dl in intact and GDX, respectively). After the end of restraint, relative to the intact group, GDX rats showed higher and sustained elevations in corticosterone above baseline up to 2 h after the onset of 30-min restraint (2 h corticosterone, 5.13 ± 1.2 and 16.1 ± 3.7 µg/dl in intact and GDX, respectively). At 4 h after the onset of restraint, corticosterone levels were not significantly different between intact and GDX male rats, nor significantly different from their prestress counterparts. This indicates that the variations in corticosterone release were related to gonadal effects on stress-induced HPA activation and were not due to departures in basal HPA function, at least during the morning period of the circadian cycle.
Fos induction
To examine the effects of gonadectomy on the pattern and strength of activational responses within different functional cell types of the paraventricular nucleus, Fos-ir cells were first counted within different subregions of the PVN as defined by the pattern of Nissl staining taken from adjacent series of sections. As shown in Fig. 2, rats that did not experience restraint displayed relatively little Fos-ir neurons in the PVN as a whole. Quantitative assessment of Fos induction in tissue obtained at the end of 30-min restraint revealed significant effects of treatment [F(1,50) = 56.4; P < 0.0001] and region [F(4,50) = 179.0; P < 0.0001] and a significant treatment x region interaction [F(4,50) = 25.5; P < 0.0001]. As shown in Fig. 4, this interaction was attributable to an overwhelming Fos response within medial parvocellular neurons that was significantly higher in GDX compared with intact rats.
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, bottom). Although the number of Fos-ir medial parvocellular neurons returned to control levels by the 4 h point in intact animals, in GDX rats the Fos response remained significantly elevated above baseline until 6 h. Regression analysis among intact rats revealed a reliable [F(1,16) = 8.2; P = 0.011] negative relationship (Fig. 4) between the number of medial parvocellular neurons induced to express Fos protein and the plasma testosterone concentration in individual rats perfused at 0.5, 1, and 2 h after the onset of restraint, which was best described using a linear fit (r2 = 0.34).
To characterize the extent to which differences in Fos activation between intact and GDX animals are represented in the recruitment profile of CRH-expressing neurons, tissue from rats perfused at 2 h after the onset of a 30-min episode of restraint were prepared for concurrent localization of Fos-ir and CRH mRNA. Restraint-induced Fos was concentrated within the dorsal aspect of the medial parvocellular part of the PVN where CRH-expressing neurons are massed (see top and middle panels in Fig. 5). However, the overlap in Fos-responding, CRH-expressing neurons was invariably higher in GDX rats; medial parvocellular CRH-expressing neurons consistently displayed Fos-ir in GDX rats, whereas CRH-positive, Fos-negative cells were more readily encountered in intact male rats (compare bottom panels, Fig. 5). Thus, differences in the number of Fos-responding medial parvocellular neurons between intact and GDX male rats are accountable to CRH-expressing cells.
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, left panels). Marked differences in expression were consistently seen under stress conditions, with GDX rats showing stronger labeling at 30 min of restraint exposure compared with intact rats (Fig. 6, right panels). Semiquantitative densitometric analysis of CRH hnRNA expression within medial parvocellular nuclei of intact and GDX rats under basal conditions and at different poststress intervals revealed significant effects of treatment [F(1,30) = 0.9; P = 0.0076] and time [F(2,30) = 17.9; P < 0.0001] and a significant treatment x time interaction [F(2,30) = 10.2; P = 0.0004]. As indicated in Fig. 7, this interaction was credited to the transience in CRH transcriptional activation that was maximal at 30 min of restraint and significant only in GDX rats. Gonad-intact rats are not incapable of mounting a parvocellular CRH transcriptional response to restraint per se, as CRH hnRNA levels are inducible by 60 min of restraint exposure (26).
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). AVP hnRNA was robustly stimulated by restraint exposure and was sensitive to gonadal status, as revealed by significant effects of treatment [F(1,30) = 19.3; P < 0.0001] and time [F(2,30) = 25.6; P < 0.001] and a significant treatment x time interaction [F(2,30) = 4.5; P = 0.0188]. Post hoc analysis indicated significantly higher and earlier elevations in medial parvocellular AVP hnRNA OD values in GDX compared with intact rats, which were maximal at 0.5 and 1 h after the beginning of restraint exposure, respectively (Fig. 9, top panel). The relative differences in the magnitude and timing of the CRH and AVP transcriptional responses were similar to those previously described in intact male rats exposed to 30-min restraint (26).
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, bottom panel). This indicates that the relative increase in AVP hnRNA in GDX rats, as revealed by population densitometry, is produced by a recruitment of parvocellular neurons induced to express the transcript and is not attributable to ectopic magnocellular neurons in the parvocellular division of the PVN. Moreover, densitometric analysis in other magnocellular cell groups revealed no significant effects of treatment and stress on AVP hnRNA expression levels in the posterior magnocellular division of the PVN or in the supraoptic nucleus (P > 0.05 in all cases). Thus, gonadal status and restraint stress do not interact on AVP transcription within magnocellular neurons, adding further credence to a selective influence of gonadal status on stress-induced AVP transcription within parvocellular hypophysiotropic neurons.
Regression analysis of gene transcription as a function of gonadal status in intact rats revealed no [F(1,4) = 2.0; P = 0.23] relationship between stress-induced levels of CRH hnRNA and plasma testosterone concentration (Fig. 10, left). A reliable [F(1,10) = 8.9; P = 0.014] negative relationship was found for AVP hnRNA expression and individual testosterone levels, which was best described using a linear fit (r2 = 0.47; Fig. 10, right).
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Discussion |
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The current study represents a logical extension of our earlier work, in which we discovered that testosterone regulation of stress-related HPA function could not be entirely predicted by actions on CRH and AVP biosynthesis, but is also related to effects mediated under stress conditions. This was suggested by a strong parallel between the inhibitory effects of testosterone on restraint-induced Fos and the magnitude of the ACTH response, executed, however, in different groups of animals at different poststress intervals (27). Chen and Herbert (16) have shown that GDX does not alter the number of Fos-ir cells in the PVN induced by restraint-immobilization. The extent to which restraint and immobilization recruit distinct pathways that are differentially sensitive to gonadal status remains unresolved (28, 29). Nonetheless, these earlier findings raise some concern as to whether a Fos-based characterization of restraint-responding neurons in the PVN can be used to reliably explore gonadal regulation of the HPA axis in males.
Here we compared more thoroughly the capacity by which PVN cells can react to acute restraint in gonad-intact vs. GDX male rats and as a function of individual plasma testosterone levels. This was approached on several fronts, by 1) comparing the relative pattern of recruitment and magnitude of Fos induction in different PVN compartments, 2) tracing the strength of this induction temporally, and 3) exploring possible functional links among stress-induced Fos activation, CRH and AVP gene transcription, and corticosterone output.
Our findings revealed a large response range in the number of medial parvocellular neurons recruited to express Fos protein between gonad-intact and GDX male rats. This range was sufficient to examine and support the view that the stress-induced activation of medial parvocellular neurons is sensitive to gonadal status and individual differences in plasma testosterone levels. In intact male rats, Fos induction was restricted to the medial parvocellular (ACTH-regulating) portion of the PVN, far exceeding the response in other compartments and was sustained until the 4 h poststress point. Fos activation was similarly preferential to medial parvocellular neurons in GDX rats; however, the magnitude of this response was significantly higher. This difference was remarkably apparent within 30 min after the onset of restraint and was maintained throughout the entire poststress period, indicating a gonadal influence on stress-induced Fos that is borne during stress exposure. This early and sustainable gonad-dependent difference in medial parvocellular activation is also reflective of an effect on restraint-induced drive to these neurosecretory motor neurons. This is in agreement with the parallel stimulatory effect of GDX on the number of neurons recruited to express Fos protein and the magnitude of the corticosterone response. We could not reliably measure plasma ACTH release due to its variable and relatively rapid response to chloral hydrate-induced anesthesia, however swift. Although one step removed from the anterior pituitary corticotroph, as the duration as well as the magnitude of the corticosterone response are dictated by ACTH (30), differences in the corticosterone release pattern shown here definitely reflect a stimulatory influence of GDX on restraint-induced ACTH release. This is consistent with our previous study, which demonstrated an androgen-reversible, stimulatory effect of GDX on restraint-induced ACTH and corticosterone release in male rats sampled via an indwelling jugular catheter (10).
In gonad-intact rats, mean plasma testosterone values did not vary between groups of animals perfused under basal conditions and at different poststress intervals. There was some indication of a decrement in testosterone release, albeit insignificantly, at 4 h after the onset of restraint exposure. This interval is too far removed to suggest that the early parvocellular Fos response is determined by changes in plasma testosterone levels at the time of restraint. We can assume, rather, that any individual differences in plasma testosterone levels proximal to restraint exposure are representative of individual, steady-state differences in plasma testosterone occurring before restraint exposure. This distinction is important, considering that the magnitude of the ACTH and corticosterone responses to restraint varies inversely as a function of basal, prestress levels of testosterone in intact males and in a dose-related manner in GDX rats replaced with static testosterone levels (10). Ideally, the range in plasma testosterone levels among gonad-intact males in the current study was broad enough to examine how individual differences in testosterone could contribute, in turn, to differences in the magnitude of the Fos response. The number of medial parvocellular neurons induced to express Fos varied strongly and negatively with plasma testosterone. Taken together with the stimulatory effect of GDX on corticosterone release and stress-induced Fos among CRH-expressing parvocellular neurons, this suggests an inhibitory effect of testosterone on the stress-induced drive to the PVN neurosecretory neurons that is responsive to individual variations in plasma testosterone concentration.
In gonad-intact males, the parvocellular PVN displayed little if any increase in CRH hnRNA levels in response to restraint. In stark contrast, GDX rats displayed a robust and transient increase in CRH hnRNA, evident at the termination of restraint. The AVP hnRNA response in intact males was relatively delayed and was elevated above control levels by 1 h post stress. GDX animals showed a significantly higher and, interestingly, earlier (30 min) rise in AVP hnRNA levels. Finer grain analysis revealed that this increased response was provided by a larger number of cells in the parvocellular PVN, indicating that more neurons were recruited to transcribe AVP in GDX rats. Together these findings reflect a general stimulatory effect of GDX on the stress-induced transcriptional activation of the CRH and AVP genes, temporally coupled to higher Fos expression in parvocellular neurosecretory neurons.
Among intact rats, an indication of an androgenic influence on stress-induced gene transcription in the PVN is suggested by a negative correlation between individual plasma testosterone levels and the magnitude of the AVP hnRNA response, but not the CRH response. Unlike CRH, a strong relationship was found for AVP due to its relatively broader temporal pattern of induction, permitting analysis over a larger range of animals showing individual differences in testosterone. Nonetheless, CRH transcription arguably appears more sensitive to GDX, given the stark difference in inducible levels of CRH hnRNA levels between intact and GDX rats, responsive, perhaps, only over the extreme lower range of plasma testosterone levels seen in intact males. In contrast, stress-induced AVP transcription varied over the wider range in circulating testosterone levels.
AVP is thought to be the key variable imparting situation-specific alterations in the magnitude of the ACTH response to stress (31). This is in line with several studies showing that alterations in the release patterns of ACTH and corticosterone conform more with changes in AVP than with parvocellular CRH activity, identified at the transcriptional, synthetic, and secretory levels (32, 33, 34, 35). Superimposing gonadal status on this system, based on our current findings it now seems reasonable to suggest that AVP may also be the more readily adaptable ACTH secretagogue to differences in testosterone secretion. Taken in this context, given the strength and range to which stress-induced activity of parvocellular neurons vary as a function of testosterone, the extent to which the HPA axis adapts to repeated stress may also very well depend on alterations in gonadal output. An example of this has been suggested by Gomez and Dallman (36), in which male rats are better capable of surviving the metabolic demands of chronic cold stress and mounting an adequate corticosterone response if normal declines in testosterone levels are allowed to occur. Thus, androgen-sensitive pathways to the PVN that are capable of registering changes in circulating testosterone levels and directed at regulating parvocellular AVP may play a critical role in maintaining continual adaptation of the HPA axis.
Although demanding of multiple markers of neuronal activation, our current findings provide a clear indication that Fos can be used to approach gonadal influences on HPA function centrally. Given the potency with which GDX alters parvocellular synaptic and cellular responses to restraint stress and the occurrence of these responses as a function of plasma testosterone levels, we are now in a position to examine more directly the androgen-reversible effects of GDX within parvocellular neurons and the extended circuitries of the PVN.
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Footnotes |
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Abbreviations: AVP, Arginine vasopressin; GDX, gonadectomized; hn, heteronuclear; HPA, hypothalamic-pituitary-adrenal; -ir, immunoreactivity; KPBS, potassium PBS; PVN, paraventricular nucleus; SSC, 0.15 M NaCl and 15 mM citric acid.
Received January 14, 2003.
Accepted for publication April 2, 2003.
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, P < 0.01 vs. intact. mpd, Medial parvocellular dorsal; pm, posterior magnocellular; pv, periventricular; dp, dorsal parvocellular; mpv, medial parvocellular ventral. n = 6/region. Quantitative assessment of Fos induction (mean total cell counts ± SEM) through the mpd part of the paraventricular nucleus. Intervals (hours) indicate time after the onset of restraint. **, P < 0.01 vs. basal; 
