The Role of the Locus Coeruleus in Corticotropin-Releasing Hormone and Stress-Induced Suppression of Pulsatile Luteinizing Hormone Secretion
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内分泌学杂志 2005年第1期
Division of Reproductive Health (J.C.M., X.F.L., L.B., K.T.O.), Endocrinology and Development, School of Medicine, New Hunt’s House, King’s College London, London SE1 1UL, United Kingdom; and Biostatistiques-Medecine de la Reproduction (J.-C.T.), Hopital Necker, 75743 Paris Cedex 15, France
Address all correspondence and requests for reprints to: Dr. K. T. O’Byrne, Division of Reproductive Health, Endocrinology and Development, School of Medicine, 2.36D New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom. E-mail: Kevin.o’byrne@kcl.ac.uk.
Abstract
Despite a wealth of evidence for CRH mediating stress-induced suppression of the hypothalamic GnRH pulse generator, and hence reproductive dysfunction, the site and mechanism of action remains elusive. The locus coeruleus (LC), a prominent noradrenergic brain stem nucleus, is innervated by CRH neurons, mediates several behavioral stress responses, and is implicated in the control of pulsatile LH secretion. The aim of this study was to test the hypothesis that LC CRH has a critical role in mediating stress-induced suppression of pulsatile LH secretion in the rat. Ovariectomized rats with 17?-estradiol or oil-filled sc capsules were implanted with bilateral LC and iv cannulae. Central administration of CRH (10 ng to 1 μg) resulted in a dose-dependent suppression of LH pulses, which was reversed by a CRH receptor antagonist (-helical CRF9–41, 1 μg). The induction of c-fos expression in glutamic acid decarboxylase67 immunostained neurons in the preoptic area suggests activation of the secretion of -aminobutyric acid in response to intracoerulear administration of CRH; 17?-estradiol further increased the percentage of glutamic acid decarboxylase67-positive neurons that expressed fos and augmented suppression of LH pulses. Furthermore, intracoerulear administration of -helical CRF9–41 completely blocked restraint stress-induced suppression of LH pulses, without affecting the inhibitory response to hypoglycemia. These results suggest that CRH innervation of the LC may play a pivotal, but differential, role in the normal physiological response of stress-induced suppression of the GnRH pulse generator and hence the reproductive system.
Introduction
CRH PLAYS A central role in stress-induced suppression of the hypothalamo-pituitary-gonadal (HPG) axis, specially the GnRH pulse generator, the central neural regulator of pituitary LH and FSH secretion, resulting in reproductive dysfunction. Intracerebroventricular administration of CRH to a variety of species including rats and monkeys, inhibits pulsatile LH release (1, 2), and central infusion of CRH antagonists reverses the LH pulse-suppressing effects of a variety of stressful stimuli (1, 3, 4, 5).
Although contacts between CRH and GnRH neurons have been described in the preoptic area of the rat (6) and human (7), the source of those CRH fibers is unknown. The CRH cells of the paraventricular nucleus (PVN), which form the central component of the hypothalamo-pituitary-adrenocortical axis appear not to be essential in stress-induced suppression of the reproductive axis because bilateral electrolytic lesions of this nucleus fail to prevent the inhibition of LH secretion in response to a variety of stressors (8). Moreover, neural tract-tracing studies, examining the major populations of CRH cells including the PVN, failed to find any CRH neurons projecting to the GnRH-rich regions of the preoptic area in the rat (9), thus suggesting that CRH may not act directly on GnRH perikarya but rather by indirect means to mediate stress-induced inhibition of pulsatile LH secretion.
CRH inputs to the brain stem noradrenergic nucleus, the locus coeruleus (LC), are involved in stress-induced activation of corticosterone release and stress-related behaviors. Anatomical studies have identified several sources of CRH innervation to the LC region, including the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and a minor innervation from the hypothalamic PVN (10, 11, 12). CRH terminals contact catecholaminergic neurons of the LC (13), and tyrosine hydroxylase immunoreactive neurons in the LC express CRH receptors (14). Furthermore, microinfusion of CRH into the LC, which increases LC neuronal activity (15), enhances secretion of ACTH-releasing hormone (16) and elicits anxiogenic behavioral responses (17), whereas blockade of CRH receptors within the LC via infusion of a CRH antagonist attenuates stress-induced anxiety behaviors (18).
The LC is also strongly implicated in the regulation of the GnRH pulse generator because bilateral electrolytic lesions of this nucleus dramatically alters the frequency of LH pulses (19) and abolished the preovulatory LH surge (20) in the rat. The present study was designed to investigate whether intracoerulear administration of CRH inhibits LH pulses and intracoerulear administration of CRH antagonists block hypoglycemic (1, 21) and restraint (22) stress-induced suppression of pulsatile LH secretion in the rat. In addition, immunocytochemical staining for c-fos protein, a marker of neuronal activation, was used to establish whether the preoptic area is implicated in the response to intracoerulear administration of CRH. Because noradrenergic interactions and interactions of the secretion of -aminobutyric acid (GABAergic) in the preoptic area are important in the regulation of the GnRH pulse generator (23), we further examined whether these fos-activated neurons were GABAergic by double labeling for glutamic acid decarboxylase (GAD67), a marker of GABAergic neurons, and fos immunoreactivity. Finally, because 17?-estradiol (E2) potentiates the inhibitory effect of intracerebroventricular administration of CRH on LH pulses in the rat (1) and sensitizes the GnRH pulse generator to the inhibitory effects of stress in a variety of species (21, 24, 25, 26), we compared the effect of intracoerulear CRH injection on fos and GAD67 expression in the preoptic area in the presence and absence of E2 and examined the relationship to the degree of LH pulse suppression.
Materials and Methods
Animals and surgical procedures
Adult female Wistar rats weighing 230–280 g (Tuck Suppliers Ltd., Battlesbridge, UK) were used. The animals were housed under controlled conditions (14-h light, 10-h dark cycle, with lights on at 0700 h; temperature 22 ± 2 C) and provided with food and water ad libitum. All animal procedures were undertaken in accordance with the United Kingdom Home Office Regulations. All surgical procedures were carried out under ketamine (100 mg/kg ip; Pharmacia and Upjohn Ltd., Crawley, UK) and Rompun (10 mg/kg ip; Bayer, Leverkusen, Germany) anesthesia. Rats were implanted with bilateral guide cannulae (22 gauge, 9.5 mm long; Plastics One Inc., Roanoke, VA) directed toward the LC. The coordinates for implantation were 1.2 mm lateral, 9.7 mm posterior to bregma, and 7.7 mm below the surface of the skull (27). The guide cannulae were secured to the skull with three stainless steel screws and dental cement (Dental Filling Ltd., Swindon, UK) and fitted with dummy cannulae (Plastics One) to maintain patency. Two weeks later the animals were bilaterally ovariectomized (ovx) and implanted with a Silastic capsule (inner diameter 1.57 mm; outer diameter 3.18 mm; Sanitech, Havant, UK) filled to a length of 25 mm with E2 (Sigma-Aldrich Ltd., Poole, UK) dissolved at a concentration of 20 μg/ml arachis oil (Sigma-Aldrich) or peanut oil alone. The E2-containing capsules should produce circulating concentrations of E2 within the range observed during the diestrous phase of the estrus cycle (38.8 ± 1.2 pg/ml) as previously described by Maeda and colleagues (28). A further 7 d later, the rats were fitted with two indwelling cardiac catheters via the jugular veins (26). The catheters were exteriorized at the back of the head and secured to a cranial attachment: the rats were fitted with a 30-cm-long metal spring tether (Instec Laboratories Inc., Boulder, CO). The distal end of the tether was attached to a fluid swivel (Instec Laboratories), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3–7 d later.
CRH infusion into the locus coeruleus and pulsatile LH secretion
On the morning of experimentation, bilateral injection cannulae (Plastics One) with extension tubing, preloaded with drug or vehicle, were inserted into the guide cannulae. The distal end of the tubing was extended outside the animal cage to allow remote infusion without disturbing the rat during the experiment. The injection cannulae, which extended 1.5 mm beyond the tip of the guide cannulae, reached the injection site, the center of the LC (27). Rats were then attached via one of the two cardiac catheters to a computed-controlled automated blood sampling system, which allows for the intermittent withdrawal of small blood samples (25 μl) without disturbing the rats (21). Experimentation commenced between 0900 and 1000 h and blood samples were taken every 5 min for 6 h. After removal of each 25-μl blood sample, an equal volume of heparinized saline (10 U/ml normal saline; CP Pharmaceuticals Ltd., Wrexham, UK) was automatically infused into the animal to maintain patency of the catheter and blood volume. Blood samples were frozen at –20 C for later assay to determine LH concentrations. After 2 h of blood sampling, CRH (Sigma-Aldrich) was bilaterally infused into the LC over 4 min. Rats with E2-filled capsules received a single dose of 10 ng, 100 ng, or 1 μg CRH in 200 nl artificial cerebrospinal fluid (aCSF) (n = 8–15 per treatment group). Control rats received 200 nl aCSF bilaterally into the LC (n = 13). Animals implanted with oil-filled capsules received 100 ng CRH (n = 13) or 200 nl aCSF (n = 5) bilaterally into the LC.
To test the receptor specificity of the CRH action, E2-replaced ovx rats were coadministered CRH (100 ng) and the CRH receptor antagonist (-helical CRF9–41, 1 μg, n = 5; Sigma-Aldrich) bilaterally (200 nl) into the LC. Control rats received either aCSF (200 nl, n = 6) or -helical CRF9–41 (1 μg, n = 4) alone.
CRH infusion into the LC and fos and GAD67 expression in the presence and absence of E2
CRH (100 ng in 200 nl aCSF) was bilaterally administered over a 4-min period into the LC of ovx E2-treated rats (n = 9) and ovx oil-treated rats (n = 6) as described above. Control animals received 200 nl aCSF (ovx E2-treated rats, n = 6; ovx oil-treated rats, n = 4). Sixty minutes later the rats were deeply anesthetized using 0.5 ml sodium pentobarbitone (60 mg/ml iv; Rhone Mérieux Ltd., Harlow, UK) and perfused transcardially with heparinized saline (5 U/ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed and immediately placed in a postfix solution of 15% sucrose in 4% paraformaldehyde in 0.1 M PB at 4 C until they sank. They were then transferred into a solution of 30% sucrose in 0.1 M PB at 4 C. Brains were stored at –80 C for later immunocytochemical detection of fos and GAD67 protein. The hypothalami were coronally sectioned (30 μm), and every fourth section was used for fos and GAD67 double immunostaining. Free-floating sections were used in all experiments. Sections were incubated for 10 min in 0.02% H2O2 and then 10 min in 2% normal goat serum (NGS). Sections were incubated in 1:100,000 polyclonal rabbit anti-c-fos primary antibody (AB-5 OSI, Oncogene Science, San Diego, CA) containing 1.25% NGS at 4 C for 36 h. Sections were processed with a biotinylated goat antirabbit secondary antibody (Stratech Scientific Ltd., Cambridge, UK) at a dilution of 1:1000 in PBS in 1.25% NGS, followed by incubation in streptavidin peroxidase tertiary reagent. Visualization of fos immunoreactivity was achieved using the diaminobenzidine reaction, intensified with nickel ammonium chloride (Vector Laboratories, Burlingame, CA). Sections were then incubated for 30 min in 0.025% H2O2, followed by a 36-h incubation at 4 C in a polyclonal rabbit anti-GAD (Chemicon International Inc., Temecula, CA) antibody at a dilution of 1:2000 in PBS. A further incubation in biotinylated donkey antirabbit antibody (Stratech Scientific) at a dilution of 1:200 in PBS, followed by incubation in Vectastain Elite ABC peroxidase system (Vector Laboratories), was carried out, and the diaminobenzidine reaction was used to visualize GAD immunoreactivity. Omission of the GAD primary antibody resulted in the absence of specific staining. Sections were mounted on slides, dehydrated, and coverslipped. Several brains from each experimental group were reacted on the same day to control for interbatch variability.
Semiquantitative analysis of immunostaining data for fos and GAD67 was carried out on an AxioVision microscope image system (Zeiss, Oberkochen, Germany). All analysis was performed on coded slides by an investigator without knowledge of experimental treatment conditions. The boundaries of regions counted were determined by comparing the Paxinos and Watson’s rat brain atlas (27) with neuroanatomical and cytoarchitectural landmarks. Sections used for the preoptic area analysis were taken from the region corresponding to bregma +0.20 to –0.40 mm. The number of immunostained cells for fos was determined bilaterally in four sections from each rat. The number of cells expressing the GAD67 protein was counted, and the percentage of these cells also displaying fos immunoreactivity was calculated. Neurons expressing fos and GAD67 immunoreactivity were counted with bright-field microscopy at x40 magnification. Fine focusing was performed to ensure counting of all immunostained cells throughout the thickness of the sections.
CRH antagonist infusion into the locus coeruleus and hypoglycemic and restraint stress-induced suppression of LH pulses
To examine the role of LC CRH in hypoglycemic stress-induced suppression of LH pulses, ovx E2-treated rats were fasted overnight. The following morning bilateral LC injection cannulae (Plastics One) with extension tubing, preloaded with -helical CRF9–41 or aCSF, were inserted into the guide cannulae as described above. Rats were then connected via one of the two cardiac catheters to the automated blood sampling system. Blood sampling commenced between 0900 and 1000 h and continued for 6 h, as described above, and assayed for LH. Every 60 min, blood samples (50 μl) were drawn manually from the second catheter to determine blood glucose concentrations, which were measured using a Reflolux S blood glucose monitor (Roche Molecular Biochemicals, Mannheim, Germany). Of the 50 μl of blood collected for each measurement of blood glucose, only 10–20 μl are required, and the remaining 30–40 μl of blood are immediately returned to the animal. The reason for collecting 50 μl of blood is to minimize potential dilution of sample during the collection procedure. After 2 h of automated blood sampling, the CRH antagonist (2 μg in 200 nl aCSF) was administered bilaterally into the LC over 4 min (n = 6). Ten minutes later, a single iv injection of 0.25 U/kg insulin (Nordisk Wellcome human insulin, Crawley, UK) in 0.2 ml saline was given. For the 60-min period after insulin administration, blood glucose concentrations were monitored every 5–10 min. Control rats were fasted overnight but received either CRH antagonist bilaterally into the LC and saline iv (n = 4) or aCSF into the LC and insulin iv (n = 5).
To determine the role of LC CRH in restraint stress-induced suppression of pulsatile LH secretion, ovx E2-treated rats were used. On the morning of experimentation, rats were connected to the preloaded LC cannulae and automated blood sampling (25 μl every 5 min for 6 h) commenced for LH measurement as described above. After 2 h blood sampling, 1 μg CRH antagonist (-helical CRF9–41; n = 5) in 200 nl aCSF was infused bilaterally into the LC over 4 min. Ten minutes later, animals were placed in a restraint device for 60 min. Additional infusions of the CRH antagonist (500 ng in 100 nl aCSF) were administered 20 and 40 min after the initial infusion. Control rats received aCSF in place of the CRH antagonists (n = 7). Additional controls were left to roam freely around their enclosure after infusion of aCSF (n = 8) or -helical CRF9–41 (n = 4) into the LC. The restraint devices were constructed from transparent plastic tubing, 18.0 cm long and 5.8 cm in diameter, with a hinged top to allow placement of the rat inside. The head of the animal was free, extending beyond a fixed collar at one end of the device. The collar diameter was 3.2 cm. At the tail end of the restraint device, an adjustable O-shaped fixture, with a central aperture (1.5 cm) to accommodate the tail of the rat, was in place to allow the length of the tube to be set to the exact length of the rat. The device was equipped with air holes.
LC cannulae placement
Cannula placement was verified histologically, and all animals with cannulae tips located outside the LC were excluded from the analysis. Figure 1, A and B, are representative examples of cannula placement within and outside the LC, respectively.
FIG. 1. Representative examples illustrating correct cannula placement within (A) and incorrect cannula placement outside (B) the LC, with the tip of the cannula lying dorsal to the fourth ventricle.
Hormone assay
A double-antibody RIA supplied by the National Institute of Diabetes and Digestive and Kidney Diseases was used to determine LH concentrations in the 25-μl whole-blood samples. The sensitivity of the assay was 0.093 ng/ml. The inter- and intraassay variations were 5.8 and 5.0%, respectively.
Statistical analysis
Detection of LH pulses was established by use of the algorithm ULTRA (29). Two intraassay coefficients of variation of the assay were used as the reference threshold for pulse detection. The effect of infusion of CRH into the LC on pulsatile LH secretion was calculated by comparing the mean LH pulse interval before and after treatment and expressed as prolongation of LH pulse interval as the percentage of the pretreatment control value (1). The significance of the effect of LC infusion of CRH in the presence and absence of E2 was also assessed. The decrease in blood glucose concentration in response to insulin was determined by comparing the mean glucose level before insulin injection with the mean blood glucose concentration during the 60-min period after insulin injection. The inhibitory effect of hypoglycemic stress on LH pulses was calculated by comparing the mean LH pulse interval before insulin with the first interval after administration and expressed as prolongation of LH pulse interval as the percentage by which the mean first interval exceeds the pretreatment control value (21, 26, 30). The inhibitory effect of restraint stress on LH pulses was calculated by comparing the mean LH pulse interval before and during restraint in each treatment group and expressed as prolongation of LH pulse interval as the percentage of the prerestraint control value. For the 2-h prerestraint and the 1-h restraint periods, the number of LH pulses detected in each of these periods was calculated and divided by the period duration in minutes to give the appropriate LH pulse interval (22). When there were no LH pulses evident during the 1-h restraint period, the LH pulse interval assigned to the restraint period was taken as the interval from the onset of restraint to the first LH pulse in the 2-h postrestraint period (22). Statistical significance was tested using one-way ANOVA and Dunnett’s test and was carried out on actual LH pulse intervals before transformation to percentage. For fos and GAD67 immunostaining, individual rat means were combined to provide group means and analyzed using ANOVA followed by the nonparametric Dunn’s test. P < 0.05 was considered statistically significant.
Results
Effects of CRH infusion into the LC on pulsatile LH release
The LH pulse intervals in the pretreatment control period for the aCSF vehicle-treated rats with and without E2 replacement were 23.7 ± 1.1 and 25.7 ± 2.5 min (mean ± SEM), respectively. The LH pulse interval in the pretreatment control period was not significantly different between the CRH and CRH antagonist-treated rats, with a group mean ± SEM of 24.2 ± 1.3 min. Administration of CRH into the LC of ovx E2-replaced rats resulted in a dose-dependent increase in LH pulse interval (Fig. 2, B–D and G; P < 0.05; 10 ng, n = 8; 100 ng, n = 11; 1 μg, n = 10). The inhibitory response was evident for the duration of the 4-h posttreatment period examined. Whereas the administration of CRH (100 ng; n = 12) into the LC of ovx non-E2-treated rats also resulted in an increase (P < 0.05) in LH pulse interval, the increase was significantly smaller (P < 0.05) than that observed in the presence of E2 (Fig. 2, F and G). Control infusion of aCSF had no effect on LH pulse frequency in either E2- (n = 7) or non-E2- (n = 5) replaced animals (P = 0.14 and 0.69, respectively; Fig. 2, A, E, and G).
FIG. 2. Representative examples illustrating the effects of bilateral infusion () of 200 nl aCSF (A), 10 ng (B), 100 ng (C), or 1 μg (D) CRH in 200 nl aCSF into the LC on pulsatile LH secretion in ovx E2-treated rat. Examples of bilateral infusion of aCSF (E) or 100 ng CRH (F) to ovx, non-E2-treated rat are also displayed (*, LH pulse). G, Summary showing the dose-dependent inhibitory effect of intracoerulear administration of CRH on pulsatile LH secretion, expressed as a prolongation of LH pulse interval, in ovx, E2-treated rat (). Note the attenuated response in the absence of E2 replacement (). *, P < 0.05 vs. aCSF infusion in the same steroid group; **, P < 0.001 vs. aCSF infusion in the same steroid group; , P < 0.05 vs. 100 ng CRH in E2-treated group.
Coadministration of CRH (100 ng) and -helical CRF9–41 (1 μg) bilaterally into the LC completely blocked the inhibitory effect of CRH (Fig. 3, B, D, and E; n = 5). Control infusion of aCSF (n = 5) and/or -helical CRF9–41 (n = 4) alone did not affect pulsatile LH secretion (Fig. 3, A, C, and E).
FIG. 3. Representative examples showing the effects of bilateral intracoerulear infusion () of 200 nl aCSF (A), 100 ng CRH (B), or 1 μg CRH antagonist -helical CRF9–41 (hCRF) (C) or coinfusion of 100 ng CRH and 1 μg -helical CRF9–41 (D) in 200 nl aCSF on pulsatile LH secretion in ovx E2-treated rat (*, LH pulse). E, Summary showing the effect of CRH receptor antagonist on CRH-induced suppression of pulsatile LH secretion. **, P < 0.001 vs. aCSF; , P < 0.05 vs. CRH infusion.
Ten rats administered CRH were considered to have cannulae located outside the LC. There was no prolongation of LH pulse interval in response to bilateral infusion of 100 ng (n = 4) or 1 μg (n = 6) CRH in these animals (0.84 ± 4.73 and 2.37 ± 5.45%; prolongation of LH pulse interval percent of pretreatment control value, respectively; mean ± SEM).
Effects of LC administration of CRH on fos and GAD67 expression
The fos immunoreactive neurons were identified by black reaction product restricted to the nucleus of the cell. In ovx, non-E2-replaced rats, very few fos-expressing neurons were detected in the preoptic area (Fig. 4, A and H) 60 min after bilateral infusion of aCSF (n = 4) into the LC. In contrast, the number of fos-positive cells increased dramatically after CRH (100 ng) administration (Fig. 4, B and H; n = 6). E2 treatment increased the number of fos-immunoreactive neurons in the aCSF-treated control rats (Fig. 4, C and H; n = 6) and further potentiated the increase in fos-positive neurons in response to infusion of CRH into the LC (Fig. 4, D and H; n = 8).
FIG. 4. Representative examples of double-labeling immunoreactivity for fos and GAD67 in neurons within the preoptic area in response to bilateral intracoerulear administration of aCSF (200 nl) or CRH (100 ng in 200 nl aCSF) in ovx rats with or without E2 replacement. A, ovx + aCSF; B, ovx + E2 + aCSF; C, ovx + CRH; D, ovx + E2 + CRH. Scale bars, 100 μm. E and F, Higher-power magnification images showing a fos and GAD67 double-labeled neuron and a GAD67 single-labeled neuron, respectively. Scale bars, 10 μm. G, Representative example of a GAD67-negative control. H, Summary showing the effect of CRH on fos expression in the preoptic area. I, Summary showing the effect of CRH on the percentage of GAD67 neurons also immunoreactive for the fos protein. *, P < 0.05 and **, P < 0.001 vs. aCSF infusion in the same steroid group; , P < 0.05 vs. ovx, non-E2-treated animals in the same infusion group.
CRH infusion (100 ng) into the LC resulted in a significant increase in the percentage of GAD67-immunoreactive neurons that expressed fos within the preoptic area (Fig. 4, A, B, and I). E2 replacement to ovx animals enhanced the percentage of GAD67-positive neurons that expressed fos in the aCSF control rats (Fig. 4, A, C, and I) and further augmented the increase in response to CRH infusion into the LC (Fig. 4, B, D, and I). There was no significant difference in the number of GAD67-expressing neurons in any of the four treatment groups (data not shown).
Effects of CRH receptor antagonists on hypoglycemic and restraint stress-induced interruption of LH pulses
Administration of insulin (0.25 U/kg) iv resulted in a significant decrease in blood glucose in both CRH antagonist-treated animals (70.3 ± 4.1%) and those that received bilateral intracoerulear infusion of aCSF (68.9 ± 2.8%) as controls (mean ± SEM, P < 0.05). The LH pulse interval in the pretreatment control period was 23.5 ± 2.3 and 25.2 ± 1.3 min (mean ± SEM) for aCSF vehicle- and CRH antagonist-treated rats, respectively. Hypoglycemia resulted in a significant interruption of LH pulses in the aCSF-treated animals (Fig. 5, A and E; P < 0.05). Bilateral intracoerulear administration of -helical CRF9–41 10 min before the injection of insulin was unable to prevent the interruption of LH pulses induced by hypoglycemic stress (Fig. 5, B and E; P < 0.05; n = 6). The administration of saline iv to control rats was without effect on either blood glucose or LH pulse interval in aCSF (Fig. 5, C and E; P = 0.86; n = 4) or -helical CRF9–41- (Fig. 5, D and E; P = 0.69; n = 4) treated animals.
FIG. 5. Representative examples illustrating the effects of bilateral intracoerulear injection () of 200 nl aCSF (A) or 1 μg CRH antagonist -helical CRF9–41 (hCRF) (B) on pulsatile LH secretion in ovx, E2-replaced rats subjected to insulin-induced hypoglycemia. The intracoerulear injection was made 10 min before the iv injection () of insulin (0.25 U/kg). C and D, Representative examples showing the effects of administration of aCSF (200 nl) or hCRF (1 μg) 10 min before saline () iv, respectively. E, Summary showing the inability of intracoerulear administered CRH antagonist to affect hypoglycemic stress-induced interruption of LH pulses. #, P < 0.05 vs. saline treatment group. *, denotes LH pulse.
The LH pulse interval in the pretreatment control period for the restraint stress study was 28.2 ± 2.3 and 27.6 ± 1.6 min (mean ± SEM) for aCSF vehicle- and CRH antagonist-treated rats, respectively. Restraint stress induced a profound suppression of pulsatile LH secretion in aCSF-treated animals (Fig. 6, A and E; P < 0.001; n = 6). The effect was immediate in onset, with most animals having no LH pulses during the 60-min restraint period and showing a normal LH pulse frequency on release from the restraint device. Bilateral intracoerulear administration of -helical CRF9–41 completely prevented the suppression of pulsatile LH secretion in response to this stress (Fig. 6, B and E; P = 0.91; n = 5). Control rats treated with aCSF (Fig. 6, C and E; P = 0.41; n = 7) or -helical CRF9–41 (Fig. 6, D and E; P = 0.69; n = 4) alone without restraint stress did not show any change in LH pulse frequency.
FIG. 6. Representative examples illustrating the effects of bilateral intracoerulear injections () of aCSF (A) or CRH antagonist -helical CRF9–41 (hCRF) (B) on pulsatile LH secretion in ovx E2-replaced rats subjected to restraint stress. The initial intracoerulear injection (1 μg hCRF or 200 nl aCSF) was made 10 min before the animals were placed in the restraint device (—) for 60 min. Additional infusions of aCSF (100 nl) or hCRF (500 ng in 100 nl aCSF) were given at 20 and 40 min after the initial injection. C and D, Representative examples showing the effects of intracerulear administration of aCSF or hCRF in the absence of restraint stress, respectively. E, Summary showing the effect of CRH receptor antagonist on restraint stress-induced suppression of pulsatile LH secretion. #, P < 0.001 vs. nonrestraint stress group. *, LH pulse.
Discussion
Administration of CRH into the LC resulted in a dose-dependent decrease in LH pulse frequency in ovx E2-treated rats. This response was specific to the LC because infusion of CRH into nearby brain stem areas was without effect. CRH specificity was also confirmed because coadministration of CRH and the CRH antagonist, -helical CRF9–41, into the LC completely blocked the inhibitory response. These data provide evidence that CRH activity within the LC can impact the HPG axis and extend previous studies implicating this noradrenergic brain stem nucleus in the control of pulsatile LH secretion (19). Moreover, the data provide the first evidence that endogenous LC CRH plays a pivotal role in the normal physiological response to stress-induced suppression of the hypothalamic GnRH pulse generator because intracoerulear administration of a CRH antagonist completely blocked restraint stress-induced suppression of LH pulses. However, it would appear that LC CRH is not involved in the normal regulation under nonstressed condition because the CRH antagonist did not affect basal pulsatile LH secretion.
The HPG-inhibitory role for the LC is consistent with its well-established function in regulating stress responses. For example, microinfusion of CRH into the LC, which increases the activity of LC neurones (15), enhances secretion of ACTH (16) and elicits anxiogenic behavioral responses (17), whereas blockade of CRH receptors within the LC via infusion of a CRH antagonist attenuates stress-induced behaviors such as freezing (31) and withdrawal (18). Moreover, lesions of the LC attenuate the ACTH and corticosterone response to restraint stress (32) and also attenuate fos activation in the central nervous system in response to restraint (33). In addition, CRH receptors are localized to the LC (14, 34, 35), and CRH neurons arising in the central nucleus of the amygdala and bed nucleus of the stria terminalis, regions that coordinate emotional responses to stress and to a much lesser extent the PVN, provide direct synaptic input to noradrenergic neurons of the LC, (10, 11, 12) thus providing an anatomical substrate whereby endogenous CRH may alter the activity of the LC neurones.
Although CRH has been shown to suppress the activity of the GnRH pulse generator (1, 2), the site(s) of action remains to be established. The PVN forms the major hypothalamic component of the hypothalamo-pituitary-adrenocortical axis, releasing CRH to drive pituitary ACTH secretion in response to stress. However, the role of PVN CRH in the control of pulsatile LH secretion is controversial. Although there is a rise in CRH mRNA expression in the PVN in response to a variety of stressors that suppress LH pulses, including hypoglycemic stress (26), and various pharmacological experiments have provided evidence that stress-activated inputs, including noradrenergic inputs, to the PVN suppress LH pulses via CRH (36, 37), bilateral electrolytic lesions of the PVN fail to prevent the inhibition of LH secretion in response to various stressors (8). Furthermore, there is a scarcity of PVN projections to the GnRH-rich regions of the preoptic area (9).
Because neurons arising in the LC project directly to the GnRH-rich regions of the preoptic area (38, 39, 40) and selective lesioning of the LC results in a decrease in noradrenaline levels within the preoptic area (41), LC noradrenergic neurons may directly alter the activity of the GnRH neural system. Numerous studies implicated noradrenaline as an important regulator of GnRH/LH release. Infusion of noradrenaline either intracerebroventricularly (42, 43) or directly into the preoptic area (44) of the rat suppresses LH pulses, as does electrical stimulation of the brain stem ascending noradrenergic pathways (45), thus suggesting that increases in noradrenergic activity results in a suppression of the GnRH pulse generator. In contrast, however, peripheral (46) or intrapreoptic area (47) administration of -adrenergic receptor antagonists also decreases the frequency of the GnRH pulse generator. The finding that both a reduction and increase in adrenergic receptor activity have the same effect on pulsatile LH secretion led Leng and colleagues (48) to propose that fluctuating patterns of adrenergic receptor activity are essential for pulsatile GnRH release, a postulate substantiated by theoretical modeling experiments. Although the majority of brain stem noradrenergic neurons that project to the GnRH-rich region of the preoptic area originate in the ventrolateral medulla and nucleus tractus solitarii, with a small component arising from the LC (40), it is interesting to note that near complete interruption of ascending noradrenergic pathways did not alter LH pulse frequency (49), whereas discrete lesions of the LC, destroying 50% or more of the nucleus, resulted in a permanent inhibition of pulsatile LH release (19). Therefore, the LC may provide a critical noradrenergic tone, with increases or decreases beyond certain threshold levels resulting in suppression of LH pulses. Hence, stress-induced activation of CRH inputs to the LC, like those arising in the limbic brain, may enhance noradrenergic activity in the preoptic area and suppress the GnRH pulse generator. Moreover, the present data suggest a differential role for CRH inputs to the LC because the CRH antagonist was affective against the inhibitory influence of the psychological/physical stressor restraint but not against the metabolic stressor hypoglycemia.
These findings are supported by previous studies showing that tyrosine hydroxylase mRNA expression is up-regulated in the LC in response to restraint (50), but not hypoglycemic stress (51), and are consistent with the heterogeneity in stressor-specific neural circuitry in the central nervous system (52).
The activation of GABAergic neurons in the preoptic area in response to intracoerulear administration of CRH, shown by the increased percentage of GAD67-immunostained neurons that expressed fos, is consistent with the possibility that preoptic noradrenergic-GABAergic interactions could potentially underlie the reduction in GnRH pulse generator frequency. Certainly, GABAergic neurons, in contrast to GnRH neurons, receive a prominent direct noradrenergic innervation (53), GABAergic terminals are relatively plentiful on GnRH neurons (54), preoptic area GABA release is enhanced by noradrenergic inputs (55), and local infusion of GABA into the preoptic area suppresses pulsatile LH release (56). Furthermore, endogenous GABA signaling through the GABAA receptor exerts a powerful net inhibitory effect on the excitability of GnRH neurons (57). However, there are conflicting data on the action of exogenously applied GABA, with both increases (58) and decreases (59) in GnRH neuron excitability evident, which may result from the use of different experimental techniques.
The present data are also of potential relevance to the issue concerning the sensitizing influence of E2 on stress-induced suppression of the GnRH pulse generator (21, 24, 25, 26). E2 not only increased the number of fos-activated GABA and non-GABAergic neurons in the preoptic area but also augmented the suppression of pulsatile LH secretion induced by intracoerulear administration of CRH. Because we have recently shown that E2 profoundly augments the suppression of LH pulses in response to intracerebroventricular administration of CRH (1), the present data suggest the LC as a plausible site for the sensitizing effects of E2 on stress-induced dysfunction of the reproductive axis. Both estrogen receptor- and -? are expressed in the LC (60, 61), and E2 up-regulates tyrosine hydroxylase mRNA expression in the LC (62), which could result in a greater releasable pool of noradrenaline in response to stress. The increased percentage of GAD67-positive neurons that express fos in the preoptic area in response to E2 per se suggests enhanced GABA activity, which is supported by previous studies demonstrating that GABA release in this region of ovx rats is increased by E2 (63). These data raise the possibility that the sensitizing effects of E2 on stress-induced suppression of pulsatile LH secretion may also be exerted directly at the level of the preoptic area by enhancing both basal and stimulated GABA release. Similarly, the increased fos expression in the preoptic area neurons of undetermined neurochemical phenotype may also be involved in the sensitizing influence of E2 on stress responsiveness.
Acknowledgments
The authors thank Dr. A. F. Parlow (NIDDK) for providing the LH RIA kit.
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Address all correspondence and requests for reprints to: Dr. K. T. O’Byrne, Division of Reproductive Health, Endocrinology and Development, School of Medicine, 2.36D New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom. E-mail: Kevin.o’byrne@kcl.ac.uk.
Abstract
Despite a wealth of evidence for CRH mediating stress-induced suppression of the hypothalamic GnRH pulse generator, and hence reproductive dysfunction, the site and mechanism of action remains elusive. The locus coeruleus (LC), a prominent noradrenergic brain stem nucleus, is innervated by CRH neurons, mediates several behavioral stress responses, and is implicated in the control of pulsatile LH secretion. The aim of this study was to test the hypothesis that LC CRH has a critical role in mediating stress-induced suppression of pulsatile LH secretion in the rat. Ovariectomized rats with 17?-estradiol or oil-filled sc capsules were implanted with bilateral LC and iv cannulae. Central administration of CRH (10 ng to 1 μg) resulted in a dose-dependent suppression of LH pulses, which was reversed by a CRH receptor antagonist (-helical CRF9–41, 1 μg). The induction of c-fos expression in glutamic acid decarboxylase67 immunostained neurons in the preoptic area suggests activation of the secretion of -aminobutyric acid in response to intracoerulear administration of CRH; 17?-estradiol further increased the percentage of glutamic acid decarboxylase67-positive neurons that expressed fos and augmented suppression of LH pulses. Furthermore, intracoerulear administration of -helical CRF9–41 completely blocked restraint stress-induced suppression of LH pulses, without affecting the inhibitory response to hypoglycemia. These results suggest that CRH innervation of the LC may play a pivotal, but differential, role in the normal physiological response of stress-induced suppression of the GnRH pulse generator and hence the reproductive system.
Introduction
CRH PLAYS A central role in stress-induced suppression of the hypothalamo-pituitary-gonadal (HPG) axis, specially the GnRH pulse generator, the central neural regulator of pituitary LH and FSH secretion, resulting in reproductive dysfunction. Intracerebroventricular administration of CRH to a variety of species including rats and monkeys, inhibits pulsatile LH release (1, 2), and central infusion of CRH antagonists reverses the LH pulse-suppressing effects of a variety of stressful stimuli (1, 3, 4, 5).
Although contacts between CRH and GnRH neurons have been described in the preoptic area of the rat (6) and human (7), the source of those CRH fibers is unknown. The CRH cells of the paraventricular nucleus (PVN), which form the central component of the hypothalamo-pituitary-adrenocortical axis appear not to be essential in stress-induced suppression of the reproductive axis because bilateral electrolytic lesions of this nucleus fail to prevent the inhibition of LH secretion in response to a variety of stressors (8). Moreover, neural tract-tracing studies, examining the major populations of CRH cells including the PVN, failed to find any CRH neurons projecting to the GnRH-rich regions of the preoptic area in the rat (9), thus suggesting that CRH may not act directly on GnRH perikarya but rather by indirect means to mediate stress-induced inhibition of pulsatile LH secretion.
CRH inputs to the brain stem noradrenergic nucleus, the locus coeruleus (LC), are involved in stress-induced activation of corticosterone release and stress-related behaviors. Anatomical studies have identified several sources of CRH innervation to the LC region, including the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and a minor innervation from the hypothalamic PVN (10, 11, 12). CRH terminals contact catecholaminergic neurons of the LC (13), and tyrosine hydroxylase immunoreactive neurons in the LC express CRH receptors (14). Furthermore, microinfusion of CRH into the LC, which increases LC neuronal activity (15), enhances secretion of ACTH-releasing hormone (16) and elicits anxiogenic behavioral responses (17), whereas blockade of CRH receptors within the LC via infusion of a CRH antagonist attenuates stress-induced anxiety behaviors (18).
The LC is also strongly implicated in the regulation of the GnRH pulse generator because bilateral electrolytic lesions of this nucleus dramatically alters the frequency of LH pulses (19) and abolished the preovulatory LH surge (20) in the rat. The present study was designed to investigate whether intracoerulear administration of CRH inhibits LH pulses and intracoerulear administration of CRH antagonists block hypoglycemic (1, 21) and restraint (22) stress-induced suppression of pulsatile LH secretion in the rat. In addition, immunocytochemical staining for c-fos protein, a marker of neuronal activation, was used to establish whether the preoptic area is implicated in the response to intracoerulear administration of CRH. Because noradrenergic interactions and interactions of the secretion of -aminobutyric acid (GABAergic) in the preoptic area are important in the regulation of the GnRH pulse generator (23), we further examined whether these fos-activated neurons were GABAergic by double labeling for glutamic acid decarboxylase (GAD67), a marker of GABAergic neurons, and fos immunoreactivity. Finally, because 17?-estradiol (E2) potentiates the inhibitory effect of intracerebroventricular administration of CRH on LH pulses in the rat (1) and sensitizes the GnRH pulse generator to the inhibitory effects of stress in a variety of species (21, 24, 25, 26), we compared the effect of intracoerulear CRH injection on fos and GAD67 expression in the preoptic area in the presence and absence of E2 and examined the relationship to the degree of LH pulse suppression.
Materials and Methods
Animals and surgical procedures
Adult female Wistar rats weighing 230–280 g (Tuck Suppliers Ltd., Battlesbridge, UK) were used. The animals were housed under controlled conditions (14-h light, 10-h dark cycle, with lights on at 0700 h; temperature 22 ± 2 C) and provided with food and water ad libitum. All animal procedures were undertaken in accordance with the United Kingdom Home Office Regulations. All surgical procedures were carried out under ketamine (100 mg/kg ip; Pharmacia and Upjohn Ltd., Crawley, UK) and Rompun (10 mg/kg ip; Bayer, Leverkusen, Germany) anesthesia. Rats were implanted with bilateral guide cannulae (22 gauge, 9.5 mm long; Plastics One Inc., Roanoke, VA) directed toward the LC. The coordinates for implantation were 1.2 mm lateral, 9.7 mm posterior to bregma, and 7.7 mm below the surface of the skull (27). The guide cannulae were secured to the skull with three stainless steel screws and dental cement (Dental Filling Ltd., Swindon, UK) and fitted with dummy cannulae (Plastics One) to maintain patency. Two weeks later the animals were bilaterally ovariectomized (ovx) and implanted with a Silastic capsule (inner diameter 1.57 mm; outer diameter 3.18 mm; Sanitech, Havant, UK) filled to a length of 25 mm with E2 (Sigma-Aldrich Ltd., Poole, UK) dissolved at a concentration of 20 μg/ml arachis oil (Sigma-Aldrich) or peanut oil alone. The E2-containing capsules should produce circulating concentrations of E2 within the range observed during the diestrous phase of the estrus cycle (38.8 ± 1.2 pg/ml) as previously described by Maeda and colleagues (28). A further 7 d later, the rats were fitted with two indwelling cardiac catheters via the jugular veins (26). The catheters were exteriorized at the back of the head and secured to a cranial attachment: the rats were fitted with a 30-cm-long metal spring tether (Instec Laboratories Inc., Boulder, CO). The distal end of the tether was attached to a fluid swivel (Instec Laboratories), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3–7 d later.
CRH infusion into the locus coeruleus and pulsatile LH secretion
On the morning of experimentation, bilateral injection cannulae (Plastics One) with extension tubing, preloaded with drug or vehicle, were inserted into the guide cannulae. The distal end of the tubing was extended outside the animal cage to allow remote infusion without disturbing the rat during the experiment. The injection cannulae, which extended 1.5 mm beyond the tip of the guide cannulae, reached the injection site, the center of the LC (27). Rats were then attached via one of the two cardiac catheters to a computed-controlled automated blood sampling system, which allows for the intermittent withdrawal of small blood samples (25 μl) without disturbing the rats (21). Experimentation commenced between 0900 and 1000 h and blood samples were taken every 5 min for 6 h. After removal of each 25-μl blood sample, an equal volume of heparinized saline (10 U/ml normal saline; CP Pharmaceuticals Ltd., Wrexham, UK) was automatically infused into the animal to maintain patency of the catheter and blood volume. Blood samples were frozen at –20 C for later assay to determine LH concentrations. After 2 h of blood sampling, CRH (Sigma-Aldrich) was bilaterally infused into the LC over 4 min. Rats with E2-filled capsules received a single dose of 10 ng, 100 ng, or 1 μg CRH in 200 nl artificial cerebrospinal fluid (aCSF) (n = 8–15 per treatment group). Control rats received 200 nl aCSF bilaterally into the LC (n = 13). Animals implanted with oil-filled capsules received 100 ng CRH (n = 13) or 200 nl aCSF (n = 5) bilaterally into the LC.
To test the receptor specificity of the CRH action, E2-replaced ovx rats were coadministered CRH (100 ng) and the CRH receptor antagonist (-helical CRF9–41, 1 μg, n = 5; Sigma-Aldrich) bilaterally (200 nl) into the LC. Control rats received either aCSF (200 nl, n = 6) or -helical CRF9–41 (1 μg, n = 4) alone.
CRH infusion into the LC and fos and GAD67 expression in the presence and absence of E2
CRH (100 ng in 200 nl aCSF) was bilaterally administered over a 4-min period into the LC of ovx E2-treated rats (n = 9) and ovx oil-treated rats (n = 6) as described above. Control animals received 200 nl aCSF (ovx E2-treated rats, n = 6; ovx oil-treated rats, n = 4). Sixty minutes later the rats were deeply anesthetized using 0.5 ml sodium pentobarbitone (60 mg/ml iv; Rhone Mérieux Ltd., Harlow, UK) and perfused transcardially with heparinized saline (5 U/ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed and immediately placed in a postfix solution of 15% sucrose in 4% paraformaldehyde in 0.1 M PB at 4 C until they sank. They were then transferred into a solution of 30% sucrose in 0.1 M PB at 4 C. Brains were stored at –80 C for later immunocytochemical detection of fos and GAD67 protein. The hypothalami were coronally sectioned (30 μm), and every fourth section was used for fos and GAD67 double immunostaining. Free-floating sections were used in all experiments. Sections were incubated for 10 min in 0.02% H2O2 and then 10 min in 2% normal goat serum (NGS). Sections were incubated in 1:100,000 polyclonal rabbit anti-c-fos primary antibody (AB-5 OSI, Oncogene Science, San Diego, CA) containing 1.25% NGS at 4 C for 36 h. Sections were processed with a biotinylated goat antirabbit secondary antibody (Stratech Scientific Ltd., Cambridge, UK) at a dilution of 1:1000 in PBS in 1.25% NGS, followed by incubation in streptavidin peroxidase tertiary reagent. Visualization of fos immunoreactivity was achieved using the diaminobenzidine reaction, intensified with nickel ammonium chloride (Vector Laboratories, Burlingame, CA). Sections were then incubated for 30 min in 0.025% H2O2, followed by a 36-h incubation at 4 C in a polyclonal rabbit anti-GAD (Chemicon International Inc., Temecula, CA) antibody at a dilution of 1:2000 in PBS. A further incubation in biotinylated donkey antirabbit antibody (Stratech Scientific) at a dilution of 1:200 in PBS, followed by incubation in Vectastain Elite ABC peroxidase system (Vector Laboratories), was carried out, and the diaminobenzidine reaction was used to visualize GAD immunoreactivity. Omission of the GAD primary antibody resulted in the absence of specific staining. Sections were mounted on slides, dehydrated, and coverslipped. Several brains from each experimental group were reacted on the same day to control for interbatch variability.
Semiquantitative analysis of immunostaining data for fos and GAD67 was carried out on an AxioVision microscope image system (Zeiss, Oberkochen, Germany). All analysis was performed on coded slides by an investigator without knowledge of experimental treatment conditions. The boundaries of regions counted were determined by comparing the Paxinos and Watson’s rat brain atlas (27) with neuroanatomical and cytoarchitectural landmarks. Sections used for the preoptic area analysis were taken from the region corresponding to bregma +0.20 to –0.40 mm. The number of immunostained cells for fos was determined bilaterally in four sections from each rat. The number of cells expressing the GAD67 protein was counted, and the percentage of these cells also displaying fos immunoreactivity was calculated. Neurons expressing fos and GAD67 immunoreactivity were counted with bright-field microscopy at x40 magnification. Fine focusing was performed to ensure counting of all immunostained cells throughout the thickness of the sections.
CRH antagonist infusion into the locus coeruleus and hypoglycemic and restraint stress-induced suppression of LH pulses
To examine the role of LC CRH in hypoglycemic stress-induced suppression of LH pulses, ovx E2-treated rats were fasted overnight. The following morning bilateral LC injection cannulae (Plastics One) with extension tubing, preloaded with -helical CRF9–41 or aCSF, were inserted into the guide cannulae as described above. Rats were then connected via one of the two cardiac catheters to the automated blood sampling system. Blood sampling commenced between 0900 and 1000 h and continued for 6 h, as described above, and assayed for LH. Every 60 min, blood samples (50 μl) were drawn manually from the second catheter to determine blood glucose concentrations, which were measured using a Reflolux S blood glucose monitor (Roche Molecular Biochemicals, Mannheim, Germany). Of the 50 μl of blood collected for each measurement of blood glucose, only 10–20 μl are required, and the remaining 30–40 μl of blood are immediately returned to the animal. The reason for collecting 50 μl of blood is to minimize potential dilution of sample during the collection procedure. After 2 h of automated blood sampling, the CRH antagonist (2 μg in 200 nl aCSF) was administered bilaterally into the LC over 4 min (n = 6). Ten minutes later, a single iv injection of 0.25 U/kg insulin (Nordisk Wellcome human insulin, Crawley, UK) in 0.2 ml saline was given. For the 60-min period after insulin administration, blood glucose concentrations were monitored every 5–10 min. Control rats were fasted overnight but received either CRH antagonist bilaterally into the LC and saline iv (n = 4) or aCSF into the LC and insulin iv (n = 5).
To determine the role of LC CRH in restraint stress-induced suppression of pulsatile LH secretion, ovx E2-treated rats were used. On the morning of experimentation, rats were connected to the preloaded LC cannulae and automated blood sampling (25 μl every 5 min for 6 h) commenced for LH measurement as described above. After 2 h blood sampling, 1 μg CRH antagonist (-helical CRF9–41; n = 5) in 200 nl aCSF was infused bilaterally into the LC over 4 min. Ten minutes later, animals were placed in a restraint device for 60 min. Additional infusions of the CRH antagonist (500 ng in 100 nl aCSF) were administered 20 and 40 min after the initial infusion. Control rats received aCSF in place of the CRH antagonists (n = 7). Additional controls were left to roam freely around their enclosure after infusion of aCSF (n = 8) or -helical CRF9–41 (n = 4) into the LC. The restraint devices were constructed from transparent plastic tubing, 18.0 cm long and 5.8 cm in diameter, with a hinged top to allow placement of the rat inside. The head of the animal was free, extending beyond a fixed collar at one end of the device. The collar diameter was 3.2 cm. At the tail end of the restraint device, an adjustable O-shaped fixture, with a central aperture (1.5 cm) to accommodate the tail of the rat, was in place to allow the length of the tube to be set to the exact length of the rat. The device was equipped with air holes.
LC cannulae placement
Cannula placement was verified histologically, and all animals with cannulae tips located outside the LC were excluded from the analysis. Figure 1, A and B, are representative examples of cannula placement within and outside the LC, respectively.
FIG. 1. Representative examples illustrating correct cannula placement within (A) and incorrect cannula placement outside (B) the LC, with the tip of the cannula lying dorsal to the fourth ventricle.
Hormone assay
A double-antibody RIA supplied by the National Institute of Diabetes and Digestive and Kidney Diseases was used to determine LH concentrations in the 25-μl whole-blood samples. The sensitivity of the assay was 0.093 ng/ml. The inter- and intraassay variations were 5.8 and 5.0%, respectively.
Statistical analysis
Detection of LH pulses was established by use of the algorithm ULTRA (29). Two intraassay coefficients of variation of the assay were used as the reference threshold for pulse detection. The effect of infusion of CRH into the LC on pulsatile LH secretion was calculated by comparing the mean LH pulse interval before and after treatment and expressed as prolongation of LH pulse interval as the percentage of the pretreatment control value (1). The significance of the effect of LC infusion of CRH in the presence and absence of E2 was also assessed. The decrease in blood glucose concentration in response to insulin was determined by comparing the mean glucose level before insulin injection with the mean blood glucose concentration during the 60-min period after insulin injection. The inhibitory effect of hypoglycemic stress on LH pulses was calculated by comparing the mean LH pulse interval before insulin with the first interval after administration and expressed as prolongation of LH pulse interval as the percentage by which the mean first interval exceeds the pretreatment control value (21, 26, 30). The inhibitory effect of restraint stress on LH pulses was calculated by comparing the mean LH pulse interval before and during restraint in each treatment group and expressed as prolongation of LH pulse interval as the percentage of the prerestraint control value. For the 2-h prerestraint and the 1-h restraint periods, the number of LH pulses detected in each of these periods was calculated and divided by the period duration in minutes to give the appropriate LH pulse interval (22). When there were no LH pulses evident during the 1-h restraint period, the LH pulse interval assigned to the restraint period was taken as the interval from the onset of restraint to the first LH pulse in the 2-h postrestraint period (22). Statistical significance was tested using one-way ANOVA and Dunnett’s test and was carried out on actual LH pulse intervals before transformation to percentage. For fos and GAD67 immunostaining, individual rat means were combined to provide group means and analyzed using ANOVA followed by the nonparametric Dunn’s test. P < 0.05 was considered statistically significant.
Results
Effects of CRH infusion into the LC on pulsatile LH release
The LH pulse intervals in the pretreatment control period for the aCSF vehicle-treated rats with and without E2 replacement were 23.7 ± 1.1 and 25.7 ± 2.5 min (mean ± SEM), respectively. The LH pulse interval in the pretreatment control period was not significantly different between the CRH and CRH antagonist-treated rats, with a group mean ± SEM of 24.2 ± 1.3 min. Administration of CRH into the LC of ovx E2-replaced rats resulted in a dose-dependent increase in LH pulse interval (Fig. 2, B–D and G; P < 0.05; 10 ng, n = 8; 100 ng, n = 11; 1 μg, n = 10). The inhibitory response was evident for the duration of the 4-h posttreatment period examined. Whereas the administration of CRH (100 ng; n = 12) into the LC of ovx non-E2-treated rats also resulted in an increase (P < 0.05) in LH pulse interval, the increase was significantly smaller (P < 0.05) than that observed in the presence of E2 (Fig. 2, F and G). Control infusion of aCSF had no effect on LH pulse frequency in either E2- (n = 7) or non-E2- (n = 5) replaced animals (P = 0.14 and 0.69, respectively; Fig. 2, A, E, and G).
FIG. 2. Representative examples illustrating the effects of bilateral infusion () of 200 nl aCSF (A), 10 ng (B), 100 ng (C), or 1 μg (D) CRH in 200 nl aCSF into the LC on pulsatile LH secretion in ovx E2-treated rat. Examples of bilateral infusion of aCSF (E) or 100 ng CRH (F) to ovx, non-E2-treated rat are also displayed (*, LH pulse). G, Summary showing the dose-dependent inhibitory effect of intracoerulear administration of CRH on pulsatile LH secretion, expressed as a prolongation of LH pulse interval, in ovx, E2-treated rat (). Note the attenuated response in the absence of E2 replacement (). *, P < 0.05 vs. aCSF infusion in the same steroid group; **, P < 0.001 vs. aCSF infusion in the same steroid group; , P < 0.05 vs. 100 ng CRH in E2-treated group.
Coadministration of CRH (100 ng) and -helical CRF9–41 (1 μg) bilaterally into the LC completely blocked the inhibitory effect of CRH (Fig. 3, B, D, and E; n = 5). Control infusion of aCSF (n = 5) and/or -helical CRF9–41 (n = 4) alone did not affect pulsatile LH secretion (Fig. 3, A, C, and E).
FIG. 3. Representative examples showing the effects of bilateral intracoerulear infusion () of 200 nl aCSF (A), 100 ng CRH (B), or 1 μg CRH antagonist -helical CRF9–41 (hCRF) (C) or coinfusion of 100 ng CRH and 1 μg -helical CRF9–41 (D) in 200 nl aCSF on pulsatile LH secretion in ovx E2-treated rat (*, LH pulse). E, Summary showing the effect of CRH receptor antagonist on CRH-induced suppression of pulsatile LH secretion. **, P < 0.001 vs. aCSF; , P < 0.05 vs. CRH infusion.
Ten rats administered CRH were considered to have cannulae located outside the LC. There was no prolongation of LH pulse interval in response to bilateral infusion of 100 ng (n = 4) or 1 μg (n = 6) CRH in these animals (0.84 ± 4.73 and 2.37 ± 5.45%; prolongation of LH pulse interval percent of pretreatment control value, respectively; mean ± SEM).
Effects of LC administration of CRH on fos and GAD67 expression
The fos immunoreactive neurons were identified by black reaction product restricted to the nucleus of the cell. In ovx, non-E2-replaced rats, very few fos-expressing neurons were detected in the preoptic area (Fig. 4, A and H) 60 min after bilateral infusion of aCSF (n = 4) into the LC. In contrast, the number of fos-positive cells increased dramatically after CRH (100 ng) administration (Fig. 4, B and H; n = 6). E2 treatment increased the number of fos-immunoreactive neurons in the aCSF-treated control rats (Fig. 4, C and H; n = 6) and further potentiated the increase in fos-positive neurons in response to infusion of CRH into the LC (Fig. 4, D and H; n = 8).
FIG. 4. Representative examples of double-labeling immunoreactivity for fos and GAD67 in neurons within the preoptic area in response to bilateral intracoerulear administration of aCSF (200 nl) or CRH (100 ng in 200 nl aCSF) in ovx rats with or without E2 replacement. A, ovx + aCSF; B, ovx + E2 + aCSF; C, ovx + CRH; D, ovx + E2 + CRH. Scale bars, 100 μm. E and F, Higher-power magnification images showing a fos and GAD67 double-labeled neuron and a GAD67 single-labeled neuron, respectively. Scale bars, 10 μm. G, Representative example of a GAD67-negative control. H, Summary showing the effect of CRH on fos expression in the preoptic area. I, Summary showing the effect of CRH on the percentage of GAD67 neurons also immunoreactive for the fos protein. *, P < 0.05 and **, P < 0.001 vs. aCSF infusion in the same steroid group; , P < 0.05 vs. ovx, non-E2-treated animals in the same infusion group.
CRH infusion (100 ng) into the LC resulted in a significant increase in the percentage of GAD67-immunoreactive neurons that expressed fos within the preoptic area (Fig. 4, A, B, and I). E2 replacement to ovx animals enhanced the percentage of GAD67-positive neurons that expressed fos in the aCSF control rats (Fig. 4, A, C, and I) and further augmented the increase in response to CRH infusion into the LC (Fig. 4, B, D, and I). There was no significant difference in the number of GAD67-expressing neurons in any of the four treatment groups (data not shown).
Effects of CRH receptor antagonists on hypoglycemic and restraint stress-induced interruption of LH pulses
Administration of insulin (0.25 U/kg) iv resulted in a significant decrease in blood glucose in both CRH antagonist-treated animals (70.3 ± 4.1%) and those that received bilateral intracoerulear infusion of aCSF (68.9 ± 2.8%) as controls (mean ± SEM, P < 0.05). The LH pulse interval in the pretreatment control period was 23.5 ± 2.3 and 25.2 ± 1.3 min (mean ± SEM) for aCSF vehicle- and CRH antagonist-treated rats, respectively. Hypoglycemia resulted in a significant interruption of LH pulses in the aCSF-treated animals (Fig. 5, A and E; P < 0.05). Bilateral intracoerulear administration of -helical CRF9–41 10 min before the injection of insulin was unable to prevent the interruption of LH pulses induced by hypoglycemic stress (Fig. 5, B and E; P < 0.05; n = 6). The administration of saline iv to control rats was without effect on either blood glucose or LH pulse interval in aCSF (Fig. 5, C and E; P = 0.86; n = 4) or -helical CRF9–41- (Fig. 5, D and E; P = 0.69; n = 4) treated animals.
FIG. 5. Representative examples illustrating the effects of bilateral intracoerulear injection () of 200 nl aCSF (A) or 1 μg CRH antagonist -helical CRF9–41 (hCRF) (B) on pulsatile LH secretion in ovx, E2-replaced rats subjected to insulin-induced hypoglycemia. The intracoerulear injection was made 10 min before the iv injection () of insulin (0.25 U/kg). C and D, Representative examples showing the effects of administration of aCSF (200 nl) or hCRF (1 μg) 10 min before saline () iv, respectively. E, Summary showing the inability of intracoerulear administered CRH antagonist to affect hypoglycemic stress-induced interruption of LH pulses. #, P < 0.05 vs. saline treatment group. *, denotes LH pulse.
The LH pulse interval in the pretreatment control period for the restraint stress study was 28.2 ± 2.3 and 27.6 ± 1.6 min (mean ± SEM) for aCSF vehicle- and CRH antagonist-treated rats, respectively. Restraint stress induced a profound suppression of pulsatile LH secretion in aCSF-treated animals (Fig. 6, A and E; P < 0.001; n = 6). The effect was immediate in onset, with most animals having no LH pulses during the 60-min restraint period and showing a normal LH pulse frequency on release from the restraint device. Bilateral intracoerulear administration of -helical CRF9–41 completely prevented the suppression of pulsatile LH secretion in response to this stress (Fig. 6, B and E; P = 0.91; n = 5). Control rats treated with aCSF (Fig. 6, C and E; P = 0.41; n = 7) or -helical CRF9–41 (Fig. 6, D and E; P = 0.69; n = 4) alone without restraint stress did not show any change in LH pulse frequency.
FIG. 6. Representative examples illustrating the effects of bilateral intracoerulear injections () of aCSF (A) or CRH antagonist -helical CRF9–41 (hCRF) (B) on pulsatile LH secretion in ovx E2-replaced rats subjected to restraint stress. The initial intracoerulear injection (1 μg hCRF or 200 nl aCSF) was made 10 min before the animals were placed in the restraint device (—) for 60 min. Additional infusions of aCSF (100 nl) or hCRF (500 ng in 100 nl aCSF) were given at 20 and 40 min after the initial injection. C and D, Representative examples showing the effects of intracerulear administration of aCSF or hCRF in the absence of restraint stress, respectively. E, Summary showing the effect of CRH receptor antagonist on restraint stress-induced suppression of pulsatile LH secretion. #, P < 0.001 vs. nonrestraint stress group. *, LH pulse.
Discussion
Administration of CRH into the LC resulted in a dose-dependent decrease in LH pulse frequency in ovx E2-treated rats. This response was specific to the LC because infusion of CRH into nearby brain stem areas was without effect. CRH specificity was also confirmed because coadministration of CRH and the CRH antagonist, -helical CRF9–41, into the LC completely blocked the inhibitory response. These data provide evidence that CRH activity within the LC can impact the HPG axis and extend previous studies implicating this noradrenergic brain stem nucleus in the control of pulsatile LH secretion (19). Moreover, the data provide the first evidence that endogenous LC CRH plays a pivotal role in the normal physiological response to stress-induced suppression of the hypothalamic GnRH pulse generator because intracoerulear administration of a CRH antagonist completely blocked restraint stress-induced suppression of LH pulses. However, it would appear that LC CRH is not involved in the normal regulation under nonstressed condition because the CRH antagonist did not affect basal pulsatile LH secretion.
The HPG-inhibitory role for the LC is consistent with its well-established function in regulating stress responses. For example, microinfusion of CRH into the LC, which increases the activity of LC neurones (15), enhances secretion of ACTH (16) and elicits anxiogenic behavioral responses (17), whereas blockade of CRH receptors within the LC via infusion of a CRH antagonist attenuates stress-induced behaviors such as freezing (31) and withdrawal (18). Moreover, lesions of the LC attenuate the ACTH and corticosterone response to restraint stress (32) and also attenuate fos activation in the central nervous system in response to restraint (33). In addition, CRH receptors are localized to the LC (14, 34, 35), and CRH neurons arising in the central nucleus of the amygdala and bed nucleus of the stria terminalis, regions that coordinate emotional responses to stress and to a much lesser extent the PVN, provide direct synaptic input to noradrenergic neurons of the LC, (10, 11, 12) thus providing an anatomical substrate whereby endogenous CRH may alter the activity of the LC neurones.
Although CRH has been shown to suppress the activity of the GnRH pulse generator (1, 2), the site(s) of action remains to be established. The PVN forms the major hypothalamic component of the hypothalamo-pituitary-adrenocortical axis, releasing CRH to drive pituitary ACTH secretion in response to stress. However, the role of PVN CRH in the control of pulsatile LH secretion is controversial. Although there is a rise in CRH mRNA expression in the PVN in response to a variety of stressors that suppress LH pulses, including hypoglycemic stress (26), and various pharmacological experiments have provided evidence that stress-activated inputs, including noradrenergic inputs, to the PVN suppress LH pulses via CRH (36, 37), bilateral electrolytic lesions of the PVN fail to prevent the inhibition of LH secretion in response to various stressors (8). Furthermore, there is a scarcity of PVN projections to the GnRH-rich regions of the preoptic area (9).
Because neurons arising in the LC project directly to the GnRH-rich regions of the preoptic area (38, 39, 40) and selective lesioning of the LC results in a decrease in noradrenaline levels within the preoptic area (41), LC noradrenergic neurons may directly alter the activity of the GnRH neural system. Numerous studies implicated noradrenaline as an important regulator of GnRH/LH release. Infusion of noradrenaline either intracerebroventricularly (42, 43) or directly into the preoptic area (44) of the rat suppresses LH pulses, as does electrical stimulation of the brain stem ascending noradrenergic pathways (45), thus suggesting that increases in noradrenergic activity results in a suppression of the GnRH pulse generator. In contrast, however, peripheral (46) or intrapreoptic area (47) administration of -adrenergic receptor antagonists also decreases the frequency of the GnRH pulse generator. The finding that both a reduction and increase in adrenergic receptor activity have the same effect on pulsatile LH secretion led Leng and colleagues (48) to propose that fluctuating patterns of adrenergic receptor activity are essential for pulsatile GnRH release, a postulate substantiated by theoretical modeling experiments. Although the majority of brain stem noradrenergic neurons that project to the GnRH-rich region of the preoptic area originate in the ventrolateral medulla and nucleus tractus solitarii, with a small component arising from the LC (40), it is interesting to note that near complete interruption of ascending noradrenergic pathways did not alter LH pulse frequency (49), whereas discrete lesions of the LC, destroying 50% or more of the nucleus, resulted in a permanent inhibition of pulsatile LH release (19). Therefore, the LC may provide a critical noradrenergic tone, with increases or decreases beyond certain threshold levels resulting in suppression of LH pulses. Hence, stress-induced activation of CRH inputs to the LC, like those arising in the limbic brain, may enhance noradrenergic activity in the preoptic area and suppress the GnRH pulse generator. Moreover, the present data suggest a differential role for CRH inputs to the LC because the CRH antagonist was affective against the inhibitory influence of the psychological/physical stressor restraint but not against the metabolic stressor hypoglycemia.
These findings are supported by previous studies showing that tyrosine hydroxylase mRNA expression is up-regulated in the LC in response to restraint (50), but not hypoglycemic stress (51), and are consistent with the heterogeneity in stressor-specific neural circuitry in the central nervous system (52).
The activation of GABAergic neurons in the preoptic area in response to intracoerulear administration of CRH, shown by the increased percentage of GAD67-immunostained neurons that expressed fos, is consistent with the possibility that preoptic noradrenergic-GABAergic interactions could potentially underlie the reduction in GnRH pulse generator frequency. Certainly, GABAergic neurons, in contrast to GnRH neurons, receive a prominent direct noradrenergic innervation (53), GABAergic terminals are relatively plentiful on GnRH neurons (54), preoptic area GABA release is enhanced by noradrenergic inputs (55), and local infusion of GABA into the preoptic area suppresses pulsatile LH release (56). Furthermore, endogenous GABA signaling through the GABAA receptor exerts a powerful net inhibitory effect on the excitability of GnRH neurons (57). However, there are conflicting data on the action of exogenously applied GABA, with both increases (58) and decreases (59) in GnRH neuron excitability evident, which may result from the use of different experimental techniques.
The present data are also of potential relevance to the issue concerning the sensitizing influence of E2 on stress-induced suppression of the GnRH pulse generator (21, 24, 25, 26). E2 not only increased the number of fos-activated GABA and non-GABAergic neurons in the preoptic area but also augmented the suppression of pulsatile LH secretion induced by intracoerulear administration of CRH. Because we have recently shown that E2 profoundly augments the suppression of LH pulses in response to intracerebroventricular administration of CRH (1), the present data suggest the LC as a plausible site for the sensitizing effects of E2 on stress-induced dysfunction of the reproductive axis. Both estrogen receptor- and -? are expressed in the LC (60, 61), and E2 up-regulates tyrosine hydroxylase mRNA expression in the LC (62), which could result in a greater releasable pool of noradrenaline in response to stress. The increased percentage of GAD67-positive neurons that express fos in the preoptic area in response to E2 per se suggests enhanced GABA activity, which is supported by previous studies demonstrating that GABA release in this region of ovx rats is increased by E2 (63). These data raise the possibility that the sensitizing effects of E2 on stress-induced suppression of pulsatile LH secretion may also be exerted directly at the level of the preoptic area by enhancing both basal and stimulated GABA release. Similarly, the increased fos expression in the preoptic area neurons of undetermined neurochemical phenotype may also be involved in the sensitizing influence of E2 on stress responsiveness.
Acknowledgments
The authors thank Dr. A. F. Parlow (NIDDK) for providing the LH RIA kit.
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