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Institute of Zoology (I.D.N., O.J.B., N.T., L.T.), University of Regensburg, 93040 Regensburg, Germany; and Division of Biomedical Sciences (A.J.D.), University of Edinburgh, Edinburgh EH8 9XD, United Kingdom
Address all correspondence and requests for reprints to: Professor Dr. Inga D. Neumann, Institute of Zoology, University of Regensburg, 93040 Regensburg, Germany. E-mail: inga.neumann{at}biologie.uni-regensburg.de.
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Introduction |
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The activity of the HPA axis during parturition itself, however, has gained very little attention until now. Recently we reported that the HPA axis is unresponsive to parturition-related events (delivery of pups) in undisturbed, chronically catheterized dams because of a strong inhibition of the HPA axis by endogenous opioids (11). Similarly, in the pig, no relationship of plasma cortisol and fetal expulsion could be found (12), and neither vaginal/cervical dilatation nor external events, e.g. space restriction of the gilt, induced cortisol secretion (13). In contrast, in humans, enhanced HPA axis secretory activity has been found during the dilatation and expulsion stages of labor (14, 15, 16). However, to which extent the HPA-axis is responsive to external, potentially disturbing stimuli during parturition is not known.
An inhibitory effect of brain oxytocin on basal and stimulated HPA axis activity has been shown in virgin female and male rats (17, 18); however, its involvement in the blunted HPA axis responsiveness in pregnancy and lactation is controversial (17, 19). Although the brain oxytocin system becomes enhanced in the last days of pregnancy in terms of increased oxytocin receptor expression and binding (20, 21, 22) and there are some reports of increased oxytocin expression (23, 24 , but see also 25, 26, 27) and content of oxytocin within the supraoptic nucleus (SON) (28), the brain oxytocin system becomes highly activated only at parturition when oxytocin firing rate (29), synthesis (25, 26), and intracerebral release (30, 31, 32) as well as Fos expression in oxytocin neurons (33) increase greatly. During parturition, oxytocin released within the hypothalamic SON and paraventricular nuclei (32) has a positive feedback action (34) and appears to be involved in the regulation of the timing of the parturition process (34). Furthermore, such intracerebrally released oxytocin may also be involved in regulating neuroendocrine stress responses during parturition.
Therefore, the aim of the present study was to compare the responsiveness of the HPA axis and oxytocin system to a mild psychological stressor (repeated exposure to airpuff) in virgin female, late pregnant, and parturient rats fitted with a chronic jugular vein catheter. We hypothesized that oxytocin released within the hypothalamus during parturition inhibits the HPA axis causing the blunted stress response, and this has been tested by intracerebroventricular (icv) administration of a selective oxytocin receptor antagonist (OTA) into the lateral cerebral ventricle before stressor exposure. Preliminary data have been published in abstract form (35).
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Materials and Methods |
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Surgery
All surgical, sampling, and behavioral procedures were approved by the Committee on Animal Health and Care of the local government of Bavaria. Four to 5 d before the beginning of the experiment, surgery was performed on virgin rats and rats on d 18 of pregnancy under halothane anesthesia (Hoechst, Frankfurt/Main, Germany) and using semiaseptic conditions.
Icv guide cannula.
Rats were fixed in a stereotaxic frame, the calvaria were exposed, and an icv guide cannula (21 gauge) was inserted stereotaxically toward the lateral ventricle (coordinates: 0.6 mm caudal to Bregma, 1.6 mm lateral to midline, 1.8 mm beneath the surface of the skull) (36), fixed to the skull and two jeweler’s screws with dental acrylic, and closed with a stylet.
Jugular vein catheter.
After implantation of the icv guide cannula, the jugular vein was exposed by blunt dissection, a catheter consisting of silicone tubing (Dow Corning Corp., Midland MI) and PE-50 polyethylene tubing was inserted approximately 3 cm into the vessel through an incision in a cardiac direction and exteriorized at the neck of the animal. The catheter was filled with sterile saline containing gentamicin (30 000 IU/ml, Centravet, Bad Bentheim, Germany). Following surgery, animals were kept individually in transparent polycarbon cages (24 x 40 x 35.5 cm) and handled carefully each day to familiarize them with the icv infusion and blood sampling procedures and reduce nonspecific stress responses during the experiments.
Experimental protocols
Exposure to airpuff and effects of icv infusion of OTA on ACTH, corticosterone, and oxytocin secretion in virgin rats.
Five days after surgery, virgin female rats were randomly divided into the following four groups: 1) vehicle-treatment (Ringer’s solution, 5 µl), no stress (ACTH/corticosterone/oxytocin: n = 10); 2) vehicle, airpuff exposure (ACTH/corticosterone/oxytocin: n = 12); 3) OTA treatment (des Gly-NH2 d(CH2)5 [Tyr(Me)2, Thr4] OVT; 0.75 µg/5 µl; Dr. M. Manning, Toledo, OH), no stress (ACTH: n = 12; corticosterone/oxytocin: n = 10); and 4) OTA, airpuff exposure (ACTH: n = 15; corticosterone/oxytocin: n = 13).
Exposure to airpuff and effects of icv infusion of OTA on ACTH secretion in d 22 pregnant rats. Four days after surgery, pregnant rats were randomly divided into the following three groups: 1) vehicle, no stress (n = 5); 2) vehicle, airpuff exposure (n = 7); and 3) OTA, airpuff exposure (n = 5).
In virgin and pregnant rats, the experiments were performed almost parallel with the parturient rats between 0800 h and 1800 h.
Two hours before blood sampling, the jugular vein catheter was attached to an extension tubing (50 cm) connected to a 1-ml syringe filled with sterile heparinized saline (30 IU/ml). The icv infusion cannula (25 gauge), filled with either vehicle or OTA and connected to a 10-µl microsyringe via PE-50 tubing (50 cm), was lowered into the icv guide cannula and fixed in place. Rats were left undisturbed for the next 2 h. Either 0.2-ml (for detection of ACTH, corticosterone), or 0.6-ml blood samples (ACTH, corticosterone, oxytocin), substituted immediately by sterile saline, were taken under basal conditions. Then either vehicle or OTA were infused icv (5 µl injected over 30 sec) without additional disturbance of the rats. Ten minutes later in their home cages, rats were exposed to repeated airpuff consisting of three blocks of 5 x 1 sec, 30 sec apart; 5, 15, and 60 min after termination of the stressor further blood samples were taken.
Exposure to airpuff and effects of icv infusion of OTA on ACTH, corticosterone, and oxytocin secretion in parturient rats.
Four to 5 d after surgery (and on the same days as the experiments with virgin and pregnant rats), parturient rats were also randomly divided into the following four groups: 1) vehicle, no stress (ACTH: n = 9; corticosterone/oxytocin: n = 6); 2) vehicle, airpuff exposure (ACTH: n = 12; corticosterone/oxytocin: n = 7); 3) OTA, no stress (ACTH/corticosterone/oxytocin: n = 9); and 4) OTA, airpuff exposure (ACTH/corticosterone/oxytocin: n = 7).
At 0800 h on d 22 and/or 23 of pregnancy, the jugular vein catheter was attached to an extension tubing connected to a 1-ml syringe, and the icv infusion cannula filled with either vehicle or OTA was lowered into the icv guide cannula as described above. Animals were carefully observed for impending birth. After delivery of the first pup, the first and after delivery of pup 2, the second blood samples were taken (pre1 and pre2, respectively). Five minutes after the second blood sample, either vehicle or OTA was injected icv, and 10 min later, rats were exposed to airpuff (see above). Further blood samples were taken 5, 15, and 60 min after termination of airpuff exposure according to the protocol. The delivery of the pups and behavior of the rats were monitored by an observer blind to the treatment. Freezing behavior in response to the stressor was noted. On the next day, the number of live pups with milk in the stomach and in the nest was counted for each litter.
Treatment of blood samples and RIAs for ACTH, corticosterone, and oxytocin
All blood samples were collected on ice in EDTA-coated tubes containing 10 µl aprotinin (Trasylol, Bayer Corp. AG, Leverkusen, Germany) and centrifuged at 4 C (4000 rpm, 5 min). Eighty microliters (ACTH), 30 µl (corticosterone), and 160 µl (oxytocin) plasma samples were stored at -20 C until assay. Oxytocin concentrations in plasma were estimated in extracted samples by a sensitive and specific RIA (detection limit: 0.3 pg/sample; cross-reactivity of the antiserum with related peptides, including vasopressin, was <0.7%) (37).
Plasma ACTH and corticosterone were measured using commercially available kits (ICN Biomedicals, Inc., Costa Mesa, CA) according to the respective protocols. Detection limits for ACTH and corticosterone were 4 pg/ml and 10 ng/ml, respectively.
Statistical analysis
Data are expressed as the means ± SEM. Statistical analysis was performed using a computer software package (GB-Stat 6.0, Dynamic Microsystems, Silver Spring, MD). Plasma values of ACTH, corticosterone, and oxytocin were analyzed using a two-way ANOVA (factors treatment x time, factors reproductive state x time) with repeated measures on the last factor followed by Newman-Keuls post hoc test. Basal plasma concentrations and plasma increments (delta) of ACTH, corticosterone, and oxytocin after antagonist treatment and stressor exposure, in virgin and parturient rats, were analyzed using a one-way or two-way (factors treatment x reproductive state) ANOVA. P less than 0.05 was considered statistically significant.
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).
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). In detail, in vehicle-treated virgin rats, exposure to airpuff significantly increased ACTH secretion, compared with basal (P < 0.01) and nonstressed virgins (P < 0.05). Compared with vehicle treatment, administration of the OTA significantly increased ACTH secretion in nonstressed virgins (P < 0.01), i.e. under basal conditions, and further elevated the stress-induced rise in ACTH secretion (P < 0.05 vs. vehicle).
In pregnant rats, ACTH plasma concentrations did not significantly change in response to airpuff exposure and/or icv OTA treatment (F4,40 0.59, P = 0.67; Fig. 1C).
Similarly, during parturition, plasma ACTH did not significantly change in response to airpuff exposure and/or antagonist treatment (F4,132 0.93, P = 0.51, Fig. 1D).
The stress-induced rise in ACTH secretion as reflected by respective delta values differed significantly between vehicle-treated virgin, pregnant, and parturient groups (P = 0.0076) with reduced responses in parturient rats, compared with virgin rats (P < 0.01); ACTH responses of pregnant rats tended to be reduced, compared with virgin (P = 0.06), but tended to be higher, compared with parturient rats (P = 0.082; Fig. 1B).
Effects of OTA treatment on the stress-induced rise in plasma ACTH concentrations were dependent on the reproductive state (P = 0.0028) with increased ACTH levels after OTA treatment only in virgin (P < 0.05) but not pregnant or parturient rats (Fig. 1B).
Corticosterone (Fig. 2).
In contrast to ACTH, plasma corticosterone concentrations before treatment did not significantly differ between virgin (mean of pretreatment samples 1 and 2: 142 ± 12.8 pg/ml) and parturient (172 ± 18.8 pg/ml, F1,74 2.43, P = 0.12) rats. Corticosterone has not been quantified in plasma samples from pregnant rats.
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During parturition, plasma corticosterone did not significantly change either in response to exposure to airpuff in vehicle-treated rats (F4,44 0.89, P < 0.48) or in response to icv OTA under basal conditions (F4,52 0.62, P < 0.65) or after airpuff exposure (F4,48 0.44, P < 0.78, Fig. 2B).
Oxytocin (Fig. 3).
In vehicle-treated virgin rats, exposure to airpuff did not significantly increase oxytocin secretion into blood (delta: 0.27 ± 0.2 pg/ml vs. delta nonstressed control: -0.18 ± 0.12 pg/ml, P = 0.06; Fig. 3A). Administration of the OTA did not change oxytocin secretion into blood either under basal conditions or in response to airpuff exposure (Fig. 3B).
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, A and B). Similarly, exposure to airpuff did not change oxytocin secretion in vehicle-treated (delta: 0.01 ± 0.84 pg/ml, Fig. 3A) or OTA-treated (delta: 0.47 ± 0.55 pg/ml; Fig. 3B) parturient rats (P > 0.05).
Effects of airpuff exposure and OTA on the timing of birth and survival of the pups
Rats delivered between 7 and 15 pups with no significant differences in the number of pups delivered among the treatment groups. There was no significant difference in the cumulative time to deliver 2–10 pups between the vehicle-treated and OTA-treated, unstressed rats (Fig. 4). In contrast, airpuff exposure significantly delayed the birth of pups 2–10 in both vehicle- and OTA-treated rats (P < 0.015).
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Neuroendocrine responses of virgin, pregnant, and parturient rats to airpuff
Exposure to airpuff has recently been established as a moderate psychological stressor and was shown to increase not only the activity of the HPA axis but also the emotional response to a subsequent defensive withdrawal procedure in male rats (38). Comparing their data with those of the virgin female rats in our study, higher ACTH concentrations were found after airpuff exposure in virgin rats, which is consistent with other studies demonstrating a generally higher HPA axis responsiveness in virgin females (18, 39). In contrast to the HPA axis, the oxytocin system did not respond with significantly increased neurohypophysial secretion. Oxytocin is released into blood in response to a variety of relevant stressors, including forced swim in both males (40, 41) and females (2, 4, 8) and maternal defeat in virgin female rats (42). However, exposure to novel environment (42) or social defeat (43) was not found to be sufficient to activate peripheral oxytocin secretion. Thus, exposure to airpuff may be considered as a relatively weak stressor that is not strong enough to consistently activate the oxytocin system.
The advantages of using airpuff exposure as a psychological stressor are that it lacks any physical pain component and avoids physical intervention so it can be applied in the home cage of the experimental rat, thereby excluding confounding effects of additional manipulation like handling procedures that in most experimental models may interfere with the actual exposure to the stressor (44). This is of particular importance during parturition to avoid moving the delivering rats out of their cages and away from their nest and newborn pups. Furthermore, in contrast to exposure to noise stress, control rats may be housed in the same room without additional apparatus (e.g. sound-isolated cages). No significant response of the HPA axis was found in unstressed control rats in this and a recent study (38), even though being indirectly exposed to the sound of released air. However, in our experiment, we avoided placing the cages of stressed and nonstressed rats side by side, whereas remaining in the same room. The tendency of increased hormone concentrations in unstressed animals should therefore be a nonspecific effect of icv treatment and/or blood sampling the more because blood samples were taken with a gap of 3–5 min between each rat. In addition to the characterization of neuroendocrine parameters in response to airpuff exposure, it is also possible to monitor the immediate behavioral reactions of the animals, which mainly comprise short jumps and freezing. Although this gives only a rough indication of the emotional state of the animal, our preliminary observations indicate that OTA treatment enhances the occurrence of freezing behavior during airpuff exposure in parturient rats (data not shown).
In contrast to virgin female rats, exposure to airpuff did not significantly activate the HPA axis in pregnant and parturient rats as reflected by unchanged ACTH and corticosterone plasma concentrations. We have recently shown that the HPA axis is unresponsive to parturition-related stimuli, i.e. in undisturbed, basal conditions, plasma ACTH and corticosterone concentrations remain low or even fall with the progression of the parturition process (11). Here we extend these findings of the unresponsive HPA axis during parturition by the demonstration that external, stressful stimuli are also unable to measurably increase ACTH and corticosterone secretion into the blood in the rat. The attenuated responsiveness of the HPA axis in the peripartum period extensively studied in pregnant and lactating rats is due to a variety of inhibitory adaptations at different HPA axis levels including, for example, reduced neuronal responses to stressor exposure in limbic brain regions and altered threshold of perception of the stressor (5), reduced basal and stress-induced CRH synthesis within the paraventricular nucleus (10, 45), reduced CRH binding at adenohypophysial corticotroph cells, and enhanced negative feedback mechanisms (4, 10). Furthermore, endogenous opioids have recently been shown to strongly attenuate the basal activity of the HPA axis in parturition (11).
In addition, in pregnant and parturient women CRH and CRH-binding protein synthesized and secreted by the placenta should play a significant role in regulating ACTH and cortisol plasma concentrations. In contrast, in the rat, an impact of CRH-binding protein on HPA axis functions is rather unlikely because it is synthesized only in the anterior pituitary and the brain with unchanged synthesis in pregnancy (Ma, S., and J. A. Russell, unpublished observations). Additionally, activation of neuropeptidergic systems such as oxytocin and prolactin, which are necessary for reproductive processes (labor, lactogenesis, maternal behavior), may be involved in the inhibition of ACTH and corticosterone secretory responses, as recently shown in male and virgin female rats (17, 18, 46). However, in pregnant and lactating rats, the presumed inhibitory action of brain oxytocin on HPA axis stress responses could not be reversed by icv application of an OTA; in lactating rats this did not depend on the presence (42) or absence (19) of the pups.
Involvement of brain oxytocin
During parturition and immediately thereafter, it has been extensively shown that the brain oxytocin system is highly activated as indicated by increased neuronal activity (25, 33, 47, 48) and increased release in brain regions in which it is relevant not only for the induction of maternal behavior (30, 31, 49) but also for the regulation of the HPA axis (18, 32). Our hypothesis of an inhibitory effect of brain oxytocin on HPA axis activity in parturition was further suggested by the findings of reduced HPA axis responses after chronic icv administration of oxytocin into virgin female rats (17). The finding of a disinhibition of basal and stress-induced HPA axis responses after icv administration of the OTA in cycling virgin female and male rats (18) was confirmed and extended. Thus, increased basal or stressor-induced secretion of ACTH and corticosterone after antagonist treatment reflects an inhibitory action of endogenous oxytocin on both basal and stress-induced HPA axis activity. This together with our recent results (18, 42) indicates that the inhibitory effects of brain oxytocin on the activity of the HPA axis in virgin female rats is independent of the kind of stressor to which the females are exposed. Stressors that have been tested in this context include exposure to a novel environment (18, 19), swimming (18), maternal defeat (42), and repeated airpuff (this study).
In contrast, during parturition blockade of brain oxytocin receptors by icv administration of OTA before airpuff exposure did not elevate ACTH or corticosterone secretion despite enhanced intracerebral release of oxytocin at this time, compared with late pregnancy (32). Similarly, under stress-free conditions, basal HPA axis activity was not increased after antagonist treatment in parturient rats. From these results we have to conclude that brain oxytocin is not significantly involved in the hyporesponsiveness of the HPA axis in the parturient rat. However, we cannot exclude that the sum of inhibitory adaptations of the HPA axis in the peripartum period mentioned above prevent, a priori, the possibility of a drug-induced disinhibition, because many other factors including prolactin and/or endogenous opioids may still act in an inhibitory, concerted manner (8, 46). Also, variations in plasma estrogen/progesterone seen peripartum may add to these neuroendocrine alterations.
Responses of the oxytocin system to airpuff and OTA in parturient rats
In the present study, the general activity of the oxytocin system of parturient rats was highly activated, reflected in the higher oxytocin plasma levels, compared with virgin rats, as might be expected (50). However, exposure to the airpuff did not further elevate oxytocin secretion into blood (as partly seen in virgins), indicating that there is a strong stimulus-dependent activation of oxytocin neurones rather than a general lowering of threshold for the responsiveness of the oxytocin system in parturition. Because the average plasma oxytocin concentration did not change but births were delayed (Fig. 4), it is likely that stress exposure inhibited the transient pulsatile secretion of oxytocin. With the timed sampling protocol used we could not directly monitor the short-lived peaks of oxytocin in blood (half-life = 1.5–2 min) but the absence of which would be detrimental to the progress of parturition (48). In this context it is of interest to note that disruption of the parturition process by placing the dam into a new cage also delays birth as a result of inhibition of peripheral oxytocin secretion by endogenous opioids (51).
With respect to an effect of brain oxytocin on neurohypophysial secretion in parturition, OTA treatment did not alter oxytocin plasma concentrations under basal or stress conditions. Therefore, we have to conclude that brain oxytocin is not obviously involved in the regulation of basal or stress-induced oxytocin secretion during the delivery process. Again, we have to consider effects of brain oxytocin on the fine-tuned pulsatile hormone secretion not detectable by the protocol used. In this context it is of interest to note that, within the SON, autoexcitatory regulation of oxytocin neurons exists during undisturbed parturition as demonstrated by direct administration of OTA into the nucleus (34). Similarly, during suckling, local autoexcitation of the oxytocin system has been described to regulate the pulsatile release of oxytocin into blood (52, 53, 54, 55). In contrast, under stress conditions, an autoinhibition of brain oxytocin on oxytocin secretion into blood has recently been reported in pregnant and nonsuckled lactating, but not virgin, rats (19, 42). Thus, the autoregulatory capacity of the brain oxytocin system seems to be strongly dependent on the reproductive state and experimental conditions.
Neither exposure to airpuff in the home cage nor acute blockade of oxytocin receptors obviously impaired the maternal behavior of the dam or onset of lactation/suckling as indicated by a similar number of alive pups with milk in their stomach and in the nest in all groups tested 24 h after birth. However, more detailed behavioral tests are needed to reveal any effects of acute psychological stressors and the region-dependent involvement of brain oxytocin in the onset of maternal behavior.
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Acknowledgments |
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Footnotes |
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Abbreviations: HPA, Hypothalamo-pituitary-adrenal; icv, intracerebroventricular; OTA, oxytocin receptor antagonist; SON, supraoptic nucleus.
Received January 8, 2003.
Accepted for publication March 3, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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