A number of changes occur in the oxytocin (OT) system during gestation, such as increases in hypothalamic OT mRNA, increased neural lobe and systemic OT, and morphological and electrophysiological changes in OT-containing magnocellular neurons, suggestive of altered neuronal sensitivity, which may be mediated by ovarian steroids. Because central norepinephrine (NE) and histamine (HA) are potent stimulators of OT release during parturition and lactation, the present study investigated the effects of central noradrenergic and histaminergic receptor activation on systemic (NE, HA) and intranuclear (NE) OT release in pregnant rats and in ovariectomized rats treated with ovarian steroids. Plasma OT levels in late gestation were significantly higher compared with all other groups, and neither adrenergic nor histaminergic receptor blockade decreased these elevated levels. Furthermore, the α-adrenergic agonist phenylephrine, but not histamine, stimulated systemic OT release to a significantly greater extent in late gestation than in midpregnant, ovariectomized, or steroid-treated females. Although basal extracellular OT levels in the paraventricular nucleus, as measured with microdialysis, were unchanged during pregnancy or steroid treatment, noradrenergic receptor stimulation of intranuclear OT release was significantly elevated in midgestation females compared with all other groups. These studies indicate that sensitivity of intranuclear and systemic OT release to noradrenergic receptor activation differentially varies during the course of gestation.
previous studies have shown that central and systemic oxytocin (OT) release is important during parturition and lactation. OT is synthesized in hypothalamic magnocellular paraventricular (PVN) and supraoptic (SON) nuclei and is released via the neurohypophysis into circulation in response to uterine contractions and mammary gland stimulation (8). OT is also released within the PVN and SON (intranuclear release) (28), which is thought to be critical for initiating and coordinating the firing activities of the magnocellular OT neurons responsible for systemic OT release during parturition and lactation (23, 27).
A number of changes in the OT system have been documented during late gestation: central OT mRNA is increased (7, 26, 33, 39, 41), morphological rearrangements of magnocellular nuclei facilitate increased connectivity of soma and dendrites in OT neurons (37), and electrophysiological changes in OT neuronal firing patterns (18) and in active membrane properties (35) are associated with increases in spontaneous bursting. As a likely consequence of these changes, OT concentrations in the neural lobe (13, 22) and systemic OT release (22) are increased during late gestation, consistent with the increased sensitivity to excitatory stimuli and neurotransmission. However, responses to excitatory neurotransmitters during late gestation have not been previously tested.
A large body of work has shown that norepinephrine (NE) plays an important role in the regulation of the central OT system during parturition and lactation. Notably, adrenergic afferents from the nucleus of the solitary tract to magnocellular nuclei (30) are activated by signals from the uterus via spinal pathways, causing OT release during labor and delivery (2, 12, 25). Central NE is increased during parturition (16) and suckling (3, 9), and stimulation of α-adrenergic receptors increases systemic and intranuclear OT release during lactation in suckled and nonsuckled rats (3, 9, 29, 38).
Histamine (HA) has also been shown to be important in the central control of OT during parturition and lactation. Histaminergic neurons make synaptic contact with OT neurons (40), and central administration of HA increases systemic OT release (4, 19), OT mRNA levels (20), and c-fos expression in OT containing neurons (21). Blockade of central H1 receptors delays delivery of rat pups during parturition (26). Suckling enhances neuronal HA synthesis, which in turn regulates systemic and intranuclear (PVN) release of OT via postsynaptic H1 and H2 receptors in the hypothalamus (5, 31).
Because of anatomic and electrophysiological changes in OT neurons suggesting increased neuronal sensitivity during late gestation and the documented importance of NE and HA in OT release, our first goal was to determine whether intranuclear or systemic OT responses are altered during the course of gestation.
Ovarian steroids have been implicated in changes in the OT system characteristic of late gestation. Specifically, estradiol and progesterone treatment followed by progesterone withdrawal in ovariectomized (OVX) females has been suggested to be a model for the changing hormonal milieu of late gestation (6) and is associated with increased OT mRNA (7), whereas maintenance of elevated progesterone in late pregnant or in estrogen- and progesterone-primed OVX females blocks the OT mRNA increase (7). Therefore, it is possible that this ovarian steroid milieu may contribute to alterations in the OT system, which could lead to elevations in systemic OT during late gestation. However, the role of ovarian steroids in altered OT release is unknown. Thus the second goal of this research was to compare the responsiveness of intranuclear and systemic OT in OVX rats treated with ovarian steroids with responses observed in rats during gestation.
MATERIALS AND METHODS
Timed-pregnant or virgin female Sprague-Dawley rats were obtained from a commercial supplier (Charles River Laboratories, Wilmington, MA). The animals were singly housed on a 12:12-h light-dark cycle with ad libitum access to food and water. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Utah. The animals used in these series of experiments included OVX virgin (OVX), midpregnant (MIDP, days 12–14), and late pregnant (LATEP, days 18–20) females. Each animal was used in one experiment only.
Virgin female rats were bilaterally ovariectomized under anesthesia with methohexital sodium (Brevital, 60 mg/kg) or 2,2,2-tribromoethanol (Avertin, 300 mg/kg). A midlateral incision was made into the body cavity, and the ovaries were removed. The body wall was sutured, and the external incision was closed.
The hormone replacement regimen used in these studies has been used extensively to mimic the late gestational elevated estradiol and progesterone withdrawal in rats (6, 7) and is associated with increased magnocellular OT mRNA levels (7). Hormone-treated females were ovariectomized a minimum of 2 days before implantation of estradiol capsules. After anesthesia, Silastic tubing (ID 1.98 mm, OD 3.175 mm) filled with estradiol (0.25 mg/ml in sesame oil) and progesterone crystals (powder) was implanted subcutaneously in the back. OVX females were implanted with estradiol capsules (30 mm) on day 1, followed by progesterone capsules (40 mm) on day 3. On day 14, progesterone implants were removed and animals were tested two days later (E+P−). In another group, progesterone implants were not removed (E+P+). Non-hormone-treated OVX females were implanted with empty capsules that were inserted and removed in identical fashion to the hormone-treated females.
Animals were implanted with a guide cannula in the brain on the day before testing in two different protocols. Animals were anesthetized and placed in a stereotaxic instrument. The scalp was incised and retracted laterally, and a 2-mm hole was drilled above the target brain site.
Acute intracerebroventricular injections.
A guide cannula (22-gauge, stainless steel tubing) was implanted in the third ventricle 0.4 mm posterior to bregma along the midline and 7.5 mm below the surface of the skull. The cannula was secured with small screws placed in the skull and dental acrylic. The animals were returned to their individual cages where they remained until testing.
Surgery and recovery were performed in identical fashion to that described above. A guide cannula (20-gauge, stainless steel tubing) was implanted immediately dorsolateral to the PVN at coordinates 1.9 mm posterior to bregma, 0.6 mm lateral from the midline, and 7.5 mm beneath the surface of the skull.
On the day before an experiment, females were anesthetized and implanted with polyethylene catheters (PE-50 anchored to PE-10 tubing, filled with 50 U/ml heparin) in a femoral vein and artery. The catheters were led subcutaneously up the back, exteriorized, and secured at the level of the scapulae.
Microdialysis Probe Construction
A concentric style microdialysis probe (32) was constructed by adhering stainless steel tubing (28 mm, 25-gauge) to PE-20 polyethylene tubing (0.3 m). Hollow silica tubing (OD 153 μm, ID 76 μm; Polymicro Technologies, Phoenix, AZ) was inserted into the stainless steel tubing and polyethylene tubing, exiting near the distal end through a small hole that was then glued. At the proximal end of the probe, a 4-mm dialysis membrane with one end occluded was inserted over the silica tubing protruding from the stainless steel tubing and was sealed to it. The dialysis membrane was Qupra-ammonium-rayon with a molecular mass cut-off of 40,000 Da and a diameter of 250 μm (Asahi Medical, Tokyo, Japan). The dimensions of the membrane exchange area at the tip of the microdialysis probe were 2.0 × 0.3 mm (length × diameter).
Neurotransmitter release of systemic OT.
before each experiment, females were weighed and placed in a test cage. All animals were allowed to equilibrate for ∼30 min and were conscious and unrestrained during the experiment. A back-filled microsyringe was connected via polyethylene tubing to a stainless steel injector (30-gauge) that was inserted into the third ventricle through the guide cannula. Initially, two baseline blood samples (200–250 μl) were collected from the femoral artery, 5 min apart. Then, a 10-μl bolus of phenylephrine, phentolamine, propranolol, histamine, chlorpheniramine and ranitidine (see Table 1), or vehicle [artificial cerebrospinal fluid (aCSF)] was injected over ∼10–15 s into the third ventricle. Blood samples were collected 1, 5, 10, 20, and 30 min after the injection. After each collection, blood was centrifuged and the plasma placed in chilled plastic tubes and subsequently stored at −80°C until assayed. Red blood cells were resuspended in saline and returned to the animal through the femoral vein catheter.
Neurotransmitter release of intranuclear OT.
All animals were conscious and unrestrained during the experiment. At the start of the experiment, the microdialysis probe was inserted into the guide cannula so that the probe tip was adjacent to the PVN at its lateral extent. The PE-20 tubing (input) was connected to a 500-μl syringe containing aCSF and bacitracin (20 μM), placed in a syringe pump. The exposed end of the silica tubing (output) was inserted into a chilled Eppendorf centrifuge tube. The infusion rate was 1 μl/min throughout the protocol. Initially, the animals were allowed to equilibrate during perfusion of aCSF for 45 min; this dialysate was discarded. After equilibration, aCSF was continued, and a 75-min control sample (CONT) was collected. The dialysis fluid was then replaced with aCSF containing phenylephrine (25 mg/ml), and two 75-min samples were collected (EXP1 and EXP2). Finally, probes were perfused with aCSF, and a 75-min recovery (REC) sample was collected.
At the termination of each experiment, animals were anesthetized with pentobarbital soidum (Nembutal, 60 mg/kg) or Avertin and perfused transcardially with 0.9% saline. The brain was removed and placed in formalin (4%) containing sucrose (30%). Brains were blocked, frozen, sectioned (40 μm), and stained with cresyl violet. The sections were examined under a light microscope to determine placement of the cannula.
OT concentrations were determined in plasma and dialysate samples (0.05 ml) with a radioimmunoassay as previously described (24), using an OT antibody from Biogenics (Brentwood, NH). This antibody cross-reacts 0.0001% with vasopressin and has a detection limit of 0.7 pg.
To evaluate differences in basal OT concentrations, a single-factor ANOVA was carried out, and individual means were compared using a Newman-Keuls a posteriori test (P < 0.05). Plasma and dialysate OT concentrations at basal levels and following adrenergic or HA receptor agonist or antagonist or vehicle (aCSF) administration were statistically evaluated using single- or two-factor ANOVAs for repeated measures. Significant differences in individual means were determined using a Newman-Keuls a posteriori test (P < 0.05). All data are expressed as means ± SE, and significance was established at P < 0.05.
Estradiol, progesterone, phenylephrine, phentolamine, propranolol, histamine, chlorpheniramine, and ranitidine were purchased from Sigma Chemical (St. Louis, MO).
Because there were no differences in systemic or intranuclear OT concentrations between OVX females and OVX females receiving empty capsules, the data were pooled within each experiment.
Figure 1 shows basal OT levels among all OVX, MIDP, LATEP, E+P−, and E+P+ females. OT levels were significantly elevated in LATEP females compared with all other groups. As shown in Fig. 2A, phenylephrine treatment caused a significant increase in plasma OT concentrations in all groups 5 and 10 min after treatment compared with preinjection. However, comparisons among the groups indicated that at 5 min postinjection, LATEP OT levels were significantly greater compared with all other conditions. OT concentrations returned to basal levels 20 min postinjection in all groups. Vehicle injections had no effect on plasma OT concentrations in OVX, MIDP, or LATEP females (data not shown).
To control for differences in basal plasma OT levels among groups, we evaluated changes in plasma OT after phenylephrine treatment by subtracting basal OT from postinjection OT concentrations. The increase in OT levels 5 min after injection was significantly greater in LATEP compared with OVX, MIDP, E+P−, and E+P+ females, as shown in Fig. 2B. Therefore, after phenylephrine administration, both the absolute OT concentration and the increase in plasma OT above control levels were greater in LATEP compared with all other experimental groups.
Figure 3 shows OT concentrations in dialysates collected before (CONT), during (EXP1 and EXP2), and after (REC) phenylephrine perfusion in microdialysis probes placed adjacent to the PVN in MIDP, LATEP, OVX, and E+P− females. There were no differences in basal PVN OT levels among groups. Phenylephrine significantly increased dialysate OT concentrations during EXP1 and EXP2 compared with CONT in all groups. Dialysate OT concentrations declined to basal levels during REC in all but the MIDP group, in which OT levels remained significantly elevated. Furthermore, between-group analysis demonstrated that the phenylephrine-induced increase in dialysate OT was significantly greater in MIDP compared with all other groups in the EXP2 period and that this difference remained during the REC period.
Figure 4A shows the effects of acute intracerebroventricular HA injections on plasma OT concentrations in MIDP, LATEP, OVX, and E+P− females. OT release was significantly increased 1 min after HA treatment but returned to control values in all animals within 10 min. Subsequent evaluation of changes in systemic OT after HA treatment by subtraction of basal OT from postinjection OT concentrations demonstrated equivalent increases across groups (Fig. 4B).
Intracerebroventricular administration of the α-adrenergic receptor antagonist phentolamine (MIDP, LATEP, and OVX), the β-adrenergic receptor antagonist propranolol (LATEP), and coadministration of HA H1 and H2 receptor antagonists chlorpheniramine and ranitidine (LATEP) did not alter basal plasma OT concentrations (data not shown).
This study evaluated the influences of central neurotransmitter receptor activation on peripheral and central OT release during gestation and ovarian steroid treatment in rats. Basal systemic OT concentrations were elevated in LATEP females. Furthermore, acute intracerebroventricular injections of phenylephrine, but not HA, caused a significantly greater increase in systemic OT release in LATEP compared with MIDP, OVX, and steroid-treated females. However, intracerebroventricular administration of NE or HA receptor antagonists did not lower plasma OT concentrations of LATEP females. Finally, although basal PVN OT concentrations were of similar magnitude in all groups, phenylephrine stimulation caused a significantly greater increase in dialysate OT concentrations in MIDP females. Taken together, these findings suggest that adrenergic sensitivity of both intranuclear and systemic OT release varies during the course of pregnancy in a differential manner.
The significantly greater increase in systemic OT release after central NE stimulation in LATEP rats, compared with MIDP, OVX, and nonpregnant, hormone-treated rats, suggests that OT neurons are highly sensitized to adrenergic input by late gestation. Because the NE system is involved in OT release during parturition and lactation (2, 3, 9, 12, 16, 25, 29, 38), it is likely that increased sensitivity of the OT system to noradrenergic receptor stimulation during LATEP will have important implications for peri- and postpartum events. Although it is not certain what causes the increased sensitivity, this may be related to alterations in electrophysiological (18, 35) and/or morphological (37) characteristics of OT magnocellular neurons present during LATEP.
The lack of increase in basal PVN OT concentrations during gestation in this study confirms the findings of other researchers (10, 28), However, we found that central α-adrenergic receptor stimulation produces a significantly greater increase in intranuclear OT release in MIDP rats compared with OVX, LATEP, and steroid-treated animals. These results are the first to identify alterations in the central OT system at MIDP. However, the functional significance and the mechanism(s) of this enhanced sensitivity of central OT to α-adrenoreceptor stimulation are currently unknown. Functionally, it is possible that activation of noradrenergic pathways during this stage of gestation increases central OT, which contributes to the anatomic (36, 37), genetic (17, 34), and/or electrophysiological (35) changes in the OT system present in LATEP, and is necessary for normal OT responses during lactation (24). This interpretation is consistent with the significant increase in magnocellular NE observed during MIDP (day 15) (14). Although the stimulus for activation of central noradrenergic pathways during MIDP has not been identified, it may originate from uterine activity, as this stimulatory pathway has been demonstrated in later stages of gestation (12).
Although intranuclear OT release to central α-adrenergic stimulation was significantly enhanced during MIDP, the systemic OT response was not altered. The mechanism of the dissociation between central and systemic OT release during α-adrenergic stimulation has not been elucidated. It is possible that adaptations in the OT system are not sufficiently developed by MIDP to tightly couple the central and systemic OT responses. Alternatively, inhibitory systems, such as opioids, which inhibit systemic OT release during MIDP (11), may restrain the systemic response to increased intranuclear OT during this stage of gestation. The precise mechanism mediating the dissociation between central and systemic OT during MIDP remains to be investigated.
A dissociation between central and systemic OT concentrations was also present in LATEP animals. However, at this stage of gestation, both basal and stimulated systemic OT secretion following central α-adrenergic stimulation were enhanced, whereas control and stimulated intranuclear OT levels were similar among groups. The mechanisms of increased basal plasma OT concentration and the dissociation of central and systemic OT during LATEP are not known. Data from the present studies demonstrate that increased basal OT concentration is not due to enhanced noradrenergic or histaminergic tone. It is possible that other excitatory neurotransmitter systems that stimulate systemic OT release without changing intranuclear concentrations, such as glutamate (15), are activated during LATEP. Another potential explanation is that the progressive increase in systemic OT concentration, without a concomitant change in intranuclear OT release, results from the fall in opioid inhibition of OT nerve terminals in the neural lobe occurring between mid- and late gestation (11). Finally, it is possible that the gains relating stimulation of central noradrenergic and/or OT receptors and systemic OT secretion are increased in LATEP by some other mechanism, such as anatomic (37) or electrophysiological (18, 35) adaptations known to be present in late gestation. However, the precise mechanism(s) mediating the dissociation between central and systemic OT during LATEP remains to be investigated.
The possibility that ovarian hormones are responsible for enhancing basal systemic OT release, as well as increasing the sensitivity of the OT system to neurotransmitter receptor activation, was not supported by results of the present study. Although elevated estradiol titers and progesterone withdrawal (E+P−), a characteristic of late gestation (6), have been implicated in increases in central OT mRNA in ovariectomized hormone-treated E+P− females (7), plasma OT concentrations were not altered in the Amico et al. (1) or the present studies. Likewise, in E+P− females in the present study, systemic OT release to NE stimulation was significantly smaller than that observed in LATEP and was equivalent to that in MIDP rats. Thus the E+P− model does not precisely mimic changes in the OT system in LATEP.
In the present study, we have assessed the roles of central neurotransmitter receptor activation in the control of intranuclear and systemic OT release during gestation. Our finding of elevated basal systemic OT concentrations during late gestation (days 18–20) supports the finding established previously (22). The present experiments extend these earlier studies by demonstrating that release of intranuclear OT to adrenergic stimulation is enhanced in MIDP compared with LATEP, whereas release of systemic OT to central NE receptor activation is increased in LATEP compared with MIDP. Therefore, sensitivity of intranuclear and systemic OT release to NE stimulation changes across gestation, in a dissimilar manner, representing a qualitatively opposite dissociation between central and systemic OT concentrations during MIDP and LATEP. However, despite the increase in sensitivity, NE receptor stimulation is not involved in maintaining elevated basal systemic OT levels evident during LATEP. In addition, the E+P− condition did not exhibit increased basal OT levels and/or NE-induced systemic OT release characteristic of LATEP rats. Therefore, this hormone treatment does not appear to mimic LATEP in these aspects of systemic OT responses. Further investigation is required to determine the functional significance and mechanisms responsible for the sequential alterations of the OT system during gestation in rats.
These studies were supported by National Institute of Child Health and Human Development Grants HD-32516 (S. L. Bealer) and HD-20074 (W. R. Crowley).
We acknowledge the expert technical assistance of Gina Ramoz, Kelsey Flake, Trevor Grubbs, Cameron Metcalf, and Natalie Knotts.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 by American Physiological Society