We hypothesized that neural structures, involved in sensing extracellular body fluid composition in adult animals during an osmotic challenge, would show similar patterns of activation in fetal sheep. Eight adult sheep [4 hypertonic saline-treated adults (HYP-A), 4 isotonic saline-treated adults] and six near-term fetal sheep [3 hypertonic saline-treated fetuses (HYP-F), 3 isotonic saline-treated fetuses; 130 days gestation] were prepared with vascular and intraperitoneal catheters. Seventy-five minutes before tissue collection, hypertonic (1.5 M) or isotonic saline was infused via an intraperitoneal catheter to adult (18 ml/kg) or fetal sheep (6 ml/kg). Brains were examined for patterns of neuronal activation (demonstrated by Fos protein expression). HYP-A and HYP-F demonstrated similar acute increases in plasma osmolality (∼10 mosmol/kgH2O) and comparable patterns of Fos expression within the organum vasculosum of the lamina terminalis (HYP-A, 67 ± 2 vs. HYP-F, 63 ± 6; means ± SE) and hypothalamic supraoptic (SON; HYP-A, 107 ± 8 vs. HYP-F, 102 ± 7) and paraventricular nuclei (PVN; HYP-A, 71 ± 18 vs. HYP-F, 124 ± 19). Fewer activated neurons were detected in HYP-A vs. HYP-F within the subfornical organ (HYP-A, 33 ± 8 vs. HYP-F, 91 ± 17) and median preoptic nucleus (HYP-A, 33 ± 5 vs. HYP-F, 70 ± 6). In adults and fetuses, counterstaining for arginine vasopressin revealed that neurons within the SON and PVN respond to osmotic challenge. These findings demonstrate that central osmoregulatory centers in adult and near-term fetal sheep are similarly activated by osmotic challenge.
- circumventricular organs
- arginine vasopressin secretion
thirst-mediated drinking behavior, in concert with arginine vasopressin (AVP)-mediated antidiuresis, are the fundamental biological systems that maintain body water volume and tonicity homeostasis. Systemically, there are two principal mechanisms for body water regulation in the adult mammal. Small increases in extracellular fluid osmolality (∼2%) stimulate AVP secretion (and urinary antidiuresis) and thirst. Somewhat larger decreases (∼10%) in circulating volume, sensed by arterial and low-pressure baroreceptors, initiate AVP secretion and thirst via stimulation of renin release and direct activation of central neural thirst systems. Studies in adult animals indicate that putative dipsogens [hypertonicity, angiotensin II (ANG II)] initiate responses via stimulation of specialized cells within neural structures lacking a blood-brain barrier, such as the anterior circumventricular organs [CVOs, i.e., organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO)], or at the level of the median preoptic nucleus (MnPO), lying within the brain barrier.
We previously demonstrated a functional systemic dipsogenic response in the near-term ovine fetus (129–133 days gestation; full term = 145–150 days), with swallowing activity stimulated in response to intravenous or intracarotid hypertonic saline infusion (29, 30). Although the near-term fetal osmotic dipsogenic response is intact, there are distinct differences from that of the adult. Whereas a 2–3% increase in plasma osmolality stimulates adult water intake (5), only a brief (5 min) stimulation of fetal swallowing activity occurs in response to acute and chronic increases in plasma osmolality above this threshold level. Similarly, in response to maternal water deprivation, chronic increases in fetal plasma osmolality (3–4% above basal) do not stimulate fetal swallowing activity (31). Thus, in contrast to adults, stimulation of fetal swallowing activity may require greater increases in osmolality. Consistent with this hypothesis, we recently calculated a threshold for fetal osmotic dipsogenic stimulation that was markedly greater than the 2–3% increase (24). Alternatively, the fetal dipsogenic response may occur acutely in response to traditional (2–3%) increases in plasma osmolality, with an enhanced regulatory mechanism capable of suppressing subsequent swallowing activity.
Immunocytochemical localization of immediate-early gene products, such as Fos, provides a powerful tool with which to demonstrate activation of neuronal populations in response to a wide variety of stimuli. Numerous studies in adult animals (e.g., rodent, sheep, vole, chicken, and nonhuman primate) have established the utility of using immunocytochemical staining to anatomically identify populations of activated neurons at the cellular level (13-15, 35), with fewer studies having been conducted in fetal or neonatal brain tissue. In adults, Fos expression has been demonstrated to increase within distinct subsets of hypothalamic neurons along the lamina terminalis in response to systemic hypertonicity (6, 8, 26) or chronic hypertonic saline loading (34). Specifically, systemic hypertonicity in rats results in a concentration-related increase in Fos expression in the OVLT, SFO, MnPO, and supraoptic (SON) and paraventricular (PVN) nuclei (26). Moreover, systemic infusion of dipsogenic ANG II has been shown to increase Fos expression in neurons throughout the lamina terminalis as well as neurosecretory sites in the hypothalamus (8, 23, 25). These findings, along with other studies, suggest that at least in the adult mammal, the CVOs, MnPO, and AVP-containing hypothalamic neurosecretory neurons are activated directly or indirectly by systemic hypertonicity and are involved in osmoregulation.
In light of the fundamental importance of fetal dipsogenic and AVP secretory maturation in utero, we sought to determine fetal neural involvement in response to an osmotic stimulus. We hypothesized that central neural structures, involved in sensing acute changes in extracellular body fluid composition in adult animals during an osmotic challenge, would show similar patterns of activation in fetal sheep. In the present study, we examined neuronal activation, as demonstrated by Fos protein expression of the immediate-early gene, c-fos, within select hypothalamic CVOs and neurosecretory regions in fetal sheep after an intraperitoneal hypertonic saline infusion, and we compared fetal with adult patterns of neuronal activation in response to a comparable osmotic challenge.
MATERIALS AND METHODS
Eight Western mixed-breed nonpregnant adult ewes [hypertonic saline-treated adults (HYP-A), n = 4; isotonic saline-treated adults (ISO-A),n = 4] and six pregnant ewes and their near-term fetuses [hypertonic saline-treated fetuses (HYP-F), n = 3; isotonic saline-treated fetuses (ISO-F), n = 3; 130 days of gestational age = 0.87 − 0.90 of gestation] were used in this study. These experiments were conducted within National Institutes of Health guidelines for animal research and were approved by the Harbor-University of California Los Angeles Animal Care Committee. Animals were housed indoors in individual steel study cages and were acclimated to a 12:12-h light-dark cycle. Water was available ad libitum, and food (alfalfa pellets) was provided on a twice-daily schedule, except during the 24-h period immediately preceding surgery.
For the surgical procedures, atropine sulfate (0.05 mg/kg im) was administered before induction of anesthesia with ketamine hydrochloride (20 mg/kg im). After endotracheal intubation, anesthesia was maintained with isoflurane (1–2%) and oxygen (1–2 l/min) through a mask. Polyethylene catheters (ID 1.0 mm, OD 1.8 mm) were placed in the fetal dorsal hindlimb vein and artery and were threaded to the inferior vena cava and abdominal aorta, respectively. The maternal femoral vein and artery were catheterized similarly (ID 1.3 mm, OD 2.3 mm) at the time the fetal catheters were implanted. Catheters were inserted in the adult nonpregnant ewe femoral artery and vein, and all experimental animals were fitted with indwelling intraperitoneal catheters (7 Fr). All fetal and maternal incisions were closed in layers, and catheters were accessed externally via the ewe flank incision.
Immediately preoperatively and twice daily during the initial 2 days of recovery, gentamicin (8 mg/kg) and oxacillin (33 mg/kg) were administered to the fetus, and gentamicin (72 mg/kg), oxacillin (1 g/kg), and chloramphenicol (1 g/kg) were administered to the ewe. Methods for maintenance of catheters and protocols employed for the sampling of blood have been detailed previously (30). All animals were allowed a minimum of 5 days for postoperative recovery before initiation of experiments.
Experimental Protocol: Osmotic Challenge
Adult nonpregnant ewes. The intraperitoneal hypertonic infusion delivered to the adult nonpregnant ewe was employed to validate the induction of Fos expression in subsets of hypothalamic neurons involved in osmoregulation. Patterns of neuronal activation were compared between adult and fetal sheep after comparable osmotic stimuli. On the day of study, the sides of the cart were moved inward so that the ewe could sit and stand but not turn around. Food and water were withheld on the morning of the study to avoid feeding- or drinking-induced alterations in the adult ewe’s plasma osmolality. Nonpregnant ewe arterial blood (5 ml) was sampled before the initiation of the study and at 15-min intervals during the study for measurement of hematocrit (Hct), hemoglobin (Hb), pH, , , plasma osmolality, Na, K, Cl, AVP, and ANG II concentrations. All blood samples were replaced with equivalent volumes of physiological saline. Immediately after baseline blood samples were obtained, either hypertonic (1.5 M) or isotonic (0.15 M) saline solution, 18 ml/kg body wt (at a rate of 80 ml/min), was infused through the indwelling intraperitoneal catheter. (The total volume of infusate, ranging from 800 to 1,200 ml, was delivered within 10–12 min.) We projected that this dosage of hypertonic saline would induce a rise in plasma osmolality of ∼10 mosmol/kgH2O, correlating to a 2–3% increase in extracellular fluid osmolality. This level of increase in plasma osmolality was demonstrated to consistently stimulate AVP secretion and thirst as well as to activate neuronal centers involved in osmoregulation in adult mammals. (The initial rise in plasma osmolality was verified at minute 15 after the completion of the infusion and was evaluated at 15-min intervals throughout the study.)
Near-term fetal sheep. Fetal sheep were studied with their unanesthetized maternal ewes. Experiments were undertaken only if the fetal arterial blood pH value was >7.3. On the day of study, either hypertonic (1.5 M) or isotonic (0.15 M) saline solution, 6 ml/kg of predicted fetal body weight (at a rate of 2 ml/min), was infused through the fetal intraperitoneal catheter (within 10 min). In contrast to the adult ewe, the fetus did not survive an infusion of 18 ml/kg of hypertonic saline. Considering this limitation, it was determined during pilot studies that a 6 ml/kg dose of hypertonic saline could be safely administered to the fetus and that this dose resulted in a rise of fetal plasma osmolality (of ∼10 mosmol/kgH2O). Atminute 15, the increase in fetal plasma osmolality was similar to that observed in the adult ewe (after the 18 ml/kg dose of hypertonic saline) and had been shown to stimulate near-term fetal swallowing (30). Although variant doses of hypertonic saline were used between adult and fetal sheep, the initial rise in the level of hypertonicity was similar between the two experimental groups.
Serial blood samples were withdrawn throughout the study from the fetuses for measurement of blood gases, electrolytes, and hormones, with an equivalent volume of heparinized maternal blood (withdrawn before the study) replaced after each sample. Maternal ewe arterial blood was sampled as described above for the adult ewes.
Adult nonpregnant ewes. Although a time-course study to determine the expression of Fos protein after a strong intraperitoneal osmotic stimulus has not been conducted in adult or fetal sheep, in general, the production of Fos protein reaches a maximum within 60–90 min after stimulation and persists for 2–5 h (34). Thus animals were killed ∼60 min after the osmotic stimulus to provide sufficient time for Fos protein expression. Seventy-five minutes after the initiation of the infusion, animals were anesthetized rapidly with ketamine hydrochloride (20 mg/kg iv) and then were ventilated through a mask with a mixture of isoflurane (2–5%) and oxygen (1–2 l/min). Within 15 min of initial ketamine administration, one common carotid artery was catheterized with a 15-gauge indwelling catheter pointed toward the brain. The nonsurgically catheterized carotid was occluded, and both jugular veins were cut. Within 30 min of the initial anesthesia, the ewe’s head was perfused with 1 liter of normal saline containing heparin (10 IU/ml) and sodium nitrite (2.0% wt/vol), followed by 1 liter of 2.5% acrolein (no. 00016; Polysciences, Warrington, PA) in buffered 4% paraformaldehyde, pH 6.8 (11). Because the appearance of Fos proteins is normally delayed by at least 30 min after the delivery of the stimulus, we were able to anesthetize, move, and catheterize the animals before death without having concerns that such handling would result in nonspecific or stress-induced Fos protein production.
Near-term fetal sheep. Seventy-five minutes after the initiation of the infusion, the dams were anesthetized rapidly with ketamine hydrochloride (20 mg/kg iv) and then were ventilated through a mask with a mixture of isoflurane (2–5%) and oxygen (1–2 l/min). A midline abdominal incision was made to gain access to the uterus. The fetal head and neck were exposed, and one fetal carotid artery was catheterized with a 15-gauge indwelling catheter pointed toward the brain. The nonsurgically catheterized carotid artery was occluded, and both jugular veins were cut (12). The fetal head was perfused with 700 ml of normal saline containing heparin (10 IU/ml) and sodium nitrite (2.0% wt/vol), followed by 700 ml of 2.5% acrolein in buffered 4% paraformaldehyde. Care was taken to begin fixation of the brain tissue within 30 min of the initial administration of anesthesia of the dam to minimize any potential confounding influence on fetal neuronal activation.
After perfusion, the brains were removed, blocked rostral to the optic chiasm and caudal to the mammillary bodies, dehydrated in 30% sucrose solution for 4–6 days, serially sectioned at 30 μm on a freezing microtome, and stored in cryoprotectant for at least 1 wk before staining (7, 39). Alternate series of 1:6 serial sections were analyzed for 1) mean number of Fos-positive cells within specific hypothalamic nuclei and2) percentage of activated AVP-containing cells double stained for Fos protein in the selected nuclei. Sections were immunostained as described previously (2, 3, 13,22, 27) using the Peroxidase Vectastain Elite ABC kit (no. PK6100; Vector Laboratories, Burlingame, CA). Final dilutions of primary antibodies were as follows: Fos 1:75,000 (no. 94012; generously donated by Dr. Philip Larsen, Copenhagen, Denmark) and AVP 1:150,000 (no. 20069; Incstar, Stillwater, MN). Brain tissue from both treatment groups of ewes and fetal sheep were immunostained simultaneously.
To evaluate the patterns of neuronal activation in response to the intraperitoneal hypertonic saline in the adult or fetal sheep, we examined the expression of the immediate-early gene product, Fos, in areas of the hypothalamus that are known to respond to changes in osmolality, in particular, the OVLT, SFO, MnPO, SON, and PVN. For adult nonpregnant ewes, four to six sections per nucleus were analyzed, whereas two to three sections per nucleus were analyzed in the fetal sheep. To analyze Fos induction, the number of Fos-positive cells was counted within a grid (400 × 400 μm) placed over each section in a standardized position. This enabled the observer to examine sections from throughout the anterior to posterior range of each nucleus. We collected data on the mean number of cells stained for Fos within the hypothalamic nuclei. To determine the percentage of activated AVP-containing cells within the SON and PVN, we stained appropriate tissue sections for Fos expression and then counterstained for AVP-containing neurons.
Plasma Na, K, and Cl concentrations were determined with a Nova 5 electrolyte analyzer (Nova Biochemical, Waltham, MA), and osmolality was determined with a Fiske 2400 Multi-sample osmometer (Norwood, MA). Blood pH, , and (0.4 ml blood) values were determined with a Nova Stat Profile Plus 3 acid-base analyzer system (Nova Biomedical).
Blood samples for AVP assays were collected in chilled heparinized glass tubes. Plasma samples for AVP were extracted by a modification of the procedures of LaRochelle et al. (16), and AVP levels were determined by radioimmunoassay (36). Sensitivity of our AVP antiserum is 0.8 pg of AVP/tube with intra-assay and interassay coefficients of variation of 6 and 9%, respectively.
Blood samples for ANG II assays were collected in chilled plastic tubes containing EDTA (1 mg/ml) and aprotinin (250 IU/ml). Samples were centrifuged immediately (4°C), and plasma aliquots were stored (−20°C) until extraction. Plasma samples for ANG II extraction were acidified with 1 N HCl and extracted on C18 Sep-Pak columns (Waters, Milford, MA) as described by Cain et al. (1). Before use, columns were washed with methanol (10 ml) followed by distilled water (10 ml). Acidified plasma samples were added slowly to the columns, and the columns were washed with 10 ml of 0.1% trifluoroacetic acid (TFA). The absorbed peptide was eluted with 3 ml of 80% methanol and 0.1% TFA, and the eluates were dried in a Speed-Vac concentrator. Dried fractions were stored (−20°C) until dissolved in radioimmunoassay buffer immediately before assay.
The ANG II radioimmunoassay procedure has been described previously (28). The standard curve ranged from 1 to 200 pg/tube; sensitivity of the assay was 2 pg/tube. The intra- and interassay coefficients of variation were 7 and 9%, respectively.
Physiological measures were analyzed for differences over time using repeated-measures analysis of variance (ANOVA;P < 0.05 considered significant). The number of Fos-positive cells counted per field was averaged to obtain a single independent number for each structure from each adult ewe or fetal sheep. Data are presented as means ± SE. Averages for each nucleus were compared between treatment groups using ANOVA.
Physiological Parameters: Adult Nonpregnant Ewes
Arterial plasma osmolality, electrolytes, AVP, ANG II, Hct, Hb, pH, , and were within normal limits for euhydrated adult nonpregnant sheep during the baseline periods (HYP-A and ISO-A; Tables 1 and2, respectively). In response to the intraperitoneal hypertonic saline infusions, arterial plasma osmolality, AVP, Na, and Cl concentrations significantly increased, and arterial plasma Hct, Hb, and K concentrations decreased in HYP-A animals. At time 15 min, adult plasma osmolality and Na (Fig. 1,A andB) concentration increased by ∼10 mosmol/kgH2O and 5 meq/l, respectively, with a continued increase demonstrated at 60 min. In response to the intraperitoneal isotonic saline infusions, arterial plasma osmolality, Hct, Hb, and AVP concentration decreased significantly in the ISO-A group. There were physiologically small, although statistically significant, changes in arterial and pH in both HYP-A and ISO-A groups.
Near-term fetal sheep. Arterial plasma osmolality, electrolytes, ANG II, Hct, Hb, pH, , and were within normal limits for euhydrated near-term fetal sheep during the baseline measurements (HYP-F and ISO-F; Tables 3 and4, respectively). In response to the intraperitoneal hypertonic saline infusions, arterial plasma Na and Cl concentrations significantly increased in HYP-F animals. Attime 15 min, fetal plasma osmolality and Na (Fig. 1, C andD) concentrations increased by ∼10 mosmol/kgH2O and 5 meq/l, respectively, similar to that noted in the adult ewes. In contrast to the adult response, the increase in fetal plasma osmolality was not significantly different from baseline levels (P < 0.07). Furthermore, fetal plasma osmolality and Na concentration did not demonstrate further increases after the 15-min sample. There were no significant fetal arterial responses to the intraperitoneal isotonic saline infusions, although there was a trend toward decreased plasma osmolality.
In the pregnant ewes of the treated fetuses, there were no significant changes in measured parameters throughout either study protocol (hypertonic saline treated: plasma osmolality, 299.8 ± 1.2 mosmol/kgH2O; AVP, 10.9 ± 2.5 pg/ml; ANG II, 17.2 ± 7.0 pg/ml; Hct, 30.6 ± 1.8%; Hb, 9.8 ± 0.5 g/dl; pH, 7.47 ± 0.02; , 112.0 ± 8.5 mmHg; , 33.6 ± 2.6 mmHg; Na, 146.6 ± 2.0 meq/l; Cl, 112.2 ± 1.3 meq/l; K, 4.2 ± 0.2 meq/l; isotonic saline treated: plasma osmolality, 301.7 ± 3.8 mosmol/kgH2O; AVP, 8.6 ± 5.0 pg/ml; ANG II, 4.3 ± 1.1 pg/ml; Hct, 27.7 ± 0.9%; Hb, 8.3 ± 0.5 g/dl; pH, 7.47 ± 0.01; , 110.9 ± 5.0 mmHg; , 34.3 ± 1.3 mmHg; Na, 147.1 ± 0.6 meq/l; Cl, 110.5 ± 1.5 meq/l; K, 4.1 ± 0.2 meq/l).
Comparison of Adult Ewes and Fetal Sheep
Comparison of HYP-A and HYP-F indicated that there were no significant changes in plasma Hb over the course of the study (see Tables 1 and 3). HYP-A animals varied significantly in their responses for plasma osmolality, AVP, Na, Cl, and compared with the HYP-F animals over the course of the study. Comparison of the ISO-A and ISO-F groups indicated similar responses in plasma osmolality and Hb (Tables 2 and 4).
Cells Stained for Fos Protein
Adult nonpregnant ewes. The hypertonic saline infusion resulted in intense Fos immunoreactivity in several areas of the hypothalamus involved in osmoregulation, which validates the use of Fos as a marker of neuronal activation after an intraperitoneal hyperosmotic challenge in the adult sheep (Fig.2). The mean number of cells stained for Fos within the OVLT, SFO, MnPO, SON, and PVN was significantly increased in the HYP-A animals compared with the ISO-A group (P < 0.05; Fig.3).
Near-term fetal sheep. Similar to the adult ewes, the hypertonic saline infusion resulted in intense Fos immunoreactivity in several areas of the hypothalamus in the fetal sheep (Fig. 4). The mean number of cells stained for Fos within the OVLT, SFO, MnPO, SON, and PVN was significantly increased in the HYP-F animals compared with the ISO-F group (P < 0.05, Fig. 3).
Comparison of Adult Ewes and Fetal Sheep
In comparison with HYP-A, we detected a greater mean number of Fos-positive neurons in the HYP-F animals within the SFO and MnPO, with similar mean numbers of Fos-positive cells in the other analyzed nuclei (Fig. 3). In comparison with the ISO-A, ISO-F animals expressed significantly greater numbers of Fos-activated neurons within the OVLT and SON, whereas other areas showed a trend toward increased numbers of Fos-activated neurons (Fig. 3).
Percentage of AVP-Containing Cells Double Stained for Fos
Adult nonpregnant ewes. In hypertonic-treated adult ewes, 63.4 ± 4.3% of the AVP-containing cells were double stained or activated in the SON compared with 0.8 ± 0.5% in the isotonic-treated adult ewes (P < 0.05). A significantly greater percentage of AVP-containing neurons in the PVN was activated in the hypertonic-treated adult ewe (55.0 ± 10.9%) when compared with the isotonic-treated ewe (1.3 ± 0.7%; Fig.5).
Near-term fetal sheep. Within the SON, the HYP-F group showed a significantly greater percentage of AVP-containing cells that were activated (66.1 ± 5.4%) compared with the ISO-F animals (16.5 ± 7.6%;P < 0.05). Moreover, within the PVN, Fos activation was detected within 65.7 ± 3.13% of AVP-containing neurons in the HYP-F animals compared with ∼12.1 ± 6.0% in the ISO-F animals (Fig. 5). Figure6 presents very typical immunocytochemical data from the PVN for both the HYP-F and ISO-F animals. In the HYP-F animals, intense Fos activation was detected in AVP-containing cells as well as in other neurons of unknown phenotype (see Fig.6 A).
Comparison of Adult Ewes and Fetal Sheep
Similar percentages of Fos-activated AVP-containing cells were detected in the HYP-A and HYP-F for the SON (Fig. 5). Although there was a tendency for Fos activation in AVP-containing neurons in the PVN to be decreased in the HYP-A compared with the HYP-F, there remained too much within-group variation for the result to be considered significant (Fig. 5). Similarly, percentages of Fos-activated AVP-containing neurons detected in ISO-A were slightly, although not significantly, reduced compared with ISO-F (Fig. 5).
Fetal swallowing is a primary route of amniotic fluid reabsorption and has significant effects on fetal gastrointestinal development and perhaps somatic growth. The near-term ovine fetus demonstrates dipsogenic responses to systemic, intracarotid, or intracerebroventricular hypertonicity (29, 30). We previously demonstrated that an intravenous hypertonic saline injection stimulates near-term fetal swallowing activity with a concomitant increase in plasma osmolality of 14 mosmol/kgH2O (30). Interestingly, whereas fetal plasma osmolality remains 2–3% above basal osmolality (6–9 mosmol), a level that typically would induce prolonged ingestive behavior in adult animals (40), fetal swallowing response is abated within minutes. Despite this brief response to hypertonicity, primary osmoregulatory mechanisms in the near-term fetus are functional (21, 24, 29, 30), and thus Fos expression may be detected within fetal osmoreceptors after hypertonic stimulation. Alternatively, during this stage of development, an enhanced regulatory mechanism, which suppresses swallowing activity, may also be involved in fetal osmoregulation. In the present study, we examined the responsiveness of osmoregulatory neural mechanisms in the near-term ovine fetus.
In this study, intraperitoneal infusion of hypertonic saline induced increases in adult and fetal plasma osmolality. Although we administered a reduced dosage in the fetuses (due to the morbidity at the higher dose), both adults and fetuses demonstrated increases in plasma osmolality and Na concentrations of ∼10 mosmol/kgH2O and 5 meq/l, respectively, within 15 min after the infusion (Fig. 1). These increases are consistent with a 2–3% change in systemic osmolality previously demonstrated to induce adult osmoregulatory responses. Adult plasma osmolality continued to increase during the following 45 min, although fetal plasma osmolality leveled off and remained elevated above baseline values. The lack of a continued and prolonged rise in fetal plasma osmolality is likely a result of transplacental exchange of water and electrolytes with the isotonic pregnant ewe. Due to the larger volume of distribution, these fluid shifts did not significantly impact maternal plasma values, regardless of the treatment to the fetus. Moreover, after the hypertonic saline infusion, both adults and fetuses demonstrated decreased Hct and Hb, consistent with plasma volume expansion; therefore, it is unlikely that hypovolemia contributed to central Fos stimulation. In response to intraperitoneal isotonic (0.15 M) saline infusion, there were small decreases in adult and fetal plasma osmolality, likely a result of the infusate osmolality (284 mosmol/kgH2O) being slightly lower than basal plasma values.
In the present study, adult ewes responded to osmotic stimulation with a marked increase in Fos expression within the OVLT, SFO, and MnPO, as well as within hypothalamic nuclei neurosecretory nuclei, the SON, and PVN (Fig. 2). These results are consistent with studies in the adult rat (8, 25, 26), employing a variety of methods to administer hypertonic stimuli and demonstrating significant increases in Fos activation within osmosensitive neurons. Furthermore, administration of hypertonic stimuli to adult rats results in Fos activation within both AVP- and oxytocin (OT)-containing neurons located in the SON and PVN parvocellular and magnocellular divisions (6, 14, 17, 18, 34) and is consistent with OT secretory responses to hypertonicity (38). Although in the present study we did not stain for OT, we did observe that, for all groups, virtually all Fos (within the SON) was located within AVP-containing neurons. The absence of osmotically induced Fos expression within SON OT neurons in our studies is likely a result of species differences or differences in the route of intraperitoneal administration of hypertonic saline (via an indwelling catheter rather than through subcutaneous injection).
In the late-gestation fetal sheep, hypertonic saline infusion induced marked Fos expression within the CVOs, the MnPO, and hypothalamic nuclei synthesizing AVP (Figs. 4 and 6). The detection of neural activation indicates that near-term fetal osmoreceptors are capable of appropriately responding to an osmotic stimulus. These results also suggest that neuronal pathways from osmotic sensing neurons within the CVOs and MnPO, and the extensive projections to the neurosecretory SON and PVN nuclei, may be intact within the near-term fetus. However, it is unknown whether the development of neural pathways to swallowing centers (i.e., motor neurons) are fully functional at this stage of fetal development.
These data are consistent with fetal swallowing stimulation and AVP secretion in response to an osmotic challenge and support a functional thirst or drinking response in the near-term ovine fetus (29, 30). However, the possible modulatory role of ascending hindbrain and/or brain stem projections to the hypothalamus in response to the osmotic challenge cannot be excluded. Furthermore, neurosecretory neurons may be directly and intrinsically osmosensitive (19, 20). Recently, in a study of the identical infusion paradigm, we detected a 10- to 20-fold increase in neuronal activation in caudal catecholaminergic brain stem neurons in hypertonic-treated fetal sheep, specifically in the commissural subnucleus of the solitary tract and ventrolateral medulla (3). Because these neurons have established projections to the hypothalamus (4, 33), it is likely that these ascending brain stem projections also play a modulatory role in hypothalamic osmotic sensing neuronal activation.
We have recently demonstrated intense Fos activation of the CVOs, SON, and PVN in the fetal sheep after a 20% mannitol infusion administered to the dam (21). The identical mannitol infusion to the dam had been demonstrated previously to increase near-term fetal plasma osmolality by 13 mosmol/kgH2O and to stimulate fetal AVP secretion, primarily by an induced fetal-to-maternal transplacental water flow. In contrast to the present experimental paradigm, maternal mannitol infusion may have induced acute fetal blood volume contraction, with secondary effects on CVOs sensing intravascular volume. Despite the different experimental paradigms, these studies described similar patterns of Fos activation in fetal sheep within most, but not all, osmosensitive hypothalamic neural regions. Specifically, mannitol-treated fetal sheep failed to demonstrate a significant increase in Fos activation within the MnPO, which may have been due to the functional differences within the MnPO nucleus. Future studies will be required to identify the phenotype of activated neurons located within the MnPO under the two paradigms.
Our results indicate similar patterns of Fos activation in the fetal and adult sheep, within the osmotic sensing neurons and neurosecretory AVP populations, in response to hypertonicity. Both adult ewes and fetuses exhibited similar osmotic responses at 15 min after the hypertonic infusion, with the adult ewes demonstrating an extended rise in plasma osmolality. Despite this prolonged osmotic stimulation in adults, fetal sheep demonstrated similar patterns of intense Fos activation in the SFO and MnPO regions. These results suggest that altered near-term fetal dipsogenic responses to osmotic stimulation may not be due to the inability to activate osmosensitive neurons but rather may imply an immaturity-related difference in regulating hypertonicity in the fetus. Moreover, although similar percentages of activation of AVP-containing neurons were observed for HYP-A and HYP-F, differences in total AVP cell number, cellular AVP synthesis, and secretion may account for the observed reduction of fetal vs. adult plasma AVP levels in response to the osmotic stimulation.
Interestingly, in ISO-F, we detected greater Fos expression within osmotic sensing neural areas, and significantly greater mean numbers of activated neurons specifically in the OVLT and SON, in comparison with the ISO-A group. These findings were unexpected, because isotonic infusions in adult mammals generally result in minimal stimulation of Fos expression in osmosensitive neural regions. Moreover, the expression of Fos in neurons is generally low, or undetectable, at basal levels of neuronal activity (10, 32). Whereas adult ewes demonstrated a reduction in plasma AVP levels after isotonic saline infusion, isotonic saline-treated fetal sheep responded with a trend toward increased AVP levels. Together, these results may suggest a potential stress-induced AVP secretion in fetal sheep and may explain the increased Fos activation in ISO-F animals. Although ISO-A and ISO-F were appropriate control groups for this study, further experiments comparing adults and fetuses without intraperitoneal infusions may be required to explain elevated basal levels of activation in the fetus.
The demonstration of intact central and systemic dipsogenic mechanisms in utero has important implications in the regulation of fetal fluid homeostasis during gestation. The normal development of maternal and thus fetal plasma hypoosmolality during human pregnancy and the potential intermittent exposure to maternal thermal exposure, dehydration, or exercise-induced water loss may have important implications for fetal and adult dipsogenic regulation. Understanding the consequences of such altered osmotic environments in modulating swallowing activity in utero and in developing adult sensitivities to thirst and AVP secretion will require further studies to elucidate the mechanisms modulating the development of central sensitivity and responsiveness to fetal body fluid composition.
In conclusion, we demonstrate neuronal activation, after a hypertonic saline infusion, of the CVOs, the MnPO, and hypothalamic AVP secretory nuclei in the late-gestation fetal sheep. These observations suggest that osmotic sensing neurons within the CVOs and MnPO, and the neurosecretory SON and PVN nuclei, may be playing a role in mediating near-term fetal central osmoregulation.
We acknowledge the generous gift of antisera essential for our studies from Dr. Philip Larsen and acknowledge Linda Day, Glenda Calvario, and James Humme for technical assistance.
Address for reprint requests: A. Caston-Balderrama, Harbor-UCLA Medical Center, Department of Obstetrics and Gynecology, 1124 West Carson St., RB-1, Torrance, CA 90502.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43311.
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. §1734 solely to indicate this fact.
- Copyright © 1999 the American Physiological Society