INTRODUCTION

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DURING IN UTERO DEVELOPMENT, fetal dipsogen-mediated swallowing responses contribute to amniotic fluid volume homeostasis and development of ingestive systems. Our previous studies suggested that swallowing and esophageal fluid flow are physically functional in the near-term fetus. During the last third of the gestation, swallowing may be stimulated in response to intravenous or intracerebroventricular injection of hypertonic saline (22, 25) and to intracerebroventricular injection of angiotensin II (ANG II) (23). However, the central mechanisms of ANG II-mediated fetal swallowing stimulation are unknown.

In adult animals, there is significant information regarding central "dipsogenic" areas and thirst mechanisms (6, 10). Intracerebroventricular ANG II has been repeatedly shown to produce a reliable thirst response in adult animals. Furthermore, directed microinjections of ANG into the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and median preoptic nucleus (MnPO) elicit water intake in conscious animals (9, 33). Intracerebroventricular administration of ANG II also induces cardiovascular and neuroendocrinological responses (e.g., vasopressin release) (6, 12, 15, 16, 30).

Despite these results, questions remain as to what central sites are essential for ANG central dipsogenic actions in the fetus. Current evidence from studies in adult models indicates that central thirst mechanisms are not dependent upon a single population of receptors but rather a multiple receptor complex is involved. The most important of these are thought to be contained in the region of the anterior third ventricle (AV3V), including the MnPO, and in the hypothalamic nuclei (6, 10, 30). Although many regions in the central nervous system (CNS) may be sensitive to central ANG II, there are three specific structures lying outside the blood-brain barrier that almost certainly act as sensors of the humoral circulatory signal. These sensory circumventricular organs are the SFO, the OVLT, and the area postrema (AP) (5, 9, 21, 30). Notably, this information has been obtained entirely from adult models. Conversely, the development of central dipsogenic centers and ANG mechanisms in the fetus is largely unknown.

The present study sought to examine the functional response of fetal central dipsogenic sites in utero. The questions we addressed included, are central fetal swallowing control areas similar to adult dipsogenic centers? Does the fetal dipsogenic center express functionality in response to stimulation of putative dipsogen ANG II? Assuming these centers are functional, are the patterns of central activation marked with c-fos expression in the fetal brain similar to that reported in the adult brain? This information may aid in understanding the ontogeny of ANG-mediated central dipsogenic mechanisms in the control of body fluid balance and may provide further evidence for development of dipsogenic neural network in the early stage of life.

	MATERIAL AND METHODS

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Ten time-dated pregnant ewes with singleton fetuses (130 ± 2 days gestation at study) were used. Animals were housed in individual study cages and in a light-controlled room (12 h light/12 h dark) with food and water provided ad libitum.

Surgical preparation. Anesthesia was induced with ketamine hydrochloride (20 mg/kg im), and general anesthesia was maintained with 1-2% isoflurane and 1 l/min oxygen. Bipolar electromyography (EMG) electrodes (model AS632, Cooner Wire, Chatsworth, CA) were placed on the fetal thyrohyoid muscle and upper and lower esophagus for determination of swallowing activity as previously reported (11, 24). Electrodes were also implanted on the parietal dura through two burr holes for the determination of fetal electrocortical activity (ECoG). Intracranial cannulas (23 gauge) were placed in the lateral ventricle and held in place with two stainless steel screws (in the skull) and dental cement. Patency of the catheter at insertion was assessed by free flow of artificial cerebrospinal fluid via gravity drainage. Polyethylene catheters were placed in the maternal and fetal femoral artery and vein and threaded to the inferior vena cava and abdominal aorta, respectively. An intrauterine catheter (Corometrics Medical System, Wallingford, CT) was inserted for measuring amniotic fluid pressure. All catheters and electrodes were externalized to the maternal flank and placed in a cloth pouch. Animals were allowed 5 days of postoperative recovery that included catheter maintenance and antibiotic administration.

Behavioral and physiological experiments. Animals were studied only if the fetal arterial pH >7.3. Studies began with a baseline period (120 to 0 min ) followed by the study period (0 to 100 min). There were two groups of animals (control n = 5; experimental n = 5). Beginning at time 0, ANG II (1.5 µg/kg, 1 ml) (Sigma, St. Louis, MO) in isotonic saline was injected (over 5 min) into the lateral ventricle of the fetus. For the control animals, isotonic (0.15 M in 1 ml icv) saline was injected into the fetus. The animals were continually observed for 100 min after injections.

Immunochemistry experiments. At the conclusion of the study, ewes were anesthetized under ketamine anesthesia (20 mg/kg im) and ventilated with a mixture of isofluorane and oxygen. A midline abdominal incision was made, and the fetal head and neck were exposed. A 16-gauge needle was inserted into one fetal carotid artery for perfusion. The fetuses were perfused via carotid artery with 0.01 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Postfixation was performed in PFA solution for 12-24 h, after which the brains were placed in 20% sucrose in 0.01 M phosphate overnight. Thirty-micrometer coronal sections of fetal brain were cut on a cryostat. Every other section of the OVLT, MnPO, and SFO, and every third section of other parts of the forebrain were used for c-fos immunoreactivity (FOS-ir) staining using the avidin-biotin-peroxidase technique. The primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was raised from rabbits against amino acids 3-16 at the amino terminus of Fos protein p62. This antibody specifically reacts with Fos p62 of mouse, rat, human, and sheep in Western blotting or immunohistochemistry. The tissue sections were incubated for 1 h at room temperature on a gentle shaker and then overnight at 4°C in the primary antibody (Fos antibody 1:10,000 with 0.3% Triton X-100). The sections were further incubated in goat anti-rabbit serum (1:500) for 1 h and then processed with the ABC kit for 1 h (Vector Labs, Burlingame, CA), both at room temperature. After the tissue was washed three times with 0.01 M PBS, the sections were treated with 1 mg/ml diaminobenzidine tetrahydrochloride (Sigma) (0.02% hydrogen peroxide). All sections were mounted on gelatinized slides, dehydrated in alcohol, and then coverslipped.

Data analysis. EMG signals were processed as previously described (28). An EMG-propagated swallow, representing a coordinated laryngeal-esophageal contraction, was defined by a time sequence of integrated EMG signals from the thyrohyoid muscle to the upper and lower nuchal esophagus (28). ECoG patterns were divided into high-voltage (HV) and low-voltage (LV) periods. Because fetal swallowing occurs predominantly during periods of LV ECoG activity (8), change in the relative duration of LV periods may influence the swallowing rate. Thus swallowing data are presented as swallows per minute of LV ECoG activity. Fetal swallows per minute during LV ECoG during the 60 min before and after intracerebroventricular injections were compared. Fetal swallowing over 10 min immediately before and after intracerebroventricular injections was also analyzed to assess the acute effect of intracerebroventricular ANG II.

	RESULTS

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ECoG, swallowing activity, and arterial values. The majority of fetal swallowing activity occurred during LV ECoG periods for both groups, and this pattern did not change with control or intracerebroventricular ANG II injections. There was no difference in the percentage of LV ECoG between control and treated groups during the basal period [F_8,1 = 0.44, P, not significant (ns)]. In response to intracerebroventricular injections, percent time in fetal LV ECoG did not change from the baseline level after intracerebroventricular ANG II (39.0 ± 8.0 vs. 45.0 ± 3.8%) (F_8,1 = 0.63; P, ns; Fig. 1). Similarly, for the control animals, no significant difference of the percent LV ECoG was observed before and after intracerebroventricular injection of vehicle (45.0 ± 2.7 vs. 44.0 ± 2.8%; t = 1.04; P, ns).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Fetal swallows per minute before and after intracerebroventricular injection of vehicle or ANG II. *P < 0.01.

Fos-ir. Histological analysis confirmed that all intracerebroventricular catheters were inserted into the ventricle. Ten fetal brains (n = 5/group) were used for Fos-ir. One control study brain was damaged during processing so that final number for the control group was four. In the control fetuses, there was little Fos-ir in the fetal forebrain and hindbrain structures that have been demonstrated as putative dipsogenic nuclei after injection with intracerebroventricular vehicle. These results are consistent with the specificity of the antibody used for sheep tissue Fos-ir. However, intracerebroventricular ANG II produced intense Fos-ir in both forebrain and hindbrain in the fetus. The areas of intense Fos-ir staining included the OVLT, MnPO (dorsal and ventral parts), and SFO in the forebrain (Fig. 2). There were significant differences of Fos-ir between intracerebroventricular vehicle- and ANG II-injected fetuses (OVLT: F = 38.0, SFO: F = 39.3, both P < 0.001; ventral MnPO: F = 8.2, P < 0.05; Fig. 3). For both control and treated animals, similar Fos-ir was observed in the pyriform cortex, as that was observed previously in the adult brains and treated as nonspecific c-fos response. In the hindbrain, no or very little Fos-ir was observed in the LPBN and AP in the control animals injected with vehicle. However, intracerebroventricular ANG II induced positive Fos-ir in the fetal AP (F = 48.9, P < 0.001) and the LPBN (F = 6.9, P < 0.05; Figs. 3 and 4).

View larger version (178K): [in this window] [in a new window]   Fig. 2.   c-fos Immunoreactivity (Fos-ir) induced by intracerebroventricular ANG II in the subfornical organ (SFO, A and B) and the organum vasculosum of lamina terminalis (OVLT, C and D) of the fetus. A and C, intracerebroventricular 0.9% NaCl as the control; B and D, intracerebroventricular ANG II. ×40.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Fos-ir induced by intracerebroventricular ANG II in the fetal forebrain and hindbrain. MnPO, median preoptic nucleus; AP, area postrema; LPBN, lateral parabrachial nucleus. *P < 0.05.

View larger version (145K): [in this window] [in a new window]   Fig. 4.   Fos-ir induced by intracerebroventricular ANG II in the LPBN (A and B) and the AP (C and D) of the fetal hindbrain. A and C: intracerebroventricular ANG II; B and D: intracerebroventricular 0.9% NaCl as the control; ×40.

	DISCUSSION

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The present study demonstrated that intracerebroventricular application of ANG II not only stimulated swallowing in the near-term ovine fetus but also increased the neural activity in the central dipsogenic centers in utero. The finding that c-fos expression was induced in the circumventricular organs, the AV3V region, and the hindbrain of the fetus is evidence that fetal dipsogenic centers are functional in response to intracerebroventricular ANG II before birth. Thus central ANG systems for control of body fluid homeostasis are intact near term.

The present experiments were designed to correlate stimulated fetal swallowing activity and central neural activation under conditions of intracerebroventricular administration of ANG II. Because a number of previous studies in adult models showed that central ANG-induced drinking reliably occurs within first 10 min after application of the octapeptide (6, 10, 14, 19), we paid special attention to the time course of fetal swallowing stimulation. Thus we not only measured fetal swallowing during LV periods of ECoG as previously reported but also analyzed the time course for stimulated swallowing immediately before and after central ANG II injection.

Stimulated fetal swallowing behavior occurs in response to dipsogenic stimulation, including intravenous and intracerebroventricular infusion of hypertonic saline and central ANG II (23, 24). In the present study, fetal swallowing was markedly increased by intracerebroventricular ANG II in the ovine fetus at 0.9 gestation, confirming our previous report of ANG II-induced swallowing stimulation (23). The dose of ANG II in the present study was slightly higher than the dose used in the previous experiment. The stimulated fetal swallowing activity was also relatively higher in the present work. This suggests that there may be a dose-dependent response for intracerebroventricular ANG II in the near-term ovine fetus. Both control and stimulated fetal swallowing activity occurred primarily in LV ECoG periods (about 50% of time was LV ECoG). The percentage of time spent in LV ECoG periods was not changed by intracerebroventricular administration of ANG II, indicating that the injected peptide did not disturb the fetal neurobehavioral state. In addition, fetal physiological status remained stable as arterial values (pH, PCO₂, PO₂, hematocrit, hemoglobin, and plasma electrolytes) were not changed. The lack of change of plasma electrolytes, particularly sodium, and osmolality indicates that systemic sodium/osmolality did not affect dipsogenic responses.

Previous studies have consistently demonstrated that adult water intake (drinking behavior) induced by intracerebroventricular ANG II starts within 10 min after injection, and a large portion of the water intake is within the first 15 min (6, 10, 19). In the present study, intracerebroventricular ANG II increased swallowing activity within the first 10 min after the injection. Thus the initiation or latency of ANG stimulated near-term fetal swallowing is similar to that of adult drinking behavior after intracerebroventricular ANG II.

One may question whether stimulated fetal swallowing is comparable to adult drinking behavior; specifically, is fetal swallowing a nonspecific or specific behavioral change to dipsogenic stimulation? Stimulated fetal swallowing is likely evidence of the development of ingestive behavior responsiveness. Fetal swallowing may be regulated by numerous systems including dipsogenic factors, appetite, amniotic fluid availability, and behavioral state (1, 2, 7, 11). Accordingly, previous physiological behavioral experiments were unable to determine whether ANG II stimulated swallowing was a direct result of dipsogenic stimulation. However, the use of both physiological and neuro-immunochemical techniques provides means to elucidate the relationship and mechanisms of putative fetal dipsogens. In the present study, we used a c-fos mapping technique to explore fetal central activity that correlated to stimulated swallowing responses. A number of studies confirming dipsogenic areas in the adult brain (3, 13, 16, 17, 32) provide a rational comparison background for us to examine fetal nuclei activity after administration of ANG II. The induced Fos-ir in the MnPO and OVLT is evidence that the AV3V region was activated, consistent with the previous adult studies. Combined with the stimulated swallowing activity in the same fetuses, the c-fos results suggest that the critical areas in the CNS for control of ANG-mediated dipsogenic mechanisms are functional during the last third of the gestation. Particularly, the SFO is replete with ANG receptors, especially AT₁ subtype receptors, which may account for the biological actions of the octapeptide (6). In our and others' previous studies of effects of ANG on c-fos expression in adult animals, neural activity marked with c-fos was detected in the SFO (18, 26, 31). In the present study, we also observed intense Fos-ir in the SFO of the ovine fetuses. These results provide evidence that stimulated fetal swallowing is a specific dipsogenic or thirst response to central ANG II.

In comparison to the numerous studies of forebrain regulation of dipsogenesis, studies of the hindbrain control of ingestive behavior are relatively limited. In rats, lesions of the AP and the immediately adjacent caudal nucleus tractus solitarii (NTS) or destruction of bilateral LPBN, to which the AP and NTS make substantial secondary projections, resulted in large increases in water and salt intake (as compared with unlesioned animals) in response to dipsogens such as subcutaneous isoproterenol or ANG II (4, 6, 20, 27). Thus removing these inhibitory neurons or pathways results in selective enhancement of drinking. Based on these observations, a hypothesis has been proposed that AP/NTS/LBPN systems comprise inhibitory mechanisms for dipsogenic responses. Xu and Herbert (31) were the first to demonstrate that c-fos expression is induced by intracerebroventricular ANG in the hindbrain in the adult animal model, thus providing cellular activation evidence that the AP/NTS/LPBN systems can be activated in response to central dipsogens. The present study is the first to demonstrate that intracerebroventricular ANG II stimulated c-fos expression not only in the fetal forebrain but also in the LPBN and AP in the fetal hindbrain. This result provides evidence that the near-term fetal hindbrain also responds to central dipsogens. However, increased blood pressure may cause c-fos expression in the brain, particularly in the hindbrain (29). Further study is needed to clarify whether c-fos expression in the hindbrain in the present experiment was caused directly by intracerebroventricular ANG II or was secondary to an increase of mean arterial pressure.

In the present experiments, fetal mean arterial pressure was increased by intracerebroventricular application of ANG II, as has been demonstrated in adult animals (30). Change of blood pressure can influence water intake behavior. It is well known that hypotension facilitates drinking behavior whereas hypertension inhibits it (10). However, fetal swallowing after central administration of ANG II was significantly increased despite the increased fetal blood pressure. This suggests that ovine fetal dipsogenic mechanisms to ANG stimulation are well developed at near-term gestation. Despite the potential inhibitory effects of increased blood pressure, the fetal central dipsogenic mechanisms still function, as evidenced by increased fetal swallowing.

Perspectives