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Research report
Brainstem catecholaminergic neurons activated by hypoxemia express
GR and are coordinately activated with fetal sheep hypothalamic
paraventricular CRH neurons
a ,
*
b bThomas J. McDonald
, Wei Wei Le , Gloria E. Hoffman
a
Laboratory for Pregnancy and Newborn Research, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA b
Department of Anatomy and Neurobiology, University of Maryland, Baltimore, MD 21201, USA
Accepted 29 August 2000
Abstract
In late gestation, challenges to fetal homeostasis are accompanied by increases in adrenocorticotropin (ACTH) concentrations in fetal peripheral plasma and Fos (c-fos protein) activation in corticotropin-releasing hormone (CRH) neurons of the fetal hypothalamic paraventricular nucleus (PVN). In adults, ventrolateral brainstem catecholaminergic (CA) neurons (A1 / C1, A2 / C2) project to the parvocellular neurons of the PVN, possess glucocorticoid receptors (GR) and are Fos activated in parallel with CRH neurons of the PVN during hypoxia. Such observations suggest a role for the aforementioned medullary neurons in the function of the hypothalamo–pituitary– adrenal axis. The present study utilized late gestation fetal sheep, stereotaxic methodology and retrograde axon tracing and immunocytochemical techniques to investigate the relationship between activation of fetal brainstem CA neurons and activation of fetal PVN CRH immunopositive neurons in response to hypoxemia. Results indicated that: (1) the largest brainstem CA projection to PVN CRH neurons is from A1 / C1 neurons, (2) brainstem neurons exhibit GR immunostaining and (3) brainstem CA neurons show a strong
2 2
correlation (A1 / C1 — r 50.894, P,0.005; A2 / C2 — r 50.848; P,0.002) of Fos activation with Fos activation in PVN CRH cells. We conclude that in late gestation the brainstem A1 / C1 and A2 / C2 areas are in position to influence the function of the hypothalamo– pituitary–adrenal axis during hypoxemic challenges to homeostasis in a fashion similar to that which has been demonstrated in the adult rat. 2000 Elsevier Science B.V. All rights reserved.
Theme: Endocrine and autonomic regulation
Topic: Hypothalamic-pituitary-adrenal regulation
Keywords: A1 / C1; A2 / C2; Fos; c-fos; Glucocorticoid; Retrograde axon tracer; Glucocorticoid receptor; HPAA
1. Introduction amine (CA) synthesizing enzymes [1,20,28]. These
brain-stem neurons are also sites of glucocorticoid negative The fetal sheep hypothalamic paraventricular nucleus feedback [15]. This innervation has been of great interest (PVN) plays a central role in adrenocorticotropin (ACTH) to neuroendocrinologists studying the control of glucocor-and glucocorticoid secretion in response to homeostatic ticoid secretion by the hypothalamo–pituitary–adrenal axis challenge e.g., hypoxemia or hypotension [27] and is also a since removal of these inputs decreases CRH levels in site for glucocorticoid negative feedback [24]. In adult rats, portal plasma [13] as well as evoked levels of corticos-studies at the light and electron microscope levels reveal terone in peripheral plasma [11] and increases glucocor-that fibers glucocor-that synapse upon the corticotropin-releasing ticoid receptor (GR) numbers in the hypothalamus [22]. hormone (CRH) neurons of the PVN originate from Interestingly this brainstem–PVN connection also ap-brainstem neurons that are immunopositive for catechol- pears to cooperate in cardiovascular control i.e., brainstem CA neurons function to varying degrees in both barorecep-tion and chemorecepbarorecep-tion and the PVN participates in *Corresponding author. Tel.: 11-607-253-3086; fax: 1
1-607-253-cardiovascular regulation via direct projections to rostral 3455.
E-mail address: [email protected] (T.J. McDonald). ventrolateral medullary sympathetic premotor neurons and
to sympathetic motor neurons of the thoracic spinal cord normoxic (NORMOX n53) treatment groups. At 120 days [9,14,21]. For example there is experimental evidence of gestational age (dGA; term¯150 dGA) a subset of the which indicates that one way in which the PVN influences fetuses (n53) underwent stereotaxic neurosurgical place-blood pressure is by modifying sympathetic nervous ment of microinjections of the retrograde axon tracer system outflow that is initiated by activation of the FluoroGold (FAu; 200 or 300 nl of 5% FAu in distilled baroreceptors [29]. The anatomy of reciprocal innervation H O, Fluorochrome Inc., Englewood, CO 80155) either2 of the PVN and the brainstem has been extensively and bilaterally (n51) or unilaterally (n52; left side) into the elegantly reviewed by L.W. Swanson [33]. PVN (in-house atlas coordinates: AP 18.0, V 12.0, H 1.5) Increases in the protein product (Fos) of the immediate under halothane general anesthesia using methods de-early gene c-fos have been used extensively as markers of scribed previously in detail [26]. All animals receiving neuronal activation in neuroendocrine systems (for review FAu injections were allowed at least 2 weeks of recovery see [18]). In previous studies, we used fetal hypoxemia as time for retrograde transport of FAu to occur. At least 5 a well characterized and repeatable stimulus for ACTH days prior to the hypoxemic challenge, six of the seven secretion and as a stimulus for the production of Fos in ewes carrying HYPOXEMIC fetuses were surgically in-CRH-immunopositive neurons of the PVN of fetal sheep strumented with one jugular and one carotid polyvinyl [17]. In adult rats, hypoxia stimulates Fos production in catheter (18-gauge) and one tracheal polyvinyl catheter CA neurons of the ventrolateral (A1 / C1) and dorsal (o.d.53.5 mm, i.d.52.5 mm) while under halothane gener-medulla (A2 / C2; [8,19]) and CRH, vasopressin (AVP) and al anesthesia, as previously published [12]. No fetuses oxytocin (OT) neurons of the PVN and supraoptic nucleus. were instrumented to keep fetal stress to a minimum. Lesions of A1 / C1 CA neurons significantly reduce Fos
expression in hypothalamic CRH, AVP and OT neurons 2.2. Hypoxemic challenge [32]. However, to our knowledge, no investigations in
fetuses of any species have determined which brainstem On the day of hypoxemic challenge, fetuses ranged in nuclei projections to the fetal PVN are developed and the age from 124–144 (HYPOX) and 125–141 (NORMOX) extent to which the cells of these brainstem nuclei exhibit dGA. Hypoxemia was induced according to the method of GR and are coactivated with hypothalamic neurons during Gleed et al. [12]. Briefly, at time50, N infusion to the2 hypoxemia. maternal tracheal catheter was begun at a rate of 12 l / min. Since the fetus often displays physiological responses In order to provide variability in the degree of hypoxemia that differ markedly from adults (e.g., unlike adults, fetuses in individual fetuses after the first 10 min, N flow was2 greatly decrease or cease their in utero breathing move- adjusted to achieve differing maternal PO values over the2 ments during hypoxemia [4]), it is impossible to extrapo- 60 min hypoxemic period. One HYPOXEMIC group ewe late from the adult condition or to determine when a was not catheterized. In this ewe, N flow was maintained2 system begins to function in utero other than by examining at 8 l / min for the last 50 min of the hypoxemic period. We the fetus itself. We have chosen to perform our studies in have shown previously that this method produces de-fetal sheep at the approximate time of gestation that: (1) creases in fetal PO of approximately 10.0–14.0 Torr and2 fetal sheep adrenals are responsive to ACTH stimulation reproducibly elicits fetal ACTH secretion [24]. At 60 min, after a mid gestation refractory period (for review see N2 flow was terminated and ewes and fetuses were [6,24])and (2) negative feedback begins to function in the immediately euthanized by exsanguination under deep fetal sheep hypothalamo–pituitary–adrenal axis [36]. The halothane anesthesia.
first aim of the present study was to determine if fetal
brainstem CA neurons project to the PVN, express GR and 2.3. Tissue collection and immunocytochemistry show Fos activation during a hypoxemic challenge. The
second aim was to see if any brainstem CA neuronal Perfusion and fixation of the fetal brain were performed activation observed correlates with Fos activation of CRH via the carotid artery with a 14-gauge indwelling catheter neurons of the fetal PVN during graded hypoxemic pointed toward the brain using techniques previously challenges, which we know from previous studies reliably described in detail [26]. Both jugular veins were cut and increase ACTH secretion in peripheral fetal sheep plasma. the anterior vena cava, aorta and pulmonary artery were clamped at the heart. The fetal brain was perfused with 500 ml of normal saline containing heparin (10 IU / ml) and
2. Materials and methods NaNO (2.0%, wt / vol) as a vasodilator followed by 10002
(30%, wt / vol) and sectioned as a 1 in 12 series at 30mm 2.4. Data analysis on a sliding microtome (Fisher Scientific, Springfield, N.J.
07081) while frozen with crushed dry ice. Sections were The average number of neurons counted in each brain-stored in cryoprotectant [35] solution at2208C to preserve stem CAergic area (e.g., A1 / C1, A2 / C2) in studies of the immunogenicity until the time of staining. percent of brainstem neurons retrogradely labeled with Double label immunocytochemical staining for Fos and FAu after FAu injection to the PVN was: A1 / C1 — 307, either CRH or tyrosine hydroxylase (TH), FAu and CRH A2 / C2 — 275, A5 — 231 and A6 — 1615. The average or TH, and GR and TH was accomplished using methods number of neurons counted in studies of the correlation of previously reported [16]. Once the staining procedures Fos activation of PVN CRH-immunoreactive neurons vs. were well established on practice tissues, one well of Fos activation of brainstem CAergic area neurons over a sections (i.e., 1 / 12th of all sections cut) of brainstem range of hypoxemia were PVN CRH — 347, A1 / C1 — and / or of hypothalamus from each animal were immuno- 651, A2 / C2 — 597 and A6 — 539.
stained for each pair of antigens listed in the previous Counting of coincidence of Fos and TH in brainstem sentence. Staining of the first antigen in double reactions A1 / C1, A2 / C2 and locus coeruleus (A6) neuronal areas was accomplished with the avidin–biotin complex (ABC) and coincidence of Fos and CRH in PVN was done under method (Elite Kits, Vector Labs, Burlingame, CA 94010) light microscopy by an observer unaware of treatment. and immunoperoxidase detection with nickel–diaminoben- Counts were expressed as a single independent value i.e., zidine (Ni–DAB) as the chromogen. The presence of percentage of total CA or CRH neurons expressing Fos per antigen was detected as a blue / black product, which could brainstem neuronal area (e.g., A1, A2) or PVN, per animal. be eliminated by substitution of normal rabbit serum for The percentages of Fos and TH coincidence for each area the primary antibody or preabsorption of the primary of CAergic brainstem neurons vs. the percent of Fos and antibody with 10 mg of the antigen per ml of antibody. CRH coincidence in neurons of the PVN were plotted (Fig. Incubations with primary antisera: CRH (polyclonal, gift 7) and subjected to the Pearson Product Moment Correla-of Dr A. J. Silverman) 1:40,000; FAu (Chemicon, poly- tion test with P-value set at 0.05. All data are expressed as clonal) 1:100,000; TH (Chemicon, monoclonal) 1:250,000; mean6S.E.M.
Fos (Oncogene Sciences, AB-2 or Dr T. Curran’s alu-Fos All procedures were approved by the Cornell University antibody) 1:50,000 and GR (gift of Dr D. DeFranco, Animal Care and Use Committee. All facilities were BUGR2) 1:70,000 were performed for 48 h at 48C. After approved by the American Association for the Accredita-development of the first antibody the tissue was rinsed and tion of Laboratory Animal Care.
the second primary antiserum was applied. Afterward the tissue was again processed for ABC immunoperoxidase staining, but the chromogen used was DAB alone. The
results obtained then had either black nuclei (GR or Fos) 3. Results or black cytoplasmic granules (FAu) with brown
cyto-plasm (CRH or TH). Sections of adult rat hypothalamus At 40 and 60 min of the hypoxemic challenge, maternal served as positive controls. All sections to be compared carotid arterial blood values for PO in the HYPOX group2 within an experiment were processed simultaneously. ewes which were catheterized (n56) ranged from 24.4– In a subset of tissues, we performed triple labeling to 55.4 and 25.3–39.2 Torr, respectively. We know from assess the presence of GR in CA neurons that also were previous experiments (T.J. McDonald, unpublished data) retrogradely labeled with FAu. In those instances, the and from the extensive characterization of the production approach used by Rinaman et al. [30] was employed. of fetal hypoxemia via maternal tracheal gas infusion [12] Briefly that strategy consisted of first staining for GR with that decreasing maternal PO2 via infusion of N2 to the ABC peroxidase and NiDAB visualization, then the sec- maternal tracheal catheter to approximately 40 Torr yields tions were reacted for the presence of TH using a final a corresponding PO2 decrease in our fetuses of 10–14 concentration of the mouse monoclonal antibody of Torr.
1:100,000. After incubation we then applied an alkaline All the TH-immunopositive neurons of the A1 / C1, A2 / phosphatase-conjugated anti-mouse secondary antibody. C2, A5 (n. olivaris superior) and A6 catecholaminergic The alkaline phosphatase was then reacted with Vector Red brainstem areas demonstrated some degree of GR immuno-(Vector Laboratories) according to package inserts. The reactivity, but while all GR immunoreactivity was found to tissue was then rinsed and reacted with the anti-FAu be nuclear, as opposed to cytoplasmic, not all nuclei were antiserum. After incubation with that antibody, biotin entirely filled with GR (Fig. 1).
Fig. 1. Micrographs of the various brainstem CA cell areas double labeled for GR (black nuclei) and TH (brown cytoplasm). In A1, A2, A5 and A6, all the TH-immunopositive neurons showed staining for GR, but not all nuclei were entirely filled with GR (arrows). Scale bar5100mm.
depicted the largest proportion were found in A1 / C1 was significantly correlated with Fos activation in A1 / C1
2 2
followed by A6, A5 and A2 / C2 CAergic areas. (r 50.894; P,0.005) and A2 / C2 (r 50.848; P,0.002), 2
In order for CAergic neurons of the brainstem to be in a but not A6 / C6 (r 50.636; P50.065) areas. position to influence the physiology of PVN neurons it was
important to establish that neurons of this phenotype also
contain GR and project fibers to the PVN. Therefore triple 4. Discussion staining of brainstem sections was performed to establish
Fig. 2. Line drawing of a unilateral injection site of FAu in a fetal sheep PVN. The section was also stained for CRH, thus indicating the parvocellular portion of the PVN. Note that the injection site encompassed the dorsal portion of the CRH neurons of the PVN.
While the presence of GR throughout the brain has been the amount and location of GR in TH-positive neurons on demonstrated in fetal rats [10], to the best of our knowl- the same slide suggesting a dynamic role for GR at these edge this is the first study to demonstrate the presence of sites.
GR in CA-immunopositive neurons of the brainstem in the Retrograde labeling of fetal sheep brainstem CA neurons fetus of any species. While immunocytochemistry is only a after FAu injection to PVN produced both similarities and semi-quantitative method, we did see large differences in differences with respect to axon tracing studies in the rat.
an effective and repeatable stimulus for ACTH secretion [24] and Fos activation of CRH neurons in the PVN [17]. In the present study, we purposely varied maternal PO2 values from normoxic to markedly hypoxic in order to provide a varied stimulus to the fetal sheep central nervous system that would allow us to investigate whether or not CA-immunopositive brainstem neurons and PVN CRH neurons are activated in concert. We chose not to instru-ment the fetuses because we know that this constitutes a stress to the fetus in its own right e.g., the hypothalamo– pituitary–adrenal axis is instrumental in initiating labor and delivery in sheep [7] and in our flock, instrumented fetuses deliver 4 to 5 days earlier than non-instrumented fetuses. Precise knowledge of the degree of hypoxemia in Fig. 4. Percent of CA-immunopositive neurons in various brainstem each fetus was less important in this paradigm because we nuclei of late gestation fetal sheep (n53) which were retrogradely labeled
were not trying to establish an exact correlation between by FAu injected into the PVN.
the degree of hypoxemia and the amount of neuronal Fos immunoreactivity. It was only necessary to provide a range The percentages of retrogradely labeled TH-immuno- of hypoxemic stimuli in the fetal brainstem and PVN to positive cells found in the A1 / C1, A2 / C2 and A6 areas in determine that correlations between Fos activation in A1 / four rats were 68, 26 and 6 [31] and in the fetal sheep were C1 and A2 / C2 neurons existed with Fos activation in PVN 40.6, 3.3 and 17.2, respectively. Clearly the variation neurons (P,0.05).
between these data sets could be due to species, de- It might be suggested that the increase in Fos activation velopmental and FAu injection parameter differences, but in various areas was due to the range in ages of the fetuses it is reassuring that both studies find the largest projection used in the study. However, the age spread in the two to be to the A1 / C1 area by a factor of approximately 2 groups of fetuses was very similar (HYPOX, 124–144 and compared to the next largest projection. The demonstration NORMOX, 125–141) while in contrast, the amount of Fos of the presence of GR specifically in CA cells of the expression seen in HYPOX fetuses went as high as 80% A1 / C1 area that project to the PVN (i.e., display FAu) for CRH cells and 60% for CA cells, but in NORMOX greatly strengthens the idea that in the late gestation fetal fetuses was never greater than 15% on either axis. In sheep, a population of brainstem and PVN neurons are addition, we have previously demonstrated that levels of working in concert to influence glucocorticoid secretion as Fos activation in CRH neurons of the PVN in unperturbed has been demonstrated in the adult rat [22]. fetal sheep at both 125 and 145 dGA were less than 5%, We have previously utilized infusion of nitrogen gas to but rose to 60% with a hypoxemic challenge similar to that maternal tracheal catheters to produce fetal hypoxemia as in the present study [18].
Fig. 6. Plots of Fos activation in CRH neurons of the PVN from three fetal sheep which displayed: (A) little Fos activation of CRH and other PVN neurons, (B) a moderate degree of Fos activation in CRH and other PVN neurons, and (C) a high degree of Fos activation in CRH-immunopositive and other PVN neurons. Panels (D–F) illustrate the actual staining patterns of A1 / C1 neurons, from the same animals seen in panels A–C, double labeled for TH (grey cytoplasmic stain) and Fos (black nuclear stain). Note: (1) the animal with little Fos activation in CRH neurons has A1 / C1 neurons devoid of Fos-immunopositive nuclei, (2) with moderate activation in PVN CRH neurons, A1 / C1 CA-immunopositive neurons show increased Fos activation, and (3) the A1 / C1 area shows further increases in Fos activation that encompasses a greater number of CA neurons during strong PVN activation. Squares indicate CRH neurons, filled squares represent Fos-activated CRH neurons and non-CRH neurons that express Fos are indicated by small dots. Scale bar515mm.
hypothalamo–pituitary–ad-neurons projecting to PVN CRH hypothalamo–pituitary–ad-neurons appear to be somewhat different from the adult rat, the present study supports the hypothesis that brainstem A1 / C1 and A2 / C2 neurons are in position to influence the function of the fetal sheep hypothalamo–pituitary–adrenal axis in response to hypoxemia during late gestation.
Acknowledgements
Supported by National Institutes of Health — HD 21350.
References
Fig. 7. Correlation of Fos activation in CRH-immunopositive neurons of
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