Introduction amine CA synthesizing enzymes [1,20,28]. These brain-

Brain Research 885 2000 70–78 www.elsevier.com locate bres Research report Brainstem catecholaminergic neurons activated by hypoxemia express GR and are coordinately activated with fetal sheep hypothalamic paraventricular CRH neurons a , b b Thomas 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 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 chemoreception and the PVN participates in Corresponding author. Tel.: 11-607-253-3086; fax: 11-607-253- cardiovascular regulation via direct projections to rostral 3455. E-mail address : tjm2cornell.edu T.J. McDonald. ventrolateral medullary sympathetic premotor neurons and 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 3 6 - X T .J. McDonald et al. Brain Research 885 2000 70 –78 71 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 either 2 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 the 2 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 was 2 greatly decrease or cease their in utero breathing move- adjusted to achieve differing maternal PO values over the 2 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 maintained 2 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 and 2 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 N flow was terminated and ewes and fetuses were 2 [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 1000