Medullary pathways for adrenocorticotropic and vasopressin secretion in rabbits Z. J. GIEROBA, M. J. FULLERTON, Centre for Neuroscience, Departments Flinders University of South Australia, and Baker Medical Research Institute,

J. W. FUNDER,

W. W. BLESSING

of Medicine and Physiology,

Bedford Park, South Australia 5042; Prahran, Victoria 3181, Australia

Gieroba, 2. J., M. J. Fullerton, J. W. Funder, and W. W. Blessing. Medullary pathways for adrenocorticotropic hormoneand vasopressinsecretion in rabbits. Am. J. Physiol. 262 (Regulatory Integrative Comp. Physiol. 31): R1047-R1056,

1992.-We determined, in urethan-anesthetized rabbits, whether pharmacologicalalteration of neuronal function in the ventrolateral medulla oblongata, including the Al area, and in the nucleustractus solitarii (NTS), alters plasma adrenocorticotropic hormone (ACTH) and vasopressinand whether inhibition of neuronal function in the ventrolateral medullaimpairs the secretion of ACTH normally observedin responseto hemorrhageor constriction of the inferior vena cava. We alsotested whether the increasein plasma ACTH and vasopressinafter pharmacologicalinhibition of neuronal function in the NTS is dependenton a pathway that synapsesin the Al area of the ventrolateral medulla. Activation of the Al area with bicuculline increasedboth ACTH and vasopressin.Inhibition of the NTS with muscimolincreasedlevels of both hormones,as did hemorrhageand constriction of the inferior vena cava. Inhibition of neuronal function within the Al area with muscimol eliminated the secretionof vasopressinbut did not significantly alter the secretion of ACTH, obtained by injecting muscimol into the NTS. Injection of muscimolinto the Al areaeliminated the secretion of both ACTH and vasopressinin responseto constriction of the inferior vena cava and, in the caseof vasopressin, in responseto hemorrhage.Although hemorrhage-initiated secretionof ACTH wassignificantly reducedby injection of muscimolinto the Al area, it was not completely eliminated by these injections or by injections of muscimol into a more rostrocaudally extensive region of the medulla oblongata. We conclude that the net output from the NTS tonically inhibits secretion of both ACTH and vasopressin, reflecting tonic baroreceptor tone. For vasopressin,the pathway from the NTS to the hypothalamus is dependenton a synapsein the Al area. For ACTH, there are pathways to the hypothalamusthat do not synapsein the Al area, but neurons in this region do have an excitatory effect on secretion of ACTH. nucleus tractus solitarii; ventrolateral medulla; hemorrhage; baroreceptors;Al catecholamineneurons;y-aminobutyric acid; excitatory amino acids SYSTEMIC HEMORRHAGE, severe enough to reduce central venous pressure, causes secretion of both adrenocorticotropic hormone (ACTH) and vasopressin by a centrally mediated reflex that depends on afferent information traveling in the ninth and tenth cranial nerves (11, 15, 27). These nerves terminate in the nucleus tractus solitarii (NTS), and the reflex is then transmitted to the appropriate hypothalamic and pituitary centers. Chemoreceptor stimulation has also been shown to initiate secretion of both ACTH and vasopressin (24, 28). The central pathway for hemorrhage-induced vasopressin secretion probably involves a projection from the NTS to the Al norepinephrine-synthesizing neurons in the caudal portion of the ventrolateral medulla, with subsequent direct activation of the magnocellular 0363-6119/92

AND

hormone

neurons in the supraoptic and paraventricular nuclei of the hypothalamus (6). The corresponding pathway for ACTH secretion has not yet been determined, but noradrenergic receptors in the hypothalamus may be involved (20). Secretion of ACTH can be affected by experimental procedures within the NTS (15), and lesioning the NTS in rats eliminates hemorrhage-induced secretion of ACTH (10). Bereiter and Gann (2) showed that electrical stimulation of the region containing the Al cells caused secretion of ACTH in the cat; Carlson and Gann (7) made electrolytic lesions in the caudal ventrolateral medulla in the rat and observed that secretion of ACTH in response to hemorrhage was impaired but not eliminated. The present study was carried out in the rabbit to determine whether pharmacological manipulation of neurons in the Al area causes secretion of ACTH as well as vasopressin in this species, whether pharmacological manipulation of the NTS causes secretion of ACTH and vasopressin, and whether pharmacological blockade of either the NTS or the Al area interferes with secretion of these hormones initiated by baroreceptor or chemoreceptor stimulation. METHODS &anesthetized

Rabbits

The first experimentswere carried out on 14 unanesthetized New Zealand White rabbits (2.5-3 kg) to determine resting ACTH values in this speciesand to measurethe responseof ACTH and vasopressinto acute reduction in central venous pressure.In a preliminary procedure, l-2 wk before the first experiment, rabbits were anesthetized with thiopental sodium (40 mg/kg iv) and intubated. General anesthesiawas maintained with l-1.5% halothane in oxygen. The animal was mechanically ventilated, and thoracotomy was performed. A constrictor cuff wasplacedaround the inferior vena cava (IVC), just abovethe diaphragm.Tubing connectedto the cuff wasleft subcutaneouslyin a dorsalposition, from which it could subsequently be retrieved. By graded inflation of the cuff it was possibleto reduce venous return, thereby lowering atria1 and arterial pressure.The surgical wound was repaired, anesthesia was discontinued, and the rabbit wasextubated. On the day of the experiment the rabbit wasplaced in a small cage.One central ear artery was cannulated (under local anesthesia) for recording of blood pressureand heart rate. Mean arterial pressure (AP) was obtained by passingthe phasic signal from a Statham P23 ID pressuretransducer through a low-passfilter, and heart rate (HR) wasmeasuredwith a GrassP4 tachometer. The signalswere displayed on a Grassmodel 7 polygraph. For radioimmunoassay of plasma ACTH and vasopressin (see below) 5 ml of blood wastaken from the central ear artery, with simultaneousreplacementinto a marginal ear vein of an equal volume of warm Ringer, containing red cellsfrom the previous sample.Plasma sampleswere obtained 15 min after ear artery cannulation, and animalsthen rested for 5 min. The IVC cuff

$2.00 Copyright 0 1992 the American Physiological

Society

R1047

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OF ACTH AND VASOPRESSIN

wasthen partially inflated for 3 or 6 min, during which time AP was reducedto -40 mmHg, with an accompanyingincreasein HR. The cuff was then deflated, and plasma sampleswere obtained after 5 and 15 min. Cannulaswere removed, and the balloon tubing was securedin its subcutaneousposition. Rabbits were then returned to the animal housefor at least 5 days before being useda secondtime in similar experiments. After return to the animal housefor at least 1 wk, rabbits were used in someof the studiesunder anesthetic describedbelow. Anesthetized

Rabbits

Experiments were carried out on 69 rabbits anesthetizedwith urethan (1.5 g/kg iv infusion over 30 min). A tracheal tube was inserted.The femoral artery wascannulated for measurementof cardiovascularvariables and sampling of blood for ACTH and vasopressin.Body temperature wasmonitored by a rectal probe and maintained between38 and 39°C. Rabbits were placedin a modified Kopf stereotaxic apparatus, paralyzed with pancuronium bromide (0.5 mg/kg iv), and artificially ventilated with oxygen-enriched air. End-tidal Pcoz was kept between 30 and 35 mmHg. In someanimalsblood gaseswere measuredin 2-ml arterial blood samples.The dorsal surface of the medulla was exposedby incision and retraction of the atlantooccipital membrane, and the degreeof neck flexion was adjusted so that the dorsal surface of the medulla was horizontal. The medullary surface was covered with warm Ringer solution. Bilateral intramedullary stereotaxic injections were made through glass micropipettes with beveled tips (OD 20-30 pm) and long shanks.The volume of each injection (100nl) wasmonitored by observingmovement of the meniscusin the pipette in response to pressuretransmitted from a hand-held syringe. Intramedullary injection sites and histological examination. In 85% of rabbits, horseradishperoxidase(HRP, final concentration 0.1%) was added to the injectate before pharmacological injections into the NTS or the ventrolateral medulla. Animals were killed with an overdose of pentobarbital sodium (100 mg/kg iv) and perfused via the heart with buffered formaldehyde-glutaraldehyde. Transversesections(50 pm) were madeof the medulla, and HRP was detected by the diaminobenzidine reaction (Fig. 1, A and B). Injections into the NTS were madebilaterally, at two rostrocaudal levels. The first level was 1 mm caudal to the rostra1 border of the area postrema, at the lateral edge of the area postrema and 0.5 mm below the dorsal surface of the medulla. The secondsite wasat the level of the nucleuscommissuralis,in the midline, 0.3 mm below the dorsal surface. Agents injected into the NTS-dorsal vagal nucleustend to spreadrostrocaudally within these nuclei without extensive spreadto nearby structures such as the hypoglossaland gracile and cuneate nuclei (Fig. lA), possibly becausethese heavily myelinated structures form diffusion boundaries. In previous experiments on control of vasopressinsecretion by neuronsin the ventrolateral medulla,we concentrated on the Al cells located just caudal to the obex level (6). The injection site for this midportion of the caudal ventrolateral medulla (called “the Al area”) was 1 mm caudal to the rostra1border of the area postrema, 3 mm lateral from the midline, and 3 mm below the dorsal surface of the medulla (6). Becausethe distribution of Al neuronsprojecting to the paraventricular nucleus is more extensive in the rostrocaudal direction (3, 4), we included, in the present ACTH experiments, some studies in which inhibitory agents were injected into the ventrolateral medulla at three rostrocaudal levels. The most rostra1bilateral injections were madeat the level of the obex, 3 mm lateral from the midline, and 3 mm below the dorsal surfaceof the medulla. The histological marker wasobservedto diffuse - 1 mm rostra1 to this level (Fig. 1). The secondbilateral injections were _ _._ made. into the Al area (1 mm caudalto the obex), and a third bilateral

Fig. 1. Photomicrographs of Nissl stains of rabbit nucleus tractus solitarii (A) and Al area (B) at rostrocaudal level 1 mm caudal to rostra1 border of area postrema. Dark areas are horseradish peroxidase reaction product indicating injection sites. C: diagrammatic representation of experimental sites showing spread of horseradish peroxidase included with injectate, at 3 rostrocaudal levels of medulla (on left side, distance from obex). Bars, 0.8 mm (A); 1.1 mm (B); and 2.0 mm (Cl. AP, area postrema; IO, inferior olive; LRN, lateral reticular nucleus; nA, nucleus ambiguus; NTS, nucleus tractus solitarii; Vsp, spinal nucleus of trigeminal nerve; XII, hypoglossal nucleus.

pair of injections wasmade2 mm caudalto the obex, also3 mm from the midline and 3 mm below the dorsal surface of the medulla. Pharmacological

agents

used in intramedullary

injections.

Agents were dissolved in Ringer and bilaterally injected in 100-m amounts. The following agents (Sigma Chemical, St Louis, MO) were used:excitatory amino acids, L-glutamate (50

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nmol), N-methyl-D-aspartic acid (NMDA, 50 pmol); antagonist for NMDA receptors, DL-2amino-5-phosphonovaleric acid (APV, 5 nmol); y-aminobutyric acid (GABA, 100nmol); GABA receptor agonist, muscimol hydrochloride (1 nmol); GABA receptor antagonist, bicuculline methiodide (100 pmol). Effects of intramedullary

injections

of

phurmacological

agents.

Plasmasampleswere obtained after insertion of the tracheotomy tube and cannulation of the femoral artery. A secondsample was obtained 5 min after completion of medullary surgery and bilateral injection of 100 nl of Ringer solution into either the NTS or the Al area. Pharmacological agents were then injected into either one of these regions, and plasma samples were collected after 5 min. In control experiments, bicuculline was injected either 1.5 mm medial or 2 mm dorsal to the Al area.In a separateseriesof experiments,muscimol(1 nmol) was injected into the Al area before muscimolwasinjected into the NTS. Injection of bicuculline into the Al area causedan acute fall in AP and HR. To ensurethat any changesin plasmahormone levels were not secondary to baroreceptor-mediated effects of the fall in AP, changesin both AP and HR were prevented by blocking the sympathetic pathways in the spinal cord and the cardiac effect of the vagus. The spinal cord was exposed by laminectomy at the secondcervical level. Scopolaminemethyl bromide was injected (50 pg/kg iv). An intravenous infusion of norepinephrine was commenced (5-10 ,ug*kg-l. min-l), and tetrodotoxin (Sigma Chemical, 20 pmol in 200 nl Ringer) was injected bilaterally into the dorsolateral funiculus to inactivate the descending sympathoexcitatory axons. Resting AP was maintained at 88 t 5 mmHg (n = 6). Ringer was injected into the Al area, and a control plasmasamplewas obtained. Bicuculline wasthen injected into the Al area, and another plasma samplewasobtained for hormonal assay5 min later. Effect of prior intramedullary injection of pharmacological agents on ACTH and vasopressin responses to IVC constriction and hemorrhage. Animals with IVC cuff occluders in place

OF ACTH

AND

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R1049

plasmasampleswere shakenwith Vycor glassfor 30 min on an IKA Vibrax-VXR agitator. Sampleswere centrifuged (3,000 rpm, 5 min), and plasma was aspirated. Pellets were resuspended in distilled water and centrifuged (3,000 rpm, 5 min), and the supernatant was aspirated. To elute peptidesfrom the Vycor glass,1 ml 80% MeOH-0.1 M HCl wasaddedand tubes were agitated for 30 min. Further centrifugation (3,000 rpm, 15 min) allowed recovery of supernatantsthat were then dried on a Savant vacuum apparatus.Dried extracts were reconstituted in radioimmunoassaybuffer just before radioimmunoassay. The antiserum (R72) was raised against human ACTH (l-24) coupled to bovine serum albumin (BSA). The assayis specific for ACTH, molar cross-reactivities of structurally related peptidesbeing: ACTH (5-24) 2.1%, a-melanocyte stimulating hormone (cu-MSH) < O.l%, corticotropin-like intermediate lobe peptide. (ACTH fragment 18-39; CLIP) < O.l%, y-MSH < O.l%, P-endorphin (EP) < 0.1%. 1251-ACTHwas generatedby the iodogenmethod, and 1251ACTH was separatedfrom the iodide on a PDlO G-25 gel filtration column eluted with 0.1% BSA-0.25 M acetic acid. Tracer wasfurther diluted in 0.1% BSA-0.25 M acetic acid and stored in frozen aliquots for up to 8 wk. Specific activity of tracer was routinely 300-400 &i/hg. The radioimmunoassay for ACTH used human ACTH 74/555 (National Institute for Biological Standards and Control, London) as standard, and sensitivity of the radioimmunoassaywas 2 pg/tube. The intraand interassay coefficients of variation were 11 and 25%, respectively. Vasopressin radioimmunoassay. Blood samples(2 ml) were heparinized, stored on ice, and centrifuged within 20 min of collection. The plasma was stored at -20°C until assay (l-4 wk). Plasmavasopressinlevels were measuredby radioimmunoassay(1, 6). Briefly, plasma sampleswere extracted onto a cation-exchangeresin, Amberlite CG-50 (Sigma),and the vasopressinwaseluted from the columnswith 75%ethanol in water, pH 1.6. Recovery varied between 70 and 100%. Vasopressin antibody, usedat a dilution of 150,000,was kindly suppliedby Dr. P. Howe (Adelaide, Australia). Iodinated vasopressinwas purchasedfrom New England Nuclear (Melbourne, Australia), and unlabeled vasopressinfrom Auspep (Melbourne). Bound vasopressin was precipitated with polyethylene glycol and human y-globulin. The effective minimum of the assaywas 1 pg/ml, and the effective maximum was 640 pg/ml. The intraand interassay coefficients of variation were 16 and 19%, respectively.

received injections of either Ringer or muscimol (1 nmol) into the Al area.The IVC wasthen occludedfor 5 min to reduceAP to between40 and 50 mmHg. The cuff was then deflated, and plasmasampleswere obtained after 5 and 15 min. Animals were then acutely hemorrhaged(15 ml/kg over 3-5 min) from the femoral artery catheter until AP stabilized at -50 mmHg, and a further plasmasamplewascollected5 min after the beginning of the hemorrhage.Removal of sufficient blood to reduce arterial pressureto 50 mmHg has been shown to reliably increase plasmaACTH and plasmavasopressin(6, 11). In other animalsRinger or muscimolwasinjected into the Al Statistical Analysis area or the NTS, and plasmasampleswereobtained 5 min after In -10% of anesthetized rabbits, resting levels of ACTH Ringer or muscimolinjection and 5 min after AP wasreducedto were~2,000 pg/ml and/or resting levelsof vasopressinwere>4O -50 mmHg by hemorrhage. pg/ml; theseanimalswere excluded from further analysis.Data Effect of hypoxia-hypercapnia on plasma ACTH and were analyzed by one- or two-way analysis of variance, with vasopressin. Theseexperiments werecarried out usingthe same repeatedmeasureson the samerabbits in a given experimental protocol for anesthesia and surgical preparation as for condition. Significance of effects was assessedwith Fisher’s intramedullary injections. Ringer was injected bilaterally into protected t test as a post hoc comparison. When variances of the Al area. Chemoreceptorswere activated by changing the sampleswere markedly different, statistics were performed on inspired gasto 10% 02-5% CO, in N2 (CommonwealthIndus- logarithmic transformations of the data. Pearson’scorrelation trial Gases,Sydney, Australia) for 5 min. Arterial blood samples was usedto comparethe changesin plasmaACTH and vasofor bloodgasand pH analysisand for hormone assayweretaken pressinin responseto someof the experimental procedures.All before, during, and 5 min after chemoreceptorstimulation. results are expressedas means* SE. ACTH radioimmunoassay. Blood samples(2 ml) were collected on ice into heparinized tubes containing 100 ~1of inhibitor mix comprising400 KIU aprotinin, 0.2 M N-ethylmaleim- RESULTS ide, and 50mM disodiumEDTA. Sampleswere centrifuged, and Injection Sites eachplasmasamplewas stored at -20°C in duplicate aliquots, Figure 1 shows the position of the injection sites in the preliminary experiments having shown rabbit ACTH to be Al area, in the more rostra1 and caudal ventrolateral unstableto repeat freeze-thawing. Plasma ACTH was measured by a modification of the medulla, and in the NTS, as demonstrated by the spread method previously described(13). In an extraction procedure, of HRP included with the injectate. The myelinated

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regions around the NTS appeared to restrict the spread of the HRP in the ventral and lateral planes, and HRP staining spread along the rostrocaudal extent of the NTS from the commissural nucleus to the region of the nucleus - 1 mm rostra1 to the rostra1 border of the area postrema. The HRP staining in the ventrolateral medulla also spread along the the rostrocaudal axis of the medulla but not further than 1 mm rostra1 to the rostra1 border of the area postrema. Control injections of bicuculline 1.5 mm medial or 2 mm dorsal to the center of the Al area did not affect AP, plasma ACTH, or plasma vasopressin. No animals were excluded on histological criteria. Intramedullary injections made through the exposed dorsal surface of the medulla can be controlled quite accurately. Injections of pharmacological agents into the ventrolateral medulla have immediate effects on AP (1,5, 6), and these changes occurred in every animal, providing reassurance that the injections were accurately made.

OF ACTH AND VASOPRESSIN

Data from -10% of animals were excluded from further analysis because the baseline plasma vasopressin was >40 pg/ml or the plasma ACTH was >2,000 pg/ml. In unanesthetized rabbits the resting level of ACTH was 276 + 70 pg/ml (n = 15) and the resting level of vasopressin was 1 + 1 pg/ml (n = 15). After urethan anesthesia, tracheal tube insertion, and femoral artery catheterization, plasma ACTH was 699 + 86 pg/ml (n = 23) and plasma vasopressin was 2 + 1 pg/ml (n = 21). After mechanical ventilation and surgical procedures on the medulla oblongata with intramedullary injection of Ringer solution, plasma ACTH was 876 + 78 pg/ml (n = 37) and plasma vasopressin was 5 + 2 pg/ml (n = 32).

2. When measured 5 min after cuff deflation, ACTH had increased from 276 +: 70 to 361 f 68 pg/ml (n = 15, P < 0.05) and vasopressin had increased from 1 -+ 1 to 17 -C 3 pg/ml (n = 15, P < 0.01). Fifteen minutes after cuff deflation, ACTH had returned to 247 f 43 pg/ml (n = 15), a value not significantly different from the basal preconstriction level. At this time plasma vasopressin was 11 k 5 pg/ml (n = 13), the relatively large standard error reflecting 2 of the 13 animals in which measured values did not return toward baseline. The correlation between the increase in ACTH and the increase in vasopressin, measured 5 min after cuff deflation, was 0.75 (n = 13, P < 0.01). Constriction of the IVC in anesthetized rabbits also decreased AP from 98 f 2 to 50 k 1 mmHg (n = 5, P < 0.01) and increased HR from 320 + 18 to 342 -t 18 beats/ min (n = 5, P < 0.05). Five minutes after cessation of constriction, plasma ACTH increased from 805 f 146 to 1,262 f 258 pg/ml (n = 5, P < 0.05) and plasma vasopressin increased from 14 + 7 to 153 + 36 pg/ml (n = 4, P < 0.05) (Fig. 2). Fifteen minutes after cessation of constriction, plasma ACTH returned to 543 + 48 pg/ml (n = 3). The increase in plasma levels of both hormones in response to IVC constriction was greater in the anesthetized animals (P < 0.01). The baseline values for ACTH were also greater in the anesthetized animals. Hemorrhage in anesthetized animals decreased AP from 95 f 5 to 41 + 3 mmHg (n = 13, P < 0.01) and increased HR from 298 f 11 to 321 f 8 beats/min (n = 12, P < 0.01). Plasma ACTH increased from 1,015 k 152 to 2,272 k 291 pg/ml (n = 13, P < 0.01) and plasma vasopressin increased from 12 -I- 6 to 345 + 49 pg/ml (n = 11, P < 0.01, Fig. 2).

Cardiovascular and Hormonal Response to WC Constriction and Hemorrhage

Cardiovascular and Hormonal Responses to Hypoxia-Hypercapnia

Constriction of the IVC for 3-6 min in unanesthetized rabbits reduced AP from 75 + 1 to 41 + 1 mmHg (n = 15, P < 0.01) and increased HR from 188 + 3 to 260 f 6 beats/min (n = 13, P < 0.01). When the cuff was deflated, AP and HR returned to preconstriction levels within 1 min. Effects on plasma hormonal levels are shown in Fig.

During ventilation with hypoxic-hypercapnic gas mixture there was a small increase in AP (from 97 + 3 to 103 + 3 mmHg, n = 7, P < 0.01) and a large decrease in HR (from 273 f 21 to 208 zk 24 beats/min, n = 6, P < 0.01). We measured the arterial blood gases at the end of the 5-min period, just before reintroduction of the normal gas

Basal Values for Plasma ACTH and Vasopressin

15

15

5

4

13

11

3

10

Fig. 2. Effect on plasma ACTH and vasopressin of various experimental procedures in unanesthetized and anesthetized rabbit. n = no. of rabbits in each group. IVC, inferior vena cava. Significantly different from control value: * P < 0.05, ** P < 0.01.

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mixture, and found that PO, fell from 401 + 18 to 40 + 3 mmHg (n = 8, P < O.Ol), PCO~ increased from 36 + 3 to 53 + 2 mmHg (n = 8, P < O.Ol), and pH fell from 7.43 +0.02 to 7.28 + 0.01 U (n = 8, P < 0.01). However, the chemoreceptor stimulation did not change either plasma ACTH (653 + 160 to 708 + 158 pg/ml; n = 3, P > 0.05) or plasma vasopressin (11 + 4 to 27 f 11 pg/ml, n = 10, P > 0.05) (Fig. 2). Effects of Pharmacological Agents Injected Into Al Area or Into Three Levels of Ventrolateral Medulla Microinjection of NMDA into the Al area decreased AP from 81+ 1 to 34 + 6 mmHg (n = 3, P < 0.05) and HR from 255 + 5 to 125 + 25 beats/min (n = 3, P < 0.05). These changes lasted for -3 min after which AP and HR returned to their preinjection values. When measured 5 min after injection of NMDA, plasma ACTH was unchanged (480 + 227 compared with 570 + 300 pg/ml, n = 3, P > 0.05), but plasma vasopressin had increased (from 2 + 1 to 147 + 78 pg/ml, n = 3, P < 0.05) (Fig. 3). We reasoned that NMDA might not act for sufficient time to alter plasma ACTH so we used bicuculline to produce an excitation of neurons in the Al area, which lasts for at least 5 min (3). Plasma ACTH, measured 5 min after injection of bicuculline into the Al region, increased from 1,077 & 265 to 4,430 f 737 pg/ml (n = 5, P < O.Ol), and plasma vasopressin increased from 2 + 1 to 238 -C 54 pg/ml (n = 4, P < 0.01). The levels were still increased from the baseline when measured 15 min after injection. Injection of bicuculline into the Al area decreased AP (92 f 2 to 37 f 6 mmHg, n = 5, P < 0.01) and HR (311 +. 13 to 225 + 38 beats/min, n = 5, P < 0.01). After vagal blockade with intravenous scopolamine methyl bromide and cervical spinal blockade with injection of tetrodotoxin into the cervical cord AP was maintained at 88 + 5 mmHg (n = 6) by intravenous infusion of norepinephrine. In these circumstances there was no fall in AP or HR when bicuculline was injected into the Al area. Indeed, AP gradually increased during the 5-min period after injection of bicuculline, from 88 + 5 to 105 f 7 mmHg (n = 6, P < 0.05). Plasma ACTH increased from 1,933 + 474 to 2,315 & 490 pg/ml (n = 6, P < O.Ol), and

OF ACTH AND VASOPRESSIN

R1051

plasma vasopressin increased from 11 f 7 to 219 f 43 pg/ml (n = 7, P < 0.01, Fig. 3). Bilateral injection of bicuculline 1.5 mm medial (n = 2) or 2 mm dorsal to the Al area (n = 2) did not change plasma ACTH or plasma vasopressin. Bilateral injection of muscimol (1 nmol) into the Al area caused AP to increase from 105 +- 3 to 126 + 4 mmHg, n = 10, P < O.Ol), with a variable change in HR. A greater increase in AP (from 111 f 3 to 171 Z!Z5 mmHg, n = 6, P < O.Ol), together with an increase in HR (from 280 f 10 to 316 +: 13 beats/min, n = 5, P < 0.05), was observed after bilateral injection of muscimol into the ventrolateral medulla at all three rostrocaudal levels shown in Fig. 1. When muscimol was injected bilaterally into the Al area, plasma vasopressin was slightly, but significantly, reduced from 14 f 6 to 8 + 3 pg/ml (n = 8, P < 0.05) (Fig. 3). This procedure produced no change in plasma ACTH (648 + 109 before and 549 -I 130 pg/ml5 min after, n = 10, P > 0.05). Similarly, there was no change in the level of ACTH when muscimol was injected bilaterally into the ventrolateral medulla at all three rostrocaudal levels (623 + 84 before and 780 + 168 pg/ml5 min after, n = 6, P > 0.05). Effect of Pharmacological Agents Injected Into NTS After NTS injections of Ringer there was no change in AP, HR, or plasma levels of ACTH or vasopressin. Injection of 50 nmol of L-glutamate did not cause any consistent change in AP or HR. This was a surprising finding, so we further investigated this result by injecting into different regions of the NTS amounts of L-glutamate and NMDA, from 1 pmol to 100 nmol, in volumes ranging from 10 to 100 nl. No consistent changes in AP or HR were observed. Injection of 50 nmol of L-glutamate did not change plasma ACTH or vasopressin (Fig. 4). Injection of GABA did not consistently affect AP, but HR increased from 276 + 25 to 348 +. 26 beats/min (n = 5, P < 0.01). GABA did not significantly alter plasma levels of ACTH but it increased plasma vasopressin from 5 + 4 to 58 f. 32 pg/ml (n = 5, P < 0.05) (Fig. 4). Injection of bicuculline into the NTS did not significantly change AP but acutely reduced HR by 55 rt 14 beats/min (n = 8, P < 0.01). Bicuculline

spinal, and cardiac vagal, blockade

T

”

3

i

i

1

6

7

10

*

Fig. 3. Effect on plasma ACTH and vasopressin of injection of N-methyl-D-aspartic acid (NMDA; 50 pmol), bicuculline (100 pmol), bicuculline after spinal and vagal blockade, and muscimol (1 nmol) into Al area. n = no. of- rabbits in -each group. Significantly different from control value: * P < 0.05, ** P < 0.01.

Fig. 4. Effect on plasma ACTH and vasopressin of injection of L-glutamate (50 nmol), GABA (100 nmol), bicuculline (100 pmol), and muscimol (1 nmol) into NTS. n = no. of rabbits in each group. Significantly different from control value: * P < 0.05, ** P < 0.01.

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AND SECRETION

injections into the NTS did not change plasma levels of ACTH or vasopressin (Fig. 4). Injection of muscimol into the NTS produced, in individual animals, large changes in AP. However, the effects were variable, and statistical analysis showed that the mean changes were not significantly different from zero. Injection of muscimol did, however, increase HR (from 288 f 10 to 308 f 10 beats/ min, n = 10, P < 0.05), and both plasma ACTH (from 1,031 + 224 to 1,471 + 296 pg/ml, n = 10, P < 0.01) and plasma vasopressin (from 18 + 6 to 316 + 48 pg/ml, n = 10, P < 0.01) (Fig. 4), measured 5 min after injection of the muscimol. Effects of Pharmacological Agents Injected Into NTS After Prior Injection of Muscimol Into Al Area

In eight rabbits, with preinjection of muscimol into the Al area, after subsequent injection of muscimol into the NTS, plasma ACTH increased from 582 rf: 157 to 762 + 168 pg/ml (P < 0.05) (Fig. 5). This increase was not significantly different from the increase observed when muscimol was injected into the NTS without prior injection of muscimol into the Al area. This result contrasted sharply with the corresponding results for plasma vasopressin. Prior injection of muscimol into the Al area completely prevented the large increase in plasma vasopressin, which previously occurred when muscimol was injected into the NTS. After blockade of the Al area with muscimol plasma vasopressin was 8 f 3 pg/ml. After subsequent injection of muscimol into the NTS plasma vasopressin was 7 + 2 pg/ml (n = 8, P > 0.05) (Fig. 5). Effect of IVC Constriction After Prior Injection of Muscimol Into Al Area

In anesthetized animals, after injection of Ringer into the Al area, AP was reduced from 98 + 2 to 50 + 1 mmHg (n = 5, P < 0.01) by inflation of the IVC cuff, a fall of 48 +: 2 mmHg. At the same time HR increased from 320 + 18 to 342 + 18 beats/min (n = 5, P < 0.05). As reported

z 3000 3 Fj 2500 2 2000

OF ACTH AND VASOPRESSIN

above, plasma ACTH increased from 805 + 146 to 1,262 + 258 pg/ml (n = 5, P < 0.05) and plasma vasopressin increased from 14 + 7 to 153 -I- 36 pg/ml (n = 4, P < 0.05). After injection of muscimol into the Al area, maximal inflation of the cuff reduced AP from 125 + 4 to 73 + 9 mmHg, a fall of 51 + 8 mmHg (n = 5) not significantly less than the AP fall caused by cuff inflation after injection of Ringer. It was not possible to reduce AP to 50 mmHg by inflation of the IVC cuff. In this situation plasma ACTH did not change (986 f 245 pg/ml before cuff inflation and 1,169 + 319 pg/ml after a 5-min period of cuff inflation, n = 5, P > 0.05). Plasma vasopressin was also unchanged (19 + 9 to 26 f 14 pg/ml, n = 5, P > 0.05). Effect of Hemorrhage After Prior Injection of APV or Muscimol Into Al Area or After Muscimol Into Three Levels of Ventrolateral Medulla

In control animals, after injection of Ringer into the Al area, hemorrhage (15 ml/kg) reduced AP (95 + 5 to 41 + 3 mmHg, n = 13, P < 0.01) and increased HR (298 + 11 to 321 f 8 beats/min, n = 12, P < 0.01). In this situation (as reported above) plasma ACTH increased from 1,015 + 152 to 2,272 +- 291 pg/ml (n = 13, P < 0.01) (Fig. 5). After injection of muscimol into the Al area it was necessary to increase the amount of the hemorrhage (20-30 ml/kg) to reduce the AP to approximately the same level (from 135 f 8 to 49 f 1 mmHg, n = 8, P < 0.01). After this was done plasma ACTH still increased from 719 f 112 to 1,194 + 199 pg/ml (n = 8, P < 0.01). In the control situation (as reported above) hemorrhage increased plasma vasopressin from 12 + 6 to 345 & 49 pg/ml (n = 11, P < 0.01). Prior injection of muscimol into the Al area completely prevented hemorrhage-induced increase in plasma vasopressin (Fig. 5). After injection of APV (5 nmol) into the Al area, AP increased from 89 f 4 to 112 + 5 mmHg, n = 5, P < 0.01. There was no change in plasma ACTH or vasopressin. Hemorrhage reduced AP to 46 + 2 mmHg, a fall of 65 f

Prior muscimol

%

f&*ml 2” $5 ug

loo0 ii e$ .2. mE

500

0 n

13

11

14

h

5

4

10

10

8

8

Fig. 5. Effect of hemorrhage (left) on plasma ACTH and vasopressin of hemorrhage, after prior injection of Ringer, muscimol(1 nmol) or DL-2-amino-5-phosphonovaleric acid (APV; 5 nmol) into Al area or into 3 rostrocaudal levels of ventrolateral medulla. Effect of injections of muscimol into NTS (right) on plasma ACTH and vasopressin, after prior injection of Ringer or muscimol (1 nmol) into Al area. n = no. of rabbits in each group. Significantly different from control value: * P < 0.05, ** P < 0.01. Significantly less than hemorrhage-induced increase after prior injection of Ringer into Al area: #XP < 0.01.

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MEDULLA

OBLONGATA

AND

SECRETION

6 mmHg (n = 5, P < 0.01). During hemorrhage it was necessary to remove more blood to reduce AP. Five minutes after hemorrhage plasma ACTH had increased from 998 t 275 to 3,431 t 1,169 pg/ml (n = 5, P c 0.05), and plasma vasopressin increased from 37 t 9 to 352 t 83 P6w b = 4, P < 0.05). The plasma vasopressin data from these rabbits have been published separately (I). Five minutes after bilateral injection of muscimol into all three rostrocaudal levels of the ventrolateral medulla AP was 164 t 3 mmHg (n = 6) and plasma ACTH was 780 t 168 pg/ml (see Effects of Pharmacological Agents Injected Into Al Area or Into Three Levels of Ventrolatera1Medulla). Hemorrhage (20-30 ml/kg) reduced the AP to 53 t 1 mmHg, a fall of 111 t 4 mmHg. Plasma ACTH,

measured 5 min after hemorrhage, was 1,222 t 164 pg/ml. This value was significantly greater then the prehemorrhage value (n = 6, P < O.Ol), but the increase was not as great as the ACTH increase observed when rabbits were hemorrhaged after injection of Ringer into the Al area. The ACTH values, basal and posthemorrhage, after injection of muscimol into all three levels were not significantly different from the values recorded after a single bilateral injection of muscimol into the Al area. We therefore combined the ACTH from these two groups. In the combined groups, after hemorrhage, plasma ACTH increased from 745 t 93 to 1,206 t 129 pg/ml (n = 14, P < 0.01) (Fig. 5). However, the increase was significantly less than the increase that occurred when the animals were hemorrhaged after injection of Ringer into the Al area, as documented by the significant interaction effect (P < 0.01) in the appropriate two-way analysis of variance, with repeated measures. DISCUSSION

Plasma Levels of ACTH and Vasopressin

We found baseline plasma ACTH levels of 276 t 70 pg/ml in unanesthetized rabbits, similar to the value of 280 t 30 pg/ml reported by Monnier and Desbals (22) in adult rabbits. Reduction of central venous volume by constriction of the IVC reversibly increased the plasma level of both ACTH and vasopressin, in accordance with the known effect of baroreceptor unloading on the secretion of these hormones (15,27). After general anesthesia with urethan, plasma levels of ACTH increased to 699 t 86 pg/ml, even though the intravenous infusion of urethan was given very slowly, at a rate that did not change plasma levels of vasopressin. This sensitivity of ACTH to anesthesia has been observed by others (10, 24), and the increased basal levels mean that our results must be interpreted with caution. Nevertheless, in the anesthetized rabbit, constriction of the IVC continued to increase plasma ACTH to higher levels than occurred in the unanesthetized animal. The degree of increase in ACTH was significantly correlated with the degree of increase in vasopressin. Plasma levels of both hormones were markedly increased by hemorrhage in the anesthetized rabbit. Thus, despite high basal ACTH levels after anesthesia and surgery, both hormones were affected in the expected manner by reasonably physiological stimuli. The process of repeated plasma sampling, with simultaneous iso-

OF ACTH

AND

VASOPRESSIN

volemic plasma and red cell replacement, plasma ACTH or vasopressin.

R1053 did not alter

Role of Neurons in Al Area and Ventrolateral Medulla in Secretion of ACTH and Vasopressin

Excitation of neuronal function in the rabbit Al area, by either NMDA or bicuculline, increased plasma levels of vasopressin, as has been previously observed (1). NMDA, an excitatory stimulus, did not change plasma levels of ACTH. This may reflect the brief duration of action of the NMDA, as reflected in the short duration (l-2 min) of the depressor response evoked by this agent when injected into the Al area. Secretion of ACTH from the adenohypophysis involves more steps than does secretion of vasopressin from the neurohypophysis, which may in part explain why ACTH stimulation requires a longer duration of neuronal excitation in the Al area. Alternatively, but less likely, the neurons responsible for increasing secretion of ACTH may not possess NMDA receptors. However, when bicuculline was used to excite neuronal function, by removing tonic GABA-mediated inhibition of cells in the Al area, both ACTH and vasopressin levels were markedly elevated, possibly reflecting the longer excitatory action (~5 min) of this agent, as judged by the duration of the accompanying depressor response. In agreement with the present findings, Bereiter and Gann (2) demonstrated that electrical stimulation of the caudal ventrolateral medulla in the cat increased plasma ACTH, and Day et al. (12) showed that stimulation in the Al region excites hypothalamic tuberoinfundibular neurons via catecholamine-containing afferents. Kannan et al. (17) also showed that either electrical or L-glutamate-induced stimulation of Al area produced an excitation of the paraventricular nucleus neurons that project to the median eminence. Anatomic studies in rats also demonstrate that some Al cells project to the region containing the relevant tuberoinfundibular neurons (26). Injection of excitatory amino acid antagonist (APV) into the Al area raised AP, but there was no inhibition of hemorrhage-induced secretion of ACTH and vasopressin. Injection of muscimol into the Al area completely prevented the secretion of vasopressin normally observed with constriction of the IVC and with hemorrhage, as previously demonstrated in the rabbit and rat (6, 16). Injection of muscimol into the Al area prevented secretion of ACTH in response to IVC constriction. In contrast, although injection of muscimol into the Al area, or into the more extensive rostrocaudal region of the ventrolateral medulla, impaired the secretion of ACTH in response to hemorrhage, it did not entirely prevent this response. A significant degree of secretion still occurred, strongly suggesting that ACTH secretion in response to hemorrhage can occur by a pathway from the NTS that reaches the hypothalamus without synapsing in the ventrolateral medulla. It might be argued that the muscimol injections did not completely abolish neuronal function in the ventrolateral medulla. Against this view is the observation that muscimol entirely prevents the secretion of vasopressin, as well as entirely preventing the AP changes normally observed with injection of L-glutamate into the caudal ventrolateral medulla (unpublished observations).

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R1054

MEDULLA

OBLONGATA

AND

SECRETION

When an extracellular recording electrode is inserted from the dorsal surface into the Al area there is a characteristic pattern of neuronal activity, depending on the depth from the dorsal surface (21). After injection of muscimol into the Al area, there is virtual electrical silence in the injected regions (unpublished observations). Muscimol injections into the NTS prevent the bradycardia that normally follows increase in AP. In comparison with IVC constriction, hemorrhage is a more potent stimulus for secretion of ACTH and vasopressin (Fig. 2). After injection of muscimol into the Al area it is difficult to reduce AP to 50 mmHg by IVC constriction. Baroreceptor-induced ACTH and vasopressin secretion are especially marked when AP and central venous pressure are reduced to the level reached in severe hemorrhage (10, 11, 15, 27). It may be that, after muscimol injections into the Al area, the different effects on ACTH secretion observed with hemorrhage and IVC constriction reflect these factors. When bicuculline was injected into the Al region it caused major falls in AP and HR, as previously noted (5). When the cardiovascular changes were entirely prevented by spinal cord and vagal blockade, the bicuculline injections still increased plasma ACTH. However, the size of the increase was reduced. Interpretation of this finding is somewhat complicated because the spinal surgery also increased the preinjection levels of ACTH. In contrast, the increase in plasma vasopressin was just as great in animals with spinal blockade. This result might suggest that the large increase in plasma ACTH, observed after injection of bicuculline into the Al area, may reflect a baroreceptor-mediated response. However, when a similar fall in blood pressure was produced by constriction of the IVC, in the anesthetized animal, the levels of ACTH reached were modest compared with the levels observed after injection of bicuculline into the Al region. It is therefore unlikely that the high plasma levels of ACTH induced by bicuculline could be explained entirely by baroreceptor mechanisms. A direct hypothalamic drive from the activated Al area neurons also contributes. There may, however, be a positive interaction between the hypotension and the stimulation of the relevant neurons in the Al area. In the animals with spinal blockade the AP actually increased during secretion of the ACTH, probably because of the concomitant secretion of vasopressin. This increase in AP may have inhibited secretion of ACTH by a baroreceptor mechanism, competing with the excitation resulting from stimulation of the Al area with bicuculline. For vasopressin there was no interaction between the level of blood pressure and the degree of increase in the level of the hormone induced by injection of bicuculline into the Al area. The different interaction with baroreceptors displayed by the two hormones is consistent with the idea that the neuroendocrine neurons in the Al area are directly linked into the central baroreceptor pathway for secretion of vasopressin but not ACTH. For vasopressin, injection of bicuculline into the Al area removes the baroreceptordependent “amino acid brake” (14), and changes in AP, in either direction, no longer affect the neurons. For ACTH, baroreceptor information reaches the hypothalamus by a

OF ACTH

AND

VASOPRESSIN

pathway that, at least in part, is independent of neurons in the Al area. In the rat, Carlson and Gann (7) found that some vasopressin could still be secreted after lesions in the Al area, but Hashemzadeh-Gargari et al. (16) found that injection of muscimol into the rat Al area completely abolished hemorrhage-induced secretion of vasopressin in this species. In the present experiments plasma ACTH still increased in response to hemorrhage after injection of muscimol into the ventrolateral medulla at three rostrocaudal levels, including the Al area, suggesting that baroreceptor-mediated secretion of ACTH is mediated, at least in large part, by a pathway that travels from the NTS to the hypothalamus without synapsing in the ventrolateral medulla. Our ACTH results are thus in substantial agreement with those of Carlson and Gann (7) . NTS Neurons

and Cardiovascular

Function

Injections of either L-glutamate or NMDA into the rabbit NTS failed to affect either AP or HR. We checked this finding carefully, injecting different volumes and different amounts of L-glutamate and NMDA in various subregions of the NTS, but were unable to elicit a consistent change in AP or HR. Even in individual rabbits there was very little change. Injections of GABA did not affect AP, although they did produce significant tachycardia. Injections of muscimol acutely increased AP in many animals, but large falls occurred in other rabbits, so that the mean change was not significant. Muscimol consistently increased HR. Bicuculline caused very little change in AP, even in individual rabbits; it did, however, consistently decrease HR. We did not determine the relative contribbtions of vagal and sympathetic mechanisms to the changes observed in HR. Obviously, our injections would have directly affected the dorsal motor nucleus of the vagus, where at least some of the cardiac preganglionic neurons may be located. The failure of our NTS injections to elicit consistent changes in AP contrasts with findings in the rat (8) and suggests either that the rabbit and rat NTS neurons have different receptors or, more likely, that the functional organization of the rabbit NTS differs from that of the rat. Neurons with opposing cardiovascular functions may be intermingled in the rabbit NTS, given the opposing cardiovascular effects, for example, of baroreceptor and chemoreceptor inputs to the NTS. NTS Neurons and Plasma Levels of ACTH and Vasopressin

The cardiovascular results, considered above, demonstrate the complexity of the NTS circuitry, thereby emphasizing the need for caution in the interpretation of the hormonal data. However, taken together, our results indicate that, in the anesthetized animal, the net output of the rabbit NTS has a tonic inhibitory effect on both secretion of ACTH from the adenohypophysis and secretion of vasopressin from the neurohypophysis. Acute removal of this tonic inhibitory output results in large increases in plasma levels of both hormones. Because baroreceptor and chemoreceptor inputs to the NTS have opposing influences on secretion of both ACTH and vasopressin, our results suggest that the net output from the

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MEDULLA

OBLONGATA

AND

SECRETION

NTS reflects tonic baroreceptor activity more than tonic chemoreceptor activity. The results for vasopressin secretion are in agreement with those reported after muscimol injections in the rat NTS (8). Darlington et al. (10) did not observe high chronic basal levels of ACTH after electrolytic lesions of the NTS, but the lesions did abolish baroreceptor-mediated secretion of ACTH. In our experiments inactivation of the NTS by injection of muscimol acutely elevated vasopressin and ACTH to such high levels that it was not possible to analyze whether any further increases could be initiated by hemorrhage. We found baroreceptor unloading to be a potent stimulus for the secretion of both ACTH and vasopressin in otherwise intact anesthetized animals. Surprisingly, in the anesthetized rabbit, stimulation of chemoreceptors with hypoxia and hypercapnia did not significantly increase plasma levels of either hormone. We know that our protocol for chemoreceptor stimulation is a very effective activator of renal sympathetic nerve activity and phrenic nerve activity (unpublished data). In addition, in electrophysiological experiments, we observed that our protocol for chemoreceptor stimulation increases the activity of neurons in the Al area, antidromically activated from the supraoptic nucleus (2 1). Others have also found that chemoreceptor stimulation in the anesthetized animal is not always a potent stimulus for secretion of ACTH and vasopressin. In the case of vasopressin, Share and Levy (28) raised the possibility that the increase in AP resulting from chemoreceptor activation might secondarily inhibit the secretion of hormone by a baroreceptor mechanism. Raff et al. (24) did obtain secretion of both hormones when a combination of hypoxia and hypercapnia was maintained for 15 min or so. It is tempting to correlate our finding that inhibition of the NTS increases plasma hormone levels with the inhibitory effects of baroreceptors on the secretion of vasopressin and ACTH. Results from our experiments on the relationship between the NTS and the ventrolateral medulla bring evidence to bear on this question. Baroreceptor-initiated secretion of vasopressin was dependent on a synapse in the Al area, as previously demonstrated (6). In a similar fashion, we have now demonstrated that the secretion of vasopressin initiated by inhibiting the NTS is also entirely prevented by prior inhibition of neuronal function in the Al area. Electrophysiological studies in the rabbit, in accordance with this view, demonstrate that a large proportion of neurons in the Al area, antidromically activated from the supraoptic nucleus, are inhibited by baroreceptor inputs (18, 21). In contrast, baroreceptor-initiated secretion of ACTH was not entirely dependent on a synapse in the ventrolateral medulla, including the Al area. Similarly, secretion of ACTH initiated by inhibiting the NTS was also substantially independent of neuronal function in the Al area. Furthermore, the secretion of ACTH initiated by excitation of neuronal function in the Al area was, as discussed above, modulated by baroreceptor activity in a manner consistent with the relevant output from the NTS reaching the hypothalamus by a pathway that does not synapse in the Al area. A schematic outline of pathways suggested by the present results is shown in Fig. 6.

OF ACTH

AND

VASOPRESSIN

R1055

Fig. 6. Schematic representation of pathways from NTS to hypothalamic nuclei involved in regulation of secretion of ACTH (broken lines) and vasopressin (solid lines). ap, anterior pituitary; mc, magnocellular neurons; pc, parvicellular neurons; pp, posterior pituitary; PVN, paraventricular nucleus; SON, supraoptic nucleus.

In the case of ACTH it is not clear whether the output from the NTS projects directly to the relevant parvicellular hypothalamic neurons, directly inhibiting these cells. In the rat there is a major norepinephrine-containing input to the corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus, which derives largely from the A2 norepinephrine-synthesizing neurons in the NTS (9, 26, 30). The available evidence, however, suggests that application of norepinephrine in the vicinity of the corticotropin-releasing factor neurons increases the secretion of ACTH (20), and the electrophysiological data suggest that norepinephrine-containing inputs from the NTS excite tuberoinfundibular neurons (12). The A2 cells receive direct inputs from the vagus nerve (29)) possibly including baroreceptor inputs, but the available electrophysiological study suggests that the A2 neurons do not have a major barorecep tor input (23). It is not clear, therefore, how output from the NTS inhibits secretion of ACTH. Perhaps there is a direct non-norepinephrine-containing inhibitory pathway, such as the one described for NTS neurons containing somatostatin and inhibin-P (25). Alternatively, the pathway may synapse en route to the hypothalamus, perhaps in the parabrachial nucleus (15, 19). It is of interest to-note that pathways from the NTS to the parabrachial nucleus and to the hypothalamus traverse the caudal ventrolatera1 medulla without synapsing (30). This anatomic situation means that hormonal results from electrical stimulation and lesion studies in the Al area are likely to be confounded by effects on these fibers of passage. Conclusion

Output from the NTS tonically inhibits secretion of both ACTH and vasopressin from the pituitary gland, activity that may reflect inputs to the NTS from peripheral baroreceptors. In the case of vasopressin the output from the NTS probably acts via an inhibitory projection to the Al cells in the caudal ventrolateral medulla. The pathway whereby output from the NTS inhibits secretion of ACTH has not yet been determined. It may be direct from the NTS to the relevant neurons in the parvicellular

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R1056

paraventricular depend entirely lateral medulla region increases tatory projection nucleus.

MEDULLA

OBLONGATA

AND

SECRETION

nucleus. If it is indirect, it does not on the Al neurons in the caudal ventroeven though activation of cells in this plasma ACTH, possibly via a direct excito the parvicellular paraventricular

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of ACTH: a homeostatic reflex.

Jamie Haupt provided technical assistance. Our study was supported by the National Health and Medical Research Council and by the National Heart Foundation of Australia. Address for reprint requests: Z. J. Gieroba, Dept. of Physiology, Flinders Medical Center, Bedford Park, SA5042, Australia. Received 10 July 1991; accepted in final form 25 November 1991. 1. Allen, A. M., W. Blessing.

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J. D.

solitary tract lectin tracing

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