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Angiotensin acts at the subfornical organ to increase plasma oxytocin concentrations in the rat
Regulatory Peptides, 23 (1988) 343-352
Elsevier
343
RPT00766
Angiotensin acts at the subfornical organ to increase plasma oxytocin concentrations in the rat A l a s t a i r V. F e r g u s o n a n d N o r m a n W. K a s t i n g Department of Physiology, Queen's University, Kingston, Ont. (Canada) and Department of Physiology, UBC, Vancouver, B.C. (Canada)
(Received 21 June 1988; revisedversion receivedand accepted 31 August 1988)
Summary We have examined the effects of systemic angiotensin II (AII) on plasma oxytocin (OXY) concentrations in freely moving male Sprague-Dawley rats. We have also examined the role of the subfornical organ (SFO) as a CNS site at which circulating All acts to influence secretion of this neurohypophysial peptide. OXY concentrations were measured by radioimmunoassay in plasma samples obtained by drawing blood samples through indwelling atrial catheters. In SFO intact animals (n = 8) All infusion (1.0 #g/kg/min) resulted in increases in plasma OXY concentrations from baseline values of 6.8 + 2.5 pg/ml to postinfusion concentrations of 44.9 + 11.9 pg/ml. In a second series o f experiments electrolytic lesions were placed in the region of the SFO prior to testing the effects of All infusion on OXY concentrations. Two further experimental groups were thus established according to the histologically verified location of lesions in either the rostral or caudal SFO. In the caudal SFO lesioned group All infusion resulted in increases in plasma OXY concentrations from control values of 6.9 4- 1.4 pg/ml to postinfusion levels of 45.1 + 9.8 pg/ml. These changes were not significantly different from the SFO intact group. In contrast rostral SFO lesions resulted in significantly elevated basal concentrations of OXY (17.4 + 3.4 pg/ml, n = 6) while postinfusion concentrations were found to be 22.8 + 4.9 pg/ml indicating that All infusion was without effect following such lesions. These data are in accordance with the hypothesis that circulating All acts at the SFO to influence SFO efferents which in turn activate OXY secreting neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. These neuroendocrine cells then release this peptide into the systemic circulation from the posterior pituitary. Oxytocin; Angiotensin; Subfornical organ Correspondence: A.V. Ferguson, Department of Physiology,Queen's University,Kingston, Ont., Canada,
K7L 3N6 0167-0115/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
344 Introduction
Although angiotensin II (AII) does not readily cross the normal blood-brain barrier circulating AII has been shown to act in the CNS to increase blood pressure [1] and plasma concentrations of vasopressin [2,3], as well as to stimulate drinking [4]. These actions of circulating AII have been attributed to a direct effect of this peptide at the subfornical organ (SFO) [1 3,5], a circumventricular structure which lacks the normal blood-brain barrier [6], and contains a high density of All receptors [7]. Anatomical studies have demonstrated extensive efferent projections from the SFO which include the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) [8,9]. Studies utilizing intracerebroventricular (i.c.v) microinjection techniques have shown that i.c.v. AII increases plasma oxytocin (OXY) concentrations [10,11], suggesting a significant role for this peptide in controlling the secretion of oxytocin from the posterior pituitary. The CNS site of action for this effect has not however been established although one structure at which AII may act is the AII receptor rich SFO [7]. Evidence in support of such a hypothesis comes from our own recent studies demonstrating that electrical stimulation in the SFO results in increased plasma concentrations of OXY [12] as well as vasopressin [13]. Electrophysiological studies have demonstrated that activation of SFO efferents increases the excitability of neurohypophysial putative OXY-secreting neurons in both the supraoptic [14,15], and paraventricular nuclei [16,17]. The suggestion that circulating AII may also increase OXY concentrations has been drawn from single unit recordings illustrating that systemic AII also increases the activity of these putative OXY neurons [18]. An SFO site of action for such effects is supported by the demonstration that such single neuronal effects of circulating AII are abolished by electrolytic lesion of the SFO [18]. The initial Studies reported here were undertaken to examine whether systemic All administration results in increased plasma concentrations of OXY in conscious freely moving animals. Evidence supporting this hypothesis led to a second series of experiments to test the hypothesis that the SFO was an essential CNS structure to the expression of these effects of circulating All on hormone secretion.
Materials and Methods
Male Sprague Dawley rats (150-300 g) provided with food and water ad libitum, and maintained on a 10:14 light:dark cycle at 20.0 + 1.0°C were used in the following experiments.
Surgery Animals were anaesthetized with sodium pentobarbital (60 mg/kg). One group were fitted with atrial cannulae, while a second group underwent this initial surgery but also had electrolytic lesions placed in the region of the SFO. Atrial cannulae (Silastic Medical Grade Tubing 0.025 i.d. x 0.047 o.d.) were inserted via the jugular
345 vein, led s.c. to the intrascapular region and exteriorized at this point [12,13]. Lesion group animals were then placed in a stereotaxic frame and a small burr hole drilled in the skull in a region immediately dorsal to the SFO according to ttie stereotaxic coordinates of Paxinos and Watson [9]. A monopolar, parylene C-coated, tungsten macroelectrode (LF01G, Micro Probe Inc.; tip diameter 0.1 mm, tip exposure 0.25 mm) was advanced ventrally using a micromanipulator toward the region of SFO and a lesion was made by passing direct current (0.5 mA) through the area for a period of 15-30 s. All animals were allowed a minimum three day recovery period following surgery.
Experimental protocol After the recovery period, those rats which maintained patent catheters were placed in an isolation box which was equipped with a one-way mirror for observation purposes. Food and water were available to the animals throughout the experimental period. A second cannula (Intramedic PE60) was attached to the exteriorized silastic tubing and heparinized saline (50 units) was administered. The animals were then allowed a 1 h equilibration period prior to initial blood sampling. Each blood sample (1.0 ml) was immediately centrifuged and the plasma was removed and frozen (-80°C). The red blood cells were resuspended in saline and returned to the animal via the atrial cannula. Immediately following the equilibration period the first blood sample (CONTROL) was drawn. After obtaining this control sample, All dissolved in saline was infused at a constant rate (1.0/~g/kg/min) over a 30 min period in a volume of 0.75 ml. These doses are in accordance with previous studies demonstrating All actions at the SFO to increase plasma vasopressin concentrations [2]. These infusions would be expected to result in circulating concentrations of AII at the high end of the physiological range. A qualitative analysis of drinking behaviour (drinking observed or not observed) during AII infusion was also carried out such that each animal could be placed in a DRINKING or NON-DRINKING group. Immediately following the infusion a second blood sample was taken. A third sample was obtained 30 min following the infusion period. Oxytocin concentrations were measured in these samples using a previously described radioimmunoassay [20] which showed less than 0.01% cross-reactivity for either vasopressin or All. Intra-assay (within), and interassay (between) variability were 8.6% and 4.9% respectively.
Histology At the completion of each experiment the animal was removed from the isolation box, given an overdose of sodium pentobarbital, and perfused with 0.9% saline followed by 10% formalin administered through the left ventricle of the heart. The brain was removed and placed in 10% formalin overnight. The following day 100 /~m sections were cut through the forebrain using a vibratome. These sections were mounted, stained with Cresyl violet and the anatomical location of lesion sites was histologically verified.
346
Statistics All OXY concentrations are expressed as mean group values + S.E.M. Initial within group statistical analyses involved comparison of control plasma concentrations with values measured immediately following All infusion. Such comparisons were made using a paired Students t-test. Further comparisons between groups where made utilised the unpaired Students t-test.
Results
Angiotens& infusion Intact animals (n = 8) had control plasma OXY concentrations (6.8 ± 2.5 pg/ml) similar to those reported in previous studies [12,20]. However, following All infusion these concentrations had risen over 500% to a mean concentration of 44.9 ± 11.9 pg/ml, a value which was significantly elevated compared to control values (P < 0.01, Fig. 1). A gradual return toward control levels occurred over the following 30 min to a mean concentration of 25.4 ± 10.5 pg/ml. The majority of animals (7/8) in this group drank during AII infusion. 75
J
50 ¸
o
25
0
30
1 60
TIME (mins)
Fig. 1. This histogram illustrates the mean ( ± S.E.M.) plasma concentrations oEOXY measured in SFO
intact animals (n = 8) before and after All infusion.The dashed line in this and all followinghistograms represents the time period during which All infusion took place. * P < 0.05 compared to preinfusion values.
SFO lesion Lesioned animals were split into one of two experimental groups according to the location and extent of SFO lesions (Fig. 2). The caudal SFO lesion group (n = 6) had histologically verified lesions which included the caudal SFO. In all these animals the rostral third of the SFO was intact (i.e. the region through which the majority of SFO efferents leave this structure [8,9]). In contrast this specific region of the SFO was completely destroyed in all animals placed in the rostral SFO lesion group (n = 6; some of these animals also had caudal SFO damage), effectively destroying all efferent fibres emanating from this structure. Control plasma OXY concentrations were found to be significantly elevated in the rostral SFO lesion group (17.4 + 3.4 pg/ml)
347
Fig. 2. These photomicrographsillustrate typical examples of rostral (A) and caudal (B) SFO lesions. In each photomicrographthe arrow indicates the lesioned tissue lying immediately ventral to the hippocampal commissure (HC), and dorsal to the third ventricle (V). Bar = 0.5 mm. when compared to both intact (6.8 q- 2.5 pg/ml P < 0.05), and caudal SFO lesioned animals (6.8 + 2.5 pg/ml P < 0.05) (see Figs. 1,3). Increases in plasma OXY concentrations were observed in the caudal SFO lesioned group in response to AII administration which were similar to the responses of intact animals (Fig. 3). In contrast no significant changes in plasma O X Y concentrations occurred in the rostral SFO lesioned group following A l l infusion (Fig. 3). Drinking was observed in response to AII in 3/6 caudal SFO lesioned, and 0/6 rostral SFO lesioned animals.
75
348
ROSTRAL SFO LESION
CAUDAL SFO LESION 75-
50-
~o ~x
o
25-
O.
25
0
0
30 60 TiME (mins)
0
30 60 TIME (rnins)
- -
Fig. 3. These histograms illustrate plasma OXY concentrations measured before and after AII infusion in rostral (n = 6) and caudal (n = 6) SFO lesioned groups. * P < 0.05 compared to preinfusion values. INTACT SFO
100/ z
i ,°
} CAUDAL SFO LESION
1 O0
/
_'Z 50
,o.
ROSTRAL SFO LESION
100 J
_7 50 ¸
/
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CONTROL
STIM.
DRINKING
CONTROL
STIM.
NO DRINKING
Fig. 4. Graphs illustrating basal (CONTROL) and AII-stimulated (STIM.) plasma OXY concentrations in individual animals from intact, caudal SFO-lesioned and rostral SFO-lesioned groups. Each line represents data from a single animal. Each data group has been further subdivided into drinking and no-drinking animals according to observed responses to All. Solid circles represent mean ( ± S.E.M.) for ~rouDed data in that category.
349
Correlation of OXY concentrations with drinking In order to examine whether AII induced increases in OXY concentrations were an essential prerequisite to drinking or vice versa we compared the endocrine responses to All administration in drinking and non-drinking animals from all three groups (Fig. 4). These data illustrate that AII may increase OXY concentrations without stimulating drinking (see caudal lesion non-drinking group), and also that in some animals drinking occurs although no clear increase in OXY concentrations was observed. These data suggest that separate mechanisms underlie these different effects of systemic All.
Discussion I.c.v. All has previously been demonstrated to increase plasma OXY concentrations in the conscious rat [11]. These studies led to the hypothesis that centrally administered AII acts directly on OXY neurons in the SON and PVN of the hypothalamus, a view supported by electrophysiological observations illustrating these neurons to be activated by iontophoretic application of this peptide [21]. Although All would not normally be expected to cross the blood-brain barrier, recent studies have demonstrated that systemic administration of this peptide also results in activation of putative OXY neurons in the hypothalamus [18]. The present studies correlate such electrophysiological findings with more classical endocrine measurements to show that systemic infusion of AII in the conscious freely moving rat increases plasma concentrations of OXY. The SFO has been demonstrated to be a site at which systemic AII acts to elicit many of its centrally mediated effects [1-3,5] including this peptide's excitatory action on OXY secreting neurons in the hypothalamus [18]. The SFO lesion experiments undertaken in the present studies were therefore designed to examine whether this circumventricular organ was essential to the mechariisms through which systemic AII increases plasma OXY concentrations. The finding that rostral SFO lesion abolished OXY release in response to All supports a mandatory role for this structure in mediating these effects. These data do not rule out the possibility that All influences the release of another circulating substance which in turn acts at the SFO to influence OXY secretion. However the demonstration that SFO neurons with identified projections to the PVN are influenced by systemic All [22] further supports a direct action within the SFO for this peptide in eliciting OXY release from the neurohypophysis. The additional observation that caudal SFO lesions were without effect on the OXY responses to AII indicate a primary role for neurons within the rostral SFO in mediating these effects. Rostral SFO lesions were also found to result in a significant increase in basal plasma concentrations of OXY indicating that SFO efferents may tonically inhibit the activity of OXY-secreting neurons. This suggestion gains support from electrophysiological recordings showing that although activation of SFO efferents has predominantly excitatory effects on hypothalamic OXY and VP neurons, a smaller proportion of these neurosecretory cells are inhibited by such stimulation [14,16,22],
350
presumably as the result of activation of separate functional pathways. Such findings are in accordance with reports of increased plasma vasopressin concentrations following SFO lesions [2]. The alternative explanation that such increases in OXY concentrations are secondary to chronic lesion induced changes in body fluids is unlikely in view of previous reports indicating SFO lesions to be without effect on electrolyte balance [23]. All infusion would be expected to result in increases in blood pressure raising the possibility that changes in OXY concentrations may occur as a secondary effect of such cardiovascular changes. Previous studies demonstrating the activity of OXY neurons to be unaffected by large changes in blood pressure [16,18,24] argue strongly against such a possibility. Drinking in response to systemic AII was frequently observed in both intact and caudal SFO-lesioned animals. However, drinking has been reported to result in decreased oxytocin and vasopressin concentrations [25,26] indicating that the observed increases in OXY concentrations were not a response to this water intake. The fact that increased OXY concentrations were observed in some animals which did not drink suggests that these two physiological responses to systemic All acting at the SFO result from actions of this peptide on separate functional populations of SFO neurons. The functional significance of this action of All at the SFO to induce release of OXY in the control of body fluid balance remains to be established. However, a number of studies have suggested a functional role for OXY in the control of sodium excretion [27-29]. In particular higher plasma concentrations of oxytocin (similar to those measured here following All) have been shown to be natriuretic [27,29]. Thus one can envisage a situation during a period of hyperosmolarity in which appropriate physiological responses would include drinking, the retention of water, and the excretion of sodium. All of these effects could result from the CNS actions of systemic All in stimulating drinking [4], the release of vasopressin [2,3] (water retention), and the release of oxytocin (sodium excretion). In conclusion the present studies have provided additional evidence supporting a significant role for oxytocin in the control of body fluid balance. Also these data further emphasise the essential roles of the circumventricular organs at the bloodbrain interface in sensing peptide hormones in the peripheral circulation and eliciting appropriate neuroregulatory responses.
Acknowledgements This work was supported by a grant from the Medical Research Council of Canada. Expert technical assistance was provided by Pauline Marcus.
References 1 Mangiapane, M.L. and Simpson, J.B., Subfornical organ lesions reduce the pressor effect of systemic angiotensin II, Neuroendocrinology, 31 (1980) 380-384.
351 2 Knepel, W., Nutto, D. and Meyer, D.K., Effect of transection of subfornical organ efferent projections on vasopressin release induced by angiotensin or isoprenaline in the rat, Brain Res., 248 (1982) 180184. 3 Iovino, M. and Steardo, L., Vasopressin release to central and peripheral angiotensin II in rats with lesions of the subfornical organ, Brain Res., 322 (1984) 365-368. 4 Simpson, J.B., Epstein, A.N. and Camardo, J.S., Localization of receptors for dipsogenic action of angiotensin II in subfornical organ of rat, J. Comp. Physiol. Psychol., 92 (1978) 581~08. 5 Mangiapane, M.L. and Simpson, J.B., Subfornical organ: forebrain site of pressor and dipsogenic action of angiotensin II, Am. J. Physiol., 239 (1980) R382-R389. 6 Dellman, H.D. and Simpson, J.B., The subfornical organ, Int. Rev. Cytol., 58 (1979) 333-421. 7 Mendelsohn, F.A.O., Quirion, R., Saavedra, J.M. and Aguilera, G., Autoradiographic localization of angiotensin II receptors in rat brain, Neurobiology, 81 (1984) 1575-1579. 8 Lind, R.W., Van Hoesen, G.W. and Johnson, A.K., An HRP study of the connections of the subfornical organ of the rat. J. Cornp. Neurol., 210 (1982) 265-277. 9 Miselis, R.R., The efferent projections of the subfornical organ of the rat: a circumventricular organ within a neural network subserving water balance, Brain Res., 230 (1981) 1-23. 10 Lang, R.E., Rascher, W., Heil, J., Unger, T. and Ganten, D., Angiotensin stimulates oxytocin release: impaired response in rats with genetic hypothalamic diabetes insipidus, Eur. J. Pharmacol., 83 (1982) 113-117. 11 Lang, R.E., Rascher, W., Heil, J., Unger, T., Wiedmann, G. and Ganten, D., Angiotensin stimulates oxytocin release, Life Sci., 29 (1981) 1425-1428. 12 Ferguson, A.V. and Kasting, N.W., Activation of subfornical organ efferents stimulates oxytocin secretion in the rat, Regul. Pept., 18 (1987) 93-100. 13 Ferguson, A.V. and Kasting, N.W., Electrical stimulation in the subfornical organ increases plasma concentrations in the conscious rat, Am. J. Physiol., 251 (1986) R712-717. 14 Sgro, S., Ferguson, A.V. and Renaud, L.P., Subfornical organ-supraoptic nucleus connections: an electrophysiological study in the rat, Brain Res., 303 (1984) 7-13. 15 Tanaka, J., Kaba, H., Saito, H., Seto, K. and Sakuma, Y., Subfornical organ neurons projecting to the hypothalamic supraoptic nucleus in the rat, Jpn. J. Vet. Sci., 48 (1986) 813-816. 16 Ferguson, A.V., Day, T.A. and Renaud, L.P., Subfornical organ efferents influence the excitability of neurohypophysial and tuberoinfundibular paraventricular nucleus neurons in the rat, Neuroendocrinology, 39 (1984) 423~128. 17 Tariaka, J., Kaba, H., Saito, H. and Seto, K., Efferent pathways from the region of the subfornical organ to hypothalamic paraventricular nucleus: an electrophysiological study in the rat, Exp. Brain Res., 62 (1986) 509-514. 18 Ferguson, A.V. and Renaud, L.P., Systemic angiotensin acts at subfornical organ to facilitate activity of neurohypophysial neurons, Am. J. Physiol., 251 (1986) R712-R717. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1982. 20 Kasting, N.W., Indomethacin, an antipyretic drug, prevents the endotoxin-induced, and potentiates the haemorrhage-induced oxytocin release into the plasma of the male rat, Neuroendocrinology, 42 (1986) 285-288. 21 Akaishi, T., Negoro, H. and Kobayasi, S., Electrophysiological evidence for multiple sites of action of angiotensin II for stimulating paraventricular neurosecretory cells in the rat, Brain Res., 220 (1981) 386-390. 22 Tanaka, J., Kaba, H., Saito, H. and Seto, K., Electrophysiological evidence that circulating angiotensin II sensitive neurons in the subfornical organ alter the activity of hypothalamic paraventricular neurohypophyseal neurons in the rat, Brain Res., 342 (1985) 361-365. 23 Thrasher, T.N., Simpson, J.B. and Ramsay, D.J., Lesions of the subfornical organ block angiotensininduced drinking in the dog, Neuroendocrinology, 35 (1982) 68-72. 24 Day, T.A., Ferguson, A.V. and Renaud, L.P., Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol. (Lond.), 355 (1984) 237-249. 25 Thrasher, T.N., Nistal-Herrera, J.F., Keil, L.C. and Ramsay, D.J., Satiety and inhibition of vasopressin secretion after drinking in dehydrated dogs, Am. J. Physiol., 240 (1981) E394-E401.
352 26 Stricker, E.M. and Verbalis, J.G., Interaction of osmotic and volume stimuli in regulation of neurohypophyseal secretion in rats, Am. J. Physiol., 250 (1986) R267-R275. 27 Balment, R.J., Brimble, M.J. and Forsling, M.L., Release of oxytocin induced by salt loading and its influence on renal excretion in the male rat, J. Physiol. (Lond.), 308 (1980) 439-449. 28 Edwards, B.R. and LaRochelle, Jr., F.T., Antidiuretic effect of endogenous oxytocin in dehydrated Brattleboro homozygous rats, Am. J. Physiol., 247 (1984) F453-F465. 29 Balment, R.J., Brimble, M.J., Forsling, M.L., Kelly, L.P. and Musabayane, C.T., A synergistic effect of oxytocin and vasopressin on sodium excretion in the neurohypophysectomized rat, J. Physiol. (Lond.), 381 (1986) 453-464.
Elsevier
343
RPT00766
Angiotensin acts at the subfornical organ to increase plasma oxytocin concentrations in the rat A l a s t a i r V. F e r g u s o n a n d N o r m a n W. K a s t i n g Department of Physiology, Queen's University, Kingston, Ont. (Canada) and Department of Physiology, UBC, Vancouver, B.C. (Canada)
(Received 21 June 1988; revisedversion receivedand accepted 31 August 1988)
Summary We have examined the effects of systemic angiotensin II (AII) on plasma oxytocin (OXY) concentrations in freely moving male Sprague-Dawley rats. We have also examined the role of the subfornical organ (SFO) as a CNS site at which circulating All acts to influence secretion of this neurohypophysial peptide. OXY concentrations were measured by radioimmunoassay in plasma samples obtained by drawing blood samples through indwelling atrial catheters. In SFO intact animals (n = 8) All infusion (1.0 #g/kg/min) resulted in increases in plasma OXY concentrations from baseline values of 6.8 + 2.5 pg/ml to postinfusion concentrations of 44.9 + 11.9 pg/ml. In a second series o f experiments electrolytic lesions were placed in the region of the SFO prior to testing the effects of All infusion on OXY concentrations. Two further experimental groups were thus established according to the histologically verified location of lesions in either the rostral or caudal SFO. In the caudal SFO lesioned group All infusion resulted in increases in plasma OXY concentrations from control values of 6.9 4- 1.4 pg/ml to postinfusion levels of 45.1 + 9.8 pg/ml. These changes were not significantly different from the SFO intact group. In contrast rostral SFO lesions resulted in significantly elevated basal concentrations of OXY (17.4 + 3.4 pg/ml, n = 6) while postinfusion concentrations were found to be 22.8 + 4.9 pg/ml indicating that All infusion was without effect following such lesions. These data are in accordance with the hypothesis that circulating All acts at the SFO to influence SFO efferents which in turn activate OXY secreting neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. These neuroendocrine cells then release this peptide into the systemic circulation from the posterior pituitary. Oxytocin; Angiotensin; Subfornical organ Correspondence: A.V. Ferguson, Department of Physiology,Queen's University,Kingston, Ont., Canada,
K7L 3N6 0167-0115/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
344 Introduction
Although angiotensin II (AII) does not readily cross the normal blood-brain barrier circulating AII has been shown to act in the CNS to increase blood pressure [1] and plasma concentrations of vasopressin [2,3], as well as to stimulate drinking [4]. These actions of circulating AII have been attributed to a direct effect of this peptide at the subfornical organ (SFO) [1 3,5], a circumventricular structure which lacks the normal blood-brain barrier [6], and contains a high density of All receptors [7]. Anatomical studies have demonstrated extensive efferent projections from the SFO which include the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) [8,9]. Studies utilizing intracerebroventricular (i.c.v) microinjection techniques have shown that i.c.v. AII increases plasma oxytocin (OXY) concentrations [10,11], suggesting a significant role for this peptide in controlling the secretion of oxytocin from the posterior pituitary. The CNS site of action for this effect has not however been established although one structure at which AII may act is the AII receptor rich SFO [7]. Evidence in support of such a hypothesis comes from our own recent studies demonstrating that electrical stimulation in the SFO results in increased plasma concentrations of OXY [12] as well as vasopressin [13]. Electrophysiological studies have demonstrated that activation of SFO efferents increases the excitability of neurohypophysial putative OXY-secreting neurons in both the supraoptic [14,15], and paraventricular nuclei [16,17]. The suggestion that circulating AII may also increase OXY concentrations has been drawn from single unit recordings illustrating that systemic AII also increases the activity of these putative OXY neurons [18]. An SFO site of action for such effects is supported by the demonstration that such single neuronal effects of circulating AII are abolished by electrolytic lesion of the SFO [18]. The initial Studies reported here were undertaken to examine whether systemic All administration results in increased plasma concentrations of OXY in conscious freely moving animals. Evidence supporting this hypothesis led to a second series of experiments to test the hypothesis that the SFO was an essential CNS structure to the expression of these effects of circulating All on hormone secretion.
Materials and Methods
Male Sprague Dawley rats (150-300 g) provided with food and water ad libitum, and maintained on a 10:14 light:dark cycle at 20.0 + 1.0°C were used in the following experiments.
Surgery Animals were anaesthetized with sodium pentobarbital (60 mg/kg). One group were fitted with atrial cannulae, while a second group underwent this initial surgery but also had electrolytic lesions placed in the region of the SFO. Atrial cannulae (Silastic Medical Grade Tubing 0.025 i.d. x 0.047 o.d.) were inserted via the jugular
345 vein, led s.c. to the intrascapular region and exteriorized at this point [12,13]. Lesion group animals were then placed in a stereotaxic frame and a small burr hole drilled in the skull in a region immediately dorsal to the SFO according to ttie stereotaxic coordinates of Paxinos and Watson [9]. A monopolar, parylene C-coated, tungsten macroelectrode (LF01G, Micro Probe Inc.; tip diameter 0.1 mm, tip exposure 0.25 mm) was advanced ventrally using a micromanipulator toward the region of SFO and a lesion was made by passing direct current (0.5 mA) through the area for a period of 15-30 s. All animals were allowed a minimum three day recovery period following surgery.
Experimental protocol After the recovery period, those rats which maintained patent catheters were placed in an isolation box which was equipped with a one-way mirror for observation purposes. Food and water were available to the animals throughout the experimental period. A second cannula (Intramedic PE60) was attached to the exteriorized silastic tubing and heparinized saline (50 units) was administered. The animals were then allowed a 1 h equilibration period prior to initial blood sampling. Each blood sample (1.0 ml) was immediately centrifuged and the plasma was removed and frozen (-80°C). The red blood cells were resuspended in saline and returned to the animal via the atrial cannula. Immediately following the equilibration period the first blood sample (CONTROL) was drawn. After obtaining this control sample, All dissolved in saline was infused at a constant rate (1.0/~g/kg/min) over a 30 min period in a volume of 0.75 ml. These doses are in accordance with previous studies demonstrating All actions at the SFO to increase plasma vasopressin concentrations [2]. These infusions would be expected to result in circulating concentrations of AII at the high end of the physiological range. A qualitative analysis of drinking behaviour (drinking observed or not observed) during AII infusion was also carried out such that each animal could be placed in a DRINKING or NON-DRINKING group. Immediately following the infusion a second blood sample was taken. A third sample was obtained 30 min following the infusion period. Oxytocin concentrations were measured in these samples using a previously described radioimmunoassay [20] which showed less than 0.01% cross-reactivity for either vasopressin or All. Intra-assay (within), and interassay (between) variability were 8.6% and 4.9% respectively.
Histology At the completion of each experiment the animal was removed from the isolation box, given an overdose of sodium pentobarbital, and perfused with 0.9% saline followed by 10% formalin administered through the left ventricle of the heart. The brain was removed and placed in 10% formalin overnight. The following day 100 /~m sections were cut through the forebrain using a vibratome. These sections were mounted, stained with Cresyl violet and the anatomical location of lesion sites was histologically verified.
346
Statistics All OXY concentrations are expressed as mean group values + S.E.M. Initial within group statistical analyses involved comparison of control plasma concentrations with values measured immediately following All infusion. Such comparisons were made using a paired Students t-test. Further comparisons between groups where made utilised the unpaired Students t-test.
Results
Angiotens& infusion Intact animals (n = 8) had control plasma OXY concentrations (6.8 ± 2.5 pg/ml) similar to those reported in previous studies [12,20]. However, following All infusion these concentrations had risen over 500% to a mean concentration of 44.9 ± 11.9 pg/ml, a value which was significantly elevated compared to control values (P < 0.01, Fig. 1). A gradual return toward control levels occurred over the following 30 min to a mean concentration of 25.4 ± 10.5 pg/ml. The majority of animals (7/8) in this group drank during AII infusion. 75
J
50 ¸
o
25
0
30
1 60
TIME (mins)
Fig. 1. This histogram illustrates the mean ( ± S.E.M.) plasma concentrations oEOXY measured in SFO
intact animals (n = 8) before and after All infusion.The dashed line in this and all followinghistograms represents the time period during which All infusion took place. * P < 0.05 compared to preinfusion values.
SFO lesion Lesioned animals were split into one of two experimental groups according to the location and extent of SFO lesions (Fig. 2). The caudal SFO lesion group (n = 6) had histologically verified lesions which included the caudal SFO. In all these animals the rostral third of the SFO was intact (i.e. the region through which the majority of SFO efferents leave this structure [8,9]). In contrast this specific region of the SFO was completely destroyed in all animals placed in the rostral SFO lesion group (n = 6; some of these animals also had caudal SFO damage), effectively destroying all efferent fibres emanating from this structure. Control plasma OXY concentrations were found to be significantly elevated in the rostral SFO lesion group (17.4 + 3.4 pg/ml)
347
Fig. 2. These photomicrographsillustrate typical examples of rostral (A) and caudal (B) SFO lesions. In each photomicrographthe arrow indicates the lesioned tissue lying immediately ventral to the hippocampal commissure (HC), and dorsal to the third ventricle (V). Bar = 0.5 mm. when compared to both intact (6.8 q- 2.5 pg/ml P < 0.05), and caudal SFO lesioned animals (6.8 + 2.5 pg/ml P < 0.05) (see Figs. 1,3). Increases in plasma OXY concentrations were observed in the caudal SFO lesioned group in response to AII administration which were similar to the responses of intact animals (Fig. 3). In contrast no significant changes in plasma O X Y concentrations occurred in the rostral SFO lesioned group following A l l infusion (Fig. 3). Drinking was observed in response to AII in 3/6 caudal SFO lesioned, and 0/6 rostral SFO lesioned animals.
75
348
ROSTRAL SFO LESION
CAUDAL SFO LESION 75-
50-
~o ~x
o
25-
O.
25
0
0
30 60 TiME (mins)
0
30 60 TIME (rnins)
- -
Fig. 3. These histograms illustrate plasma OXY concentrations measured before and after AII infusion in rostral (n = 6) and caudal (n = 6) SFO lesioned groups. * P < 0.05 compared to preinfusion values. INTACT SFO
100/ z
i ,°
} CAUDAL SFO LESION
1 O0
/
_'Z 50
,o.
ROSTRAL SFO LESION
100 J
_7 50 ¸
/
,o
CONTROL
STIM.
DRINKING
CONTROL
STIM.
NO DRINKING
Fig. 4. Graphs illustrating basal (CONTROL) and AII-stimulated (STIM.) plasma OXY concentrations in individual animals from intact, caudal SFO-lesioned and rostral SFO-lesioned groups. Each line represents data from a single animal. Each data group has been further subdivided into drinking and no-drinking animals according to observed responses to All. Solid circles represent mean ( ± S.E.M.) for ~rouDed data in that category.
349
Correlation of OXY concentrations with drinking In order to examine whether AII induced increases in OXY concentrations were an essential prerequisite to drinking or vice versa we compared the endocrine responses to All administration in drinking and non-drinking animals from all three groups (Fig. 4). These data illustrate that AII may increase OXY concentrations without stimulating drinking (see caudal lesion non-drinking group), and also that in some animals drinking occurs although no clear increase in OXY concentrations was observed. These data suggest that separate mechanisms underlie these different effects of systemic All.
Discussion I.c.v. All has previously been demonstrated to increase plasma OXY concentrations in the conscious rat [11]. These studies led to the hypothesis that centrally administered AII acts directly on OXY neurons in the SON and PVN of the hypothalamus, a view supported by electrophysiological observations illustrating these neurons to be activated by iontophoretic application of this peptide [21]. Although All would not normally be expected to cross the blood-brain barrier, recent studies have demonstrated that systemic administration of this peptide also results in activation of putative OXY neurons in the hypothalamus [18]. The present studies correlate such electrophysiological findings with more classical endocrine measurements to show that systemic infusion of AII in the conscious freely moving rat increases plasma concentrations of OXY. The SFO has been demonstrated to be a site at which systemic AII acts to elicit many of its centrally mediated effects [1-3,5] including this peptide's excitatory action on OXY secreting neurons in the hypothalamus [18]. The SFO lesion experiments undertaken in the present studies were therefore designed to examine whether this circumventricular organ was essential to the mechariisms through which systemic AII increases plasma OXY concentrations. The finding that rostral SFO lesion abolished OXY release in response to All supports a mandatory role for this structure in mediating these effects. These data do not rule out the possibility that All influences the release of another circulating substance which in turn acts at the SFO to influence OXY secretion. However the demonstration that SFO neurons with identified projections to the PVN are influenced by systemic All [22] further supports a direct action within the SFO for this peptide in eliciting OXY release from the neurohypophysis. The additional observation that caudal SFO lesions were without effect on the OXY responses to AII indicate a primary role for neurons within the rostral SFO in mediating these effects. Rostral SFO lesions were also found to result in a significant increase in basal plasma concentrations of OXY indicating that SFO efferents may tonically inhibit the activity of OXY-secreting neurons. This suggestion gains support from electrophysiological recordings showing that although activation of SFO efferents has predominantly excitatory effects on hypothalamic OXY and VP neurons, a smaller proportion of these neurosecretory cells are inhibited by such stimulation [14,16,22],
350
presumably as the result of activation of separate functional pathways. Such findings are in accordance with reports of increased plasma vasopressin concentrations following SFO lesions [2]. The alternative explanation that such increases in OXY concentrations are secondary to chronic lesion induced changes in body fluids is unlikely in view of previous reports indicating SFO lesions to be without effect on electrolyte balance [23]. All infusion would be expected to result in increases in blood pressure raising the possibility that changes in OXY concentrations may occur as a secondary effect of such cardiovascular changes. Previous studies demonstrating the activity of OXY neurons to be unaffected by large changes in blood pressure [16,18,24] argue strongly against such a possibility. Drinking in response to systemic AII was frequently observed in both intact and caudal SFO-lesioned animals. However, drinking has been reported to result in decreased oxytocin and vasopressin concentrations [25,26] indicating that the observed increases in OXY concentrations were not a response to this water intake. The fact that increased OXY concentrations were observed in some animals which did not drink suggests that these two physiological responses to systemic All acting at the SFO result from actions of this peptide on separate functional populations of SFO neurons. The functional significance of this action of All at the SFO to induce release of OXY in the control of body fluid balance remains to be established. However, a number of studies have suggested a functional role for OXY in the control of sodium excretion [27-29]. In particular higher plasma concentrations of oxytocin (similar to those measured here following All) have been shown to be natriuretic [27,29]. Thus one can envisage a situation during a period of hyperosmolarity in which appropriate physiological responses would include drinking, the retention of water, and the excretion of sodium. All of these effects could result from the CNS actions of systemic All in stimulating drinking [4], the release of vasopressin [2,3] (water retention), and the release of oxytocin (sodium excretion). In conclusion the present studies have provided additional evidence supporting a significant role for oxytocin in the control of body fluid balance. Also these data further emphasise the essential roles of the circumventricular organs at the bloodbrain interface in sensing peptide hormones in the peripheral circulation and eliciting appropriate neuroregulatory responses.
Acknowledgements This work was supported by a grant from the Medical Research Council of Canada. Expert technical assistance was provided by Pauline Marcus.
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