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Septal release of vasopressin in response to osmotic, hypovolemic and electrical stimulation in rats
Brain Research, 381 l ltl~6) 3 l-I 32 i
314 BRE 11965
Septal Release of Vasopressin in Response to Osmotic, Hypovolemic and Electrical Stimulation in Rats J. DEMOTES-MAINARD, J. CHAUVEAU, F. RODRIGUEZ, J.D. VINCENT and D.A. POULAIN 1. N. S. E. R.M. U 176, Bordeaux Cedex (France
(Accepted January 21st, 1986) Key words: vasopressin - - septum - - push-pull cannula - - hemorrhage - - hypertonic stimulation - - magnoceUular system
The central release of vasopressin was studied in anesthetized rats using push-pull perfusions and radioimmunoassay of the hormone. A basal release was observed in the lateral septum and in the lateral ventricle, whereas no vasopressin was detected in the perfusates from the caudate nucleus. Under osmotic stimulation, vasopressin release increased up to 12 and 60 times basal levels following i.p. injections of 5 ml and 10 ml/kg b.wt. of 2 M NaCI, respectively. This increase was blocked by using a calcium-free perfusion medium containing 0.1 mM EGTA. In the lateral ventricle, osmotic stimulation (5 ml/kg of 2 M NaCI i.p.) had the same effect as in the septum. In the caudate nucleus, no release was observed. Hemorrhage also increased the septal release of vasopressin in 5 out of 6 animals tested. Electrical stimulation of the pituitary stalk and of the supraoptic nucleus was used to evoke the release of vasopressin into the bloodstream. Septal release slightly decreased during pituitary stalk stimulation, whereas it did increase during stimulation of the supraoptic region. Our results show that systemic stimuli for vasopressin release evoke both a peripheral and a septal release of the hormone. The dissociation of the effects of electrical stimulation of the pituitary stalk and of the supraoptic nucleus suggests, however, that the vasopressinergic neurones responsible for septal release are distinct from those which project to the neurohypophysis.
INTRODUCTION A large body of immunocytochemical evidence has now clearly established that vasopressinergic innervation extends to several central nervous structures in addition to the h y p o t h a l a m o - n e u r o h y p o p h y sial pathway 3'33'34. This raises n u m e r o u s questions concerning the role of this extra-hypothalamic innervation, and the physiological conditions u n d e r which a central release of h o r m o n e may occur. In the lateral septum, there is an a b u n d a n t vasopressinergic innervation 4'9 and in vitro experiments have demonstrated that vasopressinergic terminals onto septal n e u r o n e s are indeed able to release h o r m o n e in response to depolarization5. We therefore examined the septal release of vasopressin in vivo using a push-pull perfusion technique. Systemic osmotic and hypovolemic stimuli were chosen in order to determine whether a central release of h o r m o n e was taking place simultaneously with the peripheral release that they are known to evoke. In addition, in order to examine
whether the hypothalamo-neurohypophysial axis has any role in septal release, we also studied such a release after electrical stimulation of the pituitary stalk and of the supraoptic (SO) region of the hypothalamus. A preliminary report of these results has been presented earlier 28. MATERIALS AND METHODS Animals
The experiments were performed on Wistar rats (280-450 g b.wt.), bred in a controlled e n v i r o n m e n t , with water and food available ad libitum. The animals were anesthetized with urethane (1.3 g/kg i.p.) and set in a stereotaxic device (Narishige), Body temperature was m a i n t a i n e d at 37 °C by a homeothermic blanket (CFP 8185, Bioscience). Push-pull p e r f u s i o n s
The stainless steel cannula that we used consisted of an external guide tube (diameter 0.6 mm) paired
Correspondence: D.A. Poulain, I.N.S.E.R.M. U 176, 1 rue Camille Saint-Saens, F-33077 Bordeaux Cedex, France.
0006-8993/86/$03.50 (~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)
315 with a sharp pointed mandrel during insertion into the central nervous system. The mandrel was then replaced by an inner double-barrelled pipe, the expelling ('push') pipe projecting 0.5 mm out of the guide tube, and the tip of the sucking ('pull') pipe resting 1 mm inside the guide tube. The cannula was connected to two peristaltic pumps (Gilson Minipulse 2) allowing a regular perfusion at a rate of 67 pl/min (i.e. = 1 ml/15 min) of an artificial cerebrospinal fluid (in mM: NaC1 126.5, NaHCO 3 27.5, KC1 2.4, KH2PO 4 0.5, CaC12 1.1, MgC12 0.83, Na2SO 4 0.5, glucose 5.9, pH adjusted at 7.3 by using an O2/CO 2 gas mixture at 95%/5% (v/v); T 37 °C). Sampling began only after a one-hour period following the onset of the push-pull perfusion to allow the stabilization of the perfusion. Then, 1 ml samples were collected every 15 rain in pre-cooled polypropylene tubes, immediately frozen at -20 °C, and subsequently assayed for arginine-vasopressin.
Arginine-vasopressin radioimmunoassay Samples were treated for extraction by addition of two volumes of dioxane. The homogenate was centrifuged at 12,000 g for 10 min. The supernatant was lyophilized, then rehydrated in 250/~1 incubation buffer containing 10 mM sodium phosphate, 10 mM sodium azide, 0.15 M sodium chloride and 1 g/l bovine serum albumin. Radioimmunological incubations were carried out by equilibrium dialysis. The 10 dialysis cells were filtered by large pore cellulose membranes (Sartorius SM 1153). Incubation time was 44 h. Before being placed on one side of this membrane, the test samples (or standards) were mixed with an equal volume of [125I]ArgS-vasopressin solution. The other side was filled with diluted antibody as described previously8'29. The cross-reactivity factor for vasopressin analogues were: arginine- and lysinevasopressin 1; arginine-vasotocin 8.9; oxytocin 3.9 x 10-4. The minimum amount of arginine-vasopressin detectable was 0.35 pg/sample.
Experimental groups Osmotic stimulation. Vasopressin release was studied in male rats after i.p. injection of hypertonic saline (NaCl 2 M) at a dose of 5 or 10 ml/kg b.wt. After craniotomy, the push-pull cannula was implanted into the lateral septum or, for controls, into the lateral ventricle or in the caudate nucleus. Samples of per-
fusate were collected during 45 min before and during at least 105 min after the i.p. injection. In a group of animals, a calcium-free medium containing 0.1 mM EGTA was used instead of the normal artificial cerebrospinal fluid. Finally, the changes in plasma osmolarity were studied in separate groups of rats similarly anesthetized with urethane. Venous blood samples were collected via a previously inserted catheter into the jugular vein, before and at different times (15, 45, 60, 90 and 120 min) after osmotic load. Blood samples were centrifuged at 4 °C and plasma osmolarity determined by the freezing point method (Fishke osmometer). Hemorrhage. In one group of male rats with a push-pull cannula implanted into the lateral septum, hemorrhage was performed by removing blood via a catheter inserted into the right atrium (12.5 ml/kg in 10 min, i.e. about 15% blood volume). Samples of perfusate were collected 45 min before and up to 120 min after hemorrhage. Subsequently, an equal volume of isotonic saline was slowly reinjected via the atrial catheter and perfusion fluid were collected for a further 60 min period. Electrical stimulation. After inserting a push-pull cannula into the lateral septum, two stimulating electrodes (SNEX 100, Rhodes Medical Instruments) were lowered, one in the supraoptic nucleus ipsilateral to the push-puU cannula, the other one in the pituitary stalk, using a dorsal approach. To allow precise implantation of the electrodes, the experiments were carried out in lactating rats. One mammary gland was cannulated in order to monitor intramammary pressure via a pressure gauge transducer (Statham) connected to a pen recorder (Racia). The electrode positions were adjusted until a short train (5 s) of electrical pulses (50 Hz, 0.5 ms, 0.5 mA) caused a rise in intramammary pressure similar to that produced by an i.v. injection of 0.5 mU oxytocin (see ref. 26). After the electrodes were correctly positioned, perfusion through the push-pull cannula was started, and after a one-hour period, three control samples were taken. The pituitary stalk was then stimulated for 45 min, and after a 45 min resting period, the SO nucleus was then stimulated for 45 rain. A phasic pattern of stimulation was applied using a burst of action potentials previously recorded from a vasopressinergic neurone (burst duration: 26 s, intraburst frequency 13 Hz, silent period duration 22 s,
316
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see Fig. 6A). Such a phasic pattern has been shown to be highly efficient for promoting hormone release 12. Histological controls. A t the end of each experiment, the animals were decapitated and the brains were fixed in a 10% formaldehyde solution. Light microscopic observation of paraffin-embedded sections allowed to locate the path and the tip of the push-pull cannula, and also the electrolytic lesion in the SO nucleus made by passing a direct current at the end of the electrical stimulation experiments.
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Effect o f osmotic stimulation The effect of osmotic stimulation by i.p. injection of 2 M NaCI on vasopressin release depended on the amount injected and on the site of perfusion. After injection of 10 ml/kg b.wt., plasma osmolarity rose from 311 + 0.6 up to 375 + 5 mOsm/kg H 2 0 (n = 5) 15 min after injection, then slowly declined down to 360 + 4 mOsm/kg H 2 0 by 120 min after injection. Under these conditions, the amount of vasopressin in the perfusates from the lateral septum increased up to 8.5 + 4.5 pg/sample at 15 min (n = 5, significantly different from control values at P < 0.01, Student's paired t-test), and then to 58 + 3 pg/sample at 105 min (Fig. 1). In the caudate nucleus (n = 3), on the other hand, vasopressin levels remained at the detection limit of our assay throughout the experiment. With a lower osmotic load (5 ml/kg b.wt.), plasma
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Basal release o f vasopressin Basal levels of vasopressin release from the lateral septum in animals under urethane anesthesia had a mean of 0.9 + 0.2 (S.D.) pg/sample (n = 7 rats); in two animals that were not stimulated, this basal release remained stable even during long periods of perfusion (Fig. 2). Basal levels of vasopressin in samples from the lateral ventricle were very similar (0.8 + 0.1, n = 4, Fig. 3). On the other hand, the amount of vasopressin in the perfusates from the caudate nucleus (Fig. 1) were at the limit of detection of our assay (35 pg/sample).
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Fig. 1. Effect of i.p. injection of a high dose of hypertonic saline on central release of vasopressin. Each point represents
the vasopressin content of a sample of push-pul! perfusate (1 ml) collected over a 15-min period; each curve represents one experiment performed in one animal. The first three points are controls. Note the large increase in vasopressin release from the lateral septum evoked by osmotic stimulation. In the caudate nucleus, vasopressin levels remained undetectable throughout the experiment. osmolarity reached 335 + 3 mOsm/kg H20 at 15 min and remained stable up to 120 min after injection. Perfusates from the lateral septum showed a substantial increase in vasopressin in each of the 4 animals
317 tested (Fig. 2). Although there were large individual variations between animals, the increase, much lower than that evoked by a higher osmotic load, reached 5.1 + 2.1 pg/sample at 15 min (P < 0.01, paired t-test, n = 4 rats) and attained a peak at 105 min (12 + 1.4 pg/sample, n.s.). In perfusates from the lateral ventricle (Fig. 3), a similar pattern of vasopressin increase was observed following i.p. injection of 5 ml of 2 M NaCI, reaching 2.0 + 1.0 pg at 30 min,
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Time (rain) Fig. 2. Release of vasopressin in the lateral septum in response to i.p. injection of 5 ml/kg 2 M NaCI in four animals. For comparison, the two bottom curves ( i t , O ) show the stability of the septal release of vasopressin in the absence of osmotic stimulation. (Note the change of scale between Figs. 1 and 2.)
and 4.1 + 1.2 pg/sample at 105 min (P < 0.01 paired t-test in both cases). In an attempt to see whether the osmotically evoked release of vasopressin in the septum was a calcium-dependent and thus presumably a synaptic process, a calcium-free medium containing 0.1 mM EGTA was used instead of the normal perfusion medium in a group of 8 animals. Osmotic stimulation (5 ml/kg) evoked no consistent pattern of vasopressin release in the septal perfusates, either from one animal to another, or from one sample to another in each animal. The vasopressin contents in the perfusates from the lateral septum were not significantly altered by injection of hypertonic saline in comparison to the control periods using either the normal or calcium-free media (Fig. 4). However, they were significantly lower in animals perfused with a calciumfree medium than in animals perfused with the normal medium in all samples taken 30 to 90 min after injection of hypertonic saline (P < 0.01, Mann-Whitney U test).
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Fig. 4. Effect of the substitution of standard artificial cerebrospinal fluid (CSF) by a calcium-free solution containing 0.1 mM EGTA on vasopressin release in the septum before and after i.p, injection of hypertonic saline in 8 animals. Vasopressin contents are expressed in percentage of control levels (2.4 pg = 100%). Note that the osmotic stimulus had no significant effect on septal release of vasopressin (compare with Fig. 2). 10_
Effect of hemorrhage The effect of hemorrhage on septal release of vasopressin was studied in six animals by removing rapidly blood from the right atrium. Plasma concentration of vasopressin, assayed in the last 2.5 ml of blood removed, reached 342 + 184 pg/ml. In 5 animals, septal release of vasopressin increased progressively, being significantly higher (P < 0.01, paired ttest) than control levels in all samples between 30 and 120 min, and reaching a peak at 75-90 min after hemorrhage (Fig. 5). At 90 min, injection of isotonic saline to restore blood volume was accompanied by a progressive decline in vasopressin release. A 6th animal in which vasopressin levels remained constantly at detection level throughout the periods of hemorrhage and saline infusion, was not included in the statistical analysis.
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Fig. 5. Septal release of vasopressin in response to changes in blood volume by progressive bleeding and by isotonic saline infusion into the right atrium (12,5 ml/kg in 10 min).
319 the supraoptic nucleus was performed in 4 lactating rats, using a phasic pattern of stimulation (see Methods). Peripheral release of neurohypophysial hormones after such stimuli was monitored by the recurrent variations in intramammary pressure (Fig. 6). In each animal, stimulation of the pituitary stalk was accompanied by a slight decrease in vasopressin levels in the perfusates from the lateral septum, from 2.5 + 1.5 pg/sample before stimulation down to 1.3 +
0.8 pg/sample 45 min after the onset of stimulation (P < 0.025, paired t-test). At the end of the 45 min recovery period, vasopressin levels were back to 2.0 + 0.2 pg/sample. Stimulation of the SO nucleus had an effect opposite to that of pituitary stalk stimulation. In each animal, there was an increased content in vasopressin (up to 5.6 + 1.3 pg/sample; P < 0.005) 45 min after the onset of stimulation. In three animals, vasopressin levels dropped back to control level after cessation of stimulation.
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Fig. 6. Effects of electrical stimulation of the pituitary stalk and of the supraoptic nucleus on the release of vasopressin from the septum. A: intramammary pressure recording (top line) during electrical stimulation of the SO nucleus using a phasic pattern of electrical stimuli (bottom line). Recurrent milk ejections provided evidence that stimulation did induce peripheral release of neurohypophysial hormones. B: vasopressin release from the lateral septum during electrical stimulation. Changes are expressed in percentage of control levels (2.5 pg = 100%). During pituitary stalk stimulation, vasopressin levels decreased significantly (P < 0.01, paired t-test on raw data). In contrast, during SO stimulation, vasopressin release increased significantly (P < 0.01).
The push-pull perfusion technique that we used in these experiments permitted us to establish that during systemic or central stimulation, not only is vasopressin released into the bloodstream, but also into central structures such as the sepl;um. Our results thus support the suggestion that the vasopressin innervation described in the lateral septum is of functional significance under certain conditions of stimulation of hormone release. They suggest, moreover, that the peripheral and central release of vasopressin are dependent upon separate pools of vasopressinergic neurones. As all the stimuli that we used led to the release of hormones into the blood, it could be argued that diffusion from the bloodstream into the septum could account for the presence of vasopressin in the septal perfusates. However, vasopressin does not cross the blood-brain barrier 1'7'2t and our own data exclude a non-specific diffusion or uptake in the brain, since perfusates from the caudate nucleus were devoid of hormone. Diffusion or uptake specifically restricted to the septum is also unlikely since vasopressin release in blood evoked by electrical stimulation of the pituitary stalk was not accompanied by an increase in vasopressin content in the septal perfusates. A more critical issue concerns the possible diffusion of vasopressin from the lateral ventricle to the septum. Release of vasopressin into the cerebrospinal fluid has been reported by several authors, and in our experiments, vasopressin increased in a similar manner in the perfusates from the lateral ventricle and from the lateral septum. Alternatively, vasopressin could also diffuse from central structures to the ventricles, or it may be that both types of release occur. None of these possibilities can be excluded. Nevertheless,
320 several lines of evidence make it highly probable that vasopressin was actually released within the septum. Vasopressin-containing terminals have been identified by electron microscopic immunocytochemistry in the lateral septum4; in vitro, vasopressin release from septal slices can be increased by depolarization with potassium or veratridine5; and finally, in the present in vivo experiments, the osmotically induced release of vasopressin was prevented when using a calcium-free medium containing 0.1 mM E G T A to reduce or block local synaptic activity. Our experiments using electrical stimulation indicate that the vasopressinergic innervation of the septum is independent of the hypothalamo-neurohypophysial axis. This is in agreement with several observations showing that the magnocellular neurones which project to the neurohypophysis do not also project to the septum. Electrophysiologically, magnocellular neurones in the SO or paraventricular nuclei cannot be antidromically invaded when electrically stimulating the septum 23-25. On morphological grounds, extensive lesions of hypothalamus do not alter the septal content in vasopressinergic fibers 9, which appear in fact to arise from the bed nucleus of the stria terminalis 36. In the present experiments, since stimulation of the pituitary stalk activates magnocellular cell bodies antidromically, a septal release of hormone should have been observed if the neurones possessed septal efferents. On the other hand, stimulation of the supraoptic region led to both a peripheral and a central release of hormone. As stated before, this cannot be attributed to supraoptic efferents to the septum. We can also exclude the possibility that SO cells would activate other vasopressinergic cells through a polysynaptic pathway, since such an anatomical arrangement would have permitted the release of hormone when stimulating the cell bodies antidromically from the pituitary stalk. The most likely hypothesis is that electrical stimulation of the SO region activated an
REFERENCES 1 Ang, V.T.Y. and Jenkins, J.S., Blood-cerebrospinal fluid barrier to arginine-vasopressin, desmopressin and desglycinamide arginine-vasopressin in the dog, J. Endocrinol., 93 (1982) 319-325. 2 Barnard, R.R. and Morris, M., Cerebro-spinal fluid vasopressin and oxytocin: evidence for an osmotic response, Neurosci. Lett., 29 (1982) 275-279. 3 Buijs, R.M., Swaab, D.F., Dogterom, J. and van Leeuwen, F.W., Intra- and extra-hypothalamicvasopressin and
afferent pathway common both to the hypothalamoneurohypophysial system and to a cell group projecting to the septum. Since osmoreceptors may be located in the vicinity of the SO nucleus 1517~37, and since osmotic stimulation induced a septal release, it is tempting to speculate that SO stimulation may have activated this osmoreceptor system. Our observation of a central release of vasopressin during hypovolemic and osmotic stimulation is in agreement with several experiments reporting a release of hormone into central structures or in the cerebrospinal fluid during h e m o r r h a g e 6"19"3~'3~,3s or osmotic stimuli 2'6"11"13"27"35"39"4°. Although there appear to be important variations in the intensity of vasopressin release, depending upon the sites and the methods of push-pull perfusions, and on the routes of administration, it is clear that homeostatic processes involved in the control of salt and water balance in the blood are simultaneously inducing a release of vasopressin in central structures. The physiological significance of such a release under these conditions has yet to be determined. The septum does participate in the control of water intake or arterial blood pressure, and therefore, at least indirectly, in the control of neurohypophysial function 2°'31. More specifically, the electrical activity of vasopressin neurones can be altered by septal stimulation ~5'3°, and septal lesions modify the release of vasopressin during osmotic stimulation x6. Conversely, electrical stimulation of the SO nucleus, which in these experiments increased vasopressin release in the septum, inhibits or excites the electrical activity of septal neurones26; microiontophoretic application of vasopressin also alters the electrical activity of septal cells 1~22. Thus, notwithstanding other functions for vasopressin in the central nervous system (see refs. 10, 14, 19. 24, 27), vasopressin release in the septum may be considered as part of a central mechanism whereby the vasopressinergic system can in turn control its own control structures.
oxytocin pathways in the rat, Cell Tiss. Res., 186 (1978) 423-433. 4 Buijs, R.M. and Swaab, D.F., Immuno-electron microscopical demonstration of vasopressin and oxytocin pathways in the rat, Cell Tiss. Res., 204 (1979) 355-365. 5 Buijs, R.M. and van Heerikhuize, J.J., Vasopressin and oxytocin release in the brain - - a synaptic event, Brain Research, 252 (1982) 71-76. 6 Burnard, D.M., Pittman, Q.J. and Veale, W.L., Increased motor disturbances in response to arginine-vasopressin following haemorrhage or hvpertonic saline: evidence for cen-
321 tral AVP release in rats, Brain Research, 273 (1983) 59-65. 7 Clark, R.G., Jones, P.M. and Robinson, I.C.A.F., Clearance of vasopressin from cerebrospinal fluid to blood in chronically cannulated Brattleboro rats, Neuroendocrinology, 37 (1983) 242-247. 8 Cupo, A., Rougon-Rapuzzi, G. and Delaage, M.A., Immunochemical detection of vasopressin precursors: artificial processing and quantification along the hypothalamoneurohypophysial axis, Eur. J. Biochem., 115 (1981) 169-174. 9 de Vries, G.J. and Buijs, R.M., The origin of the vasopressinergic and oxytocinergic innervation of the rat brain with special reference to the lateral septum, Brain Research, 273 (1983) 307-317. 10 de Wied, D., Central actions of neurohypophysial hormones. In The Neurohypophysis: Structure, Function and Control, Progr. Brain Res., 60 (1983) 155-167. I1 Doris, P.A. and Bell, F.R., Vasopressin in plasma and cerebrospinal fluid of hydrated and dehydrated steers, Neuroendocrinology, 38 (1984) 290-296. 12 Dutton, A. and Dyball, R.E.J., Phasic firing enhances vasopressin release from the rat neurohypophysis, J. Physiol. (London), 290 (1979) 433-440. 13 Epstein, Y., Castel, M., Giick, S.M., Sivan, N. and Ravid, R., Changes in hypothalamic and extra-hypothalamic vasopressin content of water-deprived rats, Cell Tiss. Res., 233 (1983) 99-111. 14 Gash, D.M. and Thomas, G.J., What is the importance of vasopressin in memory processes? Trends Neurosci., 6 (1983) 197-198. 15 Hayward, J.N. and Vincent, J.D., Osmosensitive single neurones in the hypothalamus of unanesthetized monkeys, J. Physiol. (London), 210 (1970) 947-972. 16 Iovino, M., Poenaru, S. and Annunziato, L., Basal and thirst-evoked vasopressin secretion in rats with electrolytic lesion of the medio-ventral septal area, Brain Research, 258 (1983) 123-126. 17 Jewell, P.A. and Verney, E.B., An experimental attempt to determine the site of neurohypophyseal osmoreceptors in the dog, Phil. Trans. R. Soc. London Ser. B, 240 (1957) 197-324. 18 Joels, M. and Urban, I.J.A., The effect of microiontophoretically applied vasopressin and oxytocin on single neurones in the septum and dorsal hippocampus of the rat, Neurosci. Lett., 33 (1982) 79-84. 19 Kastings, N.W., Veale, W.L., Cooper, K.E. and Lederis, K., Effect of hemorrhage on fever: the putative role of vasopressin, Can. J. Physiol. Pharmacol., 59 (1981) 324-328. 20 Lubar, J.F., Boyce, B.A. and Schaeffer, C.F., Etiology of polydipsia and polyuria in rats with septal lesions, Physiol. Behav., 3 (1968) 289-292. 21 Mens, W.B.J., Witter, A. and van Wimersma Greidanus, T.B., Penetration of neurohypophysial hormones from plasma into cerebrospinal fluid: half-times of disappearance of these neuropeptides from CSF, Brain Research, 262 (1983) 143-149. 22 Pestre, M., Arnauld, E. and Vincent, J.D., Actions of microiontophoretically applied vasopressin selective agonists and antagonists on single neurons in the lateral septum of the rat, Neurosci. Lett., (19_84)Suppl. $341. 23 Pittman, Q.J., Blume, H.W. and Renaud, L.P., Connections of the hypothalamic paraventricular nucleus with the neurohypophysis, median eminence, amygdala, septum and midbrain periaqueductal gray: an electrophysiological study in the rat, Brain Research, 215 (1981) 15-28.
24 Pittman, Q.J., Veale, W.L. and Lederis, K., Central neurohypophysial peptide pathways: interaction with endocrine and other autonomic functions, Peptides, 3 (1982) 512-520. 25 Poulain, D.A., Ellendorff, F. and Vincent, J.D., Septal connections with identified oxytocin and vasopressin neurones in the supraoptic nucleus of the rat. An electrophysiological investigation, Neuroscience, 5 (1980) 379-387. 26 Poulain, D.A., Lebrun, C.J. and Vincent, J.D., Electrophysiological evidence for connections between septal neurones and the supraoptic nucleus of the hypothalamus of the rat, Exp. Brain Res., 42 (1981) 260-268. 27 Robinson, I.C.A.F., Neurohypophysial peptides in cerebrospinal fluid. In The Neurohypophysis: Structure, Function and Control, Progr. Brain Res., 60 (1983) 129-145. 28 Rodriguez, F., Demotes-Mainard, J., Chauveau, J., Poulain, D.A. and Vincent, J.D., Vasopressin release in the rat septum in response to systemic stimuli, Soc. Neurosci. Abstr., 9 (1983) 445. 29 Rougon-Rapuzzi, G., Millet, Y.A. and Delaage, M.A., Preparation of antivasopressin antibodies using an IgA carder. Application to radioimmunoassay, Biochimie (Paris), 59 (1977) 939-942. 30 Shibuki, K., Supraoptic cells: synaptic inputs from the nucleus accumbens in the rat, Exp. Brain Res., 53 (1984) 341-348. 31 Sibole, W., Miller, J.J. and Mogenson, G.J., Effects of septal stimulations on drinking elicited by electrical stimulation of the lateral hypothalamus, Exp. Neurol., 32 (1971) 466-477. 32 Simon-Opperman, C., Gray, D., Szczepanska-Sadowska, E. and Simon, E., Vasopressin in blood and third ventricle CSF of dogs in chronic experiments, Am. J. Physiol., 245 (1983) R541-R548. 33 Sofroniew, M.V. and Weindl, A., Extrahypothalamic neurophysin-containing perikarya, fiber pathways and fiber clusters in the rat brain, Endocrinology, 102 (1978) 334-337. 34 Sofroniew, M.V., Morphology of vasopressin and oxytocin neurones and their central and vascular projections. In The Neurohypophysis: Structure, Functions and Control, Progr. Brain Res., 60 (1983) 101-114. 35 Szczepanska-Sadowska, E., Gray, D. and Simon-Opperman, C., Vasopressin in blood and third ventricle CSF during dehydration, thirst and hemorrhage, Am. J. Physiol., 245 (1983) R549-R555. 36 van Leeuwen, F. and Caffe, R., Vasopressin-immunoreactive cell bodies in the bed nucleus of stria terminalis of the rat, Cell Tiss. Res., 228 (1983) 525-534. 37 Vincent, J.D., Arnauld, E. and Nicolescu-Catargi, A., Osmoreceptors and neurosecretory cells in the supraoptic complex of the unanesthetized monkey, Brain Research, 45 (1972) 278-281. 38 Wang, B.C., Share, L., Crofton, J.T. and Kimura, T., Changes in vasopressin concentrations in plasma and cerebrospinal fluid in response to hemorrhage in anesthetized dogs, Neuroendocrinology, 33 (1981) 61-66. 39 Wang, B.C., Share, L., Crofton, J.T. and Kimura, T., Effect of intravenous and intracerebroventricular infusion of hypertonic solutions on plasma and cerebrospinal fluid vasopressin concentrations, Neuroendocrinology, 34 (1982) 215-221. 40 Zerbe, R.L. and Palkovits, M., Changes in the vasopressin content of discrete brain regions in response to stimuli for vasopressin secretion, Neuroendocrinology, 38 (1984) 285-289.
314 BRE 11965
Septal Release of Vasopressin in Response to Osmotic, Hypovolemic and Electrical Stimulation in Rats J. DEMOTES-MAINARD, J. CHAUVEAU, F. RODRIGUEZ, J.D. VINCENT and D.A. POULAIN 1. N. S. E. R.M. U 176, Bordeaux Cedex (France
(Accepted January 21st, 1986) Key words: vasopressin - - septum - - push-pull cannula - - hemorrhage - - hypertonic stimulation - - magnoceUular system
The central release of vasopressin was studied in anesthetized rats using push-pull perfusions and radioimmunoassay of the hormone. A basal release was observed in the lateral septum and in the lateral ventricle, whereas no vasopressin was detected in the perfusates from the caudate nucleus. Under osmotic stimulation, vasopressin release increased up to 12 and 60 times basal levels following i.p. injections of 5 ml and 10 ml/kg b.wt. of 2 M NaCI, respectively. This increase was blocked by using a calcium-free perfusion medium containing 0.1 mM EGTA. In the lateral ventricle, osmotic stimulation (5 ml/kg of 2 M NaCI i.p.) had the same effect as in the septum. In the caudate nucleus, no release was observed. Hemorrhage also increased the septal release of vasopressin in 5 out of 6 animals tested. Electrical stimulation of the pituitary stalk and of the supraoptic nucleus was used to evoke the release of vasopressin into the bloodstream. Septal release slightly decreased during pituitary stalk stimulation, whereas it did increase during stimulation of the supraoptic region. Our results show that systemic stimuli for vasopressin release evoke both a peripheral and a septal release of the hormone. The dissociation of the effects of electrical stimulation of the pituitary stalk and of the supraoptic nucleus suggests, however, that the vasopressinergic neurones responsible for septal release are distinct from those which project to the neurohypophysis.
INTRODUCTION A large body of immunocytochemical evidence has now clearly established that vasopressinergic innervation extends to several central nervous structures in addition to the h y p o t h a l a m o - n e u r o h y p o p h y sial pathway 3'33'34. This raises n u m e r o u s questions concerning the role of this extra-hypothalamic innervation, and the physiological conditions u n d e r which a central release of h o r m o n e may occur. In the lateral septum, there is an a b u n d a n t vasopressinergic innervation 4'9 and in vitro experiments have demonstrated that vasopressinergic terminals onto septal n e u r o n e s are indeed able to release h o r m o n e in response to depolarization5. We therefore examined the septal release of vasopressin in vivo using a push-pull perfusion technique. Systemic osmotic and hypovolemic stimuli were chosen in order to determine whether a central release of h o r m o n e was taking place simultaneously with the peripheral release that they are known to evoke. In addition, in order to examine
whether the hypothalamo-neurohypophysial axis has any role in septal release, we also studied such a release after electrical stimulation of the pituitary stalk and of the supraoptic (SO) region of the hypothalamus. A preliminary report of these results has been presented earlier 28. MATERIALS AND METHODS Animals
The experiments were performed on Wistar rats (280-450 g b.wt.), bred in a controlled e n v i r o n m e n t , with water and food available ad libitum. The animals were anesthetized with urethane (1.3 g/kg i.p.) and set in a stereotaxic device (Narishige), Body temperature was m a i n t a i n e d at 37 °C by a homeothermic blanket (CFP 8185, Bioscience). Push-pull p e r f u s i o n s
The stainless steel cannula that we used consisted of an external guide tube (diameter 0.6 mm) paired
Correspondence: D.A. Poulain, I.N.S.E.R.M. U 176, 1 rue Camille Saint-Saens, F-33077 Bordeaux Cedex, France.
0006-8993/86/$03.50 (~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)
315 with a sharp pointed mandrel during insertion into the central nervous system. The mandrel was then replaced by an inner double-barrelled pipe, the expelling ('push') pipe projecting 0.5 mm out of the guide tube, and the tip of the sucking ('pull') pipe resting 1 mm inside the guide tube. The cannula was connected to two peristaltic pumps (Gilson Minipulse 2) allowing a regular perfusion at a rate of 67 pl/min (i.e. = 1 ml/15 min) of an artificial cerebrospinal fluid (in mM: NaC1 126.5, NaHCO 3 27.5, KC1 2.4, KH2PO 4 0.5, CaC12 1.1, MgC12 0.83, Na2SO 4 0.5, glucose 5.9, pH adjusted at 7.3 by using an O2/CO 2 gas mixture at 95%/5% (v/v); T 37 °C). Sampling began only after a one-hour period following the onset of the push-pull perfusion to allow the stabilization of the perfusion. Then, 1 ml samples were collected every 15 rain in pre-cooled polypropylene tubes, immediately frozen at -20 °C, and subsequently assayed for arginine-vasopressin.
Arginine-vasopressin radioimmunoassay Samples were treated for extraction by addition of two volumes of dioxane. The homogenate was centrifuged at 12,000 g for 10 min. The supernatant was lyophilized, then rehydrated in 250/~1 incubation buffer containing 10 mM sodium phosphate, 10 mM sodium azide, 0.15 M sodium chloride and 1 g/l bovine serum albumin. Radioimmunological incubations were carried out by equilibrium dialysis. The 10 dialysis cells were filtered by large pore cellulose membranes (Sartorius SM 1153). Incubation time was 44 h. Before being placed on one side of this membrane, the test samples (or standards) were mixed with an equal volume of [125I]ArgS-vasopressin solution. The other side was filled with diluted antibody as described previously8'29. The cross-reactivity factor for vasopressin analogues were: arginine- and lysinevasopressin 1; arginine-vasotocin 8.9; oxytocin 3.9 x 10-4. The minimum amount of arginine-vasopressin detectable was 0.35 pg/sample.
Experimental groups Osmotic stimulation. Vasopressin release was studied in male rats after i.p. injection of hypertonic saline (NaCl 2 M) at a dose of 5 or 10 ml/kg b.wt. After craniotomy, the push-pull cannula was implanted into the lateral septum or, for controls, into the lateral ventricle or in the caudate nucleus. Samples of per-
fusate were collected during 45 min before and during at least 105 min after the i.p. injection. In a group of animals, a calcium-free medium containing 0.1 mM EGTA was used instead of the normal artificial cerebrospinal fluid. Finally, the changes in plasma osmolarity were studied in separate groups of rats similarly anesthetized with urethane. Venous blood samples were collected via a previously inserted catheter into the jugular vein, before and at different times (15, 45, 60, 90 and 120 min) after osmotic load. Blood samples were centrifuged at 4 °C and plasma osmolarity determined by the freezing point method (Fishke osmometer). Hemorrhage. In one group of male rats with a push-pull cannula implanted into the lateral septum, hemorrhage was performed by removing blood via a catheter inserted into the right atrium (12.5 ml/kg in 10 min, i.e. about 15% blood volume). Samples of perfusate were collected 45 min before and up to 120 min after hemorrhage. Subsequently, an equal volume of isotonic saline was slowly reinjected via the atrial catheter and perfusion fluid were collected for a further 60 min period. Electrical stimulation. After inserting a push-pull cannula into the lateral septum, two stimulating electrodes (SNEX 100, Rhodes Medical Instruments) were lowered, one in the supraoptic nucleus ipsilateral to the push-puU cannula, the other one in the pituitary stalk, using a dorsal approach. To allow precise implantation of the electrodes, the experiments were carried out in lactating rats. One mammary gland was cannulated in order to monitor intramammary pressure via a pressure gauge transducer (Statham) connected to a pen recorder (Racia). The electrode positions were adjusted until a short train (5 s) of electrical pulses (50 Hz, 0.5 ms, 0.5 mA) caused a rise in intramammary pressure similar to that produced by an i.v. injection of 0.5 mU oxytocin (see ref. 26). After the electrodes were correctly positioned, perfusion through the push-pull cannula was started, and after a one-hour period, three control samples were taken. The pituitary stalk was then stimulated for 45 min, and after a 45 min resting period, the SO nucleus was then stimulated for 45 rain. A phasic pattern of stimulation was applied using a burst of action potentials previously recorded from a vasopressinergic neurone (burst duration: 26 s, intraburst frequency 13 Hz, silent period duration 22 s,
316
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see Fig. 6A). Such a phasic pattern has been shown to be highly efficient for promoting hormone release 12. Histological controls. A t the end of each experiment, the animals were decapitated and the brains were fixed in a 10% formaldehyde solution. Light microscopic observation of paraffin-embedded sections allowed to locate the path and the tip of the push-pull cannula, and also the electrolytic lesion in the SO nucleus made by passing a direct current at the end of the electrical stimulation experiments.
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Effect o f osmotic stimulation The effect of osmotic stimulation by i.p. injection of 2 M NaCI on vasopressin release depended on the amount injected and on the site of perfusion. After injection of 10 ml/kg b.wt., plasma osmolarity rose from 311 + 0.6 up to 375 + 5 mOsm/kg H 2 0 (n = 5) 15 min after injection, then slowly declined down to 360 + 4 mOsm/kg H 2 0 by 120 min after injection. Under these conditions, the amount of vasopressin in the perfusates from the lateral septum increased up to 8.5 + 4.5 pg/sample at 15 min (n = 5, significantly different from control values at P < 0.01, Student's paired t-test), and then to 58 + 3 pg/sample at 105 min (Fig. 1). In the caudate nucleus (n = 3), on the other hand, vasopressin levels remained at the detection limit of our assay throughout the experiment. With a lower osmotic load (5 ml/kg b.wt.), plasma
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the vasopressin content of a sample of push-pul! perfusate (1 ml) collected over a 15-min period; each curve represents one experiment performed in one animal. The first three points are controls. Note the large increase in vasopressin release from the lateral septum evoked by osmotic stimulation. In the caudate nucleus, vasopressin levels remained undetectable throughout the experiment. osmolarity reached 335 + 3 mOsm/kg H20 at 15 min and remained stable up to 120 min after injection. Perfusates from the lateral septum showed a substantial increase in vasopressin in each of the 4 animals
317 tested (Fig. 2). Although there were large individual variations between animals, the increase, much lower than that evoked by a higher osmotic load, reached 5.1 + 2.1 pg/sample at 15 min (P < 0.01, paired t-test, n = 4 rats) and attained a peak at 105 min (12 + 1.4 pg/sample, n.s.). In perfusates from the lateral ventricle (Fig. 3), a similar pattern of vasopressin increase was observed following i.p. injection of 5 ml of 2 M NaCI, reaching 2.0 + 1.0 pg at 30 min,
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and 4.1 + 1.2 pg/sample at 105 min (P < 0.01 paired t-test in both cases). In an attempt to see whether the osmotically evoked release of vasopressin in the septum was a calcium-dependent and thus presumably a synaptic process, a calcium-free medium containing 0.1 mM EGTA was used instead of the normal perfusion medium in a group of 8 animals. Osmotic stimulation (5 ml/kg) evoked no consistent pattern of vasopressin release in the septal perfusates, either from one animal to another, or from one sample to another in each animal. The vasopressin contents in the perfusates from the lateral septum were not significantly altered by injection of hypertonic saline in comparison to the control periods using either the normal or calcium-free media (Fig. 4). However, they were significantly lower in animals perfused with a calciumfree medium than in animals perfused with the normal medium in all samples taken 30 to 90 min after injection of hypertonic saline (P < 0.01, Mann-Whitney U test).
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Effect of hemorrhage The effect of hemorrhage on septal release of vasopressin was studied in six animals by removing rapidly blood from the right atrium. Plasma concentration of vasopressin, assayed in the last 2.5 ml of blood removed, reached 342 + 184 pg/ml. In 5 animals, septal release of vasopressin increased progressively, being significantly higher (P < 0.01, paired ttest) than control levels in all samples between 30 and 120 min, and reaching a peak at 75-90 min after hemorrhage (Fig. 5). At 90 min, injection of isotonic saline to restore blood volume was accompanied by a progressive decline in vasopressin release. A 6th animal in which vasopressin levels remained constantly at detection level throughout the periods of hemorrhage and saline infusion, was not included in the statistical analysis.
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319 the supraoptic nucleus was performed in 4 lactating rats, using a phasic pattern of stimulation (see Methods). Peripheral release of neurohypophysial hormones after such stimuli was monitored by the recurrent variations in intramammary pressure (Fig. 6). In each animal, stimulation of the pituitary stalk was accompanied by a slight decrease in vasopressin levels in the perfusates from the lateral septum, from 2.5 + 1.5 pg/sample before stimulation down to 1.3 +
0.8 pg/sample 45 min after the onset of stimulation (P < 0.025, paired t-test). At the end of the 45 min recovery period, vasopressin levels were back to 2.0 + 0.2 pg/sample. Stimulation of the SO nucleus had an effect opposite to that of pituitary stalk stimulation. In each animal, there was an increased content in vasopressin (up to 5.6 + 1.3 pg/sample; P < 0.005) 45 min after the onset of stimulation. In three animals, vasopressin levels dropped back to control level after cessation of stimulation.
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Fig. 6. Effects of electrical stimulation of the pituitary stalk and of the supraoptic nucleus on the release of vasopressin from the septum. A: intramammary pressure recording (top line) during electrical stimulation of the SO nucleus using a phasic pattern of electrical stimuli (bottom line). Recurrent milk ejections provided evidence that stimulation did induce peripheral release of neurohypophysial hormones. B: vasopressin release from the lateral septum during electrical stimulation. Changes are expressed in percentage of control levels (2.5 pg = 100%). During pituitary stalk stimulation, vasopressin levels decreased significantly (P < 0.01, paired t-test on raw data). In contrast, during SO stimulation, vasopressin release increased significantly (P < 0.01).
The push-pull perfusion technique that we used in these experiments permitted us to establish that during systemic or central stimulation, not only is vasopressin released into the bloodstream, but also into central structures such as the sepl;um. Our results thus support the suggestion that the vasopressin innervation described in the lateral septum is of functional significance under certain conditions of stimulation of hormone release. They suggest, moreover, that the peripheral and central release of vasopressin are dependent upon separate pools of vasopressinergic neurones. As all the stimuli that we used led to the release of hormones into the blood, it could be argued that diffusion from the bloodstream into the septum could account for the presence of vasopressin in the septal perfusates. However, vasopressin does not cross the blood-brain barrier 1'7'2t and our own data exclude a non-specific diffusion or uptake in the brain, since perfusates from the caudate nucleus were devoid of hormone. Diffusion or uptake specifically restricted to the septum is also unlikely since vasopressin release in blood evoked by electrical stimulation of the pituitary stalk was not accompanied by an increase in vasopressin content in the septal perfusates. A more critical issue concerns the possible diffusion of vasopressin from the lateral ventricle to the septum. Release of vasopressin into the cerebrospinal fluid has been reported by several authors, and in our experiments, vasopressin increased in a similar manner in the perfusates from the lateral ventricle and from the lateral septum. Alternatively, vasopressin could also diffuse from central structures to the ventricles, or it may be that both types of release occur. None of these possibilities can be excluded. Nevertheless,
320 several lines of evidence make it highly probable that vasopressin was actually released within the septum. Vasopressin-containing terminals have been identified by electron microscopic immunocytochemistry in the lateral septum4; in vitro, vasopressin release from septal slices can be increased by depolarization with potassium or veratridine5; and finally, in the present in vivo experiments, the osmotically induced release of vasopressin was prevented when using a calcium-free medium containing 0.1 mM E G T A to reduce or block local synaptic activity. Our experiments using electrical stimulation indicate that the vasopressinergic innervation of the septum is independent of the hypothalamo-neurohypophysial axis. This is in agreement with several observations showing that the magnocellular neurones which project to the neurohypophysis do not also project to the septum. Electrophysiologically, magnocellular neurones in the SO or paraventricular nuclei cannot be antidromically invaded when electrically stimulating the septum 23-25. On morphological grounds, extensive lesions of hypothalamus do not alter the septal content in vasopressinergic fibers 9, which appear in fact to arise from the bed nucleus of the stria terminalis 36. In the present experiments, since stimulation of the pituitary stalk activates magnocellular cell bodies antidromically, a septal release of hormone should have been observed if the neurones possessed septal efferents. On the other hand, stimulation of the supraoptic region led to both a peripheral and a central release of hormone. As stated before, this cannot be attributed to supraoptic efferents to the septum. We can also exclude the possibility that SO cells would activate other vasopressinergic cells through a polysynaptic pathway, since such an anatomical arrangement would have permitted the release of hormone when stimulating the cell bodies antidromically from the pituitary stalk. The most likely hypothesis is that electrical stimulation of the SO region activated an
REFERENCES 1 Ang, V.T.Y. and Jenkins, J.S., Blood-cerebrospinal fluid barrier to arginine-vasopressin, desmopressin and desglycinamide arginine-vasopressin in the dog, J. Endocrinol., 93 (1982) 319-325. 2 Barnard, R.R. and Morris, M., Cerebro-spinal fluid vasopressin and oxytocin: evidence for an osmotic response, Neurosci. Lett., 29 (1982) 275-279. 3 Buijs, R.M., Swaab, D.F., Dogterom, J. and van Leeuwen, F.W., Intra- and extra-hypothalamicvasopressin and
afferent pathway common both to the hypothalamoneurohypophysial system and to a cell group projecting to the septum. Since osmoreceptors may be located in the vicinity of the SO nucleus 1517~37, and since osmotic stimulation induced a septal release, it is tempting to speculate that SO stimulation may have activated this osmoreceptor system. Our observation of a central release of vasopressin during hypovolemic and osmotic stimulation is in agreement with several experiments reporting a release of hormone into central structures or in the cerebrospinal fluid during h e m o r r h a g e 6"19"3~'3~,3s or osmotic stimuli 2'6"11"13"27"35"39"4°. Although there appear to be important variations in the intensity of vasopressin release, depending upon the sites and the methods of push-pull perfusions, and on the routes of administration, it is clear that homeostatic processes involved in the control of salt and water balance in the blood are simultaneously inducing a release of vasopressin in central structures. The physiological significance of such a release under these conditions has yet to be determined. The septum does participate in the control of water intake or arterial blood pressure, and therefore, at least indirectly, in the control of neurohypophysial function 2°'31. More specifically, the electrical activity of vasopressin neurones can be altered by septal stimulation ~5'3°, and septal lesions modify the release of vasopressin during osmotic stimulation x6. Conversely, electrical stimulation of the SO nucleus, which in these experiments increased vasopressin release in the septum, inhibits or excites the electrical activity of septal neurones26; microiontophoretic application of vasopressin also alters the electrical activity of septal cells 1~22. Thus, notwithstanding other functions for vasopressin in the central nervous system (see refs. 10, 14, 19. 24, 27), vasopressin release in the septum may be considered as part of a central mechanism whereby the vasopressinergic system can in turn control its own control structures.
oxytocin pathways in the rat, Cell Tiss. Res., 186 (1978) 423-433. 4 Buijs, R.M. and Swaab, D.F., Immuno-electron microscopical demonstration of vasopressin and oxytocin pathways in the rat, Cell Tiss. Res., 204 (1979) 355-365. 5 Buijs, R.M. and van Heerikhuize, J.J., Vasopressin and oxytocin release in the brain - - a synaptic event, Brain Research, 252 (1982) 71-76. 6 Burnard, D.M., Pittman, Q.J. and Veale, W.L., Increased motor disturbances in response to arginine-vasopressin following haemorrhage or hvpertonic saline: evidence for cen-
321 tral AVP release in rats, Brain Research, 273 (1983) 59-65. 7 Clark, R.G., Jones, P.M. and Robinson, I.C.A.F., Clearance of vasopressin from cerebrospinal fluid to blood in chronically cannulated Brattleboro rats, Neuroendocrinology, 37 (1983) 242-247. 8 Cupo, A., Rougon-Rapuzzi, G. and Delaage, M.A., Immunochemical detection of vasopressin precursors: artificial processing and quantification along the hypothalamoneurohypophysial axis, Eur. J. Biochem., 115 (1981) 169-174. 9 de Vries, G.J. and Buijs, R.M., The origin of the vasopressinergic and oxytocinergic innervation of the rat brain with special reference to the lateral septum, Brain Research, 273 (1983) 307-317. 10 de Wied, D., Central actions of neurohypophysial hormones. In The Neurohypophysis: Structure, Function and Control, Progr. Brain Res., 60 (1983) 155-167. I1 Doris, P.A. and Bell, F.R., Vasopressin in plasma and cerebrospinal fluid of hydrated and dehydrated steers, Neuroendocrinology, 38 (1984) 290-296. 12 Dutton, A. and Dyball, R.E.J., Phasic firing enhances vasopressin release from the rat neurohypophysis, J. Physiol. (London), 290 (1979) 433-440. 13 Epstein, Y., Castel, M., Giick, S.M., Sivan, N. and Ravid, R., Changes in hypothalamic and extra-hypothalamic vasopressin content of water-deprived rats, Cell Tiss. Res., 233 (1983) 99-111. 14 Gash, D.M. and Thomas, G.J., What is the importance of vasopressin in memory processes? Trends Neurosci., 6 (1983) 197-198. 15 Hayward, J.N. and Vincent, J.D., Osmosensitive single neurones in the hypothalamus of unanesthetized monkeys, J. Physiol. (London), 210 (1970) 947-972. 16 Iovino, M., Poenaru, S. and Annunziato, L., Basal and thirst-evoked vasopressin secretion in rats with electrolytic lesion of the medio-ventral septal area, Brain Research, 258 (1983) 123-126. 17 Jewell, P.A. and Verney, E.B., An experimental attempt to determine the site of neurohypophyseal osmoreceptors in the dog, Phil. Trans. R. Soc. London Ser. B, 240 (1957) 197-324. 18 Joels, M. and Urban, I.J.A., The effect of microiontophoretically applied vasopressin and oxytocin on single neurones in the septum and dorsal hippocampus of the rat, Neurosci. Lett., 33 (1982) 79-84. 19 Kastings, N.W., Veale, W.L., Cooper, K.E. and Lederis, K., Effect of hemorrhage on fever: the putative role of vasopressin, Can. J. Physiol. Pharmacol., 59 (1981) 324-328. 20 Lubar, J.F., Boyce, B.A. and Schaeffer, C.F., Etiology of polydipsia and polyuria in rats with septal lesions, Physiol. Behav., 3 (1968) 289-292. 21 Mens, W.B.J., Witter, A. and van Wimersma Greidanus, T.B., Penetration of neurohypophysial hormones from plasma into cerebrospinal fluid: half-times of disappearance of these neuropeptides from CSF, Brain Research, 262 (1983) 143-149. 22 Pestre, M., Arnauld, E. and Vincent, J.D., Actions of microiontophoretically applied vasopressin selective agonists and antagonists on single neurons in the lateral septum of the rat, Neurosci. Lett., (19_84)Suppl. $341. 23 Pittman, Q.J., Blume, H.W. and Renaud, L.P., Connections of the hypothalamic paraventricular nucleus with the neurohypophysis, median eminence, amygdala, septum and midbrain periaqueductal gray: an electrophysiological study in the rat, Brain Research, 215 (1981) 15-28.
24 Pittman, Q.J., Veale, W.L. and Lederis, K., Central neurohypophysial peptide pathways: interaction with endocrine and other autonomic functions, Peptides, 3 (1982) 512-520. 25 Poulain, D.A., Ellendorff, F. and Vincent, J.D., Septal connections with identified oxytocin and vasopressin neurones in the supraoptic nucleus of the rat. An electrophysiological investigation, Neuroscience, 5 (1980) 379-387. 26 Poulain, D.A., Lebrun, C.J. and Vincent, J.D., Electrophysiological evidence for connections between septal neurones and the supraoptic nucleus of the hypothalamus of the rat, Exp. Brain Res., 42 (1981) 260-268. 27 Robinson, I.C.A.F., Neurohypophysial peptides in cerebrospinal fluid. In The Neurohypophysis: Structure, Function and Control, Progr. Brain Res., 60 (1983) 129-145. 28 Rodriguez, F., Demotes-Mainard, J., Chauveau, J., Poulain, D.A. and Vincent, J.D., Vasopressin release in the rat septum in response to systemic stimuli, Soc. Neurosci. Abstr., 9 (1983) 445. 29 Rougon-Rapuzzi, G., Millet, Y.A. and Delaage, M.A., Preparation of antivasopressin antibodies using an IgA carder. Application to radioimmunoassay, Biochimie (Paris), 59 (1977) 939-942. 30 Shibuki, K., Supraoptic cells: synaptic inputs from the nucleus accumbens in the rat, Exp. Brain Res., 53 (1984) 341-348. 31 Sibole, W., Miller, J.J. and Mogenson, G.J., Effects of septal stimulations on drinking elicited by electrical stimulation of the lateral hypothalamus, Exp. Neurol., 32 (1971) 466-477. 32 Simon-Opperman, C., Gray, D., Szczepanska-Sadowska, E. and Simon, E., Vasopressin in blood and third ventricle CSF of dogs in chronic experiments, Am. J. Physiol., 245 (1983) R541-R548. 33 Sofroniew, M.V. and Weindl, A., Extrahypothalamic neurophysin-containing perikarya, fiber pathways and fiber clusters in the rat brain, Endocrinology, 102 (1978) 334-337. 34 Sofroniew, M.V., Morphology of vasopressin and oxytocin neurones and their central and vascular projections. In The Neurohypophysis: Structure, Functions and Control, Progr. Brain Res., 60 (1983) 101-114. 35 Szczepanska-Sadowska, E., Gray, D. and Simon-Opperman, C., Vasopressin in blood and third ventricle CSF during dehydration, thirst and hemorrhage, Am. J. Physiol., 245 (1983) R549-R555. 36 van Leeuwen, F. and Caffe, R., Vasopressin-immunoreactive cell bodies in the bed nucleus of stria terminalis of the rat, Cell Tiss. Res., 228 (1983) 525-534. 37 Vincent, J.D., Arnauld, E. and Nicolescu-Catargi, A., Osmoreceptors and neurosecretory cells in the supraoptic complex of the unanesthetized monkey, Brain Research, 45 (1972) 278-281. 38 Wang, B.C., Share, L., Crofton, J.T. and Kimura, T., Changes in vasopressin concentrations in plasma and cerebrospinal fluid in response to hemorrhage in anesthetized dogs, Neuroendocrinology, 33 (1981) 61-66. 39 Wang, B.C., Share, L., Crofton, J.T. and Kimura, T., Effect of intravenous and intracerebroventricular infusion of hypertonic solutions on plasma and cerebrospinal fluid vasopressin concentrations, Neuroendocrinology, 34 (1982) 215-221. 40 Zerbe, R.L. and Palkovits, M., Changes in the vasopressin content of discrete brain regions in response to stimuli for vasopressin secretion, Neuroendocrinology, 38 (1984) 285-289.