R. Landgraf

Central and peripheral release of vasopressin and oxytocin in the conscious rat after osmotic stimulation.

Vasopressin (AVP) and oxytocin (OXT) were measured by radioimmunoassay in push-pull perfusates and tissue samples of various brain areas, plasma and cerebrospinal fluid (CSF) of male rats in response to osmotic stimulation. Hypertonic saline caused a significant rise in plasma AVP and OXT and different changes in peptide contents, in the septum and hippocampus at 30 and 60 min after intraperitoneal injection. Push-pull perfusion (20 microliters artificial CSF/min, 30-min periods) of the septum and dorsal hippocampus of conscious, unrestrained animals revealed a significant, stimulus-evoked release of both AVP and OXT. This release was: (1) not always reflected by corresponding changes in the regional peptide content; (2) simultaneous with the peripheral release from the posterior pituitary; and (3) probably the result of synaptic/parasynaptic events as suggested by use of agents in the artificial CSF which either inhibit or facilitate the release from intact fibre terminals.

Septal and Hippocampal Release of Vasopressin and Oxytocin during Late Pregnancy and Parturition in the Rat

The push-pull perfusion technique, in conjunction with specific radioimmunoassays, was used to monitor the release of both arginine vasopressin (AVP) and oxytocin (OXT) within distinct limbic brain areas of conscious female rats. In pregnant rats near term, the release of AVP was greater than that of virgin rats in both the ventral (p less than 0.001) and mediolateral (p less than 0.001) septal areas; similarly, release of OXT increased in the ventral septal area (p less than 0.01) at this time. In contrast, no changes in the levels of either peptide occurred in the dorsal hippocampus. In parturient rats, AVP release tended to decrease in the septal areas but increased fivefold in the dorsal hippocampus (p less than 0.001) compared to pregnant animals. In contrast, OXT levels assayed in the same perfusates did not differ from those observed in pregnant animals. Plasma levels of AVP in pregnant rats (p less than 0.05) and of OXT in parturient animals (p less than 0.01) were found to be increased over levels in virgin rats. The regionally different and peptide-specific changes in release pattern of AVP and OXT in virgin, pregnant and parturient rats may be of physiological significance in antipyresis and behaviors accompanying parturition.

Vasopressin release within the supraoptic and paraventricular nuclei of the rat brain: osmotic stimulation via microdialysis

The combination of microdialysis and a highly sensitive radioimmunoassay was used in order to monitor the in vivo release of arginine vasopressin (AVP) within hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei of the rat brain. A dialysis probe was inserted into the SON or PVN area and microdialysis was performed in conscious or urethane-anesthetized animals before, during and after hypertonic artificial cerebrospinal fluid (aCSF, with 1 M NaCl) was delivered via the probe. The recovery of AVP in vitro was 1.60%, that of [3H]OH in vitro 14.2% and in vivo 8.44% (SON) and 9.26% (PVN), respectively. AVP was consistently detected in both SON and PVN dialysates; basal levels averaged 0.87 +/- 0.22 pg/30-min dialysate (SON, n = 51) and 0.80 +/- 0.24 pg/30-min dialysate (PVN, n = 6), respectively. Hypertonic aCSF given over a period of 30 min, 60 min or 90 min, resulted in an increased AVP release within the SON which, however, reached its peak (to 8.86-10.27 pg/sample; P less than 0.001 as compared to basal) only in the poststimulation period, i.e. after replacement of hypertonic with isotonic aCSF. An identical osmotic stimulus given 150-210 min after the first one produced similar, though slightly declined, changes in AVP release. In the PVN, AVP release patterns prior to and in response to the first hypertonic pulse were similar to those in the SON; a possible functional difference between the two nuclei is indicated by the lack of a rebound increase in AVP release following the second stimulation. The physiological significance of intranuclearly released AVP remains to be shown.(ABSTRACT TRUNCATED AT 250 WORDS)

Microdialysis administration of vasopressin and vasopressin antagonists into the septum during pole-jumping behavior in rats

Wistar rats (n = 95) were trained in a pole-jumping apparatus (10 trials/session/day) to investigate the involvement of centrally and peripherally released endogenous AVP in their acquisition rate and to examine the feasibility of the microdialysis technique for the administration of peptides during a behavioral test. After session 1, a microdialysis probe was implanted into the septum; during sessions 2 and 3 the probe was perfused with artificial cerebrospinal fluid (aCSF) alone or containing either AVP (delivered amount via the probe: 0.2 ng) or the V1 (d(CH2)5Tyr(Me)AVP, 5.0 ng) or the V2/V1 (d(CH2)5-D-Tyr(Et)VAVP, 5.0 ng) antagonist. Administration of AVP via microdialysis into the septum failed to alter the acquisition rate of pole jumping. Also, ip application of both hypertonic saline and the AVP V1 antagonist (10 micrograms) in another experiment failed to show a significant effect upon behavior. Septal administration of the V1 or the V2/V1 antagonist via microdialysis, however, produced a significantly impaired performance. The results indicate that AVP release within the septum is involved in the acquisition of pole-jumping behavior probably mediated by the V1 receptor subtype. An additional involvement of the V2 receptor subtype, however, cannot be entirely excluded. The microdialysis technique proved to be a potent tool to administer substances concomitantly with behavioral tests.

Push‐pull Perfusion and Microdialysis Studies of Central Oxytocin and Vasopressin Release in Freely Moving Rats during Pregnancy, Parturition, and Lactation

Oxytocin (0T)-containing neurons are localized predominantly in the compact magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) and in the supraoptic nucleus (SON). Unlike arginine vasopressin (AVP) neurons, OT-producing cell bodies appear to be absent in extrahypothalamic brain areas. Magnocellular hypothalamic neurons project to the neurohypophysis. Following peripheral release, OT is primarily associated with smooth muscle contraction of the female reproductive system, and AVP with water reabsorption by the kidney, vasoconstriction in certain vascular beds, and glycogenolysis in the liver. Additionally, parvocellular neurons of the PVN (OT, AVP) and of extrahypothalamic sources (AVP) project to central targets, especially limbic areasZ known to be involved in learning, memory, and behavioral performance. Since the transport of endogenous nonapeptides across the blood-brain barrier is severely re~tricted,~ central effects should be the result of centrally released rather than peripherally circulating OT and AVP, respectively. Such central effects include defervescence (AVP)4 and selective behavioral regulation (OT, AVP).5,6 The fact that different cell populations project peripherally to the neurohypophysis and centrally to limbic brain targets implies that a stimulus which provokes a release of OT and AVP into the bloodstream does not necessarily provoke a release within the

Chapter 6: Central release of vasopressin: stimuli, dynamics, consequences

Publisher Summary This chapter focuses on push–pull perfusion and microdialysis techniques to monitor central peptide release in vivo , its changes in response to various stimuli, simultaneous changes in central receptor characteristics, feedback phenomena and consequences on autonomic and behavioral functions. Experiments with applied synthetic arginine vasopressin (AVP) or analogs have produced evidence for AVP involvement in a variety of central neural functions, including learning, memory, behavior, antipyresis, and cardiovascular regulation. However, the need to work with exogenously applied peptide has made identification of the role of endogenous AVP in each of these paradigms difficult. Evidence has been accumulated indicating that endogenous AVP circulating in the plasma does not cross the blood–brain barrier in physiologically significant amounts. Although plasma AVP is believed to contribute indirectly to central effects by altering the blood–brain barrier permeability to essential substrates, AVP released centrally rather than plasma AVP should be primarily involved in the regulation of central nervous functions. Microdialysis and push–pull perfusion are appropriate in vivo techniques to monitor basal and evoked AVP release within hypothalamic nuclei and limbic and other brain areas. AVP is released centrally under basal conditions and in response to a variety of stimuli, including electrical and osmotic stimulation, fever, pregnancy, and parturition.

Vasopressin administration via microdialysis into the septum interferes with the acquisition of spatial memory in rats

The role of vasopressin (AVP) in the septohippocampal system in spatial memory was studied in 27 male hooded rats of the Long-Evans strain. The rats were implanted with a septal microdialysis probe and assigned to 3 groups. Two days later, they were trained on 3 consecutive days (12 daily trials) to locate the hidden underwater platform in the Morris water maze (MWM) while the probes were perfused with either artificial cerebrospinal fluid (aCSF) or aCSF containing vasopressin or the V1 antagonist d(CH2)5Tyr(Me)AVP (AAVP). Another group of rats (n = 8) remained untreated. Groups receiving microdialysis of aCSF or AAVP acquired the MWM task at the same rate as untreated animals. On the other hand, place navigation learning was significantly impaired by microdialysis of AVP during all sessions. The results indicate that endogenous AVP (at least that affecting the V1 receptor subtype) is not indispensable for the acquisition of spatial memories in the MWM, whereas excessive presence of synthetic AVP interferes with spatial learning.

Does the release of vasopressin within the supraoptic nucleus of the rat brain depend upon changes in osmolality and Ca2+/K+?

Microdialysis in conjunction with a highly sensitive radioimmunoassay was used to monitor the in vivo release of arginine vasopressin (AVP) within the supraoptic nucleus (SON) of the rat brain (n = 70). Thirty-min dialysates were collected in urethane-anesthetized animals before, during and after hypertonic or hypotonic pulses were delivered via the probe. As compared to artificial cerebrospinal fluid (aCSF)-perfused controls, 1M aCSF given over a period of 210 min resulted in an increased intranuclear AVP release which, however, reached its peak only in the post-stimulation period, i.e. after replacement of hypertonic with isotonic aCSF again (rebound phenomenon). Hypertonic (0.5 M, 1 M or 2 M) pulses given 150 min after the first (1 M) pulse resulted in 3 different rebound responses: a marked decrease (to 25.3%, P less than 0.001), no change or slight increase (132%, n.s.). As shown by the response to correction of the hypertonicity to normal as well as by perfusion of hypotonic aCSF (0.01 M), release of AVP within the SON appears more responsive to a reduction than to an elevation in the osmolality of aCSF. Omission of Ca2+ from and addition of EGTA to the aCSF decreased the osmotically stimulated, but not the basal AVP release. If K(+)-hypertonic aCSF was used, however, basal AVP levels increased significantly; in contrast, the rebound increase failed to differ from aCSF-perfused controls. It is concluded from these findings that release of AVP from intact neuronal structures in the SON is responsive to changes in osmolality with an apparent greater sensitivity to decrease in osmolality.

Naloxone increases the release of oxytocin, but not vasopressin, within limbic brain areas of conscious parturient rats: a push-pull perfusion study.

The influence of naloxone on the release in limbic brain areas of both oxytocin (OXT) and vasopressin, measured by radioimmunoassay, was studied in conscious parturient rats. Three consecutive 30-min push-pull perfusions (20 microliters artificial CSF/min) were made, via previously implanted guide cannulae, within the medio-lateral septum and dorsal hippocampus of parturient animals given saline or naloxone hydrochloride (5 mg/kg body weight) after delivery of the second pup. OXT release in the hippocampus, but not in the septum, was increased during parturition, compared to day 1 post partum. During the first 30-min collection period following naloxone administration, release of OXT was significantly elevated within the septum (44% compared to saline controls, p less than 0.002), but not in the dorsal hippocampus; vasopressin release was not affected. In contrast, on day 1 post partum, naloxone, administered 5 min after starting two consecutive perfusions failed to alter OXT release in septum or hippocampus in conscious rats. Naloxone, known to increase the release of OXT also from the posterior pituitary during parturition, speeded the parturition process significantly between the birth of pups 4 and 8 during push-pull perfusion of septum or hippocampus. The data suggest that endogenous opioid inhibition is involved in the regulation of central OXT release, but not vasopressin release, during parturition. Together with previous studies on OXT release from the posterior pituitary, it seems that during parturition there is coordinated endogenous opioid action on the release of OXT both into blood and into the brain.

Chapter 8: Endogenous opioids regulate intracerebral oxytocin release during parturition in a region-specific manner

Publisher Summary During parturition, the nonapeptide oxytocin (OXT) plays an important role in promoting labor. It is established that the release of OXT from the posterior pituitary into blood during the parturition process is under the inhibitory influence of endogenous opioids. OXT and the related nonapeptide arginine vasopressin (AVP) are released intracerebrally within limbic targets and hypothalamic sites of origin in response to a variety of stimuli. OXT and AVP fibres projecting for example to septal or hippocampal targets, and different structures within the hypothalamic supraoptic nucleus (SON), provide the morphological basis for central nonapeptide release. This chapter discusses if: (1) the intracerebral release of OXT and AVP by neurones projecting to the medio-lateral septum and dorsal hippocampus is inhibited by endogenous opioids during parturition, and also whether opioids act at the level of oxytocinergic structures within the target region, (2) OXT release within the SON during parturition is regulated by endogenous opioids, and (3) peripheral and septal administration of NLX, respectively, hasten the parturition process during push–pull perfusion.