INTRODUCTION

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AS ONE OF THE CIRCUMVENTRICULAR organs of the brain, the subfornical organ (SFO) located in the dorsorostral extension of the third ventricular wall lacks a blood-brain barrier, and its neurons are consequently accessible to circulating messenger molecules. Neurons in the SFO play a transducer role in the central dipsogenic activity of circulating angiotensin II (ANG II; 7, 31), and it has long been thought that the renal water-conserving action of the circulating antidiuretic hormone arginine vasopressin (AVP) might be complemented by a similarly mediated central stimulation of water intake (6). Early studies in which high doses of the AVP were tested in rats (19) and dogs (27) produced unclear results, and this topic has since received little attention (8, 11). The hypertension induced by high doses of peripherally applied AVP, however, leads to baroreceptor activation that conflicts with its putative influence on drinking. The effects of high circulating levels of the avian antidiuretic hormone, arginine vasotocin (AVT), on water intake were therefore recently studied in domestic ducks, in which AVT does not exert hypertensive actions, even at pharmacological plasma levels (21). That study revealed that AVT indeed stimulated water intake and that it did so at plasma levels similar to those that activated neurons recorded in tissue slices from the SFO of ducks. The exclusively excitatory effect of AVT on responsive SFO neurons in ducks had a threshold concentration similar to that of ANG II and was predominantly observed on neurons excited by ANG II (20).

Because the dipsogenic and neuron-activating concentrations of AVT in these studies were greater than those occurring naturally in the plasma, the observed drinking was not thought to indicate a physiological function of circulating AVT but rather to be a result of pharmacological concentrations mimicking the modulatory influence of a vasotocinergic innervation of SFO neurons that play a transducer role in the central dipsogenic activity of circulating ANG II. Similarly, AVP released centrally in the SFO of mammals might modulate signals generated locally by neurons responsive to circulating ANG II. High-affinity binding sites for AVP as well as for its degradation product AVP_(4-9) have been found in the SFO of rats (12). The mammalian SFO contains vasopressinergic fiber endings, presumably from cells in the parvocellular (24) or magnocellular (33) portions of the neurosecretory hypothalamic nuclei, and the presence of AVP mRNA in the SFO implies that AVP is formed as well as released there (15). In addition, calcium-imaging studies on dissociated neurons from the SFO of rats have indicated that these neurons respond to AVP (13). There is, as far as we know, only a single report describing the influence of AVP on the electrical activity of a small sample of rat SFO neurons: some were excited, a few were inhibited, and others were not influenced (34). Nothing, however, is known about the dose-response relationships for AVP, about the types of receptors involved, nor about the quantitative relationships between responsiveness to AVP and responsiveness to ANG II. We have therefore recorded the activity of neurons of the rat SFO in a slice preparation to examine the (excitatory or inhibitory) effect of AVP, its dose dependence and receptor pharmacology, and also to evaluate the relation between AVP sensitivity and ANG II sensitivity in identical neurons. Correlating the AVP responsiveness with the ANG II responsiveness of individual SFO neurons should allow conclusions as to whether AVP is able to affect SFO mediated water intake in rats, as it has been shown to do in ducks.

	METHODS

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Adult male Wistar rats with body weights between 170 and 300 g were decapitated. The skull was opened quickly, and the brain was rinsed with ice-cold oxygenated artificial cerebrospinal fluid (aCSF). The brain was quickly removed and transferred to a dish filled with ice-cold aCSF. The slices were trimmed to contain only the SFO and 1-2 mm of surrounding tissue. Preparation time in the ice-cold aCSF was 6-10 min. After preincubation in oxygenated aCSF at 35°C for at least 1 h, the slice was put into a recording chamber, where it was fixed with a small platinum weight. The gold-plated recording chamber had a volume of 0.7 ml, was made of solid brass, and was kept at 37°C. The aCSF, which was warmed to 37°C before flowing through the chamber at 1.6 ml/min, was aerated with 95% O₂-5% CO₂ (pH 7.4), and had the following composition (in mM): 124 NaCl, 5 KCl, 1.3 MgSO₄, 1.2 CaCl₂, 1.2 NaH₂PO₄, 26 NaHCO₃, 10 glucose, with an osmolality of 290 mosmol/kg. The aCSF used to block synaptic transmission contained less CaCl₂ (0.3 mM) and more MgSO₄ (9.0 mM). The following drugs (all from Sigma, Deisenhofen, Germany) were used in the study: AVP, the V₂-receptor agonist [deamino-Cys¹,D-Arg⁸]vasopressin (dDAVP) the V₁-receptor antagonist Manning compound, and ANG II (human type). Stock solutions with concentrations of 10⁴-10³ M were stored at 25°C in aliquots of 50-100 µl. For each experiment, the experimental drug solutions were freshly prepared by dilution with sterile aCSF and kept at +4°C until immediately before addition to the chamber perfusion line. The concentration of the drug in the recording chamber reached 95% of its steady-state value within 2 min after it was added to the perfusion, and the 60-s delay between the time the drug infusion began and the time the drug reached the chamber was taken into account when the neuronal responses were evaluated. Agonists were infused for 2-5 min, and the antagonist was infused for 10 min before the agonist infusion began. To induce synaptic blockade, the slice was perfused with the low-Ca²⁺/high-Mg²⁺ aCSF for at least 10 min before the agonist infusion began, and superfusion was maintained for 5 min after the agonist infusion had been stopped. The minimum interval between agonist infusions was 10 min.

Extracellular recordings were made using glass-coated Pt-Ir metal electrodes with tip diameters of ~2 µm. A hydraulic microdrive device was used to insert the electrode into the SFO under visual control using a surgical microscope. The SFO could be easily identified because of its protrusion into the third ventricular space. Average spike amplitude and signal-to-noise ratio of the 228 recorded neurons were 227 ± 42 µV (means ± SE) and 20:1. The signals were amplified (DAM 50 differential amplifier, World Precision Instruments, Sarasoto, FL), displayed on a storage oscilloscope (Gould, Valley View, OH) together with the corresponding normalized transistor-transistor-logic pulses obtained by passing the signal through a window discriminator and were computer stored for online evaluation with the Spike-2 program (Cambridge Electronic Design, Cambridge, UK).

A drug infusion was started only after a stable recording had been obtained for at least 5 min under control conditions, and a control period of at least 10 min elapsed before the next drug infusion. Excitation and inhibition to drug application were deemed to have occurred whenever the discharge rate determined with a 30-s bin width changed by more than 0.5 spikes/s and more than 20% of the control discharge rate during the 60 s preceding the drug perfusion. Responsiveness changes due to an antagonist or to synaptic blockade were deemed to have occurred if the average discharge rate during the entire response time was reduced by more than 0.5 spikes/s and 15% from the discharge rate during the preceding agonist infusion or if the discharge rate determined for a 30-s bin width was reduced by more than 1.5 spikes/s and 20% from that during the preceding agonist infusion.

The statistical significance of differences between the properties of groups of neurons was evaluated using the unpaired t-test. Numbers of neurons exhibiting particular properties (Table 2) were analyzed for differences with the ² test. Dose effects of AVP-induced excitations or inhibitions were analyzed with the sign test. Average values are presented as means with standard errors (SE).

	RESULTS

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Altogether 159 neurons were tested for responsiveness to AVP at 10⁶ M, and 26% (n = 41) were excited, 24% (n = 39) were inhibited, and the remaining 50% were insensitive. Several parameters characterizing changes in discharge rate during 2-min AVP infusions could be calculated for 33 of the excited neurons and 26 of the inhibited neurons (Table 1). Neurons excited or inhibited by AVP did not show significant differences in their mean spontaneous discharge rate, latency, or duration of the response. The impression of a difference between the absolute percent changes of discharge rate during excitation and inhibition (Table 1) could not be confirmed statistically (P = 0.082, t-test). There was no indication of desensitization when infusions were repeated.

View this table: [in this window] [in a new window]   Table 1.   Basal and response parameters of SFO neurons excited or inhibited by AVP (106 M)

Dose-response relationships. Figure 1 presents examples showing that both the excitatory and the inhibitory responses to AVP superfusions were dose dependent. The insets illustrate the average dose-response relationships. For both excitation and inhibition, the threshold concentration was between 10⁹ and 10⁸ M, that is about three orders of magnitude above the range of physiological AVP plasma concentrations (10¹² to 10¹¹ M). In neurons excited by AVP, the change in discharge rate when raising the AVP concentration in the perfusate by a factor of 10 was observed in 10 instances, and in 9 instances, the rise in concentration caused a stronger activation (P < 0.01, sign test). In neurons inhibited by AVP, the change in discharge rate when raising the AVP concentration in the perfusate by a factor of 10 was observed on 12 instances, and in 11 instances, the rise in concentration caused a stronger inhibition (P < 0.01, sign test).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Discharge rates of 2 subfornical organ (SFO) neurons recorded in vitro from slice preparations in the course of repeated slice superfusions with arginine vasopressin (AVP) at increasing doses (bars). Top: excitatory response. Bottom: inhibitory response. Insets: average dose-response relationships from 4 individual neurons, which could be tested repeatedly with different doses (means ± SEs, sample size in parentheses). Inset at top left: segments of original spike recording during control conditions (1) and during excitation (2) with AVP 107 M.

Relationship between responsiveness to ANG II and AVP. Of the 146 neurons tested for responsiveness to ANG II at 10⁷ M, 66% were excited and the rest were insensitive. The same neurons were tested for responsiveness to AVP at a standard concentration of 10⁶ M. Figure 2 presents examples showing that neurons excited by ANG II could be excited as well as inhibited by AVP. The results are summarized in Table 2. Of the ANG II-responsive neurons, AVP excited 26% and inhibited 30%; that is, just over one-half of the ANG II-sensitive neurons were also sensitive to AVP. Of the ANG II-insensitive neurons, AVP excited 29% and inhibited 19%. These fractions were not significantly different from those found among the ANG-sensitive neurons (² test), nor did they differ from the above-mentioned relationship found for all neurons tested with AVP.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Discharge rates of 2 SFO neurons recorded in vitro from slice preparations in the course of successive infusions of ANG II and AVP. Top: neuron excited by both ANG II and AVP. Bottom: neuron excited by ANG II but inhibited by AVP.

View this table: [in this window] [in a new window]   Table 2.   Numbers of SFO neurons responsive to ANG II (107 M) and to AVP (106 M)

Influence of low-Ca²⁺/high-Mg²⁺ aCSF. As illustrated by the example shown in Fig. 3, top, the excitatory response to AVP persisted during synaptic blockade, despite considerable changes in spontaneous discharge rate. The excitatory response to AVP persisted in each of the five neurons tested with blocking solution, and the mean excitatory response to AVP in these neurons (60 ± 28%) was statistically not different (P = 0.7; paired t-test) from the AVP response of these neurons during synaptic blockade (76 ± 56%). In contrast, an inhibitory effect of AVP could only be observed in 2 of 12 neurons tested again during synaptic blockade. In the remaining 10 neurons, AVP did not cause an inhibitory response anymore during superfusion with low-Ca²⁺/high-Mg²⁺ solution (Fig. 3, bottom), despite strong changes in spontaneous activity. The mean inhibitory response of these neurons (n = 12) decreased significantly from 34 ± 2% before to 4 ± 4% during synaptic blockade (P < 0.0001; paired t-test). In 6 of 12 neurons, the initial inhibitory response was converted to a small excitatory response, with a maximum increase of +11%.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Discharge rates of 2 SFO neurons recorded in vitro from slice preparations showing excitatory (top) and inhibitory (bottom) AVP effects, as influenced by synaptic blockade. Top: excitatory effect of AVP persists in blocking solution. Bottom: inhibitory effect of AVP is lost in blocking solution.

Receptor analysis. The examples in Fig. 4 show that both the excitatory and the inhibitory effects of AVP on SFO neurons could be suppressed by Manning compound. This V₁-specific antagonist abolished activation by AVP in each of the eight neurons tested and abolished inhibition by AVP in five of the six neurons tested. As illustrated in Fig. 4, top, application of the V₂-specific agonist dDAVP was usually ineffective. Of the nine neurons excited by AVP, only one was excited by dDAVP and none were inhibited by it.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Discharge rates of 2 SFO neurons recorded in vitro from slice preparations showing superfusions with different drugs and the effect of the V1-antagonist Manning compound (MC). Top: this neuron is excited by AVP, whereas the V2-agonist dDAVP has no effect, but superfusion with MC abolishes the AVP effect. Bottom: neuron is excited by ANG II but inhibited by AVP while superfusion with MC abolishes the AVP effect.

	DISCUSSION

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This study shows that 26% of all neurons recorded from rat SFO slices were excited and 24% were inhibited by AVP. The excitatory as well as the inhibitory effects of AVP were both dose dependent and reversible and had similar threshold concentrations. Both types of AVP-induced responses were blocked by a V₁-receptor antagonist, were not mimicked by a V₂-agonist, and were not correlated with the exclusively excitatory effect of ANG II on the same neurons.

In a previous investigation on slices from the SFO of domestic ducks, ANG II and AVT caused only excitatory effects on the same neurons (20), and when neuronal responsiveness for AVP was tested, the mammalian AVP also consistently excited SFO neurons in this bird. From these results, the hypothesis was derived that AVP, like ANG II, could stimulate SFO neurons involved in the control of water intake. However, although the excitatory effect of ANG II on neurons in vitro was obtained at concentrations close to its physiological range in plasma, the effective concentrations of AVP were clearly above the physiological range. Therefore, it was suggested that the dipsogenic effect induced by intravenous AVT in the duck mimicked the effect of AVT released locally from vasopressinergic hypothalamic nerve fibers, which might influence the function of SFO neurons responding to circulating ANG II.

The presence of high-affinity binding sites for both AVP and its active metabolite AVP_(4-9) (13, 32), as well as the presence of AVP in biochemical extracts (26), and evidence for a vasopressinergic innervation of the rat SFO (24, 33) are consistent with the notion that neurally released AVP modulates the function of ANG II-sensitive SFO neurons. However, the present study showed that the uniformly excitatory action of ANG II on rat SFO neurons is not associated with a similarly consistent action of AVP, which excited about as many SFO neurons as it inhibited. Superfusion with a medium low in Ca²⁺ and high in Mg²⁺, which is known to block synaptic transmission in slices, showed that the excitatory effect of AVP did not depend on synaptic input. The inhibitory effects of AVP, however, seem to depend largely on local inhibitory synaptic interactions, because 83% of the neurons inhibited by AVP lost their responsiveness to AVP in blocking solution.

The fact that AVP had an inhibitory effect on many SFO neurons in rats, but never in ducks, does not exclude the possibility that the fraction of rat SFO neurons that was excited by ANG II as well as by AVP serves a similar (dipsogenic) function in rats as it most likely does in ducks. The present study also suggests that the functional organization of the SFO is more com- plex in rats than in birds. As emphasized previously, this may be related to the fact that the phylogenetically old cluster organization of functionally similar neurons in hypothalamic nuclei still exists in birds, whereas only remnants of this "primitive" cytoarchitecture are preserved in mammals (17). The presence of AVP-immunoreactive neurons as well as fibers in the rat SFO (4), in contrast to only fibers in the bird SFO (33), provides further evidence for a more complex organization of vasopressinergic control in the SFO of mammals.

Excitatory actions of ANG II and AVP on the same SFO neuron would support the idea of a stimulatory action of both peptides on water intake. In the rat, no such effect of AVP has been found (19). It was shown, however, that a small dose of pittressin infused into dogs decreased the threshold for osmotically induced drinking, whereas a larger dose increased the threshold (27). Whether this dose dependence of the dipsogenic action of systemic AVP application reflects an integration of the consistently excitatory action of ANG II with both the excitatory or inhibitory actions of AVP on fractions of SFO neurons cannot be decided on the basis of the present results, since we did not find a difference in the threshold AVP concentrations for excitation or inhibition of SFO neurons. However, the data suggest, in any case, bidirectional AVP actions at the level of the SFO.

No clearly defined physiological function can presently be associated with SFO neurons that are excited by ANG II but inhibited by AVP. One possibility might be that those SFO neurons, which are excited by circulating ANG II and activate PVN neurons to release AVP from the hypophysis (25), are inhibited by a retrograde neuronal vasopressinergic input from the PVN to the SFO. A negative feedback effect of circulating AVP on AVP release was also inferred in a study in which the concentration of the cosecreted neurophysin was measured (3); however, this feedback effect involved V₂-receptors, which in the present study were involved in neither the inhibitory nor excitatory effects of AVP.

The heterogeneity of the AVP responses of SFO neurons in vitro may also be responsible for the diverging effects of AVP on blood pressure, when AVP is applied directly into the SFO. Although electrical stimulation or injection of excitatory substances such as ANG II into the SFO is known to cause exclusively increases in blood pressure in rats (1), one study reported a rise (30) and an another study reported a fall in blood pressure (23) after microapplication of AVP into the SFO. The fact that electrostimulation of the SFO revealed predominantly excitatory effects on neurons in the supraoptic (2) and paraventricular (1, 29) nuclei does not exclude the possibility that some SFO neurons are inhibited by vasopressinergic innervation originating in the PVN.

Although AVP injected into the cerebral ventricle is not likely to affect those SFO neurons that are outside the blood-brain barrier and accessible to blood-borne ANG II, it is interesting to note that conflicting data have been reported for the influence of AVP on water intake when acting on intracerebral targets. Stimulation of drinking observed in dogs given AVP intracerebroventricularly was partially reversed when the dose was increased (28). A later study, however, found that intraventricular infusions of AVP had no significant effect on water intake (5). On the other hand, preliminary observations suggest that intracerebroventricular application of either AVP-antiserum or a V₁-antagonist reduces water intake in genetically polydipsic mice (14). Evidence for a bidirectional action of centrally applied AVP was also obtained in experiments in which AVP and various AVP receptor agonists and antagonists were infused into the third cerebral ventricle of dogs (16). Applications of AVP or a V₁-agonist elicited increases in arterial pressure, although with no clear dose-response relationships in the range of 0.01-100 ng/min, but application of the V₁-antagonist dEt2Tyr(Me)DAVP caused also a rise in arterial pressure when infused at 100 ng/min, whereas V₂-agonists and V₂-antagonists were without effect at any dose level.

In in vivo recordings from the area postrema (AP), another circumventricular organ that lacks a blood-brain barrier, AVP caused excitatory as well as inhibitory effects in similar proportions (22), whereas circulating ANG II was found to be predominantly excitatory (18). When AVP and ANG II were microinjected, however, both consistently elicited increases in blood pressure and only the V₂-agonist dDAVP had a depressor action when it was microinjected (22). The pharmacological evidence thus indicates that responsiveness of AP neurons to AVP is, similar to that of SFO neurons, mediated by V₁-receptors. As pointed out in a recent study of the effects of circulating ANG II and AVP in the nucleus of the solitary tract, which exhibits some degree of capillary leakiness (9), the mixed influence of these peptides does not clearly correlate with the reported attenuation and enhancement of the baroreflex by circulating ANG II and AVP, respectively (10).

Perspectives