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Sodium deficiency enhances the behavioral responses to centrally administered vasopressin in rats
Peptides 23 (2002) 1427–1432
Sodium deficiency enhances the behavioral responses to centrally administered vasopressin in rats Francis W. Flynn∗ Department of Zoology and Physiology, Graduate Neuroscience Program, University of Wyoming, Laramie, WY 82071, USA Received 14 January 2002; accepted 11 March 2002
Abstract The ability of sodium deficiency to stimulate vasopressin (VP) release was examined by determining if sodium deficiency sensitizes the animal to the behavioral disruption caused by intraventricular injections of VP. In sodium-replete rats, intraventricular injections of 50 ng VP on Day 1 had no effect on behavior, but this dose elicited abnormal behaviors (barrel rolls, hind-limb extensions) when administered on Day 2, indicating a sensitization phenomenon. In separate experiments, the first intraventricular injection of 50 ng VP in sodium-deficient but not in sodium-replete rats also elicited barrel rotations followed by hind-limb extension. Intraventricular injection of VP also disrupted motor behavior in sodium-replete rats that had multiple prior experiences with sodium deficiency but not in naive rats. These results show that sodium deficiency can mimic the effect of central injections of VP in sensitizing the brain to the behavioral effects of exogenous VP. This suggests that sodium deficiency induces the central release of VP. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptides; Convulsions; Sensitization; Neurohypophyseal; Salt appetite
1. Introduction A number of species increase their ingestion of salty solutions when faced with sodium deficiency [5]. Sodium deficiency can be aroused by adrenalectomy, placing rats on low sodium diets for an extended number of days, or it can be induced rapidly by combined treatments of a diuretic and injections of deoxycorticosterone acetate (DOCA) [4]. In addition, the ingestion of hypertonic NaCl can be enhanced even in the absence of an actual sodium deficiency. For example, the combined administration of furosemide and low doses of angiotensin-converting enzyme inhibitors stimulate salt ingestion. These treatments are thought to mimic the natural sequence of events that are activated by sodium loss. In particular, the combined treatments affect plasma renin and angiotensin I levels, and the resulting formation of central angiotensin II [6]. Common to all of these procedures is that following the treatment rats ingest normally avoided, hypertonic saline. In addition to the renin–angiotensin system a number of other brain neurochemical systems are involved in the arousal of salt appetite. Interestingly, the neurohypophyseal hormones, oxytocin and vasopressin (VP) are associated with the control of sodium balance [15]. In regards ∗
Tel.: +1-307-766-2926; fax: +1-307-766-6446. E-mail address: [email protected] (F.W. Flynn).
to VP, the treatments identified above that arouse the ingestion of salt solutions also stimulate the release of VP into the blood [14]. In addition to a peripheral release, sodium deficiency may also stimulate the neuronal release of VP. Adrenalectomized rats show an elevated preference for hypertonic NaCl and hypothalamic levels of VP are elevated in adrenalectomized rats [8]. These results would seem to link brain VP and sodium deficiency. Whether brain VP played a role in the behavioral signature of sodium deficiency, the ingestion of hypertonic salt, was uncertain. The involvement of central VP in the arousal of salt appetite was tested by measuring the effects of intraventricular injections of selective VP receptor antagonists on NaCl intake by sodium-deficient rats. Intraventricular injections of selective V1, but not V2, receptor antagonists reduced NaCl intake without affecting sucrose intake [7]. These results suggest that VP is released centrally to modulate ingestive behavior stemming from sodium deficiency. The purpose of the present experiment was to further evaluate the notion that endogenous brain VP neurotransmission is enhanced by sodium deficiency. Behavioral tests were used that relied on the documented effect of VP on motor behavior and that the behavioral disturbance caused by intraventricular injections of VP increase in severity upon subsequent intraventricular injections of the peptide [2,3,10,17]. If sodium deficiency stimulates the central release of VP, then
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sodium deficiency should mimic the effects of exogenous injection of VP on motor behavior. Experiment 1 showed that the motor disturbance following the second intraventricular injection of VP is greater than after the initial injection, demonstrating a sensitization phenomenon and replicating other reports. Additional experiments showed that sodium deficiency mimicked the effect of an exogenous VP injection and sensitized the rat to the behavioral effects of a subsequent VP administration. 2. Methods
the baseline period and following the injections. Behaviors were scored every 30 s as one of three behaviors indicating abnormal behavior: (1) severe motor disturbance (convulsions, barrel rotations (rotation along the long axis of the body)); (2) sprawled out posture with hind-limb extension and motor difficulty; (3) myoclonic jerks or excessive scratching; or one of three normal behaviors: (1) grooming the body in a rostral–caudal body orientation; (2) locomotion (exploration); or (3) inactivity (resting) [3]. The frequency of abnormal and normal behaviors was then tabulated and expressed as a percentage of the total behavior. 2.4. Procedure
2.1. Animals and surgery Male Sprague–Dawley rats were housed in individual suspended, steel mesh cages in a temperature-controlled colony room with a 12:12 h light:dark cycle. All rats were maintained with ad libitum access to Purina chow and tap water except when indicated. Prior to surgery, rats were anesthetized with ketamine hydrochloride (70 mg/kg) and acepromazine maleate (10 mg/kg). The rat’s head was secured in a stereotaxic instrument and the skull was exposed through a midline incision. The skull was made level between bregma and lambda, and a hole was drilled through the skull at 1.0 mm posterior to bregma and 1.5 mm lateral to the midline. A stainless steel cannula (Plastics One, Roanoke, VA, USA) was lowered 4.5 mm ventral to the dura and cemented to four screws that were anchored to the skull. This positioned the cannula in the anterior horn of the lateral ventricle. The cannula was then sealed with an obturator. Following surgery, rats were administered penicillin G benzathine (10,000 units i.m.) on 2 successive days. 2.2. Sodium appetite Sodium deficiency was produced by injecting the rats with deoxycorticosterone acetate (DOCA, 5.0 mg, s.c.) and furosemide (7.5 mg, s.c.). A second furosemide injection (7.5 mg) was administered 2 h later [4,7]. The rats were then placed in clean cages and given access to a low sodium diet (ICN Co., Aurora, OH, USA) and tap water overnight. The next day, the low sodium diet and tap water were removed from the cages and each rat was administered an intraventricular injection of isotonic saline (3 l). Rats were then placed in a clear plexiglass chamber (23 cm×24 cm×22 cm) for 20–25 min. Sodium balance was restored by giving the rats access to Purina chow and water in their home cages. 2.3. Behavioral assessment Rats were removed from the home cages and placed in the plastic chambers and observed (baseline period). After 10 min the rats were removed, administered their assigned injection, and returned to the plastic chamber for another 10 min. An experimenter scored each rat’s behavior during
2.4.1. VP sensitization A group of naive male rats (n = 9) with intraventricular cannula were adapted to the plexiglass chamber on 2 days. Rats were assigned to either the saline (n = 4) or 50 ng VP groups (n = 5). Baseline behaviors were scored for 10 min, the rats were administered their assigned intraventricular injection (3 l), and returned to the chamber for another 10 min. Behaviors were scored as described above. The following day, injections were repeated and behavior was scored. 2.4.2. Sensitization to VP in sodium-deficient rats Male rats (n = 8) with lateral ventricular cannula were adapted to the test chamber on 2 successive days. Following adaptation rats were administered injections of DOCA and furosemide, and the sodium-containing Purina chow was replaced with a low sodium diet. On the next day, sodium-deficient rats were randomly assigned to either the intraventricular saline or 50 ng VP injection conditions (n = 4/injection). Baseline behavior was recorded, the rats were then administered their assigned injection, and returned to the plastic chambers. Behaviors were scored every 30 s prior to and following the intraventricular injection. Rats were then returned to their home cages with access to Purina chow and water. 2.4.3. Multiple episodes of sodium deficiency Naive male rats were assigned to either the sodium deficiency (n = 11) or control group (n = 5). Control rats were maintained on Purina chow and water. Rats in the sodium-deficient group had a total of eight experiences with sodium deficiency. Each episode was aroused using the procedures described above. This procedure was repeated weekly for 8 weeks. After the eighth experience with sodium deficiency rats were maintained exclusively on Purina chow and water. Rats in both groups were also adapted to the plastic test chambers. One week after the last sodium deficiency behavioral testing began, sodium-replete rats from both groups were removed from their home cages and placed into the plastic chambers and baseline behaviors were scored for 10 min. Rats were then administered either an intraventricular
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injection (3 l) of isotonic saline or 50 ng VP and behavior was scored every 30 s for another 10 min. Rats were then returned to their home cages with Purina chow and water. Four days later, the test procedure was repeated using a crossover design. 2.5. Cannula patency and histology After completion of the experiment, the patency of each cannula was verified using behavioral and histological criteria. First, non-deprived rats were administered lateral ventricular injections (3 l) of angiotensin II (50 ng/rat) and given access to tap water. Intake was measured after 30 min. The criterion for a patent cannula was that non-deprived rats had to drink a minimum of 5 ml of water [7]. Subsequently, rats were anesthetized and administered an intraventricular injection (3 l) of India ink through the cannula. The rat was perfused successively with isotonic saline and 10% formalin solution. The brain was extracted and cut in the coronal plane at the level where the cannula entered the brain. The spread of ink into the ventricular system was taken as a second criterion that the cannula was patent.
3. Results 3.1. Histology All of the rats that served in the experiments met the water-drinking criterion and drank over 5 ml following intraventricular injection of angiotensin. At the conclusion of the experiment, ink injections showed that the cannula were similarly positioned in all of the rats. The cannula were positioned in the anterior portion of the lateral ventricle and ink was deposited into the lateral ventricle of all of the rats.
Fig. 1. Mean percentage of abnormal and normal behaviors prior to (baseline) and following the first and second intraventricular injection of saline (upper panels) and vasopressin (VP) (lower panels) (injections were administered on successive days. Asterisk indicates a significant difference compared to the baseline behavior and the behavior of saline-treated rats (P < 0.003).
extension and immobility, barrel rotations were observed in one of the VP-treated rats.
3.2. VP sensitization
3.3. Single episode of sodium deficiency
Fig. 1 shows that on the day of the first injection all of the rats showed only normal behaviors during the baseline period and following intraventricular injections of saline and 50 ng VP. As such, the initial injection of 50 ng VP was below the threshold for eliciting abnormal behaviors in sodium-replete rats. On the following day, baseline behavior of the two groups of rats was normal and none of the rats displayed any behaviors that were scored as abnormal. However, following the injection, rats treated with 50 ng VP showed a significant increase in the frequency of abnormal behaviors compared to the baseline behavior, t (4) = 5.0, P < 0.007, and the saline-treated rats, t (7) = 4.5, P < 0.003. Also, whereas none of the saline-treated rats showed any abnormal behaviors following the second saline injection, all five rats of the VP-treated rats showed abnormal behaviors. The predominant abnormal behavior was hind-limb
During the baseline observation period, sodium-deficient rats explored the chamber and then either groomed and/or were inactive. After the baseline period rats were administered saline or VP and returned to the chamber. Sodium-deficient rats administered isotonic saline again explored the chamber and then were inactive (sleeping). None of the saline-treated sodium-deficient rats showed any type of motor disturbance. In contrast to the inactivity (rest) of saline-treated rats, all four of the sodium-deficient rats administered VP displayed barrel rotations that appeared within 1–2 min of the VP injection. As shown in Fig. 2, motor disturbances accounted for approximately 85% of the behaviors of sodium-deficient rats administered VP. After the barrel rotations had subsided, these rats assumed a sprawled out posture with hind-limb extension. There was a corresponding significant reduction in the percentage
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Fig. 2. Normal and abnormal behavior of sodium-deficient rats is expressed as a percentage of the total behavior. Intraventricular injections of vasopressin (VP) caused an increase in abnormal behavior and a decrease in normal behavior compared to saline-treated rats (P < 0.001).
of normal behavior compared to the saline treatment, P < 0.0001. 3.4. Multiple episodes of sodium deficiency The frequency of each behavior during the baseline and post injection time were tabulated for each rat. Fig. 3 shows the percentage of the total behaviors that were categorized as normal or indicating a motor disturbance. Not surprisingly, prior to the injections, control rats explored the chamber and were then inactive. Following the intraventricular injections of saline or 50 ng VP, the control rats continued to show normal behaviors and no behaviors indicating a motor disturbance were scored. This result shows that in naive rats with no prior history of sodium deficiency, intraventricular injection of 50 ng VP has no adverse effect on behavior. The behavior of sodium deficiency-experienced rats prior to the VP injection was identical to that of control rats. Prior to the injection, experienced rats explored the chamber and rested. However, following the intraventricular injection of 50 ng VP, 9/11 of the rats that had prior experience with sodium deficiency showed a significant increase in the frequency of motor disturbances and a reciprocal decrease in the frequency of normal behaviors, t (10) = 4.2, P < 0.002. The motor disturbances were body jerking and also hind-limb extensions; none of the rats displayed convulsions. The experienced rats given VP showed significantly fewer normal behaviors than did the control rats after the same 50 ng VP injection, t (14) = 2.8, P < 0.01. 3.5. Comparison of VP effects on behavior All the figures show that the magnitude of the behavioral disruption caused by intraventricular injection of VP was
Fig. 3. Comparison of the behavioral effects of intraventricular injections of saline and vasopressin (VP) in rats with no prior experience with sodium deficiency (upper panels) and rats with prior experience with sodium deficiency (lower panels). Asterisk indicates significant differences compared to no prior experience group (P < 0.01).
not similar across the conditions. Indeed, injections of VP in sodium-deficient rats resulted in significantly more abnormal behaviors than when VP was injected on 2 consecutive days or when VP was administered in sodium-replete rats that had multiple experiences with sodium deficiency, F (2, 17) = 5.1, P < 0.01. The disruption caused by repeated VP injection was similar to that caused by prior experience with sodium deficiency, P = 0.8.
4. General discussion The results of each of the experiments show that in naive, sodium-replete rats, injections of 50 ng VP was below the threshold required to elicit motor disturbances. Wurpel et al. [17] also reported that 50 ng VP failed to elicit barrel rotation in male rats and others have estimated
F.W. Flynn / Peptides 23 (2002) 1427–1432
that the effective dose for eliciting barrel rotation in 50% of the rats to be approximately 200 ng [11]. While the first injection of 50 ng VP had no effect on behavior, a second injection 1 day later did result in motor disturbances in 5/5 rats tested. Several previous studies have also shown a sensitization effect to repeated central injections of VP [2,3,10,16,17]. In previous studies, the initial dose of VP (1 g) injected into the lateral ventricle was often sufficient to induce some degree of motor disturbance [2,3,10,17]. Sensitization was shown when the motor disturbances following a second intraventricular injection of either the same dose or a lesser dose (500 ng VP) was greater than that in naive rats [2,3,17]. Also, intraventricular injection of 10 ng VP may elicit motor disturbances in rats that had been treated 2 days earlier with 1 g VP [10]. The present results show that low doses of VP that do not initially elicit motor disturbance also sensitize the brain to the behavioral effects of VP. After the low-dose injection, a subsequent injection of the sub-threshold dose of VP caused motor disturbances. The aim of the present experiment was to evaluate further whether brain VP neurotransmission was enhanced by sodium deficiency that was aroused by injections of furosemide and DOCA. We previously reported that intraventricular injections of selective V1 receptor antagonists, but not V2 receptor antagonists, suppressed salt intake by sodium-deficient rats. This led to the hypothesis that brain vasopressinergic transmission is enhanced during sodium deficiency and that brain VP arouses the ingestion of hypertonic NaCl [7]. A behavioral assay of brain VP transmission was used in the present experiments. As discussed above, central injections of VP disrupt motor performance and prior injections of VP sensitize the animal to the behavioral effects of exogenous VP injection. That is, the behavioral disruption caused by central injections of VP increases in severity upon subsequent intraventricular VP injections. Furthermore, using this strategy Burnard et al. [2] showed that hemorrhage and hypertonic saline stimulate the central release of VP. In their study, the severity of the motor disturbance caused by intraventricular injection of VP was enhanced by prior hemorrhage or hypertonic saline treatment and mimicked the effect of central injections of VP. The sensitizing effect of these treatments was attributed to a common mechanism, that these stimuli cause the central release of VP [2]. The results of the present experiment show that sodium deficiency mimics the effects of intraventricular injection of VP in sensitizing the rat to the behavioral effects of VP. While ineffective in sodium-replete rats, injection of 50 ng VP in sodium-deficient rats caused severe motor disturbances, including barrel rotation and hind-limb paralysis. The sensitizing action of sodium deficiency suggests that sodium deficiency elicits the endogenous release of VP. The combined action of the endogenous VP release and exogenously administered VP was sufficient to disrupt motor behavior. Poulin and Pittman [12] demonstrated that the
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magnitude of the VP sensitization was a function of the dose and the time separating the injections. Doses as low as 1 ng could produce a sensitization effect and that the sensitizing action of central VP injections decayed over days. Injections of VP given 6 days after the initial injection produced less of a sensitization than if the injections were separated by 1 day. The present results follow that trend and showed that the sensitizing effect of sodium deficiency decayed over time. Injections of VP caused less of a motor disturbance 1 week after the last experience with sodium deficiency than when administered to sodium-deficient rats. Thus, the smaller sensitizing effect observed in the sodium deficiency-experienced rats may be because sodium deficiency was aroused weekly, and thus the sensitization effect may have dissipated during the interval. Additional support for the proposal that sodium deficiency stimulates the central release of VP comes from direct measurement of VP in brain. Injections of DOCA, which arouse a salt appetite, increase vasopressin mRNA levels in the paraventricular nucleus of the hypothalamus, indicating that the vasopressin gene is a target of the central effects of DOCA [1,9]. Also, DOCA treatment increases the number of neuronal VP binding sites [13]. The present finding that intraventricular injections of VP disrupt behavior in sodium-replete rats that had prior experience with sodium deficiency may reflect an enduring increase in the number of binding sites. These results along with the present data and the finding that intraventricular injection of V1 receptor antagonist reduces salt appetite [7] all suggest that VP is released centrally to modulate adaptive responses stemming from sodium deficiency.
Acknowledgments The author is grateful to Stephen Newton for his technical assistance in conducting the experiment. Steven Newton and Amanda Roberts provided helpful comments on the manuscript. This research was supported by grants DK50586 and National Institute of Research Resources P20 RR15640.
References [1] Ashen MD, Hartman RD, Barraclough CA, Peterse SL, Hamlyn JM. Vasopressin gene transcripts in mineralocorticoid hypertension: an in situ study. J Hypertens 1992;10:1317–26. [2] Burnard DM, Pittman QJ, Veale WL. Increased motor disturbance in response to arginine vasopressin following hemorrhage or hypertonic saline: evidence for central AVP release in rats. Brain Res 1983;273: 59–65. [3] Burnard DM, Veale WL, Pittman QL. Prevention of arginine vasopressin-induced motor disturbances by a potent vasopressor antagonist. Brain Res 1986;362:40–6. [4] Cruz CE, Perelle IB, Wolf G. Methodological aspects of sodium appetite: an addendum. Behav Biol 1977;20:96–103.
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[5] Denton, D. The hunger for salt: an anthropological, physiological and medical analysis. Berlin: Springer–Verlag, 1984. [6] Fitts DA, Masson DB. Forebrain sites of action for drinking and salt appetite to angiotensin or captopril. Behav Neurosci 1989;103: 865–72. [7] Flynn FW, Kirchner TR, Clinton ME. Brain vasopressin and salt appetite. Am J Physiol 2002;282:R1236–44. [8] Franco-Bourland RE. Vasopressinergic, oxytocinergic, and somatostatinergic neuronal activity after adrenalectomy and immobilization stress. Neurochem Res 1998;23:695–701. [9] Grillo CA, Saravia F, Ferrini M, Piroli G, Roig P, Garcia SI, et al. Increased expression of magnocellular vasopressin mRNA in rats with deoxycorticosterone acetate induced salt appetite. Neuroendocrinology 1998;68:105–15. [10] Kasting NW, Veale WL, Cooper KE. Convulsive and hypothermic effects of vasopressin in the brain of the rat. Can J Physiol Pharmacol 1980;58:316–9. [11] Krause H, Van Wimersa Greidanus B, De Weid D. Barrel rotation induced by vasopressin and related peptides. Pharmacol Biochem Behav 1977;7:311–3.
[12] Poulin P, Pittman QJ. Arginine vasopressin-induced sensitization in brain: facilitated inositol phosphate production without changes in receptor number. J Neuroendocrinol 1993;5:23–31. [13] Swords BH, Wyss JM, Berecek KH. Central vasopressin receptors are upregulated by deoxycorticosterone acetate. Brain Res 1991;559: 10–6. [14] Thunhorst RL, Morris M, Johnson AK. Endocrine changes associated with a rapidly developing sodium appetite in rats. Am J Physiol 1994;267:R1168–73. [15] Verbalis JG, Blackburn RE, Hoffman GE, Stricker EM. Establishing behavioral and physiological functions of central oxytocin: Insights from studies of oxytocin and ingestive behaviors. In: Ivell R, Russell J, editors. Oxytocin. New York: Plenum Press, 1995. p. 209–25. [16] Wilcox BJ, Poulin P, Veale WL, Pittman QL. Vasopressin-induced motor effects: localization of a sensitive site in the amygdala. Brain Res 1992;596:58–64. [17] Wurpel JND, Dundore RL, Barbella YR, Balaban CD, Kel LC, Severs WB. Barrel rotations evoked by intracerebroventricular injections in conscious rats. Part I. Description and general pharmacology. Brain Res 1986;365:21–9.
Sodium deficiency enhances the behavioral responses to centrally administered vasopressin in rats Francis W. Flynn∗ Department of Zoology and Physiology, Graduate Neuroscience Program, University of Wyoming, Laramie, WY 82071, USA Received 14 January 2002; accepted 11 March 2002
Abstract The ability of sodium deficiency to stimulate vasopressin (VP) release was examined by determining if sodium deficiency sensitizes the animal to the behavioral disruption caused by intraventricular injections of VP. In sodium-replete rats, intraventricular injections of 50 ng VP on Day 1 had no effect on behavior, but this dose elicited abnormal behaviors (barrel rolls, hind-limb extensions) when administered on Day 2, indicating a sensitization phenomenon. In separate experiments, the first intraventricular injection of 50 ng VP in sodium-deficient but not in sodium-replete rats also elicited barrel rotations followed by hind-limb extension. Intraventricular injection of VP also disrupted motor behavior in sodium-replete rats that had multiple prior experiences with sodium deficiency but not in naive rats. These results show that sodium deficiency can mimic the effect of central injections of VP in sensitizing the brain to the behavioral effects of exogenous VP. This suggests that sodium deficiency induces the central release of VP. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptides; Convulsions; Sensitization; Neurohypophyseal; Salt appetite
1. Introduction A number of species increase their ingestion of salty solutions when faced with sodium deficiency [5]. Sodium deficiency can be aroused by adrenalectomy, placing rats on low sodium diets for an extended number of days, or it can be induced rapidly by combined treatments of a diuretic and injections of deoxycorticosterone acetate (DOCA) [4]. In addition, the ingestion of hypertonic NaCl can be enhanced even in the absence of an actual sodium deficiency. For example, the combined administration of furosemide and low doses of angiotensin-converting enzyme inhibitors stimulate salt ingestion. These treatments are thought to mimic the natural sequence of events that are activated by sodium loss. In particular, the combined treatments affect plasma renin and angiotensin I levels, and the resulting formation of central angiotensin II [6]. Common to all of these procedures is that following the treatment rats ingest normally avoided, hypertonic saline. In addition to the renin–angiotensin system a number of other brain neurochemical systems are involved in the arousal of salt appetite. Interestingly, the neurohypophyseal hormones, oxytocin and vasopressin (VP) are associated with the control of sodium balance [15]. In regards ∗
Tel.: +1-307-766-2926; fax: +1-307-766-6446. E-mail address: [email protected] (F.W. Flynn).
to VP, the treatments identified above that arouse the ingestion of salt solutions also stimulate the release of VP into the blood [14]. In addition to a peripheral release, sodium deficiency may also stimulate the neuronal release of VP. Adrenalectomized rats show an elevated preference for hypertonic NaCl and hypothalamic levels of VP are elevated in adrenalectomized rats [8]. These results would seem to link brain VP and sodium deficiency. Whether brain VP played a role in the behavioral signature of sodium deficiency, the ingestion of hypertonic salt, was uncertain. The involvement of central VP in the arousal of salt appetite was tested by measuring the effects of intraventricular injections of selective VP receptor antagonists on NaCl intake by sodium-deficient rats. Intraventricular injections of selective V1, but not V2, receptor antagonists reduced NaCl intake without affecting sucrose intake [7]. These results suggest that VP is released centrally to modulate ingestive behavior stemming from sodium deficiency. The purpose of the present experiment was to further evaluate the notion that endogenous brain VP neurotransmission is enhanced by sodium deficiency. Behavioral tests were used that relied on the documented effect of VP on motor behavior and that the behavioral disturbance caused by intraventricular injections of VP increase in severity upon subsequent intraventricular injections of the peptide [2,3,10,17]. If sodium deficiency stimulates the central release of VP, then
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 0 9 1 - 8
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sodium deficiency should mimic the effects of exogenous injection of VP on motor behavior. Experiment 1 showed that the motor disturbance following the second intraventricular injection of VP is greater than after the initial injection, demonstrating a sensitization phenomenon and replicating other reports. Additional experiments showed that sodium deficiency mimicked the effect of an exogenous VP injection and sensitized the rat to the behavioral effects of a subsequent VP administration. 2. Methods
the baseline period and following the injections. Behaviors were scored every 30 s as one of three behaviors indicating abnormal behavior: (1) severe motor disturbance (convulsions, barrel rotations (rotation along the long axis of the body)); (2) sprawled out posture with hind-limb extension and motor difficulty; (3) myoclonic jerks or excessive scratching; or one of three normal behaviors: (1) grooming the body in a rostral–caudal body orientation; (2) locomotion (exploration); or (3) inactivity (resting) [3]. The frequency of abnormal and normal behaviors was then tabulated and expressed as a percentage of the total behavior. 2.4. Procedure
2.1. Animals and surgery Male Sprague–Dawley rats were housed in individual suspended, steel mesh cages in a temperature-controlled colony room with a 12:12 h light:dark cycle. All rats were maintained with ad libitum access to Purina chow and tap water except when indicated. Prior to surgery, rats were anesthetized with ketamine hydrochloride (70 mg/kg) and acepromazine maleate (10 mg/kg). The rat’s head was secured in a stereotaxic instrument and the skull was exposed through a midline incision. The skull was made level between bregma and lambda, and a hole was drilled through the skull at 1.0 mm posterior to bregma and 1.5 mm lateral to the midline. A stainless steel cannula (Plastics One, Roanoke, VA, USA) was lowered 4.5 mm ventral to the dura and cemented to four screws that were anchored to the skull. This positioned the cannula in the anterior horn of the lateral ventricle. The cannula was then sealed with an obturator. Following surgery, rats were administered penicillin G benzathine (10,000 units i.m.) on 2 successive days. 2.2. Sodium appetite Sodium deficiency was produced by injecting the rats with deoxycorticosterone acetate (DOCA, 5.0 mg, s.c.) and furosemide (7.5 mg, s.c.). A second furosemide injection (7.5 mg) was administered 2 h later [4,7]. The rats were then placed in clean cages and given access to a low sodium diet (ICN Co., Aurora, OH, USA) and tap water overnight. The next day, the low sodium diet and tap water were removed from the cages and each rat was administered an intraventricular injection of isotonic saline (3 l). Rats were then placed in a clear plexiglass chamber (23 cm×24 cm×22 cm) for 20–25 min. Sodium balance was restored by giving the rats access to Purina chow and water in their home cages. 2.3. Behavioral assessment Rats were removed from the home cages and placed in the plastic chambers and observed (baseline period). After 10 min the rats were removed, administered their assigned injection, and returned to the plastic chamber for another 10 min. An experimenter scored each rat’s behavior during
2.4.1. VP sensitization A group of naive male rats (n = 9) with intraventricular cannula were adapted to the plexiglass chamber on 2 days. Rats were assigned to either the saline (n = 4) or 50 ng VP groups (n = 5). Baseline behaviors were scored for 10 min, the rats were administered their assigned intraventricular injection (3 l), and returned to the chamber for another 10 min. Behaviors were scored as described above. The following day, injections were repeated and behavior was scored. 2.4.2. Sensitization to VP in sodium-deficient rats Male rats (n = 8) with lateral ventricular cannula were adapted to the test chamber on 2 successive days. Following adaptation rats were administered injections of DOCA and furosemide, and the sodium-containing Purina chow was replaced with a low sodium diet. On the next day, sodium-deficient rats were randomly assigned to either the intraventricular saline or 50 ng VP injection conditions (n = 4/injection). Baseline behavior was recorded, the rats were then administered their assigned injection, and returned to the plastic chambers. Behaviors were scored every 30 s prior to and following the intraventricular injection. Rats were then returned to their home cages with access to Purina chow and water. 2.4.3. Multiple episodes of sodium deficiency Naive male rats were assigned to either the sodium deficiency (n = 11) or control group (n = 5). Control rats were maintained on Purina chow and water. Rats in the sodium-deficient group had a total of eight experiences with sodium deficiency. Each episode was aroused using the procedures described above. This procedure was repeated weekly for 8 weeks. After the eighth experience with sodium deficiency rats were maintained exclusively on Purina chow and water. Rats in both groups were also adapted to the plastic test chambers. One week after the last sodium deficiency behavioral testing began, sodium-replete rats from both groups were removed from their home cages and placed into the plastic chambers and baseline behaviors were scored for 10 min. Rats were then administered either an intraventricular
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injection (3 l) of isotonic saline or 50 ng VP and behavior was scored every 30 s for another 10 min. Rats were then returned to their home cages with Purina chow and water. Four days later, the test procedure was repeated using a crossover design. 2.5. Cannula patency and histology After completion of the experiment, the patency of each cannula was verified using behavioral and histological criteria. First, non-deprived rats were administered lateral ventricular injections (3 l) of angiotensin II (50 ng/rat) and given access to tap water. Intake was measured after 30 min. The criterion for a patent cannula was that non-deprived rats had to drink a minimum of 5 ml of water [7]. Subsequently, rats were anesthetized and administered an intraventricular injection (3 l) of India ink through the cannula. The rat was perfused successively with isotonic saline and 10% formalin solution. The brain was extracted and cut in the coronal plane at the level where the cannula entered the brain. The spread of ink into the ventricular system was taken as a second criterion that the cannula was patent.
3. Results 3.1. Histology All of the rats that served in the experiments met the water-drinking criterion and drank over 5 ml following intraventricular injection of angiotensin. At the conclusion of the experiment, ink injections showed that the cannula were similarly positioned in all of the rats. The cannula were positioned in the anterior portion of the lateral ventricle and ink was deposited into the lateral ventricle of all of the rats.
Fig. 1. Mean percentage of abnormal and normal behaviors prior to (baseline) and following the first and second intraventricular injection of saline (upper panels) and vasopressin (VP) (lower panels) (injections were administered on successive days. Asterisk indicates a significant difference compared to the baseline behavior and the behavior of saline-treated rats (P < 0.003).
extension and immobility, barrel rotations were observed in one of the VP-treated rats.
3.2. VP sensitization
3.3. Single episode of sodium deficiency
Fig. 1 shows that on the day of the first injection all of the rats showed only normal behaviors during the baseline period and following intraventricular injections of saline and 50 ng VP. As such, the initial injection of 50 ng VP was below the threshold for eliciting abnormal behaviors in sodium-replete rats. On the following day, baseline behavior of the two groups of rats was normal and none of the rats displayed any behaviors that were scored as abnormal. However, following the injection, rats treated with 50 ng VP showed a significant increase in the frequency of abnormal behaviors compared to the baseline behavior, t (4) = 5.0, P < 0.007, and the saline-treated rats, t (7) = 4.5, P < 0.003. Also, whereas none of the saline-treated rats showed any abnormal behaviors following the second saline injection, all five rats of the VP-treated rats showed abnormal behaviors. The predominant abnormal behavior was hind-limb
During the baseline observation period, sodium-deficient rats explored the chamber and then either groomed and/or were inactive. After the baseline period rats were administered saline or VP and returned to the chamber. Sodium-deficient rats administered isotonic saline again explored the chamber and then were inactive (sleeping). None of the saline-treated sodium-deficient rats showed any type of motor disturbance. In contrast to the inactivity (rest) of saline-treated rats, all four of the sodium-deficient rats administered VP displayed barrel rotations that appeared within 1–2 min of the VP injection. As shown in Fig. 2, motor disturbances accounted for approximately 85% of the behaviors of sodium-deficient rats administered VP. After the barrel rotations had subsided, these rats assumed a sprawled out posture with hind-limb extension. There was a corresponding significant reduction in the percentage
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Fig. 2. Normal and abnormal behavior of sodium-deficient rats is expressed as a percentage of the total behavior. Intraventricular injections of vasopressin (VP) caused an increase in abnormal behavior and a decrease in normal behavior compared to saline-treated rats (P < 0.001).
of normal behavior compared to the saline treatment, P < 0.0001. 3.4. Multiple episodes of sodium deficiency The frequency of each behavior during the baseline and post injection time were tabulated for each rat. Fig. 3 shows the percentage of the total behaviors that were categorized as normal or indicating a motor disturbance. Not surprisingly, prior to the injections, control rats explored the chamber and were then inactive. Following the intraventricular injections of saline or 50 ng VP, the control rats continued to show normal behaviors and no behaviors indicating a motor disturbance were scored. This result shows that in naive rats with no prior history of sodium deficiency, intraventricular injection of 50 ng VP has no adverse effect on behavior. The behavior of sodium deficiency-experienced rats prior to the VP injection was identical to that of control rats. Prior to the injection, experienced rats explored the chamber and rested. However, following the intraventricular injection of 50 ng VP, 9/11 of the rats that had prior experience with sodium deficiency showed a significant increase in the frequency of motor disturbances and a reciprocal decrease in the frequency of normal behaviors, t (10) = 4.2, P < 0.002. The motor disturbances were body jerking and also hind-limb extensions; none of the rats displayed convulsions. The experienced rats given VP showed significantly fewer normal behaviors than did the control rats after the same 50 ng VP injection, t (14) = 2.8, P < 0.01. 3.5. Comparison of VP effects on behavior All the figures show that the magnitude of the behavioral disruption caused by intraventricular injection of VP was
Fig. 3. Comparison of the behavioral effects of intraventricular injections of saline and vasopressin (VP) in rats with no prior experience with sodium deficiency (upper panels) and rats with prior experience with sodium deficiency (lower panels). Asterisk indicates significant differences compared to no prior experience group (P < 0.01).
not similar across the conditions. Indeed, injections of VP in sodium-deficient rats resulted in significantly more abnormal behaviors than when VP was injected on 2 consecutive days or when VP was administered in sodium-replete rats that had multiple experiences with sodium deficiency, F (2, 17) = 5.1, P < 0.01. The disruption caused by repeated VP injection was similar to that caused by prior experience with sodium deficiency, P = 0.8.
4. General discussion The results of each of the experiments show that in naive, sodium-replete rats, injections of 50 ng VP was below the threshold required to elicit motor disturbances. Wurpel et al. [17] also reported that 50 ng VP failed to elicit barrel rotation in male rats and others have estimated
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that the effective dose for eliciting barrel rotation in 50% of the rats to be approximately 200 ng [11]. While the first injection of 50 ng VP had no effect on behavior, a second injection 1 day later did result in motor disturbances in 5/5 rats tested. Several previous studies have also shown a sensitization effect to repeated central injections of VP [2,3,10,16,17]. In previous studies, the initial dose of VP (1 g) injected into the lateral ventricle was often sufficient to induce some degree of motor disturbance [2,3,10,17]. Sensitization was shown when the motor disturbances following a second intraventricular injection of either the same dose or a lesser dose (500 ng VP) was greater than that in naive rats [2,3,17]. Also, intraventricular injection of 10 ng VP may elicit motor disturbances in rats that had been treated 2 days earlier with 1 g VP [10]. The present results show that low doses of VP that do not initially elicit motor disturbance also sensitize the brain to the behavioral effects of VP. After the low-dose injection, a subsequent injection of the sub-threshold dose of VP caused motor disturbances. The aim of the present experiment was to evaluate further whether brain VP neurotransmission was enhanced by sodium deficiency that was aroused by injections of furosemide and DOCA. We previously reported that intraventricular injections of selective V1 receptor antagonists, but not V2 receptor antagonists, suppressed salt intake by sodium-deficient rats. This led to the hypothesis that brain vasopressinergic transmission is enhanced during sodium deficiency and that brain VP arouses the ingestion of hypertonic NaCl [7]. A behavioral assay of brain VP transmission was used in the present experiments. As discussed above, central injections of VP disrupt motor performance and prior injections of VP sensitize the animal to the behavioral effects of exogenous VP injection. That is, the behavioral disruption caused by central injections of VP increases in severity upon subsequent intraventricular VP injections. Furthermore, using this strategy Burnard et al. [2] showed that hemorrhage and hypertonic saline stimulate the central release of VP. In their study, the severity of the motor disturbance caused by intraventricular injection of VP was enhanced by prior hemorrhage or hypertonic saline treatment and mimicked the effect of central injections of VP. The sensitizing effect of these treatments was attributed to a common mechanism, that these stimuli cause the central release of VP [2]. The results of the present experiment show that sodium deficiency mimics the effects of intraventricular injection of VP in sensitizing the rat to the behavioral effects of VP. While ineffective in sodium-replete rats, injection of 50 ng VP in sodium-deficient rats caused severe motor disturbances, including barrel rotation and hind-limb paralysis. The sensitizing action of sodium deficiency suggests that sodium deficiency elicits the endogenous release of VP. The combined action of the endogenous VP release and exogenously administered VP was sufficient to disrupt motor behavior. Poulin and Pittman [12] demonstrated that the
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magnitude of the VP sensitization was a function of the dose and the time separating the injections. Doses as low as 1 ng could produce a sensitization effect and that the sensitizing action of central VP injections decayed over days. Injections of VP given 6 days after the initial injection produced less of a sensitization than if the injections were separated by 1 day. The present results follow that trend and showed that the sensitizing effect of sodium deficiency decayed over time. Injections of VP caused less of a motor disturbance 1 week after the last experience with sodium deficiency than when administered to sodium-deficient rats. Thus, the smaller sensitizing effect observed in the sodium deficiency-experienced rats may be because sodium deficiency was aroused weekly, and thus the sensitization effect may have dissipated during the interval. Additional support for the proposal that sodium deficiency stimulates the central release of VP comes from direct measurement of VP in brain. Injections of DOCA, which arouse a salt appetite, increase vasopressin mRNA levels in the paraventricular nucleus of the hypothalamus, indicating that the vasopressin gene is a target of the central effects of DOCA [1,9]. Also, DOCA treatment increases the number of neuronal VP binding sites [13]. The present finding that intraventricular injections of VP disrupt behavior in sodium-replete rats that had prior experience with sodium deficiency may reflect an enduring increase in the number of binding sites. These results along with the present data and the finding that intraventricular injection of V1 receptor antagonist reduces salt appetite [7] all suggest that VP is released centrally to modulate adaptive responses stemming from sodium deficiency.
Acknowledgments The author is grateful to Stephen Newton for his technical assistance in conducting the experiment. Steven Newton and Amanda Roberts provided helpful comments on the manuscript. This research was supported by grants DK50586 and National Institute of Research Resources P20 RR15640.
References [1] Ashen MD, Hartman RD, Barraclough CA, Peterse SL, Hamlyn JM. Vasopressin gene transcripts in mineralocorticoid hypertension: an in situ study. J Hypertens 1992;10:1317–26. [2] Burnard DM, Pittman QJ, Veale WL. Increased motor disturbance in response to arginine vasopressin following hemorrhage or hypertonic saline: evidence for central AVP release in rats. Brain Res 1983;273: 59–65. [3] Burnard DM, Veale WL, Pittman QL. Prevention of arginine vasopressin-induced motor disturbances by a potent vasopressor antagonist. Brain Res 1986;362:40–6. [4] Cruz CE, Perelle IB, Wolf G. Methodological aspects of sodium appetite: an addendum. Behav Biol 1977;20:96–103.
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F.W. Flynn / Peptides 23 (2002) 1427–1432
[5] Denton, D. The hunger for salt: an anthropological, physiological and medical analysis. Berlin: Springer–Verlag, 1984. [6] Fitts DA, Masson DB. Forebrain sites of action for drinking and salt appetite to angiotensin or captopril. Behav Neurosci 1989;103: 865–72. [7] Flynn FW, Kirchner TR, Clinton ME. Brain vasopressin and salt appetite. Am J Physiol 2002;282:R1236–44. [8] Franco-Bourland RE. Vasopressinergic, oxytocinergic, and somatostatinergic neuronal activity after adrenalectomy and immobilization stress. Neurochem Res 1998;23:695–701. [9] Grillo CA, Saravia F, Ferrini M, Piroli G, Roig P, Garcia SI, et al. Increased expression of magnocellular vasopressin mRNA in rats with deoxycorticosterone acetate induced salt appetite. Neuroendocrinology 1998;68:105–15. [10] Kasting NW, Veale WL, Cooper KE. Convulsive and hypothermic effects of vasopressin in the brain of the rat. Can J Physiol Pharmacol 1980;58:316–9. [11] Krause H, Van Wimersa Greidanus B, De Weid D. Barrel rotation induced by vasopressin and related peptides. Pharmacol Biochem Behav 1977;7:311–3.
[12] Poulin P, Pittman QJ. Arginine vasopressin-induced sensitization in brain: facilitated inositol phosphate production without changes in receptor number. J Neuroendocrinol 1993;5:23–31. [13] Swords BH, Wyss JM, Berecek KH. Central vasopressin receptors are upregulated by deoxycorticosterone acetate. Brain Res 1991;559: 10–6. [14] Thunhorst RL, Morris M, Johnson AK. Endocrine changes associated with a rapidly developing sodium appetite in rats. Am J Physiol 1994;267:R1168–73. [15] Verbalis JG, Blackburn RE, Hoffman GE, Stricker EM. Establishing behavioral and physiological functions of central oxytocin: Insights from studies of oxytocin and ingestive behaviors. In: Ivell R, Russell J, editors. Oxytocin. New York: Plenum Press, 1995. p. 209–25. [16] Wilcox BJ, Poulin P, Veale WL, Pittman QL. Vasopressin-induced motor effects: localization of a sensitive site in the amygdala. Brain Res 1992;596:58–64. [17] Wurpel JND, Dundore RL, Barbella YR, Balaban CD, Kel LC, Severs WB. Barrel rotations evoked by intracerebroventricular injections in conscious rats. Part I. Description and general pharmacology. Brain Res 1986;365:21–9.