Arginine vasotocin: Effects on open field behavior, whole brain monoamines and plasma corticosterone

Peptides, Vol. 2, pp. 437--440, 1981.Printedin the U.S.A.

Arginine Vasotocin: Effects on Open Field Behavior, Whole Brain Monoamines and Plasma Corticosterone P E T E R G O L U S A N D M A U R I C E G. K I N G

D e p a r t m e n t o f Psychology, The University o f N e w c a s t l e N e w South Wales 2308, Australia R e c e i v e d 5 J a n u a r y 1981 GOLUS, P. AND M. G. KING. Arghdne Vasotocin: Effects on open field behavior, whole brahl monoamines and plasma corticosterone. PEPT1DES 2(4) 437--440, 1981.--Two experiments were performed to evaluate the effects of Arginine Vasotocin (AVT) on open field behavior, whole brain monoamines and plasma corticosterone. In Experiment I rats treated with AVT compared to controls displayed an increased initial latency to move, less postural freezing and increased exploration of the central area of the open field..In Experiment 2 it was found that AVT significantly elevated whole brain 5-HT levels (Experiment 2A) without altering levels of dopamine or norepinephrine. Plasma corticosterone was significantly decreased by AVT treatment (Experiment 2B). Arginine vasotocin

Open-field

Monoamines

Corticosterone

T H E mammalian pineal gland has been reported to secrete a neuropeptide known as Arginine Vasotocin (AVT) [19]. While the effects of the administration of AVT are well documented with respect to certain physiological and endocrinological parameters [19,20], to date only a few studies have examined the behavioral effects of AVT [10,33]. We have reported that a very small dose of AVT (1 ng/kg) administered intraperitoneally to rats significantly reduced the initial step down latency in a novelty box [10], whereas doses of 2 ng/kg and 20/zg/kg produced no behavioral effect. Since step down latency has been considered as an index of fear/emotiona.lity produced by a novel (stressful) environment [6,27], one interpretation offered for this finding was that AVT reduced the animal's emotional response to novelty [10]. However, an alternative proposition is that AVT activates the animal and this is reflected in the lower step down latency of AVT treated animals. The purpose of the first experiment was to assess the validity of the above hypotheses employing the open field as the behavioral test. Should AVT affect motor activity this would be evidenced in a higher activity score of AVT treated rats. Alternatively, should AVT decrease emotionality/arousal in response to novelty this would be indicated in the measures typically employed to assess emotionality in the open field test, e.g. latency to move from the center after initial placement, center field penetration, defecation. EXPERIMENT 1 METHOD

Animals Male Wistar rats (n= 16) aged 90 to 1I0 days at the begin-

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ning of the experiment were used. Upon arrival in the laboratory the animals were individually housed in wire mesh cages with free access to food and water. The light cycle was 12:12 hour light:dark with light off at 06.00 hr. Prior to testing the rats were assigned randomly to either AVT treatment or AVT control treatment such that each group had 8 animals.

Apparatus The apparatus was based on the open field described by Broadhurst [3] and consisted of a circular enclosure painted white and constructed of sheet metal which was secured onto a white wooden base. The enclosure was 80 cm in diameter with the wall 30 cm in height. The floor was subdivided by three concentric circles: 9cm, 25 cm and 40 cm in diameter, with radiating lines from the center dividing this surface into 19 segments. Testing occurred under dim red light (illumination open field=0.7 lux) between the 5th and 7th hrs of darkness. The apparatus was housed in a sound lagged air-conditioned cubicle (temperature: 23__.I~ adjoining the housing room.

Procedure After three weeks adaptation to the laboratory conditions testing of the animals commenced. AVT (1 ng/kg) or its vehicle (distilled water) was administered intraperitoneally ( I P ) 30 mins prior to testing in keeping with previous p r a c t i c e [ 10]. All injections and removal of the animal from the housing room to the test cubicle were carded out under dim red ' light. Duration of open field testing was 30 mins for each animal. The following measures were recorded: latency (see) to move from the center of the field after initial placement;

C o p y r i g h t 9 1981 A N K H O I n t e r n a t i o n a l Inc.--0196-9781/81/040437-04500.90/0

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defecation (the number of fecal boluses deposited); rearing frequency (the number of times the animal stood on its hindlegs); freezing (remaining motionless for a minimum of a 5 see period, in accordance with previous procedures [8,12]; central crossings (the number of grids traversed in the central area of the field).

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RESULTS AND DISCUSSION

15

The data obtained from each of the recorded measures were analyzed by the t-test. AVT treated rats did not differ significantly from control animals in total activity (i.e., central plus peripheral crossings), defecation or rearing (p's > 0.05). Utilizing the central and peripheral crossing scores a proportional measure was employed [2, 10, 12] to assess the pattern of exploration. The measure was [ Number of Central Crossings 1 ._ Exploration Index= i [ ; i . o t a l ~ ~ ~ h e r a l ) ] X 100 Statistical testing revealed that there was a highly significant difference between the groups with this measure, t(14)=3.6, p<0.05. The AVT treated group explored the central area of the open field more than the control group (see Fig. 1). AVT treatment also had a significant effect on latency, t(14)=3.18, p<0.005, and freezing, t(14)=2.82, p<0.01. Treatment with AVT decreased in both cases, latency and freezing (see Fig. 1). Since AVT did not increase the total activity of the animals in the present study the hypothesis that the lower step down latency of AVT treated animals reflects an activated state induced by AVT may be rejected. However, the alternative interpretation that AVT treatment results in a lower level of emotional reactivity to novel stimuli is supported by the present findings. AVT was found to significantly decrease latency (see Fig. 1) which is in agreement with the previously, reported behavioral effect of AVT administration [10]. The freezing response was also found to be modified such that the control rats remained motionless more than the AVT animals (see Fig. 1). Also AVT modified the pattern of locomotor activity within the open field. This is seen in the higher exploration index of AVT animals (see Fig. 1) demonstrating that the animals explored the central area of the field more than the control animals. Decreased latency and decreased freezing have been viewed as reflecting a lower degree of emotional reactivity to novel stimuli [8, 12, 15]. Increased exploration of the central area of the open field has been considered in the same sense [8, 12, 14]. Thus, the most parsimonious explanation for the behavioral effects observed in this study is that AVT treatment results in the animal being less emotional/aroused in response to novelty.

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The results of the previous experiment combined with that of the findings of Golus et al. [10] demonstrate that the intraperitoneal administration of AVT at a dose of 1 ng/kg produces behavioral effects in rats exposed to a novel environment. These behavioral changes induced by AVT administration have been interpreted as a decreased level of emotionality to novel stimuli. Considering the evidence that the monoamines dopamine (DA), norepinephrine (NE) and 5-HT, have been implicated in emotional/stress responses [5,

FIG. I. Effect o f Arginine Vasotocin (AVT) on open field behavior. Freezing is remaining motionless for a minimum 5 see period; latency (in sees) is time to move from center after initial placement; and the exploration index is percent o f central/total crossings..

Control

29, 31, 32] the first part of the study (2A) reported here examined the effects of IP administration of AVT (1 ng/kg) on the whole brain resting levels of these monoamines. Several studies indicate that the pineal exerts an inhibi-

439

A R G I N I N E VASOTOCIN A N D B E H A V I O R tory influence on the adrenal gland (e.g., [7, 16, 18, 30]) and AVT has been shown to be intimately involved in this inhibition of the adrenal [21,23]. Intraventricular administration of AVT prevents compensatory adrenal hypertrophy in mice [23] and decreases cortisol levels in cats [21]. However, to date there has been no report that AVT influences in vA'o the resting levels of 1 l-hydroxycorticosterone (1 I-OHCS) in the rat. The aim of the second part o f the experiment (2B) was to examine the effects of i.p. administration of AVT upon plasma I I-OHCS levels.

TABLE 1 MEAN AND STANDARD ERROR FOR WHOLE BRAIN NE AND DA (ng/g WET TISSUE)

NE AVT Control

468.6 473.5

DA 14.7 19.4

926.6 877.3

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METHOD

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Thirty two male Wistar rats were used aged 90 to I l0 days (300-390 g) at the beginning of the experiments. On arrival in the laboratory the animals were housed individually in wire mesh cages with free access to food and water. They were maintained on a 12:12 light:dark cycle with light off at 06.00 hr.

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Procedure After three weeks adaptation to the laboratory conditions 16 rats were assigned to each of the two experiments (2A and 2B). Within each experiment eight animals received AVT at a dose o f 1 ng/kg while the remaining control ariimals received an equivalent volume of the diluent. Lyophilized AVT (Calbiochem) was dissolved in distilled water to a concentration of 1 ng/ml just prior to administration. All injections were by the IP route. Animals were sacrificed by decapitation 30 mins after the IP injection. For experiment 2A the brain was removed and treated as previously described by Datta and King [5]. Determination of whole brain monoamine levels followed the fiuorometric methods of Haggendal [I1] and Hinterberger [13] for DA and N E , and the method of Steele and Hinterberger [28] for 5-HT. F o r Experiment 2B trunk blood was collected following decapitation, centrifuged and the plasma assayed for IIOHCS using the fluorometric method developed by Mattingly [17]. Injections and decapitation of all animals occurred between the 5th and 7th hr of darkness under dim red light conditions. RESULTS AND DISCUSSION

Experiment 2A Table 1 gives the mean and standard error for the results of whole brain NE and DA determinations. Student t-test performed on the data revealed no significant differences. However, a highly significant increase in whole brain 5-HT was found, t(14)=6.02, p<0.001 (see Fig. 2).

Experiment 2B Figure 2 depicts the results of the 1 I-OHCS determinations. A V T treatment significantly decreased mean plasma level of I1-OHCS as compared to that of the control, t(14)=1.97, p<0.05. The present experiments demonstrate that AVT significantly increased whole brain 5-HT levels and reduced plasma 1 I-OHCS 30 rains after IP administration. The rise in 5-HT is consistent with previous reports where it was found that IV administration of AVT raised hypothalamic 5-HT [21,22]. This result also adds further support to the notion

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FIG. 2. Effect of AVT (1 ng/kg) on whole brain 5-HT and plasma I I-OHCS, 30 min after IP injection. that the action o f AVT is mediated by the serotonergic system [20]. Brain 5-HT has been implicated in the suppression of the pituitary-adrenocortical system [29, 31, 32]. The decrease in plasma 1 I-OHCS levels found herein may therefore be due to the increased level of brain 5-HT. GENERAL DISCUSSION Arginine vasotocin has been shown to fulfill a number of functions in the central nervous system [19]. For example Pavel and co-workers have reported that the IV administration of subpicogram doses of A V T produces an inhibition of gonadotrophin release [24] and an enhancement of slow wave sleep [25] in cats. The present experiments demonstrate that a low dose of AVT (1 ng/kg) administered IP has profound

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behavioral, neurochemical and endocrinological effects in rats. T h e findings o f Experiment 1 have striking similarity to the results o f a recent report in which melatonin was administered to rats which then underwent open field testing [8]. Melatonin treatment resulted in a significant decrease in freezing and a significant increase in the exploration index [8]. These results were in line with previous research with melatonin in which it was considered that melatonin reduced emotional responses [4, 5, 9]. Pavei [19] has demonstrated that A V T mimics many o f the physiological and endocrinological effects o f melatonin and, since melatonin administration results in a release of A V T from the pineal it has been argued that melatonin is the releasing hormone for

A V T [20]. The similarity between the behavioral effects o f A V T , as found in this study, and that of previous research with melatonin [8] may further support the hypothesis o f melatonin induced release of AVT. It is worth noting that Romijn [26] viewed one of the many functions o f the pineal organ to be that of a "general tranquilizing organ on behalf of homeostatic equilibrium, in close relationship with changing environmental conditions" [26]. The results o f Experiment 1 add credence to previous behavioral research with A V T and melatonin demonstrating a decreased level o f emotionality/arousal produced by these substances. Considering that both AVT (Experiment 2) and melatonin [1] increase brain 5-HT levels the decrease in emotionality/arousal may be attributed to these pineal substances acting upon the serotonergic system.

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