The involvement of arginine vasotocin in the maturation of the kitten brain

Peptides. Vol. 5, pp. 25--28, 1984. ~ Ankho International Inc. Printed in the U.S.A.

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The Involvement of Arginine Vasotocin in the Maturation of the Kitten Brain R. G O L D S T E I N Institute o f Endocrinology, Bucharest, R o m a n i a

R e c e i v e d 22 N o v e m b e r

1982

GOLDSTEIN, R. The involvement of arg,inine vasotocin in the maturation of the kitten brain. PEPTIDES 5(!) 25-28, 1984.--1n order to investigate the effects of the nonapeptide hormone arginine vasotocin (AVT) on the maturation of the brain, the following developmental data were tabulated between 5 and 21 days of postnatal life, in kittens, after the daily intraperitoneal administration of 10-~ mg synthetic AVT: sleep, daily increase of body weight and locomotor, and investigative activities (LIA). Likewise, the day of the eye opening was noted and the brain weight as well as the total lipid levels within the brain in the day of sacrifice (21 days of age) were measured. The daily administration of AVT induced: (1) an increase of the total amount as well as of the intensity of active sleep (AS); (2) a decrease of the LIA; (3) a decrease of the total lipid levels within the brain and (4) a retardation of the eye opening. These effects appeared to be specific because neither arginine vasopressin, nor oxytocin, in the same doses (10-~ mg), were able to reproduce the effects of AVT. The present results demonstrate that chronic administration of AVT is associated with a retardation of brain maturation. Whether AVT induces this effect by an unique mechanism or there are different mechanisms for the reported developmental data that were affected by AVT, is unknown. However, the present results suggest that the pineal gland, by its effector within the brain, AVT, is involved by an inhibitory pathway in the brain maturation and the hypothesis is advanced that the decrease of AVT content of fetal and neonatal brain could represent a hormonal signal for triggering the beginning of the brain maturation phenomena. Arginine vasotocin

Arginine vasopressin

Oxytocin

Active sleep

T H E nonapeptide hormone arginine vasotocin (AVT) is synthetized both in fetal and adult brain of mammals [2, 3, 4, 6, 14, 25] by specialized ependymal cells of the pineal recess and subcommissural organ [14]. There is a large decrease of AVT content of the pineal gland [14] and of cerebrospinal fluid of man [15] as well as a reduction of the ependymal cells of pineal recess and subcommissural organ [13] from fetal to adult age. These phenomena take place simultaneously with decrease of active sleep (AS) [24], the ontogenetic precursor of adult rapid eye movements (REM) sleep [l]. The effects of A V T on sleep in adult mammals are still controversial, varying from enhancement of nonrapid eye movements (NREM) sleep and suppression of REM sleep in cats [18] to decrease of REM sleep latency and increase o f REM sleep amounts in man [19] and from reduction of REM sleep latency [12] or reduction in the power of the delta-band of electrocorticograms [28] to reduction of REM sleep in the light-phase [7,23] and suppression o f REM sleep with increase o f N R E M sleep in the dark-phase of the day in rats [7]. Nevertheless, the acute administration of A V T increases AS in all newborn mammals so far investigated, including cat [9], rat [8], man [17] and guinea pig (Goldstein, unpublished results), and since it is supposed that AS [24], pineal gland [20, 21, 22] and AVT [9, 17, 19] are involved in the maturation of the brain, we investigated the effects of chronic administration of synthetic AVT on some developmental measures related to brain maturation [29] in newborn kittens.

Total brain iipids

Brain maturation

used in the present experiments. At three days of age, under ether anesthesia, they were implanted for monitoring sleep with six electrodes (2 for cortical, 2 for ocular and 2 for nuchal muscular activities), according to the method of Jouvet-Mounier et al. [10]. After the completion of the surgery, the animals were returned to their cages, where they stayed with the mother and their iittermates all times, including the sleep sessions. Between 5 and 21 days o f age, the following developmental data were recorded: weight, locomotor and investigative activities (LIA) and sleep. Likewise, the day of eyes opening was noted. L I A were recorded by placing the animals in a 60 by 30 cm cardboard arena, having a 2.5 cm high board and by computing the number of the head (HCA) or whole body (BCA) climbing activities during a session of one hour. An animal was placed in the middle o f arena and its behavior w~s subsequently watched. Usually, the kitten starts to move slowly towards the board of arena and when it reaches it, he lifts the head and climbs out of the board with the head. This was computed as a HCA. Sometimes the animal further climbs the board with the whole body and when he is out of arena with at least its anterior paws is computed as a BCA and returned to the middle o f arena. If the kitten did not climb with the whole body out o f arena during a session, this was computed as OBCA. After the ending o f L I A recording session, the animals were returned to their cages and were recorded for sleep in a 3-4 hr long session. All experiments were performed between 10:00 and 16:00 hr. At the end of the sleep session, animals were injected by an intraperitoneal way with 10-e mg synthetic AVT (Organon, Holland, T H 223AK)

METHOD Thirty two newborn kittens, coming from eight cats, were

25

26

GOLDSTEIN

TABLE 1 THE DAILY INCREASE OF THE BODY WEIGI-rr (W/DAY), BRAIN WEIGHT AT 21 DAYS OF AGE AND TIlE DAY OF AGE AT WHICH THE EYES ARE OPENED IN KrFrENS DAILYINJECTEDWITH0.5 ml SALINE OR 104 mg AVT, AVP OR OT Substance Saline AVT AVP OT ANOVA

W/day (g ± SE)

Brain weight (g --+ SE)

11.8 ± 1.63 12 ± i.65 11.1 -+ 1.54 11.4 _ 1.63

14.3 _ 12.8 ± 14.1 ± 14.6 ±

F(3,28)= 1.24 p >0.25.

0.8 0.4 0.6 0.8

F(3,28)= 1.71 p <0.25

Day of eyes opening (+-SE) 10 ± 14 ± ll _ l0 ±

0.2 0.7* 0.4 0.3

F(3,28)=3.05 p <0.025

TABLE 2 THE LOCOMOTORAND INVESTIGATIVEACTIVITIES(LIA) EXPRESSED BY BCA AND HCA DURINGONE HOUR. IN KITTENS DAILY INJECTED WITH0.5 ml SALINEOR 10-6 rag AVT, AVP OR OT LIA Substance Saline AVT AVP OT ANOVA

BCA 30 3 29 33

-+ 6.6 - 0.4* - 5.7 --- 6.3

F(3,28)= 28.4 p<0.001

HCA 16.9 -5 = 17.8 = 19.2 -

1.7 0.8* 1.9 2.0

F(3,28) = 10.4 p<0.001

*Greater than in all other groups, p=0.01 (Duncan's multiple comparison test).

The values represent means _ SE. *Less than in all other groups, p =0.01 (Duncan's multiple comparison test).

(8 kittens), 10-6 mg synthetic arginine vasopressin (AVP) (Organon, Holland, TH 891AK) (8 kittens) or 10-~ mg oxytocin (OT) (Orgnnon, Holland, TH 644AK) (8 kittens). The peptides were diluted in a 0.5 ml of saline. The controls (8 kittens) received the same volume of saline. The vigilance states were classified after the criteria of Jouvet-Mounier e t al. [101 in wakefulness, active sleep (AS) and quiet sleep (QS). At 21 days of age, the kittens were sacrificed by decapitation, the brains removed, weighed and the total lipid levels determined gravimetrically after the previous extraction in a chloroform/methanol (2/!, v/v) solution, as described by Folch et al. [5]. The values of total lipid levels were expressed as rag/100 mg wet brain tissue. All numerical data were statistically analysed by the one-way analysis of variance (ANOVA), followed, in the case of significant results, by Duncan's multiple comparison test. For OBCA the results were analysed by X" test.

TABLE 3 THE OBCA EXPRESSED AS PERCENTAGEFROMTOTAL LIA RECORDING SESSIONS IN KITTENSDAILY INJECTED WITH 0.5 ml SALINE OR 10-6 rag AVT, AVP OR OT

RESULTS As can be seen from Table 1, no peptide treatment was able to change the daily increase of the body weight. Although there was a tendency of AVT to decrease the brain weight in comparison with all other groups (Table l), this was not statistically significant. However, AVT induced a significant delay of eyes opening (Table 1). The daily administration of AVT induced a marked decrease of LIA measured either by HCA or BCA, when compared with all other groups (Table 2). From BCA point of view, only after AVT administration, an increase of OBCA was observed (Table 3). Moreover, while in saline as well as in AVP and OT treated groups there was a tendency towards a decrease of OBCA with age, in AVT treated animals this tendency did not appear (Fig. I). An increase of total sleep and AS, expressed as percentages from total recording time, as well as an increase of the number of rapid eye movements during an AS episode were observed after AVT administration (Table 4). Although between 5 and 21 days of age there was no difference between the precentage of QS after peptides or saline treatments (Table 4), in the second week of registration (I 3-21 days of age) there was a tendency of AVT to increase the QS amount, although this increase was not statistically significant, F(3,28)--1.93, p>0.25, ANOVA (Fig. 2).

Substance Saline AVT AVP OT

Number of LIA recording sessions

OBCA (%)

128 128 128 128

40 (31.2%) 80 (62.5%)* 46 (35.9%) 42 (32.8%)

*xz test, p<0.001.

As can be seen from Table 5, there was a decrease of total lipid levels within the brain in AVT treated group when compared with all other groups. DISCUSSION The present results, showing an AS increase after AVT administration, confirm the data obtained by acute administration of synthetic AVT in kittens [9], rat pups [8], infants [17] and newborn guinea pigs (Goldstein, unpublished results). These demonstrate that, in spite of the controversy regarding the effects of AVT on sleep in adult mammals [7, 12, 18, 19, 23, 28], in newborn mammals, AVT produces similar effects, mainly an enhancement of AS. Since in the present experiment, AVT was injected at the end of the sleep sessions, the AS increasing activity cannot be ascribed to an immediate but to a long acting effect on sleep. It also was shown that AVT is able to exert significant endocrine effects in mice at 3 or even at 7 days after a single dose [ 14]. Since REM sleep and its ontogenetic precursor, AS [l], were implicated in neural and behavioral development of mammals [24], pinealectomy, that removes the main source of AVT [14], performed in neonatal animals, produces an enhancement of the exploratory behavior in open field test [26] as well as an impairment of brain myelination [22] and since AVT increased quantitatively and qualitatively AS, inhibited behavior and lowered the brain lipid levels, it is tempting to speculate that the reported behavioral and biochemical

VASOTOCIN AND BRAIN MATURATION

27

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[--ISalk~ ~AVP

%OBCA

~AVP

nor

DoT

3O

NAVT

mArT

25

IO0

20

75.

15

50"

10

2~

5'

g.

E!

A

A

FIG. 1. The percentage of OBCA from total number of OBCA in first (a) and second (B) week of observation in kittens daily injected with 0.5 ml saline or 10-~ mg AVT, AVP or OT.

E!

FIG. 2. The evolution of the percentage of QS from total recording time (%QS/T) in first (A) and second (B) week of observation in kittens daily injected with 0.5 ml saline or l0 -~ mg AVT, AVP or OT. (Bars represent the S.E.).

TABLE 4 THE TOTAL SLEEP (TS), ACTIVE SLEEP (AS) AND QUIET SLEEP (QS) EXPRESSED AS PERCENTAGE FROM TOTAL RECORDING TIME (T) AS WELL AS THE NUMBER OF RAPID EYE MOVEMENTS PER EPISODE OF ACTIVE SLEEP (REM/AS) IN KITTEN S DAILY 1NJECTED WITH 0.5 ml SALINE OR l0-6 mg AVT, AVP OR OT Substance Saline AVT AVP OT ANOVA

TS/T 53 77 52 56

± ± -

3.4 4.7* 3.2 3.7

F(3,28)= 1 3 . 7 p <0.001

As/r 42 65 41 45

± ±

QS/T

2.7 4.3* 2.6 3.0

F(3,28)=8.7 p <0.005

11 13 11 11

± ±

1.5 1.7 1.2 1.3

F(3,28)= 1.3 p >0.25

REM/AS 43 110 50 56

-+ 4.5 ± 8.7* - 5.3 - 4.8

F(3,28)= 13 p <0.001

The values represent means _+ SE. *Greater than in all other groups, p=0.01 (Duncan's multiple comparison test).

changes o b s e r v e d after A V T , possibly could be due to the changes o f AS. E x c e p t for the daily increase of the body weight and of the brain weight, all d e v e l o p m e n t a l data tested w e r e affected by chronic administration o f A V T , and to the e x t e n t to which these data are manifestations o f brain maturation [29], A V T could be c o n s i d e r e d as an inhibitory h o r m o n e o f the maturational p h e n o m e n a o f the brain. The idea o f the inhibitory significance o f A V T on brain maturation is supported not only by the present results demonstrating: (1) inhibition o f behavioral p a r a m e t e r s , m e a s u r e d by the reduction o f L I A and increase o f O B C A ; (2) retardation o f the e y e s opening; (3) d e c r e a s e o f total lipid levels within the brain and (4) inc r e a s e o f the a m o u n t and intensity o f A S after chronic administration o f A V T , but also by p r e v i o u s l y r e p o r t e d data showing that the brain immaturity is associated with: (1) immaturity o f the m o t o r b e h a v i o r due to the immaturity o f m u s c u l a r and neural systems [29]; (2) immaturity of the sensitive and sensorial s y s t e m s , including the u n o p e n i n g o f the eyelids [29]; (3) low levels o f brain lipids [30], (4) high percentages o f A S [24,29] and (5) high c o n c e n t r a t i o n s o f A V T levels within the brain [14]. Why a chronic administration o f A V T is able to p r o d u c e a delay of brain maturation is unknown. H o w e v e r , since there

TABLE 5 THE TOTAL LIPID LEVELS WITHIN THE BRAIN FROM KITTENS OF 21 DAYS OF AGE, DAILY INJECTED WITH 0.5 ml SALINE OR 10-6 nag AVT, AVP OR OT Substance

Total lipids

Saline AVT AVP OT

4.38 4.05 4.48 4.41

ANOVA

-~ 0.05 ± 0.04* -+ 0.07 _~ 0.06

F(3,28)= 15 p<0.001

The values represent mg/100 mg wet brain _ SE. *Less than in all other groups, p=0.01 (Duncan's multiple comparison test).

is a tight paralellism b e t w e e n (1) the d e c r e a s e o f A S [24], (2) the increase o f total lipid levels within the brain [30] and (3) the d e c r e a s e o f brain A V T c o n t e n t [14] during the c o u r s e of brain maturation, it m a y be s u p p o s e d that all are related to an unique m e c h a n i s m and that pineal gland, by its effector

28

GOLDSTEIN

within the brain, AVT, plays an important role, by an inhibitory way, in the triggering of this mechanism. AVT effects were highly specific since neither AVP, nor OT, in the doses used (10-~ mg), were able to mimic these effects. This demonstrates that the substitution of isoleucine for 3-phenyl-alanine in the ring of AVP or of arginine for 8-1eucine in the side chain of OT are critical for the effects of AVT on developmental phenomena, as demonstrated for AVT effects on sleep [15] and on serotonin metabolism within the brain [15] in adult cats. The present reported tendency of AVT to increase QS in kittens in the second week of observation (13-21 days of age), but not in the first (5-12 days of age), resembling the NREM sleep increasing activity of AVT in adult cats [18], may reflect a change of AVT related mechanisms of sleep around the 10--13th day of postnatal life. Otherwise, this age seems to be clue of both sleep [1] and of brain myelination [29], coinciding with the eyelids opening in kittens. We

presume that this age corresponds to the appearance of the other pineal hormone, the releasing factor of AVT [ 14], the indole melatonin. The reversing effect of a pineal extract, but not of melatonin on the open field test in neonatally pinealectomized rats [26], in a period when pineal seems to be unable to synthesize melatonin [11], further support the above assumption. Until now, it is unknown what initiates the brain maturation processes [27], but the above data suggest that the decrease of AVT levels to a threshold could initiate the maturation of brain structures, including myelination. The pineal involvement in the development of some mental retardation states [21], supports this hypothesis. ACKNOWLEDGEMENT The author expresses gratitude to Dr. M. Popa (Institute of Endocrinology, Department of Endocrinopediatry, Bucharest, Romania) for the useful help given for the statistical evaluation of the results.

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