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Abnormal behavioural changes associated with vasopressin-induced barrel rotations
Brain Research, 326 (1985) 65-70 Elsevier
65
BRE 10485
Abnormal Behavioural Changes Associated with Vasopressin-Induced Barrel Rotations R. J. BOAKES, J. M. EDNIE, J. A. EDWARDSON, A. B. KEITH, A. SAHGAL and C. WRIGHT Medical Research Council Neuroendocrinology Unit, Newcastle General Hospital, Newcastle Upon Tyne N E4 6BE ( U. K.)
(Accepted May 1st, 1984) Key words: vasopressin - - barrel rotations - - abnormal behaviour - - rats
Arginine-8-vasopressin (AVP) was injected into the cerebral ventricles of rats in order to characterize the dose-response relations of the convulsant actions of AVP and to obtain a detailed description of other acute behavioural effects. The incidence of barrel rotations, a violent and apparently uncontrolled motor activity during which rats rotate about their long axis, was found to be dose dependent, with a threshold of between 1 and 10 ng per rat. Other behavioural effects of AVP including immobility, titubation, ataxia, backward walking, and inhibition of exploratory activities and of grooming were seen at doses as low as 100 pg. These behavioural effects occurred within 9 rain after injection, and thus have the same time course as barrel rotations. These acute actions of AVP may be significant in interpreting the effects of AVP on cognitive processes and memory and should also be taken into consideration in the clinical use of AVP as an anti-amnestic. INTRODUCTION It has been widely claimed that the intracerebral administration of vasopressin ( A V P ) improves certain types of cognitive b e h a v i o u r in animals (for recent reviews, see refs. 2 and 19) and such findings have led to the clinical use of this p e p t i d e or analogues in m e m o r y deficient states 1,t7. H o w e v e r , during investigations on the effects of A V P in a passive avoidance and conflict schedule p a r a d i g m we observed that the intra-cerebroventricular (i.c.v.) injection of A V P , in doses (1-200 ng) which have been r e p o r t e d to enhance m e m o r y , induced barrel rotations and even death in some rats. Barrel rotations were first r e p o r t e d to follow i.c.v, injection of somatostatin 6 and are a violent m o t o r disturbance in which the animal rotates about its longitudinal axis. Barrel rotations have been r e p o r t e d to follow central administration of A V P and oxytocin 12,14, substance p l l and muscarinic antagonists 5. A l t h o u g h these rotations have been described as convulsions 12 m o r e recent studies involving e l e c t r o e n c e p h a l o g r a p h i c recordings during this b e h a v i o u r have shown that it is not associated with paroxysmal epileptiform activ-
ity 3. In behavioural studies of the m n e m o n i c effects of A V P , the occurrence of barrel rotation or of other disruptive effects which might accompany barrel rotations or occur i n d e p e n d e n t l y after i.c.v, administration of A V P has been largely ignored. W e have therefore analyzed in detail the acute behavioural effects of i.c.v, injections of A V P in rats and quantified both the barrel rotation and other disruptive effects. Two series of experiments were carried out: the first series involved injections of A V P into the cerebral ventricles through the foramen m a g n u m of rats briefly anaesthetized with ether, in o r d e r to establish d o s e - r e s p o n s e characteristics for A V P - i n d u c e d barrel rotations and to obtain information on any other, acute, effects of A V P . In the second series of experiments, injections were m a d e through cannulae permanently implanted into the lateral ventricle, to study in more detail the acute effects of i.c.v. A V P which occur even in the absence of barrel rotations, and also to d e t e r m i n e whether changes in sensitivity to A V P develop. W e confirm that A V P causes barrel rotation in some animals but also find it consistently alters the pattern of b e h a v i o u r after i.c.v, injections of amounts as small as 100 pg.
Correspondence: R. J. Boakes, Medical Research Council, Neuroendocrinology Unit, Newcastle General Hospital, Westgate Road, Newcastle Upon Tyne, NE4 6BE, U.K.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V.
66 recording
box: &I);\ .:. 3. 7 and ii. i.c.\.
lnlectlon
saline followed by 35 min in the recording Experimental
animals
(male
Wistar
albino
rats)
were obtained from Bantin and Kingman U.K. The rats were housed in groups of 6, with uniimited access
to food
and water
(lights on between through
under
diurnal
conditions
07.00 and 10.00 h). For injections
the foramen
briefly anaesthetized
magnum
individual
rats were
with ether and a 25 g needle in-
serted into the IVth ventricle.
AVP was dissolved
in
9, 10 and 11. pcptide
followed
cording box. The rats received
no treatment
an s-switch keyboard;
an appropriate
switch was de-
pressed while the rat under observation abnormal.
Eight is thought to be the maximum
simultaneously
tween
14.00 and 16.00 h. The animals
were then
tape. The occurrence abnormal behaviours
of barrel rotations was noted. Since
and other death oc-
normal behaviour
walking; mained
landmarks (from bregma, lateral 1.5 mm and caudal 1 mm). Cannulae (Plastic Products Inc., VA) were inserted into the left ventricle (4.5 mm deep) and cemented in position: methylene blue injection after the experiments
confirmed
in every case that the can-
nulae were correctly positioned. After surgery the rats were individually housed, and allowed to recover for at least 7 days during which time the cannulae were closed with a stylet and a screw top. Peptides were obtained from Bachem (CA, U.S.A.) or Peninsula Laboratories (U.K.) and their purity was confirmed by high performance liquid chromatography (HPLC). The i.c.v. injections were carried out as follows: 4 rats were tested simultaneously: each rat was injected using a Hamilton microsyringe with a pushbutton dispenser. with 2.0 !11 peptide dissolved in sterile saline or saline alone, and then placed in one of 4 black plastic boxes (II) x 35 x 23 cm) which had one clear side; the activity of the 4 rats was then recorded on videotape simultaneously. All testing took place between 14.00 and 14.30 h. The injection protocol was as follows: days 1 and 2. habituation to the
in
The 4 categories
were: locomotion; The abnormal
num-
be monitored of
grooming;
rear-
behaviours
most
frequently seen after i.c.v. vasopressin administration and classified in this analysis were: strange locomotion, including ataxic movements and backwards
ther 4 animals were injected as described and killed 6 min after injection and a post-mortem examination performed. particular attention being paid to the
animals were anaesthetized with Nembutal. positioned in a stereotaxic frame, the scalp incised and burr holes drilled in the skull with reference to surface
by an observer”!.
ing; and inactivity.
curred in one animal in this series (see Results) a fur-
condition of the lungs, bronchi and trachea. For permanent cannula implantation, 1X0-220 g
engaged
one of 8 specified activities, 4 defined as normal and J
100 ng in 10~1 per rat. Injections
placed in a plastic aquarium (100 x SO X 50 cm) and their behaviour observed, and recorded on video-
on days
5 and 6. Analysis of taped records was carried out b_~ two observers (C.W. and J.M.E.) each equipped with
ber of categories which can successfully
be-
box:: da>,
by 25 min in the rc-
sterile saline and injected in doses of between U. 1 and were performed
OT
crouching, immobile
during
which the animals
in a crouching
position
re-
but ap-
peared alert and exhibited a full righting reflex; preand post-rotation activity. when the rats showed rotational movement of the limbs or tail but the trunk remained immobile: and barrel rotation. The kcyboards were connected to an Acorn microcomputerX programmed to read each keyboard independently as an X-bit word every 0.2 s and store the words on disc for numerical analysis. For the presentation of results in this paper the first 15 min of each recording session were divided into 5 3 min periods: the total time spent by every animal in each of the 8 activities was added to produce a total score. The information statistic (2i)l”. ;I test similar to the ~2 test was used for statistical analyses. RESULTS
A total of 37 rats received injections through the cisterna magna. Animals injected with saline recovered from the light ether anaesthesia within 30 s and showed normal movements and exploratory behaviour. Injection of AVP through the cisterna magna was followed by peripheral vasoconstriction, immobility, ataxia and inhibition of the normal exploratory behaviours such as rearing and sniffing. The incidence of barrel rotation in these animals is shown in Table I and Fig. 1. Death occurred only once in this group of animals and was accompanied by severe lung oedema. Post-mortem examination of J rats
67 treated similarly revealed lung oedema in one animal 6 rain after injection. A total of 23 rats were implanted with cannulae and received i.c.v. AVP or oxytocin, and their behaviour analyzed in detail as described above. The occurrence of barrel rotation in these cannulated animals is shown in Table I and Fig. 1. The results of the detailed analysis of the behaviour following i.c.v, peptide injection are shown in Fig. 2. The behaviour categories described accounted for almost all the behaviours observed; thus after i.c.v. saline, in the first 3 min analysis period 163 s of the available 180 s were taken up in either grooming, inactivity, rearing or locomotion. Equivalent figures for the groups receiving AVP were: 200 ng, 155 s; 100 ng, 163 s; 2/~g oxytocin, 140 s. This categorization accounted for a greater proportion of the time in subsequent analysis periods (mean + S.D., 4 periods and 4 groups of animals = 170 + 5 s, equivalent to 94% of the time accounted for by the categories described). The agreement of the two observers in classifying the activities of the rats was adequate (see legend to Fig. 2). In the group of animals videotaped for subsequent analysis, 6 rats received 100 ng AVP, 9 rats received 200 ng and 8 rats received 2/~g oxytocin. The times spent in the various activities described above by the rats in the 4 drug and saline groups are shown in Fig. 2 A - H . In all groups, grooming occupied most of the time; after saline, grooming took 443 s of the total 900 s analysis period, starting at 89 s in the first 180 s analysis period and ending slightly higher at 104 s in the last period. Less time
was spent grooming after peptide injections than after saline. Inactivity took a large and variable proportion of the analysis period, while the exploratory behaviours rearing and locomotion decreased; after saline injection the animals spent 14 s of the first 180 s in locomotor activity and only 1 s in the last period, rearing decreased from 121 s to 8 s. No abnormal behaviours were observed after i.c.v, saline injections. After peptide injection all animals engaged in activity categorized as pre- or post-rotational movements as described above (Fig. 2E). This activity usually reached a peak during the second 180 s analysis period; thus during the second analysis period animals injected with 100 ng vasopressin showed this activity for 61 s, 200 ng vasopressin, 56 s, and 2 pg oxytocin, 63 s. Immobile crouching was seen after 200 ng vasopressin and 2/zg oxytocin; this activity was only a minor part of the total duration of the analysis period except during the second 180 s period. Barrel rotation occurred after peptide injection in all groups and in almost all cases occurred in the first 360 s, as did the pre- or post-rotation activity and immobile crouching. Strange locomotion differed from the other abnormal activities in that it decreased throughout the analysis and was not seen in the last period. After peptide injections the animals also engaged in the normal activities shown after saline, but the total time spent in these normal activities was less; grooming and rearing were less in all peptide treated groups than after saline. A total of 27 rats was injected i.c.v, on 3 successive days with AVP; the occurrence of barrel rotation in
TABLE I
Occurrence of barrel rotation after intra-ventricular A VP
tr~ n-<
Ventricle
Dose o f A VP
Number of rats showing barrel rotation
IVth
100 ng 50 ng 10 ng 1 ng 0.1 ng saline
5/6 3/5 4/9 2/4 0/6 0/7
200 ng 100 ng 20 ng 2/xg oxytocin saline
4/6 3/9 1/12 1/8 0/23
lateral
A 60-
•
•
:
mO
U,. I1~
0 LU (.9 Z ,,, a
4020-
0"1 i 10 100 VASOPRESSIN (ng) Fig. 1. Dose-response relations for AVP and the proportions of rats (expressed as percent) exhibiting barrel rotation. (I--I), intracisternal AVP; (& ..... &), intraventricular AVP. Linear regression lines were calculated according to the method of Wiggans and Sahga123; both regression lines were significant, P < 0.05.
14o-
A.
GROOMING
B.
INACTIVITY
C.
REARING
D.
LOCOMOTION
H.
STRANGE LOCOMOTION
!
1OO
i Ill
0
--
u
bE.
6o
.
PPC ~
F.
BARREL ROTATIONS
G.
CROUCHING
40-
o
o.
-
-
-
TIME PERIODS (3 mins) Fig. 2. The graphs A - H show the total number of seconds that each activity was exhibited by the animals in the 4 drug or saline groups
during the 900 s analysis period. The data are cumulated times during successive analysis periods of 3 rain and are not vector quantities, but are more clearly represented as such, and are plotted against the analysis periods. The normal activities grooming, inactivity, rearing and locomotion are shown in the top 4 graphs (A-D) and the abnormal, drug-induced activities in the lower 4 graphs (E-H). (@~---Q), data for saline groups; (B ~), 200 ng AVP; ( C ] - - D ) , 100 ng AVP; (& A), 2/~g oxytocin. It can be seen that the normal activities of grooming and inactivity occupied a large, variable proportion of the total analysis period, whereas the exploratory activities rearing and locomotion decreased during observation, irrespective of the i.c.v, injection. By contrast, the abnormal activities peaked in the first two periods, except for strange locomotion, the time course of which resembled locomotion. The peptide induced activity which took the largest amount of time was the pre- and post-rotational category (E). Addition of the cumulative times allows the comprehensiveness of the analysis to be assessed. Thus, out of 900 s analysis of the activities of saline-injected rats, 836 s arc accounted within Fig. 2: for 200 ng AVP, 847 s arc accounted, for 100 ng AVP. 787 s and for 2,ug oxytocin. 892 s. The total time accounted for by the two observers never differed by more than 11% (observer l/observer 2 × 100).
those animals is shown in Table II. The decrease in numbers of animals injected on the successive days of
killed 6 min later revealed no lung or tracheal oedema.
the e x p e r i m e n t s is due to d e a t h o r dislocation of the cannulae. T h e direction of r o t a t i o n of 60 c a n n u l a t e d animals was n o t e d to find if t h e r e was a c o r r e l a t i o n b e t w e e n
TABLE II Occurrence of barrel rotation after repeated injections of A VP
the side of c a n n u l a t i o n and the d i r e c t i o n of rotation. T h e results are s h o w n in T a b l e III, and s h o w that the direction of r o t a t i o n did not d e p e n d on the side of
Day
Proportion of animals rotating at different doses 20 ng
c a n n u l a t i o n (2I = 0.29, df = 1, P > 0.5). P o s t - m o r -
1
l / 12
tern e x a m i n a t i o n of 7 rats c a n n u l a t e d as d e s c r i b e d and i n j e c t e d 1 w e e k later i.c.v, with 200 ng A V P and
2 3
10/12 9/1 I
100 ng
200 ng
3/9 2/'8 31~
4,/6 3/6 2/6
69 TABLE III
Direction of rotation and side of cannulation Direction of rotation L R Total
Side of cannulation L 14 11 25
R 22 13 35
Total 36 24 60
DISCUSSION
It can be seen from these results that AVP disrupts normal behaviour at very low doses. Barrel rotations were consistently induced by i.c.v, injection of 10 ng AVP, an effect in good agreement with the results of previous studiesl2,14. Thus, vasopressin appears to be approximately 1000-fold more potent than substance pH, chlorpromazine methiodide 5 or the oxytocin used in the present study. The effective dose of AVP (10 ng) can be compared with the 200-600 ng AVP stored in the pituitary gland in rats and cerebrospinal fluid levels of 11.5 pg/ml (ref. 8). Even in the absence of barrel rotations, i.c.v, administration of the peptide produced marked behavioural changes. Some of these changes, such as titubation, and circular tail movements, may be directly related to movements which occur during barrel rotation while others such as crouching and immobility are not obviously connected with barrel rotations. All the rats injected with peptides i.c.v, showed abnormal behaviours to some degree. These abnormal behaviours are not typical of sedative compounds, as has been suggested 15 but are more likely to reflect a state of overarousaP 9,20.21. The time course of these effects is similar to that observed for barrel rotations and thus may reflect a common action of AVP on neural substrates. These effects of AVP reduce the time available for normal behaviour such as rearing and grooming during the period of study. For example, the amount of time spent grooming by rats injected i.c.v, with saline was very high; grooming did not occur at all in animals which received only one injection of vasopressin either i.c.v, or into the cisterna magna. The higher amounts of time spent grooming presumably therefore reflect the successful habituation of the animals in this group to the recording procedures.
Vasopressin is released centrally when rats convulse during hyperthermia and rats which have so convulsed are subsequently sensitized to i.c.v. AVP a2. In the present study there was some evidence to suggest that rats anaesthetized with ether immediately prior to i.c.v. AVP convulsed more than rats with permanent cannulae and habituated to the observation chambers (Table I and Fig. 1) and that successive i.c.v, injections of AVP elicited increasing disruption (Table II, column 1). The ability of vasopressin and the other peptides T R H and substance P to cause barrel rotations do appear to be relatively specific, since a wide range of unrelated compounds including strychnine and picrotoxin do not elicit barrel rotationsL The lack of correlation between the cannulation side and the direction of rotation argues against an 'imbalance' basis similar to the basis of circling after unilateral nigro-striatal lesions 22. The greater effectiveness of AVP injected through the cisterna magna in producing barrel rotations and other behavioural disruption may indicate that the central structure involved is more accessible from the IVth ventricle. This is in agreement with the results of recent studies 4. Many of the findings in support of the mnemonic effects of AVP are based upon measures such as the increased time taken to re-enter a chamber in which the animal previously experienced shock. It is clear from the present study that such data should be re-assessed in the light of these various abnormal patterns of behaviour which occur in response to identical doses of vasopressin. The clinical implications of these results are serious in the light of the use of vasopressin as an anti-amnestic drug; convulsions can be precipitated in epileptic children by systemic administration of Pitressin 18 and schizophrenic patients become 'agitated and aggressive' after treatment with lysine vasopressin 13. Hypomania and other mood changes have also been reported to follow AVP treatment in brain damaged subjects (see ref. 9, and for review ref. 19). The present findings, together with these clinical observations, suggest that the administration of AVP or related compounds to patients with a previous history of neurological or psychiatric illness should be undertaken with extreme care.
7~ REFERENCES 1 Beckwith, B. E., Petros, T., Kanaan-Beckwith, S., Couk, D. I. and Haug, R. J., Vasopressin analog (DDAVP) facilitates concept learning in human males, Peptides. 3 (19821 627-630. 2 Burbach, J. P. H. and DeWied. D., Memory effects and brain proteolysis of neurohypophyseat hormones. In D. H. Schlesinger (Ed.), Neurohypophyseal Hormones and Other
Biologically Active Peptides. Develop. Endocrinol., Vol. 13, Elsevier/North Holland, Amsterdam, 1981, p. 69. 3 Burke, R. E. and Fahn, S., Electroencephalographic studits of chlorpromazine methiodide and somatostatin-induced barrel rotation, Exp. Neurol.. 79 (1983) 704-713. 4 Burke, R. E. and Fahn, 8., Studies of somatostatin-induced barrel rotation in rats, Regular. Pept., 7 (1983) 207-220. 5 Burke, R. E., Fahn, S., Wagncr, H. R. and Smeal, M., Chlorpromazine methiodide-induced barrel rotation: an anti-muscarinic effect, Brain Research, 250 (1982) 133-142. (~ Cohn, M. L, and ('ohm M., 'Barrel rotation' induced by somatostatin in the non-lesioned rat, Brain Research, 96 (1975) 138-141. 7 Dogterom, T., Van Wimersma Greidanus, Tj. B. and Swaab, D. F., Evidence for the release of vasopressin and oxytocin in cerebrospinal fluid: measurement in plasma and CSF of intact and hypophysectomized rats, Neuroendocrinology, 24 (1977) 108-118. 8 Fray, P. J., ONLIBASIC, a system for experimental control, Trends Neurosci., 3 (1980) XIII-XIV. 9 Gold, P. W., Goodwin, F. K., Ballenger, J. C., Weingartner. H., Robertson, G. L. and Post, R. M., Central vasopressin junction in affective illness. In D. de Wied and P. A. van Keep (Eds.), Hormones and the Brain. MTP Press, Lancaster, 1980, p. 241. 10 Green. D. M. and Swets, J. A., Signal Detection Theorv and Psychophysics, Wiley, New York, 1966. 11 James, T. A. and Starr, M. S., Effects of substance P injected into the substantia nigra, BriL J. Pharmacol., 65 (1979) 423-429,
12 Kasting, N. Vv., Vealc, W. L. and (ooper, R. l;., (onvuisire and hypothermic effects of wtsopressin m the brain ~,! the rat, Canad. J. Phvsiol. Pharmacol., 58 (19801 316 ~ 1¢~ 13 Korsgaard, S., Cascy, D. E., Pedersen, N. F. l)., Jorgcnsen, A. and Gerlach, J., Vasopressm in anergic schizophrenia: a cross-over studv with lysinc-g-vasopressin and placebo, Psychopharmacology, 74 ( 1981 / 379-382 14 Krusc, t|., Wimersma Greidanus, Ij. B. and De Wicd, D., Barrel rotation induced by vasopressin and related peptides m rats, Pharmacol. Biochem. Behav., 7 (1977) 31 ~- 313. 15 Krejci, l., Kupkova, B., Metys, J., Barth, q. and Jost, K. Vasopressin analogs: sedative properties and passive avoidance behaviour in rats. ['.'arop. ,I. t'harmacol. 5(~ 11979) 347-353. 16 Kullback, S., ln#)rmation Theory and Statistics. Dover, New York, 1968. 17 Legros, J. J., Gitot, P., Seron, X., Claessens, J., Adam, A., Moeglen, J. M., Audibert, A. and Berchier, P., Influence of vasopressin on learning and memory, Lancet. {i) 11978) 41-42. 18 McQuarrie, I. and Peeler, D. B., The effects of sustained pituitary antidiuresis and forced water drinking in epileptic children. A diagnostic and etiologic study, J. Clin. lnvest., 10 (1931) 915-940. 19 Sahgal, A., A critique of the vasopressin memory hypothesis, Psychopharmacology, in press. 20 Sahgal, A., Keitz, A. B., Wright, C. and Edwardson, J. A., Failure of vasopressin to enhance memory in a passive avoidance task, NeuroscL Lett., 28 (1982) 87-92. 21 Sahgal, A. and Wright, C., A comparison of the effects of vasopressin and oxytocin with amphetamine and chlordiazepoxide on passive avoidance behaviour in rats, Psychopharmacology, 80 11982) 88-92. 22 Ungerstedt, U,, Postsynaptic supersensitiviD after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Acta physiol, stand., 82, Suppl. 367 (1971) 69-93. 23 Wiggans, G. N. and Sahgat, A., BIREG: a basic program to estimate regression lines for bivariate data sets, Lab. Pract., 32 (1983) 77-79.
65
BRE 10485
Abnormal Behavioural Changes Associated with Vasopressin-Induced Barrel Rotations R. J. BOAKES, J. M. EDNIE, J. A. EDWARDSON, A. B. KEITH, A. SAHGAL and C. WRIGHT Medical Research Council Neuroendocrinology Unit, Newcastle General Hospital, Newcastle Upon Tyne N E4 6BE ( U. K.)
(Accepted May 1st, 1984) Key words: vasopressin - - barrel rotations - - abnormal behaviour - - rats
Arginine-8-vasopressin (AVP) was injected into the cerebral ventricles of rats in order to characterize the dose-response relations of the convulsant actions of AVP and to obtain a detailed description of other acute behavioural effects. The incidence of barrel rotations, a violent and apparently uncontrolled motor activity during which rats rotate about their long axis, was found to be dose dependent, with a threshold of between 1 and 10 ng per rat. Other behavioural effects of AVP including immobility, titubation, ataxia, backward walking, and inhibition of exploratory activities and of grooming were seen at doses as low as 100 pg. These behavioural effects occurred within 9 rain after injection, and thus have the same time course as barrel rotations. These acute actions of AVP may be significant in interpreting the effects of AVP on cognitive processes and memory and should also be taken into consideration in the clinical use of AVP as an anti-amnestic. INTRODUCTION It has been widely claimed that the intracerebral administration of vasopressin ( A V P ) improves certain types of cognitive b e h a v i o u r in animals (for recent reviews, see refs. 2 and 19) and such findings have led to the clinical use of this p e p t i d e or analogues in m e m o r y deficient states 1,t7. H o w e v e r , during investigations on the effects of A V P in a passive avoidance and conflict schedule p a r a d i g m we observed that the intra-cerebroventricular (i.c.v.) injection of A V P , in doses (1-200 ng) which have been r e p o r t e d to enhance m e m o r y , induced barrel rotations and even death in some rats. Barrel rotations were first r e p o r t e d to follow i.c.v, injection of somatostatin 6 and are a violent m o t o r disturbance in which the animal rotates about its longitudinal axis. Barrel rotations have been r e p o r t e d to follow central administration of A V P and oxytocin 12,14, substance p l l and muscarinic antagonists 5. A l t h o u g h these rotations have been described as convulsions 12 m o r e recent studies involving e l e c t r o e n c e p h a l o g r a p h i c recordings during this b e h a v i o u r have shown that it is not associated with paroxysmal epileptiform activ-
ity 3. In behavioural studies of the m n e m o n i c effects of A V P , the occurrence of barrel rotation or of other disruptive effects which might accompany barrel rotations or occur i n d e p e n d e n t l y after i.c.v, administration of A V P has been largely ignored. W e have therefore analyzed in detail the acute behavioural effects of i.c.v, injections of A V P in rats and quantified both the barrel rotation and other disruptive effects. Two series of experiments were carried out: the first series involved injections of A V P into the cerebral ventricles through the foramen m a g n u m of rats briefly anaesthetized with ether, in o r d e r to establish d o s e - r e s p o n s e characteristics for A V P - i n d u c e d barrel rotations and to obtain information on any other, acute, effects of A V P . In the second series of experiments, injections were m a d e through cannulae permanently implanted into the lateral ventricle, to study in more detail the acute effects of i.c.v. A V P which occur even in the absence of barrel rotations, and also to d e t e r m i n e whether changes in sensitivity to A V P develop. W e confirm that A V P causes barrel rotation in some animals but also find it consistently alters the pattern of b e h a v i o u r after i.c.v, injections of amounts as small as 100 pg.
Correspondence: R. J. Boakes, Medical Research Council, Neuroendocrinology Unit, Newcastle General Hospital, Westgate Road, Newcastle Upon Tyne, NE4 6BE, U.K.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V.
66 recording
box: &I);\ .:. 3. 7 and ii. i.c.\.
lnlectlon
saline followed by 35 min in the recording Experimental
animals
(male
Wistar
albino
rats)
were obtained from Bantin and Kingman U.K. The rats were housed in groups of 6, with uniimited access
to food
and water
(lights on between through
under
diurnal
conditions
07.00 and 10.00 h). For injections
the foramen
briefly anaesthetized
magnum
individual
rats were
with ether and a 25 g needle in-
serted into the IVth ventricle.
AVP was dissolved
in
9, 10 and 11. pcptide
followed
cording box. The rats received
no treatment
an s-switch keyboard;
an appropriate
switch was de-
pressed while the rat under observation abnormal.
Eight is thought to be the maximum
simultaneously
tween
14.00 and 16.00 h. The animals
were then
tape. The occurrence abnormal behaviours
of barrel rotations was noted. Since
and other death oc-
normal behaviour
walking; mained
landmarks (from bregma, lateral 1.5 mm and caudal 1 mm). Cannulae (Plastic Products Inc., VA) were inserted into the left ventricle (4.5 mm deep) and cemented in position: methylene blue injection after the experiments
confirmed
in every case that the can-
nulae were correctly positioned. After surgery the rats were individually housed, and allowed to recover for at least 7 days during which time the cannulae were closed with a stylet and a screw top. Peptides were obtained from Bachem (CA, U.S.A.) or Peninsula Laboratories (U.K.) and their purity was confirmed by high performance liquid chromatography (HPLC). The i.c.v. injections were carried out as follows: 4 rats were tested simultaneously: each rat was injected using a Hamilton microsyringe with a pushbutton dispenser. with 2.0 !11 peptide dissolved in sterile saline or saline alone, and then placed in one of 4 black plastic boxes (II) x 35 x 23 cm) which had one clear side; the activity of the 4 rats was then recorded on videotape simultaneously. All testing took place between 14.00 and 14.30 h. The injection protocol was as follows: days 1 and 2. habituation to the
in
The 4 categories
were: locomotion; The abnormal
num-
be monitored of
grooming;
rear-
behaviours
most
frequently seen after i.c.v. vasopressin administration and classified in this analysis were: strange locomotion, including ataxic movements and backwards
ther 4 animals were injected as described and killed 6 min after injection and a post-mortem examination performed. particular attention being paid to the
animals were anaesthetized with Nembutal. positioned in a stereotaxic frame, the scalp incised and burr holes drilled in the skull with reference to surface
by an observer”!.
ing; and inactivity.
curred in one animal in this series (see Results) a fur-
condition of the lungs, bronchi and trachea. For permanent cannula implantation, 1X0-220 g
engaged
one of 8 specified activities, 4 defined as normal and J
100 ng in 10~1 per rat. Injections
placed in a plastic aquarium (100 x SO X 50 cm) and their behaviour observed, and recorded on video-
on days
5 and 6. Analysis of taped records was carried out b_~ two observers (C.W. and J.M.E.) each equipped with
ber of categories which can successfully
be-
box:: da>,
by 25 min in the rc-
sterile saline and injected in doses of between U. 1 and were performed
OT
crouching, immobile
during
which the animals
in a crouching
position
re-
but ap-
peared alert and exhibited a full righting reflex; preand post-rotation activity. when the rats showed rotational movement of the limbs or tail but the trunk remained immobile: and barrel rotation. The kcyboards were connected to an Acorn microcomputerX programmed to read each keyboard independently as an X-bit word every 0.2 s and store the words on disc for numerical analysis. For the presentation of results in this paper the first 15 min of each recording session were divided into 5 3 min periods: the total time spent by every animal in each of the 8 activities was added to produce a total score. The information statistic (2i)l”. ;I test similar to the ~2 test was used for statistical analyses. RESULTS
A total of 37 rats received injections through the cisterna magna. Animals injected with saline recovered from the light ether anaesthesia within 30 s and showed normal movements and exploratory behaviour. Injection of AVP through the cisterna magna was followed by peripheral vasoconstriction, immobility, ataxia and inhibition of the normal exploratory behaviours such as rearing and sniffing. The incidence of barrel rotation in these animals is shown in Table I and Fig. 1. Death occurred only once in this group of animals and was accompanied by severe lung oedema. Post-mortem examination of J rats
67 treated similarly revealed lung oedema in one animal 6 rain after injection. A total of 23 rats were implanted with cannulae and received i.c.v. AVP or oxytocin, and their behaviour analyzed in detail as described above. The occurrence of barrel rotation in these cannulated animals is shown in Table I and Fig. 1. The results of the detailed analysis of the behaviour following i.c.v, peptide injection are shown in Fig. 2. The behaviour categories described accounted for almost all the behaviours observed; thus after i.c.v. saline, in the first 3 min analysis period 163 s of the available 180 s were taken up in either grooming, inactivity, rearing or locomotion. Equivalent figures for the groups receiving AVP were: 200 ng, 155 s; 100 ng, 163 s; 2/~g oxytocin, 140 s. This categorization accounted for a greater proportion of the time in subsequent analysis periods (mean + S.D., 4 periods and 4 groups of animals = 170 + 5 s, equivalent to 94% of the time accounted for by the categories described). The agreement of the two observers in classifying the activities of the rats was adequate (see legend to Fig. 2). In the group of animals videotaped for subsequent analysis, 6 rats received 100 ng AVP, 9 rats received 200 ng and 8 rats received 2/~g oxytocin. The times spent in the various activities described above by the rats in the 4 drug and saline groups are shown in Fig. 2 A - H . In all groups, grooming occupied most of the time; after saline, grooming took 443 s of the total 900 s analysis period, starting at 89 s in the first 180 s analysis period and ending slightly higher at 104 s in the last period. Less time
was spent grooming after peptide injections than after saline. Inactivity took a large and variable proportion of the analysis period, while the exploratory behaviours rearing and locomotion decreased; after saline injection the animals spent 14 s of the first 180 s in locomotor activity and only 1 s in the last period, rearing decreased from 121 s to 8 s. No abnormal behaviours were observed after i.c.v, saline injections. After peptide injection all animals engaged in activity categorized as pre- or post-rotational movements as described above (Fig. 2E). This activity usually reached a peak during the second 180 s analysis period; thus during the second analysis period animals injected with 100 ng vasopressin showed this activity for 61 s, 200 ng vasopressin, 56 s, and 2 pg oxytocin, 63 s. Immobile crouching was seen after 200 ng vasopressin and 2/zg oxytocin; this activity was only a minor part of the total duration of the analysis period except during the second 180 s period. Barrel rotation occurred after peptide injection in all groups and in almost all cases occurred in the first 360 s, as did the pre- or post-rotation activity and immobile crouching. Strange locomotion differed from the other abnormal activities in that it decreased throughout the analysis and was not seen in the last period. After peptide injections the animals also engaged in the normal activities shown after saline, but the total time spent in these normal activities was less; grooming and rearing were less in all peptide treated groups than after saline. A total of 27 rats was injected i.c.v, on 3 successive days with AVP; the occurrence of barrel rotation in
TABLE I
Occurrence of barrel rotation after intra-ventricular A VP
tr~ n-<
Ventricle
Dose o f A VP
Number of rats showing barrel rotation
IVth
100 ng 50 ng 10 ng 1 ng 0.1 ng saline
5/6 3/5 4/9 2/4 0/6 0/7
200 ng 100 ng 20 ng 2/xg oxytocin saline
4/6 3/9 1/12 1/8 0/23
lateral
A 60-
•
•
:
mO
U,. I1~
0 LU (.9 Z ,,, a
4020-
0"1 i 10 100 VASOPRESSIN (ng) Fig. 1. Dose-response relations for AVP and the proportions of rats (expressed as percent) exhibiting barrel rotation. (I--I), intracisternal AVP; (& ..... &), intraventricular AVP. Linear regression lines were calculated according to the method of Wiggans and Sahga123; both regression lines were significant, P < 0.05.
14o-
A.
GROOMING
B.
INACTIVITY
C.
REARING
D.
LOCOMOTION
H.
STRANGE LOCOMOTION
!
1OO
i Ill
0
--
u
bE.
6o
.
PPC ~
F.
BARREL ROTATIONS
G.
CROUCHING
40-
o
o.
-
-
-
TIME PERIODS (3 mins) Fig. 2. The graphs A - H show the total number of seconds that each activity was exhibited by the animals in the 4 drug or saline groups
during the 900 s analysis period. The data are cumulated times during successive analysis periods of 3 rain and are not vector quantities, but are more clearly represented as such, and are plotted against the analysis periods. The normal activities grooming, inactivity, rearing and locomotion are shown in the top 4 graphs (A-D) and the abnormal, drug-induced activities in the lower 4 graphs (E-H). (@~---Q), data for saline groups; (B ~), 200 ng AVP; ( C ] - - D ) , 100 ng AVP; (& A), 2/~g oxytocin. It can be seen that the normal activities of grooming and inactivity occupied a large, variable proportion of the total analysis period, whereas the exploratory activities rearing and locomotion decreased during observation, irrespective of the i.c.v, injection. By contrast, the abnormal activities peaked in the first two periods, except for strange locomotion, the time course of which resembled locomotion. The peptide induced activity which took the largest amount of time was the pre- and post-rotational category (E). Addition of the cumulative times allows the comprehensiveness of the analysis to be assessed. Thus, out of 900 s analysis of the activities of saline-injected rats, 836 s arc accounted within Fig. 2: for 200 ng AVP, 847 s arc accounted, for 100 ng AVP. 787 s and for 2,ug oxytocin. 892 s. The total time accounted for by the two observers never differed by more than 11% (observer l/observer 2 × 100).
those animals is shown in Table II. The decrease in numbers of animals injected on the successive days of
killed 6 min later revealed no lung or tracheal oedema.
the e x p e r i m e n t s is due to d e a t h o r dislocation of the cannulae. T h e direction of r o t a t i o n of 60 c a n n u l a t e d animals was n o t e d to find if t h e r e was a c o r r e l a t i o n b e t w e e n
TABLE II Occurrence of barrel rotation after repeated injections of A VP
the side of c a n n u l a t i o n and the d i r e c t i o n of rotation. T h e results are s h o w n in T a b l e III, and s h o w that the direction of r o t a t i o n did not d e p e n d on the side of
Day
Proportion of animals rotating at different doses 20 ng
c a n n u l a t i o n (2I = 0.29, df = 1, P > 0.5). P o s t - m o r -
1
l / 12
tern e x a m i n a t i o n of 7 rats c a n n u l a t e d as d e s c r i b e d and i n j e c t e d 1 w e e k later i.c.v, with 200 ng A V P and
2 3
10/12 9/1 I
100 ng
200 ng
3/9 2/'8 31~
4,/6 3/6 2/6
69 TABLE III
Direction of rotation and side of cannulation Direction of rotation L R Total
Side of cannulation L 14 11 25
R 22 13 35
Total 36 24 60
DISCUSSION
It can be seen from these results that AVP disrupts normal behaviour at very low doses. Barrel rotations were consistently induced by i.c.v, injection of 10 ng AVP, an effect in good agreement with the results of previous studiesl2,14. Thus, vasopressin appears to be approximately 1000-fold more potent than substance pH, chlorpromazine methiodide 5 or the oxytocin used in the present study. The effective dose of AVP (10 ng) can be compared with the 200-600 ng AVP stored in the pituitary gland in rats and cerebrospinal fluid levels of 11.5 pg/ml (ref. 8). Even in the absence of barrel rotations, i.c.v, administration of the peptide produced marked behavioural changes. Some of these changes, such as titubation, and circular tail movements, may be directly related to movements which occur during barrel rotation while others such as crouching and immobility are not obviously connected with barrel rotations. All the rats injected with peptides i.c.v, showed abnormal behaviours to some degree. These abnormal behaviours are not typical of sedative compounds, as has been suggested 15 but are more likely to reflect a state of overarousaP 9,20.21. The time course of these effects is similar to that observed for barrel rotations and thus may reflect a common action of AVP on neural substrates. These effects of AVP reduce the time available for normal behaviour such as rearing and grooming during the period of study. For example, the amount of time spent grooming by rats injected i.c.v, with saline was very high; grooming did not occur at all in animals which received only one injection of vasopressin either i.c.v, or into the cisterna magna. The higher amounts of time spent grooming presumably therefore reflect the successful habituation of the animals in this group to the recording procedures.
Vasopressin is released centrally when rats convulse during hyperthermia and rats which have so convulsed are subsequently sensitized to i.c.v. AVP a2. In the present study there was some evidence to suggest that rats anaesthetized with ether immediately prior to i.c.v. AVP convulsed more than rats with permanent cannulae and habituated to the observation chambers (Table I and Fig. 1) and that successive i.c.v, injections of AVP elicited increasing disruption (Table II, column 1). The ability of vasopressin and the other peptides T R H and substance P to cause barrel rotations do appear to be relatively specific, since a wide range of unrelated compounds including strychnine and picrotoxin do not elicit barrel rotationsL The lack of correlation between the cannulation side and the direction of rotation argues against an 'imbalance' basis similar to the basis of circling after unilateral nigro-striatal lesions 22. The greater effectiveness of AVP injected through the cisterna magna in producing barrel rotations and other behavioural disruption may indicate that the central structure involved is more accessible from the IVth ventricle. This is in agreement with the results of recent studies 4. Many of the findings in support of the mnemonic effects of AVP are based upon measures such as the increased time taken to re-enter a chamber in which the animal previously experienced shock. It is clear from the present study that such data should be re-assessed in the light of these various abnormal patterns of behaviour which occur in response to identical doses of vasopressin. The clinical implications of these results are serious in the light of the use of vasopressin as an anti-amnestic drug; convulsions can be precipitated in epileptic children by systemic administration of Pitressin 18 and schizophrenic patients become 'agitated and aggressive' after treatment with lysine vasopressin 13. Hypomania and other mood changes have also been reported to follow AVP treatment in brain damaged subjects (see ref. 9, and for review ref. 19). The present findings, together with these clinical observations, suggest that the administration of AVP or related compounds to patients with a previous history of neurological or psychiatric illness should be undertaken with extreme care.
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