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Opposite effects of oxytocin and vasopressin on avoidance behaviour and hippocampal theta rhythm in the rat
OPPOSITE EFFECTS OF OXYTOCIN AND VASOPRESSIN ON AVOIDANCE BEHAVIOUR AND HIPPOCAMPAL THETA RHYTHM IN THE RAT WIMERSMA GREIDANUS and D. DE WED
B~XA BOHUS.* 1. URBAN, TJ. B. VAN Rudolf
Magnus
Institute
for Pharmacology, Medical Faculty, Utrecht. The Netherlands (Accepted
16 May
University
of Utrecht,
Vondellaln
6,
19773
Summary-- Intracerebroventricular administration of oxytocin and arginine-Svasopressin (AVP) immediately after a single learning trial resulted in opposite effects on retention of passive avoidance behaviour. Passive avoidance behaviour was attenuated by oxytocin and facilitated by AVP in a dosedependent manner. Reverse effects were found after neutralizing the centrally circulating oxytocin and vasopressin by intraventricular injection of specific antiserum to these peptides. Administration of oxytotin into a lateral ventricle resulted in a decrease of the peak frequency of hippocampal theta rhythm generated during paradoxical sleep episodes. Conversely, oxytocin antiserum increased the incidence of higher frequencies in the theta rhythm. Peripheral administration of oxytocm mimicked the effect of AVP on extinction of pole-jumping avoidance behaviour. Resistance to extinction rather than facilitation of extinction was observed when higher doses of oxytocin were given. Passive avoidance behaviour appeared to be either facilitated or attenuated after subcutaneous oxytocin administration. It is concluded that centrally released oxytocin may be a naturally occurring amnesic neuropeptide.
Vasopressin or antidiuretic hormone (ADH) of hypothalamo-neurohypophyseal origin is involved in the formation and maintenance of adaptive behaviour. Removal of the neural lobe of the pituitary gland interferes with the maintenance of a shuttle-box avoidance response (de Wied, 1965). That rats with hereditary hypothalamic diabetes insipidus, which lack the ability to synthetize vasopressin (Valtin and Schroeder, 1964), show a deficit in acquiring a shuttiebox avoidance response (Celestian, Carey and Miller, 1975; Miller, Barranda, Dean and Brush, 1976) and an impaired maintenance of active and passive avoidance behaviour (Bohus, van Wimersma Greidanus and de Wied, 1975: de Wied, Bohus and van Wimersma Greidanus, 1975). corroborates the view that vasopressin is physiologically involved in learning and memory processes. In intact rats, vasopressin and analogues of this polypeptide. which are practically devoid of pressor or anti-diuretic activities, increase resistance to extinction of active (de Wied. 1971; de Wied, Greven, Lande and Witter, 1972; Schulz, Kovacs and Telegdy, 1976) and passive avoidance responses (Ader and de Wied, 1972; Bohus, Ader and de Wied, 1972). Furthermore, these peptides protect against puromycill- (Lande, Flexner and Flexner, 1972; Watter, Hoffman, Flexner and Flexner, t97.5) or CO,-induced amnesia in mice and rats (Rigter, van Riezen and de Wied, 1974). On the other hand, neutralizing the centrally circulating arginine-vasopressin
* To whom correspondence should be addressed. Key words: oxytocin. avoidance behaviour, rhythm,
theta
arginine-8-vasopressin. 239
(AVP) by specific antiserum administered intracerebroventricularly impairs passive avoidance and results in a facilitated extinction of a pole-jumping active avoidance response in the rat (van Wimersma Greidanus, Dogterom and de Wied, 1975). Oxytocin (OXT), the other neurosecretory product of the hypothalamo-neurohypophyseal system fails to affect avoidance behaviour and to prevent amnesia when administered in doses equimolar to the minimally effective amount of AVP (de Wied, 1971: Lissak and Bohus, 1972; Walter et ni., 1975). Higher doses of OXT however mimic the effect of vasopressin on the maintenance of conditioned avoidance behaviour (de Wied and Gispen, 1977). In contrast to these observations, Schulz er ul. (1976) have recently reported that small doses of OXT influence extinction behaviour opposite to that of vasopressin. Facilitation of active avoidance extinction in non-deprived but particularly in water-deprived rats was observed. In order to resolve the apparent contradictions the present experiments were aimed to investigate in more detail the central effects of oxytocin by comparing the influence of this peptide and that of its specific antiserum on active and passive avoidance behaviour, with the effects of AVP and its antiserum after peripheral ,and intracerebroventricular administration. in addition, the effects of intracerebroventricularly administered oxytocin and OXT antiserum on hippocampal theta activity generated during paradoxical sleep (PS) episodes were investigated. It has been suggested that some of the behavioural effects of vasopressin and related peptides may be due to an increase in the excitability of midbrain limbic struc-
240
BELA BOHUS ef al.
tures. Lower peak frequencies in hippocampal theta activity during PS episodes were found in rats with hereditary hypothalamic diabetes insipidus as compared to their heterozygous littermates (Urban and de Wied, 1975). In the intact rat, administration of desgly~in~ide lysine-vasopressin, which is a behaviourally active analogue of vasopressin, increases the proportion of higher frequency components in the hippocampal theta activity. ~ntraventricularly administered AVP antiserum. on the other hand, induces changes in the theta activity which resemble those of the diabetes insipidus rats (Urban and de Wied, 1978). METHODS Subjects
Male wistar rats of an inbred strain (Cpb, TNG, Zeist. The Netheriands) were used. They were kept under controlled light-dark conditions (light on between 5.00 and 19.00 hr) and had free access to food and water. The subjects were housed 5-6 per cage but rats implanted with a permanent cannula for intraventricular injection or those bearing electrodes for recording hippocampal electrical activity were kept in jndividual cages. Behaviourul procedures Active avoidance behaviour was studied in a polejumping situation (de Wied, 1966). Briefly, the rats were conditioned to avoid the unconditioned stimulus {US) of a scrambled electric floor shock (0.20 mA. a.c.) by jumping onto a pole located in the centre of the apparatus. The conditioned stimulus (CS) was a light signal above the pole. The US was applied if an avoidance response had not occurred within Ssec after the onset of CS. Ten acquisition trials were given daily with variable intertrial intervals averaging 60 sec. Acquisition training was followed by extinction sessions. Ten trials were presented daily in which the CS was terminated if the rat had jumped onto the pole within S set or after 5 sec. when avoidance did not occur. The US was never applied during the extinction sessions. Passive avoidance behaviour was studied by using a one-trial learning paradigm in a step-through type situation as described by Ader, Weijnen and Moleman (1972). Briefly, the rats were adapted to the apparatus consisting of a large dark compartment equipped with a grid floor and mesh-covered extensively Iit elevated runway, attached to the front centre of the dark chamber. Adaptation training was followed by a trial in which the rat was placed on the runway facing away from the dark compartment and allowed to enter the dark. Three more trials were given on the next day with an intertrial interval of 5 min. The last of these trials was followed by a single 2 set unavoidable scrambled footshock (0.25 mA, a.c.) immediately after entering the dark compartment. The retention of the passive avoidance response was
tested 24 and 48 hr after the single learning trial. The subject was placed on the elevated runway and the latency to reenter the dark compartment was recorded up to a maximum of 300 sec. Drugs and routes of admi~iscrafion Highly purified synthetic argjnine-8-vasopressin (AVP; pressor activity 273 Ujmg) and oxytocin (OXT: uterotropic activity 512 Ujmg) were used. Peptide solutions were freshly prepared before administrations by dissolving them in 0.Q saline (0.1 ml N/100 HCI was added to increase the stability of the peptide). The concentration of the peptides in the solutions was adjusted to a final volume of 0.5 ml for subcutaneous and 1~1 for intracerebroventricular administration. Physiologi~ai saline containing 0.1 ml NjlOOHCl was used as control. Arginine-vasopressin and oxytocin antisera were prepared by immunizing male rabbits as described by van Wimersma Greidanus et al. (1975). Undiluted AVP antiserum in the quantity of 1~1 binds approximately 2.5 ng of AVP while the binding capacity of the OXT antiserum was approximately l-l.5 ng of OXT. Normal rabbit serum was used in the control rats. For intracerebroventricular administration of the peptides and antisera a polyethylene cannula was implanted in a lateral ventricle under anesthesia as described earlier (de Wied, 1976). A 10~1 microsyringe (Unimetrics, Co.) with adjusted length of needle fitting in the lumen and exactly reaching the tip of the preimplanted cannula was used to administer the substances in the ventricle. The unanaesthetized rats were kept in hand while administering the test material. The localization of the tip of the cannula was checked at the termination of the experiments by injecting Evans blue. The staining was then inspected macrostopically in formaldehyde fixed brain sections. Only those injections were regarded as successful in which the walls of both lateral, of the third and fourth ventricles were stained blue. Experimentai designs The three main bodies of the behavioural experiments were as follows: (i) peripheral administration of OXT and AVP; (ii) intraventricular administratjon of OXT and AVP; (iii) intraventricular administration of OXT and AVP antisera. A total of 82 rats were used to study the effects of peripheral administration of OXT and AVP on active and passive avoidance behaviour. Forty-two animals, weighing l30-I4Og at the onset of the experiments were trained to acquire the pole-jumping conditioned avoidance response. Five acquisition sessions were followed by 5 extinction days. In the first series of the experiments 24 rats were trained and then divided randomly into 4 equal groups. Oxytocin in doses of 0.2, 0.02 and 0.002 pg per rat was administered subcutaneously 1 hr prior to the first 3 extinction sessions. Saline-treated subjects served as con-
241
Oxytocin. vasopressin and avoidance behaviour trols. Eighteen rats, randomly divided into 3 equal groups, were used in the second study. The subjects received either AVP or OXT in a dose of 0.02 c(g per rat subcutaneously 1 hr prior to each acquisition and extinction session. Control rats received saline daily. Forty rats, weighing 160-l?Og, were used in the passive avoidance paradigm. Randomly divided equal groups of rats received a single subcutaneous injection of OXT (0.1 or 0.5 pg/rat) or AVP (0.5 pg/rat) immediately after the learning trial. Retention of the passive avoidance response was studied 24 and 48 hr later. The effects of intracerebroventricular administration of OXT and AVP on active and passive avoidance behaviour were studied in 136 rats. Thirty-six rats, weighing 14@150g, were implanted with a permanent can&a in a lateral ventricle. Five days later they were trained to acquire a pole-jumping avoidance response. Three acquisition sessions were foilowed by 2 extinction days of 10 trials each. Randomly selected groups received either 1.0 ng of OXT or AVP or physiological saline. These treatments were given immediately after each acquisition session. Injection of Evans blue through the permanent cannula at the termination of the experiments indicated successful intraventricular treatment of 32 rats. A hundred rats, weighing 16&18Og, were also implanted with a permanent intraventricular cannula. Five days later, they were trained in the step-through passive avoidance situation. Oxytocin or AVP in doses of 0.05, 0.1, 1.0 and lO.Ong or physiological saline were given intraventricularly immediately after the single learning trial. Retention of the passive avoidance response was studied 24 and 48 hr thereafter. Localization of the cannula at the termination of the experiments indicated successful injection in 90 rats. A total of 90 male rats was used to investigate the influence of OXT and AVP antisera on the avoidance behaviour. Fifty rats, weighing 14@-150 g, were implanted with a permanent cannula for intraventricular antiserum administration. Five days later the rats were trained to acquire a pole-jumping avoidance response as described earlier. Randomly selected groups received either 2~1 of OXT antiserum or 1 fd of AVP antiserum 30 min before each acquisition session. Normal rabbit serum (2 ~1) were given to the controls. Observations on 35 rats were then analysed after checking the cannula placement. Forty rats, weighing 160-180 g, were also implanted with a permanent cannula in a lateral ventricle. Five days later they were trained in the passive avoidance situation. Oxytocin, or AVP antiserum or normal rabbit serum (1~1) was administered intraventricularly to randomly selected rats immediately after the single learning trial. Retention of the passive avoidance response was tested 24 and 48 hr later. Successful intraventricular injections were found in 31 rats.
Five male Wistar rats. weighing 2OO-220g at the time of the operation, were used for the electrophysiological experiments. Animals equipped with a permanent intracerebroventricular cannula were anaesthetized (Nembutal~) and a pair of electrodes (100~ in diameter, insulated except for the tip) was stereotaxitally implanted in the CA, field of the dorsal hippocampus (~o”ordinates: A = 0.4 mm, L = 2.5 mm and 2.5 mm from the cortex according to Albe-Fessard, Stutinsky and Libouban, 1966). Another pair of electrodes was attached to the neck muscles to record the electromyogram. Two weeks after the surgery, the rats were placed on 12 hr light-dark schedule in sound-attenuated cages with food and water ad libiturn. Hippocampal and myographic electrical activities, recorded throughout 8 hr sessions (between 9.00 and 17.00 hr of the light cycle) via impedance transformers, were stored on magnetic tape and polygraphic paper for later analysis. The first five recording sessions served for adaptation of the rats to recording conditions. The amount of PS and the frequency pattern of the hippocampal theta rhythm during PS were determined. In the subsequent sessions OXT (5 and 20 ng dissolved in 1 ~1 of physiological saline), OXT antiserum (1~1, undiluted) or placebos (saline or normal rabbit serum) were randomly injected intraventricularly. The minimal interval between 2 consecutive OXT or OXT antiserum injections was 48 hr. Each treatment was replicated 3 times. The effect of OXT was investigated in 3 rats whereas other three animals out of the 5 subjects were selected to study the influence of OXT antiserum. The power spectral analysis was used to quantify hippocampal electrical activity. Hippocampal records during PS of the first 180min following intraventricular injections were divided into lo-set analysis epochs. filtered (high-frequency cut-off at 30 c/s), digitalized (100 c/s sampling rate) and a power spectrum for each epoch was computed and stored. Subsequently, an average spectrum per 100 set was computed and its resolution of 0.1 c/s was reduced to 0.5 c/s. The resulting spectrum was converted into 100,
E x+,nr+,an,
days
Fig. 1. Effects of various doses of oxytocin (OXT) administered subcutaneously I hr prior to the first 3 extinction sessions on the maintenance of a conditioned pole-jumping avoidance response.
percentages of power per OSc/s component. The effect of OXT and OXT antiserum on hippocampal activity was estimated by comparing the means and standard errors of percentages of power per 0.5 c/s components of experimental sessions with corresponding means of placebo sessions. The total amount of PS per session was determined from the polygraphic records.
Two-tailed r-test, analysis of variance, the non-parametric statistics of Mann-Whitney (U-test) and the x*-test were used to analyse parametric. non-parametric and frequency distribution data.
‘, I
2
3
4
Acquisition
5
I
2
3
4
SAL
5
Extinctson Days
RESULTS .FJkts
oj’
~~ri~~z~ral ad~inistrat~otz of oxytocin and
arllirlitle-r’asopressin 011 active and passive woidance hehaciour As shown in Figure 1, administration of OXT prior to the first 3 extinction sessions resulted in a delay of extinction of the conditioned avoidance response. Cessation of the treatment was followed by a decline of avoidance performance particularly in rats which had been treated with 0.2 pg of OXT, but the peptidetreated rats displayed more avoidance responses than the controls. One-way analysis of variance of the total number of avoidance responses scored during extinction training indicated a significant effect of OXT (P < 0.01; FJ,20 = 5.3916). Administration of 0.02 pg of OXT or AVP throughout the acquisition and extinction sessions failed to influence the acquisition of the pole-jumping avoidance response (Fig. 2). Extinction of the response was however markedly delayed in rats treated with AVP. The total number of avoidances during extinction was significantly higher (P < 0.001) in rats treated with AVP (39.8 & 1.4) than in those which received OXT (24.8 -f 2.1) or saline (22.3 rfr 2.7). Oxytocin treatment had no effect on the rate of extinction. Retention of passive avoidance behaviour as defined by the latency to re-enter the dark compartment was enhanced by post-learning administration of AVP (Table I). Although the median avoidance latency of rats which received OXT was nearly the same as in Table
I. Effects of oxytoein
Treatment Oxytocin Arginine-vasopressin Saline
the controls, the individual latencies appeared to show a bimodal distribution. The largest percentage of rats treated with OXT displayed longer or shorter latencies than the controls. Analysis of the distribution pattern with the aid of x2-test indicated significant differences between the control group and that which received 0.1 pg of OXT immediately after the learning trial (P < 0.001, x2 = 14.286). Administration of 0.5 rug of AVP resulted in an increase of avoidance latency and most of the rats displayed longer latencies than the controls. The distribution of latenties appeared to be significantly different (P < 0.01; x2 = 9.899) when the AVP-treated group was compared to the controls. i@cts gf intracerebrouentricular~~*administered oxytotin and arg~n~~e ~~lso~ressin on active and passice avoidance behaciour Results on acquisition and extinction of the conditioned pole-jumping avoidance response in rats treated intraventricularly with OXT (n = 12). AVP (tt = 8) or saline (n = 12) imm~iately after each acquisition session are depicted in Fig. 3. Administration of OXT slightly affected acquisition and extinction behaviour of the rats. Oxytocin-treated rats
and arginine-vasopressin administered subcutaneously immediately on the retention of the passive avoidance response in the rat
Dose (/kg) 0.1 0.5 0.5
Distribution of avoidance latencies” 41-120 set > 120sec < 40 set 50 30 0 0
“Expressed as percentage of rats displaying ‘P < 0.01 (peptide vs saline: Mann--Whitney
LP < 0.05. ( ) Number of rats.
Fig. 2. Comparative effects of arginine vasopressin (AYP) and oxytocin (OXT) administered subcutaneously during both acquisition and extinction on conditioned pole-jumping avoidance behaviour.
0 30 10 80 the given test).
50 40 90 20 iatencies
during
Median
avoidance (see)
after the learning
latency
24 hr
48 hr
117.0 54.5 222.0” 71.0
74.0 39.0 186.5 30.0
the retention
trial
test at 24 hr
(10) (10) (10) (10)
Oxytocin.
vasopressin
and avoidance
243
behaviour
ANTI-OXT
Se
AVP
NORMAL
Se
OXT \,
SAL
ANTI-AVP
Se
Fig. 4. Effects of intraventricular administration of oxytotin (OXT) or arginine-vasopressin antiserum 30 min prior to each acquisition session conditioned pole-jumping avoidance behaviour of the rat. Fig. 3. Pole-jumping avoidance behaviour of rats after intraventricular administration of oxytocin (OXT) or arginine vasopressin (AVP) immediately after each acquisition session.
scored fewer avoidance responses than the controls during the third acquisition and the first extinction session but the difference appeared to be significant only on the third acquisition session (P < 0.02; t = 2.755). Treatment with AVP did not affect acquisition behaviour but despite the cessation of treatment these rats were more resistant to extinction than the controls. A significant difference between AVP-treated and control rats was found during the second extinction session (P < 0.02; t = 2.579). Intraventricular administration of OXT and AVP immediately after the single learning trial profoundly influenced the retention of the passive avoidance response (Table 2). Significantly shorter avoidance latencies were observed in rats which received OXT in doses of 0.1 or 1.0 ng as compared to controls at the 24 hr retention test. Administration of 0.05 ng of
Table 2. Etfects of intraventricular administration of oxytocin (OXT) and arginine-vasopressin (AVP) immediately after the learning trial on the retention of a one trial passive avoidance response Dose
Median
avoidance
Treatment
(ng)
24 hr
48 hr
OXT
0.05 0.1 1.0 10.0 0.05 0.1 1.0 10.0
65.0 22.0h 29.0 39.0 72.C 88.0 110.5’ 135.5 64.0
45.0 7.0 19.0 42.0 32.0 26.0 32.0 95.5” 24.5
Saline
* P < 0.05 (peptide vs saline; b P < 0.02. E P < 0.01. ( ) Number of rats.
Effects of inrracerebroventricular administration ofoxytocin and arginine vasopressin antiserum on active and passive avoidance behaviour Intraventricular administration of both OXT and AVP antiserum prior to each acquisition session resulted in a facilitation on avoidance acquisition in the pole-jumping test (Fig. 4). The total number of conditioned avoidance responses scored by the rats receiving either OXT (20.5 Ifr 1.0; n = 11) or AVP antiserum (19.4 + 1.2; n = IO) appeared to be significantly higher (P < 0.01 and P < 0.05) than that
latency
(set)
AVP
OXT failed to influence passive avoidance behaviour. Although the median of the latencies of rats which received 10.0 ng of OXT was not different from that of the controls, the distribution pattern appeared to be significantly different (P < 0.01, x2 = 8.892). The majority of the latencies was shorter (60%) respectively longer (30:;) than the controls. No significant differences were observed in retention behaviour 48 hr after the learning trial. In contrast to OXT, intraventricular administration of AVP in doses of I.0 and 10.0 ng led to a significant increase in passive avoidance latencies. The effect of 10.0 ng of AVP appeared to be long-lasting. Passive avoidance latencies were still augmented at the 48 hr retention test.
Mann-Whitney
(9) (8) (13) (10) (9) (7) (12) (10) (12)
Table 3. Effects of intraventricular administration of oxytotin- or arginine-vasopressin antiserum immediately after learning trial on the retention of a one trial passive avoidance response
Treatment Oxytocin antiserum Arginine-vasopressin antiserum Normal rabbit serum
Median avoidance 24 hr
latency (set) 48 hr
I 50.0h
58.0
(11)
25.5
17.0
(10)
48.5
36.5
(11)
test). a P < 0.05 (Mann-Whitney b P < 0.01. ( ) Number of rats.
test).
BELA BOHUS et al.
244
~ntraventricul~ administration of OXT antiserum immediately after the single learning trial was followed by longer avoidance latencies at the 24 hr retention test than that found in controls (Table 3). Administration of AVP antiserum resulted in significantly shorter latencies. No differences were observed between treated and control rats at the 48 hr retention test. ESJcts of oxytocin and oxyrociu antiserum campal theta actiuit>
cm hippo-
As compared to saline treatment, intraventricular administration of 5 or 20ng of OXT was followed by a decrease of the peak frequency from 7.5 to
4
5
6
.
.
7 Hz B
I)
l
.
9
II
IO
.
Fig. 5. Changes in power spectrum of hippocampai theta activity during PS episodes O-90 min after intraventricular administration of 5 ng (upper graph) or 20 ng (lower graph) of oxytocin (OXT). Significant differences treated vs control sessions are indicated by asterisks (P -=z0.01). scored by the controls (15.0 & 1.4). Extinction behaviour was, however, affected in an opposite manner by the administration of the two antisera. Rats which received OXT antiserum during acquisition showed resistance to extinction and the total number of avoidances (14.7 & 1.3) was significantly higher (P < 0.05) than that of the controls (10.6 + 1.4). In contrast, a more rapid extinction took place in rats which had received AVP antiserum and the total number of avoidances (4.1 + 0.9) was significantly less (P < 0.001) than that of the controls.
NORMAL
Se
*
*
7.Oc/s, a decrease of the percentage of the higher frequency components whereas the proportion of the low theta frequencies in the hippocampal activity was slightly but significantly increased (Fig. 5). Conversely, intraventricular administration of OXT antiserum resulted in an increased proportion of the higher theta frequencies (7.5-10Hz) and a decreased amount of the low-frequency components (Fig. 6). The total amount of PS during the 8-hr observation periods was not affected by the administration of OXT or OXT antiserum. DISCUSSION
The present experiments suggest that besides the well-documented effects of vasopressin of hypothalamic neurosecretory origin on behaviour and electrical activity of the brain (de Wied. van Wimersma Greidanus, Bohus, Urban and Gispen, 1976), oxytocin, which is also produced by the hypothalamic neurosecretory cells, influences brain functions in a way opposite to that of vasopressin. Opposite effects on avoidance behaviour and hippocampal theta activity during paradoxical sleep episodes were particularly observed after intracerebroventricular administration of oxytocin. Attenuation of passive avoidance behaviour followed post-learning administration of oxyto-
ANTI-OXT
*
*
**
Se
*
*
Fig. 6. Effect of intraventricular administration of oxytocin antiserum on hippocampal theta activity during PS episodes. Significant differences treated vs control sessions are indicated by asterisks (P < 0.01).
Oxytocin. vasopressin and avoidance behaviour tin and the peptide appeared to cause a decrease in dominant frequencies of hippocampaf theta activity when administered intraventricularly. Vasopressin facilities passive avoidance behaviour and increases the incidence of higher frequency components of hippocampal theta activity. That oxytocin may play a physiological role in the regulation of certain brain functions is suggested by observations with specific antibodies against this peptide. Neutralizing the centrally circulating oxytocin by specific antiserum is followed by behavioural changes and alterations in hippocampal RSA which are opposite to that of the effect of oxytocin. Evidence has already been furnished on opposite effects of vasopressin and oxytocin on nerve functions. Vasopressin facilitates while oxytocin suppresses spontaneous nerve activity of Periplaneta americana (Schulz, 1970). Oxytocin shortens the reaction time to an acoustic stimulus in the rat while lysine-vasopressin (LVP) has an opposite effect in this respect (Schwarzberg and Unger, 1970). Furthermore, Schulz et al., 1976, reported that, contrary to LVP, oxytocin facilitates extinction of a conditioned active avoidance response. More recently, van Ree and de Wied (1977) found that oxytocin and oxytocin fragments facilitate while and vasopressin fragments vasopressin attenuate heroin self-administration behaviour of the rats. Oxytocin administration was also found to cause changes in the electrical activity of the brain (Faure, 1957; Schgker, Klingberg, Sterba and Pickenheim, 1966; Schwarzberg, Unger and Schulz, 1973). It is not yet clear, however, whether this peptide possesses inherent neurotropic activity which is opposite to that of vasopressin or whether it acts as an antagonist of vasopressin by occupying putative receptor sites and therefore preventing vasopressin effects. An opposite biological effect of oxytocin and vasopressin is not an unique phenomenon. Small doses of oxytocin induce diuresis (Pickford, 1961) and inhibit the antidiuretic responses to vasopressin in the rat (Brunner, Kuschinsky and Peters, 1956; Sawyer and Valtin, 1965). Recent observations on the binding of these peptides to specific pig kidney membrane receptors indicate that although oxytocin has a lower affinity to these receptors than vasopressin, it competes with lysine-vasopressin for the binding sites (Roy, Barth and Jard, 1975). Vasopressin affects memory processes by facilitating both storage and retrieval of information (de Wied et a/., 1976). Because of the reverse effects of oxytocin when given intraventricularly after training, one may assume that this peptide is a naturally occurring amnesic neuropeptide. This assumption is obviously premature until time-gradient effects, which are typical for amnesic’treatments (McGaugh, Zornetzer, Gold and Landfield, 1972), are demonstrated. Changes in the power spectrum of hippocampal theta activity during PS episodes moreover sup_ port a view that oxytocin attenuates memory processes.
245
It may be more than a coincidence that the activity of the hippocampal theta-generating system correlates with the retention of passive avoidance behaviour. Low activity of this system and impaired passive avoidance behaviour were observed in hereditary diabetes ~~s~~~dus rats. Administration of AVP normalizes theta activity and the behaviour of these animals (de Wied et al., 1975; Urban and de Wied. 1975). In normal rats AVP facilitates passive avoidance behaviour (Ader and de Wied, 1972; Bohus et al., 1972) while AVP antiserum impairs it (van Wimersma Greidanus et al., 1975). The activity of the hippocampal RSA generating system is facilitated by AVP and attenuated by AVP antiserum (Urban and de Wied, 1978). The present experiments demonstrate that oxytocin and oxytocin antiserum affect passive avoidance behaviour and the activity of the theta-generating system in an opposite manner. Landfield, McGaugh and Tusa (1972) have postulated that the theta activity in the post-training period may be a correlate of a brain state which is optimal for information storage. Drugs which facilitate memory storage increase the occurrence of theta activity in the post-training period (Long0 and Loizzo, 1973). Electroconvulsive shock, which is effective in disrupting information storage. decreases the incidence of theta activity (Landfield and McGaugh, 1972). Paradoxical sleep during the post-training period has been regarded as an important requisite of memory storage (Leconte, Hennevin and Block, 1973, 1974). Accordingly, it may well be that facilitation or attenuation of memory processes by vasopressin and oxytocin respectively are due to changes in the activity of the theta generating system. One cannot yet, however, exclude the possibility that hippocampat theta activity simply reflects brain states which are more or less optimal for information storage. The present studies suggest that oxytocin when administered centrally influences brain functions in a way opposite to vasopressin. However, we have failed to replicate the observation of Schulz et at. (1976) using peripheral administration of OXT. Subcutaneously administered OXT, whenever it was effective, appeared to mimic the effect of vasopressin. An increased resistance to extinction rather than a facilitated extinction was observed. Different rates of biotransformation of the oxytocin preparations may partially account for the contradictory observations. Oxytocin without metabolic stabilization is rapidly metabolized in the blood through both N- and C-terminal breakdown while biotransformation in the brain is slower and related to C-terminal breakdown (Marks and Stern, 1974). Rapid biotransformation of OXT into shorter peptides may yield sequences which simulate the effect of vasopressin in particular in high quantities. It has already been shown that fragments of vasopressin like pressinamide or the C-terminal tripeptide Pro-Arg-Gly increase resistance to extinction like AVP when administered peripherally in high quantities (de Wied, 1976). It may, however, be that
B&LA Bostus
246
behavioural variables are responsible for the differences between the present observations and those of Schulz er ul. (1976). The active avoidance paradigm in the pole-jumping situation appeared to be less appropriate to demonstrate an opposite effect of OXT and AVP even after intracerebroventricular administration. Experiments in the passive avoidance situation suggest that the effect of OXT is more readily demonstrable in this one-trial learning paradigm with post-training treatment. Accordingly, OXT clearly affects certain brain functions in an opposite way than are vasopressin but further behavioural studies needed
to resolve
the apparent
contradictions.
ArknowIPdgemenrs---The authors acknowledge with gratitude the excelleni technical assistance of Miss Anja ter Avest, Miss Gerda Croiset and Miss Andrea Siepmann. The peptides were supplied by Organon B. V.. Oss, The Netherlands. Oxytocin antiserum was prepared and kindly supplied by the Netherlands Institute for Brain Research. Amsterdam. The Netherlands.
REFERENCES Ader. P. and Wied. D. de (1972). Effects of lysine vasopressin on passive avoidance learning. Psychonom. Sci. 29: 46-48. Ader. R., Weijnen. J. A. W. M. and Mofeman, P. (1972). Retention of a passive avoidance response as a function of the intensity and duration of electric shock. Ps.vchonom. Sci. 26: 125-I 28. Albe-Fessard, D., Stutinsky, F. and Libouban. S. (1966). Atlas stlreotaxique du diendphale du rat blanc. Editions C.N.R.S., Paris. Bohus, 8.. Ader. R. and Wied, D. de (1972). Effects of vasopressin on active and passive avoidance behavior. Worm. Be&r. 3: 191-197. Bohus. 8.. Wimersma Greidanus, Tj.B. van and Wied. D. de (1975). Behavioral and endocrine responses of rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain). Physiol. Behar. 14: 609.-615. Brunner. H., Kuschinsky, G. and Peters, G. (1956). Der Einfluss von Oxytocin auf die renale Wasser- und Salzausscheidung der Ratte. NaunJ7t_Schr,iir~debirhergs Arch. at-‘. Path. Phi~ri~~u~.22s: 457473. Celestian. J. F., Carey, R. J. and Miller. M. (1975). Unimpaired maintenance of a conditioned avoidance response in the rat with diabetes insipidus. Physiol. Behat!. 15: 707-711. Faure, J. (1957). De l’influence de l’oxytocine sur le noyau amy~dalien (portion cortico-midiane) et sur la come d’Ammon. J..‘Physiol., Paris 49: 154-i 57. Lande, S., Flexner, J. 8. and Flexner. L. B. (1972). EtTect of corticotropin and desglycinamide’-lysine vasopressin on suppression of memory by puromycine. Proc. natn. Acad. Sci. U.S.A. 69: 558-560. Landfield. P. W. and McGaugh, J. L. (1972). Effects of eiectroconvulsive shock and brain stimulation on EEG cortical theta rhythms in rats. Behau. Biol. 7: 271-278. Landfield. P. W.. McGaugh, J. L. and Tusa, R. J. (1972). Theta rhythm: A temporal correlate of memory storage processes in the rat. Science 175: 87-89. Leconte. P.. Hennevin, E. and Bloch. V. (1973).Analyse des effects d’un apprcntissage et de son niveau d’acquisition sur le sommeil paradoxal consecutif. Brain Res. 4% 367-3 79. Leconte, P.. Hennevin. E. and Bloch. V. (1974). Duration
et ul of paradoxical
ditioned
sleep necessary
avoidance
in the
for the acquisition of conrat. Physiol. Behat. 13:
675-681. Lissak, K. and
Bohus. B. (1972). Pituitary hormones and avoidance behavior of the rat. Ini. J. Ps~cbobjo~. t: 103-115. Longo, V. G. and Lo&o, A. (1973). Effects of drugs on the hippocampal O-rhythm. Possible relationships to learning and memory processes. In: Pharmacology and Future of Man--Proceedings q/’ rite 5th International Congress of‘ Pharmacology (Bloom, F. E. and Acheson. G. H., Eds), Vol. 4.. Brain. N~wea and S~nnpse.s. pp. 46-54. Karger, Base]. Marks, N. and Stern. F. (1974). Novel enzymes involved in the inactivation of hypothalamo -hypophyseal hormones. In Psychoneuroendocrirl~~/~~g~, (Hatotani, N.. Ed.). pp. 276284. Karger, Base]. McGaugh, J. L., Zornetzer, S. F.. Gold. P. E. and Landfield. P. W. (1972). Modi~cation of memory systems: some neurobioIogi~a1 aspects. Q. Rec. Biophys. 5: 1633186. Miller. M., Barranda. E. G., Dean, M. C. and Brush. F. R. (1976). Does the rat with herednary hypothalamic diabetes insipidus have impaired avoidance learning and/or performance. Pharmac. Biochem. Behar. 5: Suppl. 1. 35-40. Pickford, M. (1961). Some extra-uterme actions of oxytotin. In: Oxytocin {Caldeyro-Barcia. R. and He&r. H. Eds). pp. 68-79. Pergamon Press, Oxford. Ree. J. M. van. and Wied. D. de (1977). Modulation of heroin self-administration by neurohypophyseal principles. Eur. J. Pharmac. 43: 199-202. Rigter, H., Riezen. H. van and Wied, D. de (1974). The effects of ACTH- and vasopressin-analogues on CO*induced retrograde amnesia in rats. Pizwiol. &bar. 13: 381-388. Roy, C.. Barth, T. and Jard. S. ft 975). Vasopressin-sensttive kidney adenylate cyctase. Structural requirements for attachment to the receptor and enzyme activation: studies with oxytocin analogues. J. biol. Chet71. 250: 3157-3168. Sawyer. W. H. and Valtin, H. (1965). Inhibition of vasopressin antidiuresis by extracts of pituitaries from rats with hereditary hypothalamic diabetes insipidus and by oxytocin. Endocrinology 76: 999-1001. Schaker. W., Klingberg, F., Sterba, G. and Pickenheim. L. (1966). Der Einfluss von Oxytocm auf zentralnervose Funktionen bei der Ratte im chronische Experiment. Pfiigers Arch. ges. Phrsioi. 288: 322-331. Schulz, H. (1970). Der Einfluss von Vasopressin und Oxytocin auf die N~rvent~tigkeit von Peripfoneta Am~ric~lnu L. .4rchs int. Phurmtrcodw. Thk. 186: 108--f 19. Schulz, H., Kovacs, G. L. and Telegdy. G. (1976). The effect of vasopressin and oxytocin on avoidance behaviour in rats. In: Cellular and Molecular Buses of Neuroendocrine Processes (Endroczi. E. Ed.), pp. 555 564. Akademiai Kiado, Budapest. Schwarzberg, H. and Unger. f-1. (1970). Anderun~ der Reaktianszeit von Ratten nach Apptikation von Vasopressin, Oxytozin und Na-thioglykolat. Actor hiol. meci. germ. 24: 507-516. Schwarzberg, H., Unger, H. and Schulz, H. (1973). Die wirkung van Oxytozin auf das Na-Glutamat verlnderte EEG des Kaninchens. Acta bioi. med. germ. 30: 203-208. Urban, I. and Wied, D. de (1975). Inferior quality of RSA durine oaradoxical sleeu in rats with hereditary diabetes insipi&. Brain Rrs. 9?: 362-366. Urban, I. and Wied, D. de (1978). Neuropeptides: Effects on paradoxical sleep and theta rhythm in rats. P/tarmac. Biochem. Behav. In press. Valtin. H. and Schroeder. H. A. (1964). Familial hypothalamic diabetes insipidas in rats (8rattleboro strain). ,4m. J. Phrsiof. 206: 425 430.
Oxytocin,
vasopressin
Walter, R., Hoffman, P. L., Flexner, J. 9. and Flexner, L. 9. (1975). Neurohypophyseal hormones, analoga and fragments: Their effect on puromycin-induced amnesia. Proc. mtn. Acad. Sci. 72: 418&4184. Wied. D. de (1965). The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response in rats. Nature, Lond. 232: 5860. Wied. D. de (1966) Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior. Proc. Sot. exp. Biol. 122 28-32. Wied, D. de (1971). Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature, Land. 232: 58-60. Wied, D. de (1976). Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments. L$e Sci. 1% 685690. Wied. D. de and Gispen. W. H. (1977). Behavioral effects
and avoidance
behaviour
247
of peptides. In: Peprides in Neurobiology. (Gainer. H. Ed.). pp. 397448. Plenum Press, New York. Wied, D. de. Bohus. 9. and Wimersma Greidanus, Tj. 9. van (1975). Memory deficit in rats with hereditary diabetes insipidus. Bruin Rex 85: 152-156. Wied. D. de. Greven. H. M., Lande, S. and Witter, A. (1972). Dissociation of the behavioural and endocrine effects of lysine vasopressin by tryptic digestion, Br. J. Pharmac. 45: I 18-l 22. Wied. D. de. Wimersma Greidanus, Tj. 9. van, Bohus, 9.. Urban, I. and Gispen. W. H. (1976). Vasopressin and memory consolidation. In: Perspectioes in Brain Reseurch (Corner. M. A. and Swaab, D. F. Eds). Proyress in Brain Research. Vol. 45, pp. 181-191. Elsevier. Amsterdam. Wimersma Greidanus. Tj. 9. van. Dogterom, J. and Wied, D.de(1975). lntraventricular administration ofantivasopressin serum inhibits memory in rats. Life Sci. 16: 637-644.
B~XA BOHUS.* 1. URBAN, TJ. B. VAN Rudolf
Magnus
Institute
for Pharmacology, Medical Faculty, Utrecht. The Netherlands (Accepted
16 May
University
of Utrecht,
Vondellaln
6,
19773
Summary-- Intracerebroventricular administration of oxytocin and arginine-Svasopressin (AVP) immediately after a single learning trial resulted in opposite effects on retention of passive avoidance behaviour. Passive avoidance behaviour was attenuated by oxytocin and facilitated by AVP in a dosedependent manner. Reverse effects were found after neutralizing the centrally circulating oxytocin and vasopressin by intraventricular injection of specific antiserum to these peptides. Administration of oxytotin into a lateral ventricle resulted in a decrease of the peak frequency of hippocampal theta rhythm generated during paradoxical sleep episodes. Conversely, oxytocin antiserum increased the incidence of higher frequencies in the theta rhythm. Peripheral administration of oxytocm mimicked the effect of AVP on extinction of pole-jumping avoidance behaviour. Resistance to extinction rather than facilitation of extinction was observed when higher doses of oxytocin were given. Passive avoidance behaviour appeared to be either facilitated or attenuated after subcutaneous oxytocin administration. It is concluded that centrally released oxytocin may be a naturally occurring amnesic neuropeptide.
Vasopressin or antidiuretic hormone (ADH) of hypothalamo-neurohypophyseal origin is involved in the formation and maintenance of adaptive behaviour. Removal of the neural lobe of the pituitary gland interferes with the maintenance of a shuttle-box avoidance response (de Wied, 1965). That rats with hereditary hypothalamic diabetes insipidus, which lack the ability to synthetize vasopressin (Valtin and Schroeder, 1964), show a deficit in acquiring a shuttiebox avoidance response (Celestian, Carey and Miller, 1975; Miller, Barranda, Dean and Brush, 1976) and an impaired maintenance of active and passive avoidance behaviour (Bohus, van Wimersma Greidanus and de Wied, 1975: de Wied, Bohus and van Wimersma Greidanus, 1975). corroborates the view that vasopressin is physiologically involved in learning and memory processes. In intact rats, vasopressin and analogues of this polypeptide. which are practically devoid of pressor or anti-diuretic activities, increase resistance to extinction of active (de Wied. 1971; de Wied, Greven, Lande and Witter, 1972; Schulz, Kovacs and Telegdy, 1976) and passive avoidance responses (Ader and de Wied, 1972; Bohus, Ader and de Wied, 1972). Furthermore, these peptides protect against puromycill- (Lande, Flexner and Flexner, 1972; Watter, Hoffman, Flexner and Flexner, t97.5) or CO,-induced amnesia in mice and rats (Rigter, van Riezen and de Wied, 1974). On the other hand, neutralizing the centrally circulating arginine-vasopressin
* To whom correspondence should be addressed. Key words: oxytocin. avoidance behaviour, rhythm,
theta
arginine-8-vasopressin. 239
(AVP) by specific antiserum administered intracerebroventricularly impairs passive avoidance and results in a facilitated extinction of a pole-jumping active avoidance response in the rat (van Wimersma Greidanus, Dogterom and de Wied, 1975). Oxytocin (OXT), the other neurosecretory product of the hypothalamo-neurohypophyseal system fails to affect avoidance behaviour and to prevent amnesia when administered in doses equimolar to the minimally effective amount of AVP (de Wied, 1971: Lissak and Bohus, 1972; Walter et ni., 1975). Higher doses of OXT however mimic the effect of vasopressin on the maintenance of conditioned avoidance behaviour (de Wied and Gispen, 1977). In contrast to these observations, Schulz er ul. (1976) have recently reported that small doses of OXT influence extinction behaviour opposite to that of vasopressin. Facilitation of active avoidance extinction in non-deprived but particularly in water-deprived rats was observed. In order to resolve the apparent contradictions the present experiments were aimed to investigate in more detail the central effects of oxytocin by comparing the influence of this peptide and that of its specific antiserum on active and passive avoidance behaviour, with the effects of AVP and its antiserum after peripheral ,and intracerebroventricular administration. in addition, the effects of intracerebroventricularly administered oxytocin and OXT antiserum on hippocampal theta activity generated during paradoxical sleep (PS) episodes were investigated. It has been suggested that some of the behavioural effects of vasopressin and related peptides may be due to an increase in the excitability of midbrain limbic struc-
240
BELA BOHUS ef al.
tures. Lower peak frequencies in hippocampal theta activity during PS episodes were found in rats with hereditary hypothalamic diabetes insipidus as compared to their heterozygous littermates (Urban and de Wied, 1975). In the intact rat, administration of desgly~in~ide lysine-vasopressin, which is a behaviourally active analogue of vasopressin, increases the proportion of higher frequency components in the hippocampal theta activity. ~ntraventricularly administered AVP antiserum. on the other hand, induces changes in the theta activity which resemble those of the diabetes insipidus rats (Urban and de Wied, 1978). METHODS Subjects
Male wistar rats of an inbred strain (Cpb, TNG, Zeist. The Netheriands) were used. They were kept under controlled light-dark conditions (light on between 5.00 and 19.00 hr) and had free access to food and water. The subjects were housed 5-6 per cage but rats implanted with a permanent cannula for intraventricular injection or those bearing electrodes for recording hippocampal electrical activity were kept in jndividual cages. Behaviourul procedures Active avoidance behaviour was studied in a polejumping situation (de Wied, 1966). Briefly, the rats were conditioned to avoid the unconditioned stimulus {US) of a scrambled electric floor shock (0.20 mA. a.c.) by jumping onto a pole located in the centre of the apparatus. The conditioned stimulus (CS) was a light signal above the pole. The US was applied if an avoidance response had not occurred within Ssec after the onset of CS. Ten acquisition trials were given daily with variable intertrial intervals averaging 60 sec. Acquisition training was followed by extinction sessions. Ten trials were presented daily in which the CS was terminated if the rat had jumped onto the pole within S set or after 5 sec. when avoidance did not occur. The US was never applied during the extinction sessions. Passive avoidance behaviour was studied by using a one-trial learning paradigm in a step-through type situation as described by Ader, Weijnen and Moleman (1972). Briefly, the rats were adapted to the apparatus consisting of a large dark compartment equipped with a grid floor and mesh-covered extensively Iit elevated runway, attached to the front centre of the dark chamber. Adaptation training was followed by a trial in which the rat was placed on the runway facing away from the dark compartment and allowed to enter the dark. Three more trials were given on the next day with an intertrial interval of 5 min. The last of these trials was followed by a single 2 set unavoidable scrambled footshock (0.25 mA, a.c.) immediately after entering the dark compartment. The retention of the passive avoidance response was
tested 24 and 48 hr after the single learning trial. The subject was placed on the elevated runway and the latency to reenter the dark compartment was recorded up to a maximum of 300 sec. Drugs and routes of admi~iscrafion Highly purified synthetic argjnine-8-vasopressin (AVP; pressor activity 273 Ujmg) and oxytocin (OXT: uterotropic activity 512 Ujmg) were used. Peptide solutions were freshly prepared before administrations by dissolving them in 0.Q saline (0.1 ml N/100 HCI was added to increase the stability of the peptide). The concentration of the peptides in the solutions was adjusted to a final volume of 0.5 ml for subcutaneous and 1~1 for intracerebroventricular administration. Physiologi~ai saline containing 0.1 ml NjlOOHCl was used as control. Arginine-vasopressin and oxytocin antisera were prepared by immunizing male rabbits as described by van Wimersma Greidanus et al. (1975). Undiluted AVP antiserum in the quantity of 1~1 binds approximately 2.5 ng of AVP while the binding capacity of the OXT antiserum was approximately l-l.5 ng of OXT. Normal rabbit serum was used in the control rats. For intracerebroventricular administration of the peptides and antisera a polyethylene cannula was implanted in a lateral ventricle under anesthesia as described earlier (de Wied, 1976). A 10~1 microsyringe (Unimetrics, Co.) with adjusted length of needle fitting in the lumen and exactly reaching the tip of the preimplanted cannula was used to administer the substances in the ventricle. The unanaesthetized rats were kept in hand while administering the test material. The localization of the tip of the cannula was checked at the termination of the experiments by injecting Evans blue. The staining was then inspected macrostopically in formaldehyde fixed brain sections. Only those injections were regarded as successful in which the walls of both lateral, of the third and fourth ventricles were stained blue. Experimentai designs The three main bodies of the behavioural experiments were as follows: (i) peripheral administration of OXT and AVP; (ii) intraventricular administratjon of OXT and AVP; (iii) intraventricular administration of OXT and AVP antisera. A total of 82 rats were used to study the effects of peripheral administration of OXT and AVP on active and passive avoidance behaviour. Forty-two animals, weighing l30-I4Og at the onset of the experiments were trained to acquire the pole-jumping conditioned avoidance response. Five acquisition sessions were followed by 5 extinction days. In the first series of the experiments 24 rats were trained and then divided randomly into 4 equal groups. Oxytocin in doses of 0.2, 0.02 and 0.002 pg per rat was administered subcutaneously 1 hr prior to the first 3 extinction sessions. Saline-treated subjects served as con-
241
Oxytocin. vasopressin and avoidance behaviour trols. Eighteen rats, randomly divided into 3 equal groups, were used in the second study. The subjects received either AVP or OXT in a dose of 0.02 c(g per rat subcutaneously 1 hr prior to each acquisition and extinction session. Control rats received saline daily. Forty rats, weighing 160-l?Og, were used in the passive avoidance paradigm. Randomly divided equal groups of rats received a single subcutaneous injection of OXT (0.1 or 0.5 pg/rat) or AVP (0.5 pg/rat) immediately after the learning trial. Retention of the passive avoidance response was studied 24 and 48 hr later. The effects of intracerebroventricular administration of OXT and AVP on active and passive avoidance behaviour were studied in 136 rats. Thirty-six rats, weighing 14@150g, were implanted with a permanent can&a in a lateral ventricle. Five days later they were trained to acquire a pole-jumping avoidance response. Three acquisition sessions were foilowed by 2 extinction days of 10 trials each. Randomly selected groups received either 1.0 ng of OXT or AVP or physiological saline. These treatments were given immediately after each acquisition session. Injection of Evans blue through the permanent cannula at the termination of the experiments indicated successful intraventricular treatment of 32 rats. A hundred rats, weighing 16&18Og, were also implanted with a permanent intraventricular cannula. Five days later, they were trained in the step-through passive avoidance situation. Oxytocin or AVP in doses of 0.05, 0.1, 1.0 and lO.Ong or physiological saline were given intraventricularly immediately after the single learning trial. Retention of the passive avoidance response was studied 24 and 48 hr thereafter. Localization of the cannula at the termination of the experiments indicated successful injection in 90 rats. A total of 90 male rats was used to investigate the influence of OXT and AVP antisera on the avoidance behaviour. Fifty rats, weighing 14@-150 g, were implanted with a permanent cannula for intraventricular antiserum administration. Five days later the rats were trained to acquire a pole-jumping avoidance response as described earlier. Randomly selected groups received either 2~1 of OXT antiserum or 1 fd of AVP antiserum 30 min before each acquisition session. Normal rabbit serum (2 ~1) were given to the controls. Observations on 35 rats were then analysed after checking the cannula placement. Forty rats, weighing 160-180 g, were also implanted with a permanent cannula in a lateral ventricle. Five days later they were trained in the passive avoidance situation. Oxytocin, or AVP antiserum or normal rabbit serum (1~1) was administered intraventricularly to randomly selected rats immediately after the single learning trial. Retention of the passive avoidance response was tested 24 and 48 hr later. Successful intraventricular injections were found in 31 rats.
Five male Wistar rats. weighing 2OO-220g at the time of the operation, were used for the electrophysiological experiments. Animals equipped with a permanent intracerebroventricular cannula were anaesthetized (Nembutal~) and a pair of electrodes (100~ in diameter, insulated except for the tip) was stereotaxitally implanted in the CA, field of the dorsal hippocampus (~o”ordinates: A = 0.4 mm, L = 2.5 mm and 2.5 mm from the cortex according to Albe-Fessard, Stutinsky and Libouban, 1966). Another pair of electrodes was attached to the neck muscles to record the electromyogram. Two weeks after the surgery, the rats were placed on 12 hr light-dark schedule in sound-attenuated cages with food and water ad libiturn. Hippocampal and myographic electrical activities, recorded throughout 8 hr sessions (between 9.00 and 17.00 hr of the light cycle) via impedance transformers, were stored on magnetic tape and polygraphic paper for later analysis. The first five recording sessions served for adaptation of the rats to recording conditions. The amount of PS and the frequency pattern of the hippocampal theta rhythm during PS were determined. In the subsequent sessions OXT (5 and 20 ng dissolved in 1 ~1 of physiological saline), OXT antiserum (1~1, undiluted) or placebos (saline or normal rabbit serum) were randomly injected intraventricularly. The minimal interval between 2 consecutive OXT or OXT antiserum injections was 48 hr. Each treatment was replicated 3 times. The effect of OXT was investigated in 3 rats whereas other three animals out of the 5 subjects were selected to study the influence of OXT antiserum. The power spectral analysis was used to quantify hippocampal electrical activity. Hippocampal records during PS of the first 180min following intraventricular injections were divided into lo-set analysis epochs. filtered (high-frequency cut-off at 30 c/s), digitalized (100 c/s sampling rate) and a power spectrum for each epoch was computed and stored. Subsequently, an average spectrum per 100 set was computed and its resolution of 0.1 c/s was reduced to 0.5 c/s. The resulting spectrum was converted into 100,
E x+,nr+,an,
days
Fig. 1. Effects of various doses of oxytocin (OXT) administered subcutaneously I hr prior to the first 3 extinction sessions on the maintenance of a conditioned pole-jumping avoidance response.
percentages of power per OSc/s component. The effect of OXT and OXT antiserum on hippocampal activity was estimated by comparing the means and standard errors of percentages of power per 0.5 c/s components of experimental sessions with corresponding means of placebo sessions. The total amount of PS per session was determined from the polygraphic records.
Two-tailed r-test, analysis of variance, the non-parametric statistics of Mann-Whitney (U-test) and the x*-test were used to analyse parametric. non-parametric and frequency distribution data.
‘, I
2
3
4
Acquisition
5
I
2
3
4
SAL
5
Extinctson Days
RESULTS .FJkts
oj’
~~ri~~z~ral ad~inistrat~otz of oxytocin and
arllirlitle-r’asopressin 011 active and passive woidance hehaciour As shown in Figure 1, administration of OXT prior to the first 3 extinction sessions resulted in a delay of extinction of the conditioned avoidance response. Cessation of the treatment was followed by a decline of avoidance performance particularly in rats which had been treated with 0.2 pg of OXT, but the peptidetreated rats displayed more avoidance responses than the controls. One-way analysis of variance of the total number of avoidance responses scored during extinction training indicated a significant effect of OXT (P < 0.01; FJ,20 = 5.3916). Administration of 0.02 pg of OXT or AVP throughout the acquisition and extinction sessions failed to influence the acquisition of the pole-jumping avoidance response (Fig. 2). Extinction of the response was however markedly delayed in rats treated with AVP. The total number of avoidances during extinction was significantly higher (P < 0.001) in rats treated with AVP (39.8 & 1.4) than in those which received OXT (24.8 -f 2.1) or saline (22.3 rfr 2.7). Oxytocin treatment had no effect on the rate of extinction. Retention of passive avoidance behaviour as defined by the latency to re-enter the dark compartment was enhanced by post-learning administration of AVP (Table I). Although the median avoidance latency of rats which received OXT was nearly the same as in Table
I. Effects of oxytoein
Treatment Oxytocin Arginine-vasopressin Saline
the controls, the individual latencies appeared to show a bimodal distribution. The largest percentage of rats treated with OXT displayed longer or shorter latencies than the controls. Analysis of the distribution pattern with the aid of x2-test indicated significant differences between the control group and that which received 0.1 pg of OXT immediately after the learning trial (P < 0.001, x2 = 14.286). Administration of 0.5 rug of AVP resulted in an increase of avoidance latency and most of the rats displayed longer latencies than the controls. The distribution of latenties appeared to be significantly different (P < 0.01; x2 = 9.899) when the AVP-treated group was compared to the controls. i@cts gf intracerebrouentricular~~*administered oxytotin and arg~n~~e ~~lso~ressin on active and passice avoidance behaciour Results on acquisition and extinction of the conditioned pole-jumping avoidance response in rats treated intraventricularly with OXT (n = 12). AVP (tt = 8) or saline (n = 12) imm~iately after each acquisition session are depicted in Fig. 3. Administration of OXT slightly affected acquisition and extinction behaviour of the rats. Oxytocin-treated rats
and arginine-vasopressin administered subcutaneously immediately on the retention of the passive avoidance response in the rat
Dose (/kg) 0.1 0.5 0.5
Distribution of avoidance latencies” 41-120 set > 120sec < 40 set 50 30 0 0
“Expressed as percentage of rats displaying ‘P < 0.01 (peptide vs saline: Mann--Whitney
LP < 0.05. ( ) Number of rats.
Fig. 2. Comparative effects of arginine vasopressin (AYP) and oxytocin (OXT) administered subcutaneously during both acquisition and extinction on conditioned pole-jumping avoidance behaviour.
0 30 10 80 the given test).
50 40 90 20 iatencies
during
Median
avoidance (see)
after the learning
latency
24 hr
48 hr
117.0 54.5 222.0” 71.0
74.0 39.0 186.5 30.0
the retention
trial
test at 24 hr
(10) (10) (10) (10)
Oxytocin.
vasopressin
and avoidance
243
behaviour
ANTI-OXT
Se
AVP
NORMAL
Se
OXT \,
SAL
ANTI-AVP
Se
Fig. 4. Effects of intraventricular administration of oxytotin (OXT) or arginine-vasopressin antiserum 30 min prior to each acquisition session conditioned pole-jumping avoidance behaviour of the rat. Fig. 3. Pole-jumping avoidance behaviour of rats after intraventricular administration of oxytocin (OXT) or arginine vasopressin (AVP) immediately after each acquisition session.
scored fewer avoidance responses than the controls during the third acquisition and the first extinction session but the difference appeared to be significant only on the third acquisition session (P < 0.02; t = 2.755). Treatment with AVP did not affect acquisition behaviour but despite the cessation of treatment these rats were more resistant to extinction than the controls. A significant difference between AVP-treated and control rats was found during the second extinction session (P < 0.02; t = 2.579). Intraventricular administration of OXT and AVP immediately after the single learning trial profoundly influenced the retention of the passive avoidance response (Table 2). Significantly shorter avoidance latencies were observed in rats which received OXT in doses of 0.1 or 1.0 ng as compared to controls at the 24 hr retention test. Administration of 0.05 ng of
Table 2. Etfects of intraventricular administration of oxytocin (OXT) and arginine-vasopressin (AVP) immediately after the learning trial on the retention of a one trial passive avoidance response Dose
Median
avoidance
Treatment
(ng)
24 hr
48 hr
OXT
0.05 0.1 1.0 10.0 0.05 0.1 1.0 10.0
65.0 22.0h 29.0 39.0 72.C 88.0 110.5’ 135.5 64.0
45.0 7.0 19.0 42.0 32.0 26.0 32.0 95.5” 24.5
Saline
* P < 0.05 (peptide vs saline; b P < 0.02. E P < 0.01. ( ) Number of rats.
Effects of inrracerebroventricular administration ofoxytocin and arginine vasopressin antiserum on active and passive avoidance behaviour Intraventricular administration of both OXT and AVP antiserum prior to each acquisition session resulted in a facilitation on avoidance acquisition in the pole-jumping test (Fig. 4). The total number of conditioned avoidance responses scored by the rats receiving either OXT (20.5 Ifr 1.0; n = 11) or AVP antiserum (19.4 + 1.2; n = IO) appeared to be significantly higher (P < 0.01 and P < 0.05) than that
latency
(set)
AVP
OXT failed to influence passive avoidance behaviour. Although the median of the latencies of rats which received 10.0 ng of OXT was not different from that of the controls, the distribution pattern appeared to be significantly different (P < 0.01, x2 = 8.892). The majority of the latencies was shorter (60%) respectively longer (30:;) than the controls. No significant differences were observed in retention behaviour 48 hr after the learning trial. In contrast to OXT, intraventricular administration of AVP in doses of I.0 and 10.0 ng led to a significant increase in passive avoidance latencies. The effect of 10.0 ng of AVP appeared to be long-lasting. Passive avoidance latencies were still augmented at the 48 hr retention test.
Mann-Whitney
(9) (8) (13) (10) (9) (7) (12) (10) (12)
Table 3. Effects of intraventricular administration of oxytotin- or arginine-vasopressin antiserum immediately after learning trial on the retention of a one trial passive avoidance response
Treatment Oxytocin antiserum Arginine-vasopressin antiserum Normal rabbit serum
Median avoidance 24 hr
latency (set) 48 hr
I 50.0h
58.0
(11)
25.5
17.0
(10)
48.5
36.5
(11)
test). a P < 0.05 (Mann-Whitney b P < 0.01. ( ) Number of rats.
test).
BELA BOHUS et al.
244
~ntraventricul~ administration of OXT antiserum immediately after the single learning trial was followed by longer avoidance latencies at the 24 hr retention test than that found in controls (Table 3). Administration of AVP antiserum resulted in significantly shorter latencies. No differences were observed between treated and control rats at the 48 hr retention test. ESJcts of oxytocin and oxyrociu antiserum campal theta actiuit>
cm hippo-
As compared to saline treatment, intraventricular administration of 5 or 20ng of OXT was followed by a decrease of the peak frequency from 7.5 to
4
5
6
.
.
7 Hz B
I)
l
.
9
II
IO
.
Fig. 5. Changes in power spectrum of hippocampai theta activity during PS episodes O-90 min after intraventricular administration of 5 ng (upper graph) or 20 ng (lower graph) of oxytocin (OXT). Significant differences treated vs control sessions are indicated by asterisks (P -=z0.01). scored by the controls (15.0 & 1.4). Extinction behaviour was, however, affected in an opposite manner by the administration of the two antisera. Rats which received OXT antiserum during acquisition showed resistance to extinction and the total number of avoidances (14.7 & 1.3) was significantly higher (P < 0.05) than that of the controls (10.6 + 1.4). In contrast, a more rapid extinction took place in rats which had received AVP antiserum and the total number of avoidances (4.1 + 0.9) was significantly less (P < 0.001) than that of the controls.
NORMAL
Se
*
*
7.Oc/s, a decrease of the percentage of the higher frequency components whereas the proportion of the low theta frequencies in the hippocampal activity was slightly but significantly increased (Fig. 5). Conversely, intraventricular administration of OXT antiserum resulted in an increased proportion of the higher theta frequencies (7.5-10Hz) and a decreased amount of the low-frequency components (Fig. 6). The total amount of PS during the 8-hr observation periods was not affected by the administration of OXT or OXT antiserum. DISCUSSION
The present experiments suggest that besides the well-documented effects of vasopressin of hypothalamic neurosecretory origin on behaviour and electrical activity of the brain (de Wied. van Wimersma Greidanus, Bohus, Urban and Gispen, 1976), oxytocin, which is also produced by the hypothalamic neurosecretory cells, influences brain functions in a way opposite to that of vasopressin. Opposite effects on avoidance behaviour and hippocampal theta activity during paradoxical sleep episodes were particularly observed after intracerebroventricular administration of oxytocin. Attenuation of passive avoidance behaviour followed post-learning administration of oxyto-
ANTI-OXT
*
*
**
Se
*
*
Fig. 6. Effect of intraventricular administration of oxytocin antiserum on hippocampal theta activity during PS episodes. Significant differences treated vs control sessions are indicated by asterisks (P < 0.01).
Oxytocin. vasopressin and avoidance behaviour tin and the peptide appeared to cause a decrease in dominant frequencies of hippocampaf theta activity when administered intraventricularly. Vasopressin facilities passive avoidance behaviour and increases the incidence of higher frequency components of hippocampal theta activity. That oxytocin may play a physiological role in the regulation of certain brain functions is suggested by observations with specific antibodies against this peptide. Neutralizing the centrally circulating oxytocin by specific antiserum is followed by behavioural changes and alterations in hippocampal RSA which are opposite to that of the effect of oxytocin. Evidence has already been furnished on opposite effects of vasopressin and oxytocin on nerve functions. Vasopressin facilitates while oxytocin suppresses spontaneous nerve activity of Periplaneta americana (Schulz, 1970). Oxytocin shortens the reaction time to an acoustic stimulus in the rat while lysine-vasopressin (LVP) has an opposite effect in this respect (Schwarzberg and Unger, 1970). Furthermore, Schulz et al., 1976, reported that, contrary to LVP, oxytocin facilitates extinction of a conditioned active avoidance response. More recently, van Ree and de Wied (1977) found that oxytocin and oxytocin fragments facilitate while and vasopressin fragments vasopressin attenuate heroin self-administration behaviour of the rats. Oxytocin administration was also found to cause changes in the electrical activity of the brain (Faure, 1957; Schgker, Klingberg, Sterba and Pickenheim, 1966; Schwarzberg, Unger and Schulz, 1973). It is not yet clear, however, whether this peptide possesses inherent neurotropic activity which is opposite to that of vasopressin or whether it acts as an antagonist of vasopressin by occupying putative receptor sites and therefore preventing vasopressin effects. An opposite biological effect of oxytocin and vasopressin is not an unique phenomenon. Small doses of oxytocin induce diuresis (Pickford, 1961) and inhibit the antidiuretic responses to vasopressin in the rat (Brunner, Kuschinsky and Peters, 1956; Sawyer and Valtin, 1965). Recent observations on the binding of these peptides to specific pig kidney membrane receptors indicate that although oxytocin has a lower affinity to these receptors than vasopressin, it competes with lysine-vasopressin for the binding sites (Roy, Barth and Jard, 1975). Vasopressin affects memory processes by facilitating both storage and retrieval of information (de Wied et a/., 1976). Because of the reverse effects of oxytocin when given intraventricularly after training, one may assume that this peptide is a naturally occurring amnesic neuropeptide. This assumption is obviously premature until time-gradient effects, which are typical for amnesic’treatments (McGaugh, Zornetzer, Gold and Landfield, 1972), are demonstrated. Changes in the power spectrum of hippocampal theta activity during PS episodes moreover sup_ port a view that oxytocin attenuates memory processes.
245
It may be more than a coincidence that the activity of the hippocampal theta-generating system correlates with the retention of passive avoidance behaviour. Low activity of this system and impaired passive avoidance behaviour were observed in hereditary diabetes ~~s~~~dus rats. Administration of AVP normalizes theta activity and the behaviour of these animals (de Wied et al., 1975; Urban and de Wied. 1975). In normal rats AVP facilitates passive avoidance behaviour (Ader and de Wied, 1972; Bohus et al., 1972) while AVP antiserum impairs it (van Wimersma Greidanus et al., 1975). The activity of the hippocampal RSA generating system is facilitated by AVP and attenuated by AVP antiserum (Urban and de Wied, 1978). The present experiments demonstrate that oxytocin and oxytocin antiserum affect passive avoidance behaviour and the activity of the theta-generating system in an opposite manner. Landfield, McGaugh and Tusa (1972) have postulated that the theta activity in the post-training period may be a correlate of a brain state which is optimal for information storage. Drugs which facilitate memory storage increase the occurrence of theta activity in the post-training period (Long0 and Loizzo, 1973). Electroconvulsive shock, which is effective in disrupting information storage. decreases the incidence of theta activity (Landfield and McGaugh, 1972). Paradoxical sleep during the post-training period has been regarded as an important requisite of memory storage (Leconte, Hennevin and Block, 1973, 1974). Accordingly, it may well be that facilitation or attenuation of memory processes by vasopressin and oxytocin respectively are due to changes in the activity of the theta generating system. One cannot yet, however, exclude the possibility that hippocampat theta activity simply reflects brain states which are more or less optimal for information storage. The present studies suggest that oxytocin when administered centrally influences brain functions in a way opposite to vasopressin. However, we have failed to replicate the observation of Schulz et at. (1976) using peripheral administration of OXT. Subcutaneously administered OXT, whenever it was effective, appeared to mimic the effect of vasopressin. An increased resistance to extinction rather than a facilitated extinction was observed. Different rates of biotransformation of the oxytocin preparations may partially account for the contradictory observations. Oxytocin without metabolic stabilization is rapidly metabolized in the blood through both N- and C-terminal breakdown while biotransformation in the brain is slower and related to C-terminal breakdown (Marks and Stern, 1974). Rapid biotransformation of OXT into shorter peptides may yield sequences which simulate the effect of vasopressin in particular in high quantities. It has already been shown that fragments of vasopressin like pressinamide or the C-terminal tripeptide Pro-Arg-Gly increase resistance to extinction like AVP when administered peripherally in high quantities (de Wied, 1976). It may, however, be that
B&LA Bostus
246
behavioural variables are responsible for the differences between the present observations and those of Schulz er ul. (1976). The active avoidance paradigm in the pole-jumping situation appeared to be less appropriate to demonstrate an opposite effect of OXT and AVP even after intracerebroventricular administration. Experiments in the passive avoidance situation suggest that the effect of OXT is more readily demonstrable in this one-trial learning paradigm with post-training treatment. Accordingly, OXT clearly affects certain brain functions in an opposite way than are vasopressin but further behavioural studies needed
to resolve
the apparent
contradictions.
ArknowIPdgemenrs---The authors acknowledge with gratitude the excelleni technical assistance of Miss Anja ter Avest, Miss Gerda Croiset and Miss Andrea Siepmann. The peptides were supplied by Organon B. V.. Oss, The Netherlands. Oxytocin antiserum was prepared and kindly supplied by the Netherlands Institute for Brain Research. Amsterdam. The Netherlands.
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