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Elective tolerance to behavioral effects of neurotensin
Physiology&Behavior,Vol. 31, pp. ,167--470.PergamonPress Ltd., 1983. Printed in the U.S.A.
Elective Tolerance to Behavioral Effects of Neurotensin G. J. E. RINKEL, E. C. H O E K E A N D TJ. B. VAN W I M E R S M A G R E I D A N U S 1
Rudolf Magnus Institute for Pharmacology, Medical Faculty, State University of Utrecht Vondellaan 6, 3521 GD Utrecht, The Netherlands R e c e i v e d 10 J a n u a r y 1983 RINKEL, G. J. E., E. C. HOEKE AND TJ. B. VAN WIMERSMA GREIDANUS. Electivetolerance to behavioral effects ofneurotensin. PHYSIOL BEHAV 31(4) 467-470, 1983.--Neurotensin reduces locomotor activity in an open field situation as well as responsiveness to electric footshock. Repeated injections of neurotensin in combination with repeated testing of locomotor activity in the open field as well as repeated injections of neurotensin without such testing in the open field do not result in a reduction of locomotor activity. These findings make development of tolerance to this behavioral effect of neurotensin likely. Such a development of tolerance to neurotensin is not observed for the effect on responsiveness to electric footshock. Neurotensin
Tolerance
Open field behavior
Locomotor activity
N E U R O T E N S I N , a brain and gastro-intestinal tridecapeptide, exerts various behavioral effects [5, 6, 10], such as a reduction of locomotor activity [6,7] and a decreased responsiveness to aversive stimuli [2,3]. These effects are almost exclusively found after central administration, probably due to the poor permeability of the blood-brain barrier to penetration by this peptide [7]. In a previous series of preliminary experiments we studied the effect of neurotensin on open field behavior and on the responsiveness to an electric footshock of rats [10]. It was found that with respect to open field behavior the decrease in locomotor activity induced by neurotensin disappeared after repeated administration of the peptide and testing. This phenomenon of disappearance of a behavioral effect of neurotensin after multiple administration and testing was not observed using the responsiveness to an electric footshock as behavioral parameter. It was not clear from the previous experiments whether or not the ineffectiveness of neurotensin to reduce locomotor activity after repeated administration was due to development of tolerance to this effect of the peptide or to habituation of the animals to the behavioral test situation. Therefore the present study was designed to investigate whether development of tolerance occurs to the behavioral effects of neurotensin. METHOD Male rats of an inbred Wistar strain, weighing 140-160 g, were used. A polyethylene cannula was placed in the right ventricle of the brain for intracerebroventricular (ICV) administration of neurotensin (R 4322, Bachem, Torrance, CA) or of artificial cerebrospinal fluid (CSF) [artificial CSF contains (mM): NaCI 154, KCI 2.8, CaC12 1.1, MgSO4 0.8 and
Responsiveness to electric footshock
inulin (80 mg/100 ml)] (see [1]), which was used as placebo. The cannulation was performed 3-4 days prior to behavioral experimentation. All injection volumes were 2/xl. Locomotor activity of the animals was studied in a circular open field (diameter 75 cm) [8] during 3 min sessions. The behavioral parameters used during the sessions were ambulation (frequency of crossings of lines drawn on the floor of the open field), rearing, grooming and defecation. Two series of experiments were performed: one consisted of a daily observation session of locomotor activity in the open field for 3 consecutive days. On days 1 and 2 rats were ICV injected with 2/zl CSF one hr prior to observation. On day 3 animals received either placebo or 10 p.g neurotensin 1 hr prior to observation of the rats in the open field. In addition one group of animals received 10/zg neurotensin prior to each open field session for 3 consecutive days. In a second series of experiments animals received each day either placebo or 10/zg neurotensin during 3 consecutive days. A third group of animals received CSF for 2 days and 10/xg neurotensin on the third day. On days 1 and 2 animals were not subjected to the open field. One hr after the injection on the third day animals were tested for the various parameters in the open field. A dose of 10.0 p.g was chosen since this dose has been found in previous studies [10] to reduce locomotor activity in the open field effectively. Responsiveness to electric footshock (EFS) was observed in a 40×40×40 cm perspex box equipped with a grid floor. The responsiveness was determined by scoring the percentages of jerk/run/jump reactions, flinches and no responses to various shock levels, ranging from 0.031 mA to 0.187 mA [4]. Twelve different shock levels were used and each shock level was presented twice. The 24 E F S ' s were presented in two series of twelve shock levels. During each series the
~Requests for reprints should be addressed to TJ. B. van Wimersma Greidanus.
Copyright © 1983 Pergamon Press Ltd.--0031-9384/83/100467-04503.00
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IABLE 1 THE EFFECT OF NEUROTENSIN (N'IL l0/,tg, ICV) ON OPEN F1ELD BEHAVIOR OF RATS AFTER PRETREATMENT WITH EITHER NEUROTENSIN (NT) OR PLACEBO (CSFI DURING 2 CONSECUTIVE DAYS WITHOUT PRIOR TESTING Pretreatment
Treatment
CSF CSF NT
CSF NT NT
(12) (10) (12)
Ambulation
Rearing
Grooming
Defecation
79.1 z 4.8+ 52.7 + 4.7* 84.9 ± 5.5
12.1 ~_ 1.8 5.6 ± 0.9* 10.8 + 1.3
1.5 ::~ 0.8 0.9 ± 0.4 0.6 ± 0.2
2.7 ~ 0.8 1.9 ± 0.8 2.7 ~ 0.8
( ) Number of animals/group. *p <0.05 (versus group pretreatment: CSF/treatment:CSF as well as versus group pretreatment: neurotensin/treatment: neurotensin). tmean ± S.E.M. TABLE 2 THE EFFECT OF NEUROTENSIN (NT, 10/~g, ICV) ON OPEN FIELD BEHAVIOR DURING 3 CONSECUTIVE DAYS Treatment
Day
Ambulation
Rearing
Grooming
Defecation
CSF (14) CSF (14) CSF (14)
I II III
86.6 ± 5.2t 64.7 _+ 3.5 51.2 _+ 4.3
9.3 ± 1.3 5.0 ± 1.0 3.5 ± 0.8
1.6 ± 0.4 2.9 ± 0.4 1.5 ± 0.3
3.9 + 0.6 3.4 _+ 0.7 3.4 ± 0.6
CSF (9) CSF (9) NT (9)
I 1I Ili
79.7 ± 3.3 56.8 + 2.6 35.6 ± 1.7"
10.7 ± 1.4 4.6 ± 0.5 1.5 ± 0.4*
1.3 ± (I.2 2.5 ± 0.4 0.5 ± 0.2*
4.3 ± 0.6 3.3 ± 0.6 t.8 ± 0.5*
NT NT NT
1 II II1
64.0 ± 6.2* 38.5 ± 7.5* 42.4 ± 6.3
6.0 ± 0.7* 3.3 ± 0.7 1.9 ± 0.4
0.4 ± 0.2* 0.5 ± 0.2* 1.4 ± 0.4
1.7 ± 0.9* 0.8 ± 0.8* 0.3 ± 0.3*
(7) (7) (7)
( ) Number of animals/group. *p<0.05 (vs. CSF on the same day). tmean _+ S.E.M. t w e l v e s h o c k levels w e r e p r e s e n t e d in a r a n d o m l y fixed order. T h e d u r a t i o n o f e a c h E F S p r e s e n t a t i o n w a s 1 sec a n d t h e i n t e r v a l b e t w e e n p r e s e n t a t i o n s w a s 15 sec. S c o r i n g o f m o t o r r e s p o n s i v e n e s s o f rats to E F S in a situation in w h i c h t h e v a r i o u s s h o c k levels are p r e s e n t e d in an a s c e n d i n g o r d e s c e n d i n g o r d e r h a s b e e n f o u n d to give r e s u l t s w h i c h are i n f l u e n c e d b y t h e e x p e c t a n c y o f t h e a n i m a l s to r e c e i v e a n E F S . G e n e r a l l y a n i m a l s r e s p o n d less i n t e n s e l y to a c e r t a i n s h o c k level w h e n a n a s c e n d i n g o r d e r o f s h o c k levels is u s e d a n d m o r e i n t e n s e l y w h e n a d e s c e n d i n g o r d e r is used. T h i s m a k e s a p r e c i s e d e t e r m i n a t i o n o f t h r e s h o l d levels o f E F S at w h i c h e i t h e r f l i n c h e s o r j e r k s / j u m p s / r u n s o c c u r difficult. F o r t h e s e r e a s o n s a r a n d o m l y fixed o r d e r o f E F S l e v e l s h a s b e e n c h o s e n . F o r similar r e a s o n s c a l c u l a t i o n o f t h e percentages of "no responses," flinches and jerks/jumps / r u n s is s u p p o s e d to b e m o r e a p p r o p r i a t e t h a n c a l c u l a t i o n o f t h r e s h o l d levels for f l i n c h e s a n d / o r i n t e n s e m o t o r r e s p o n s e s as j e r k s / j u m p s / r u n s [4,9]. D e v e l o p m e n t o f t o l e r a n c e to the effect o f n e u r o t e n s i n o n E F S r e s p o n s i v e n e s s was s t u d i e d b y I C V i n j e c t i o n ' o f n e u r o t e n s i n (3.0 p,g) 1 h r b e f o r e e a c h daily s e s s i o n o f 24 E F S p r e s e n t a t i o n s for 3 c o n s e c u t i v e d a y s . O t h e r g r o u p s o f a n i m a l s r e c e i v e d e i t h e r C S F for 3 c o n s e c u t i v e d a y s o r C S F d u r i n g t w o d a y s f o l l o w e d b y n e u r o t e n s i n (3.0/xg) o n t h e t h i r d day. All i n j e c t i o n s w e r e p e r f o r m e d 1 h r p r i o r to b e h a v i o r a l observations. I n a d d i t i o n , a n e x p e r i m e n t w a s p e r f o r m e d in w h i c h t h e
rats w e r e p r e t r e a t e d w i t h e i t h e r C S F or n e u r o t e n s i n (3.0 k~g I C V p e r day) d u r i n g 2 c o n s e c u t i v e d a y s w i t h o u t t e s t i n g in the E F S situation. T r e a t m e n t c o n s i s t e d o f e i t h e r n e u r o t e n s i n o," C S F o n t h e t h i r d d a y a n d was p e r f o r m e d 1 h r p r i o r to t e s t i n g o f t h e r e s p o n s i v e n e s s to E F S . T h e d o s e o f 3.0 ~ g w a s c h o s e n s i n c e this d o s e h a s b e e n f o u n d to i n d u c e a m a r k e d r e d u c t i o n o f l o c o m o t o r r e s p o n s i v e n e s s to E F S [10]. A l t h o u g h a tenfold l o w e r d o s e (300 n g ) i n d u c e s this r e d u c t i o n as welt [10], t h e 3.0 /~g d o s e w a s c h o s e n in o r d e r to m a k e a fair c o m p a r i s o n w i l h t h e d o s e o f 10.0 ~ g u s e d in the o p e n field e x p e r i m e n t . T h e d o s e s c a n b e r e g a r d e d as equally s u b m a x i m a l for the two b e h a v i o r a l effects studied. Statistical e v a l u a t i o n o f the d a t a w a s p e r f o r m e d b y a n a l y s i s o f v a r i a n c e ( A N O V A ) a n d Stud e n t ' s t-test. RESULTS R e p e a t e d I C V i n j e c t i o n o f n e u r o t e n s i n (10 ~g) w i t h o u t o b s e r v a t i o n in the o p e n field followed b y a d m i n i s t r a t i o n of this p e p t i d e p r i o r to o b s e r v a t i o n o f l o c o m o t o r a c t i v i t y rev e a l e d t h a t in this s c h e d u l e n e u r o t e n s i n did n o t i n d u c e a reduction of locomotor activity, whereas neurotensin admini s t r a t i o n p r i o r to o p e n field t e s t i n g o f a n i m a l s p r e t r e a t e d w i t h C S F r e s u l t e d in r e d u c e d l o c o m o t o r activity ( T a b l e 1). Also, r e p e a t e d t e s t i n g o f l o c o m o t o r a c t i v i t y in t h e o p e n field o f rats u n d e r p l a c e b o ( C S F ) a d m i n i s t r a t i o n for t w o d a y s , followed b y a n i n j e c t i o n o f n e u r o t e n s i n 1 h r p r i o r to t h e o p e n field o b s e r v a t i o n o n the t h i r d day r e s u l t e d in a significant
TOLERANCE
TO NEUROTENSIN
469 TABLE 3
THE EFFECT OF NEUROTENSIN (NT; 3 p,g, ICV) ON RESPONSIVENESS TO ELECTRIC FOOTSHOCK (EFS) OF RATS PRETREATED WITH EITHER PLACEBO (CSF) OR NT FOR 2 CONSECUTIVE DAYS WITHOUT PRIOR TESTING FOR EFS RESPONSIVENESS
% No responses
% Flinches
% Jerks/ runs/jumps
% Vocalization
Pretreatment Treatment
CSF CSF (5)
20.9 ± 2.9¢
18.8 ± 2.7
60.4 ± 1.2
43.8 ± 4.0
Pretreatment Treatment
CSF NT (8)
43.1 ± 1.0t
11.6 ± 1.5"
45.4 ± 1.5t
20.8 ± 3.8*
Pretreatment Treatment
NT NT (8)
37.0 ± 2.3*
15.1 ± 2.4
47.9 ± 1.4t
22.4 ± 5.7*
( ) Number of animals/group. *p<0.05 (vs. CSF/CSF). tp<0.01 (vs. CSF/CSF). Smean ± S.E.M. TABLE 4 THE EFFECT OF NEUROTENSIN (NT; 3.0/~,g)ON RESPONSIVENESS TO ELECTRIC FOOTSHOCK DURING 3 CONSECUTIVE DAYS
Treatment
Day
% No responses
% Flinches
< Jerks/ runs/jumps
% Vocalization
CSF (9) CSF (9) CSF (9)
I 1I III
26.4 _+ 3.2~t 22.7 ± 3.7 23.1 ± 3.0
19.5 _+ 2.9 22.2 --- 2.5 24.5 _ 2.2
54.2 _ 2.2 55.1 ± 2.2 52.3 ± 1.6
42.1 ± 2.9 48.2 ± 3.0 45.8 ± 3.0
CSF (10) CSF (10) NT (10)
1 II III
28.8 ± 2.4 22.5 ÷ 2.2 48.7 ± 2.5§
20.0 ± 2.2 21.7 ± 2.0 14.2 ± 2.4*
51.3 ± 1.9 55.8 ± 1.9 37.1 ± 1.8"
33.0 ± 4.5 41.7 ± 3.5 41.3 _+ 4.3
NT NT NT
I II III
47.2 ± 2.9§ 54.6 ± 4.1§ 43.5 ± 4.5*
20.8 ± 3.0 13.4 ± 2.2* 14.2 ± 2.5*
33.3 ± 2.5§ 31.9 ± 2.7§ 42.6 ± 3.0*
15.9 ± 3.2§ 20.8 ± 3.1§ 29.9 ± 3.8"t
(9) (9) (9)
( ) Number ol animals/group. *p<0.05 (vs. CSF on same day). tp<0.05 (vs. NT on day 1). ~mean ± S.E.M. §p<0.01 (vs. CSF on same day).
n e u r o t e n s i n i n d u c e d r e d u c t i o n o f a m b u l a t i o n , rearing a n d g r o o m i n g b e h a v i o r , as c o m p a r e d to C S F t r e a t e d rats (Table 2). In c o n t r a s t , t r e a t m e n t w i t h n e u r o t e n s i n 1 h r p r i o r to e a c h o p e n field s e s s i o n d u r i n g 3 c o n s e c u t i v e d a y s r e s u l t e d in red u c e d l o c o m o t o r a c t i v i t y o n t h e first a n d the s e c o n d d a y , b u t n o t o n t h e third d a y , as c o m p a r e d to t h e C S F / C S F / C S F g r o u p ( T a b l e 2). R e s p o n s i v e n e s s to e l e c t r i c f o o t s h o c k was m a r k e d l y red u c e d a f t e r n e u r o t e n s i n a d m i n i s t r a t i o n (3 /xg), as c a n b e c o n c l u d e d f r o m the i n c r e a s e d p e r c e n t a g e o f n o r e s p o n s e s and the decreased percentages of flinches and jerks/runs / j u m p s (Tables 3, 4). T h i s r e d u c e d r e s p o n s i v e n e s s to E F S d u e to n e u r o t e n s i n a d m i n i s t r a t i o n w a s p r e s e n t in a n i m a l s p r e t r e a t e d with e i t h e r C S F o r n e u r o t e n s i n ( T a b l e 3). Repeated injection of the peptide and repeated testing r e s u l t e d in a similar r e d u c t i o n o f t h e m o t o r i c r e s p o n s i v e n e s s to E F S o n t h e first, s e c o n d a n d t h i r d d a y ( T a b l e 4). A l s o a d e c r e a s e in v o c a l i z a t i o n w a s o b s e r v e d a f t e r n e u r o t e n s i n a d m i n i s t r a t i o n , w h i c h w a s still p r e s e n t at the t h i r d d a y , albeit
t h a t o n this last d a y o f t e s t i n g this r e d u c t i o n o f v o c a l i z a t i o n was less p r o n o u n c e d t h a n o n t h e first day ( T a b l e 4). H o w e v e r , i n j e c t i o n o f n e u r o t e n s i n into rats w h i c h r e c e i v e d C S F p r i o r to p r e v i o u s t e s t s e s s i o n s did n o t r e s u l t in a r e d u c t i o n o f v o c a l i z a t i o n (Table 4). In c o n t r a s t , n e u r o t e n s i n r e d u c e d m o t o r r e s p o n s i v e n e s s to E F S as well as v o c a l i z a t i o n in a n i m a l s p r e t r e a t e d w i t h e i t h e r C S F or n e u r o t e n s i n w i t h o u t p r i o r t e s t i n g (Table 3). DISCUSSION T h e p r e v i o u s finding [10] that n e u r o t e n s i n a d m i n i s t r a t i o n p r i o r to e a c h s e s s i o n in the o p e n field d u r i n g 3 c o n s e c u t i v e d a y s r e d u c e s l o c o m o t o r activity o n d a y s 1 a n d 2, b u t does n o t r e d u c e l o c o m o t o r a c t i v i t y o n d a y 3, is c o n f i r m e d in t h e p r e s e n t study. T h e o b s e r v a t i o n t h a t n e u r o t e n s i n is effective in r e d u c i n g l o c o m o t o r a c t i v i t y in a n i m a l s w h i c h r e c e i v e d p l a c e b o p r i o r to t w o p r e v i o u s s e s s i o n s in t h e o p e n field, m a y b e e x p l a i n e d by d e v e l o p m e n t o f t o l e r a n c e to this effect o f n e u r o t e n s i n o n o p e n field b e h a v i o r . H o w e v e r , n o significant
470
RINKi:]
difference exists between the groups of rats treated with neurotensin on day 3 and pretreated during previous sessions with either C S F or neurotensin (Table 2). Since this difference has to be present in case of tolerance development, the results of this experiment do not prove that development of tolerance to this effect of neurotensin takes place. On the other hand habituation to the open field as indicated by the gradual reduction of locomotor activity in the control rats over the 3 days of testing may mask a difference in locomotor activity in tolerant and non-tolerant rats. Repeated injections of 10/zg neurotensin without subjecting the animals to the open field after the first two injections results in ineffectiveness of the peptide on ambulation and rearing, as tested on the third day (Table 1). However, when neurotensin is administered for the first time to rats pretreated with C S F and subjected to the first observation in the open field, the peptide causes a significant reduction in locomotor activity (ambulation, rearing; Table I), as compared to rats pretreated with either CSF or neurotensin. This result makes development of tolerance to this behavioral effect of neurotensin likely. As can be concluded from the effect of neurotensin on the percentages of " n o responses," flinches and jerks/runs /jumps in reaction to E F S presentation, the peptide markedly decreases the responsiveness to EFS. Since this effect is still present on the third day of administration and observation, no development of tolerance occurs during this time period. These results are in agreement with previous findings with a lower dose (0.3/zg) of the peptide [10]. However, the dose of 3 /zg as used in the present study results also in a reduction of vocalization, which was not found previously [101. Interestingly, the neurotensin-induced reduction of vocalization was less pronounced on the third day of observation (Table 3), suggesting a slight development of tolerance of neurotensin to this effect of the peptide. However, neurotensin did not reduce vocalization in animals subjected
t t ~,~
previously to the EFS situation and treated wit h (?SF ( Iablc 4). Thus, it may be that these animals experienced the aver siveness to the EFS during the previous session in such a way that they vocalize as soon as they experience the f:.FS again, although at the same time their motor responsiveness is markedly reduced by neurotensin. Consequently, the observation that the effect of neurotensin on vocalization is less pronounced on the third day than on the first day (Table 4) is rather due to experience of the animals to the experimental situation than to tolerance 1o neurotensin. This is also supported by the finding that pretreatment with nettrotensin without prior testing does not affect the influence of neurotensin on vocalization. In fact, thus treated rats respond to EFS in a similar way as animals pretrealed with CS F (Table 3). Preliminary experiments in the hot plate situation reveal that neurotensin (0.3/xg) induced an increase in the latency for paw lifting when the peptide was administered for the first time. This neurotensin-induced increase was observed in naive animals as well as in rats which received CSF prior to previous test sessions on the hot plate. However, after pretreatment with neurotensin an injection of neurotensin did not cause prolonged reaction time for this response, suggesting that also to this effect of neurotensin tolerance is developing. In conclusion we observe a development of tolerance to some, but certainly not all effects of neurotensin. This development of tolerance to neurotensin is observed on locomotor activity in the open field following ICV administration of a high dose (10/zg) of the peptide, but not on responsiveness to EFS after administration of 3/xg of neurotensin.
ACKNOWLEDGEMENTS The authors thank Dr. Ch. B. Nemeroff for his generous supply with neurotensin and Dr. J. M. van Ree for his stimulating comments during the preparation of this manuscript.
REFERENCES I. Bradbury, M. W. B. and H. Darson. The transport of urea, creatinine and certain monosaccharides between blood and fluid perfusing the cerebral ventricular system of rabbits. J Physiol 170: 195-211, 1964. 2. Clineschmidt, B. V. and J. C. McGuffin. Neurotensin administered intracisternally inhibits responsiveness of mice to noxious stimuli. Eur J Pharmaeol 46: 395-396, t977. 3. Clineschmidt, B. V., J. C. McGuffin and P. B. Bunting. Neurotensin: Antonocisponsive action in rodents. Eur J Pharmacol 54: 12%139, 1979. 4. Gispen, W. H., A. M. van der Poel and Tj. B. van Wimersma Greidanus. Pituitary-adrenal influences on behavior: Responses to test situations with or without electric footshock. Physiol Behav 10: 345-350, 1973. 5. Luttinger, D., Ch. B. Nemeroffand A. J. Prange, Jr. The effects of neuropeptides on discrete-trial conditioned avoidance responding. Brain Res 237: 183-192, 1982. 6. Nemeroff, Ch. B. Neurotensin: Perchance an endogenous neuroleptic? Biol Psychiatry 15: 283-302, 1980.
7. Nemeroff, Ch. B., G. Bissette, AI J. Prange, Jr., P. T. Loosen, T. S. Barlow and M. A. Lipton. Neurotensin: Central nervous system effects of a hypothalamic peptide. Brain Res 128: 485496, 1977. 8. Weijnen, J. A. W. M. and J. L. Slangen. Effects of ACTHanalogues on extinction of conditioned behavior. Pro~ Brain Res 32: 221-235, 1970. 9. Wimersma Greidanus, Tj. B. van, J. M. van Ree and D. H. G. Versteeg. Neurohypophyseal peptides and avoidance behavior: The involvement of vasopressin and oxytocin in memory processes. In: Neuropeptides and Neural Transmission, edited by C. A. Marsan and W. Z. Traczyk. New York: Raven Press, 1980, pp. 293-300. 10. Wimersma Greidanus, Tj. B. van, M. C. G. van Praag, R. Kalmann, G. J. E. Rinkel, G. Croiset, E. C. Hoeke, M. A. H. van Egmond and M. Fekete. Behavioral effects of neurotensin. Ann N Y Acad Sci 41111:31%329, 1982.
Elective Tolerance to Behavioral Effects of Neurotensin G. J. E. RINKEL, E. C. H O E K E A N D TJ. B. VAN W I M E R S M A G R E I D A N U S 1
Rudolf Magnus Institute for Pharmacology, Medical Faculty, State University of Utrecht Vondellaan 6, 3521 GD Utrecht, The Netherlands R e c e i v e d 10 J a n u a r y 1983 RINKEL, G. J. E., E. C. HOEKE AND TJ. B. VAN WIMERSMA GREIDANUS. Electivetolerance to behavioral effects ofneurotensin. PHYSIOL BEHAV 31(4) 467-470, 1983.--Neurotensin reduces locomotor activity in an open field situation as well as responsiveness to electric footshock. Repeated injections of neurotensin in combination with repeated testing of locomotor activity in the open field as well as repeated injections of neurotensin without such testing in the open field do not result in a reduction of locomotor activity. These findings make development of tolerance to this behavioral effect of neurotensin likely. Such a development of tolerance to neurotensin is not observed for the effect on responsiveness to electric footshock. Neurotensin
Tolerance
Open field behavior
Locomotor activity
N E U R O T E N S I N , a brain and gastro-intestinal tridecapeptide, exerts various behavioral effects [5, 6, 10], such as a reduction of locomotor activity [6,7] and a decreased responsiveness to aversive stimuli [2,3]. These effects are almost exclusively found after central administration, probably due to the poor permeability of the blood-brain barrier to penetration by this peptide [7]. In a previous series of preliminary experiments we studied the effect of neurotensin on open field behavior and on the responsiveness to an electric footshock of rats [10]. It was found that with respect to open field behavior the decrease in locomotor activity induced by neurotensin disappeared after repeated administration of the peptide and testing. This phenomenon of disappearance of a behavioral effect of neurotensin after multiple administration and testing was not observed using the responsiveness to an electric footshock as behavioral parameter. It was not clear from the previous experiments whether or not the ineffectiveness of neurotensin to reduce locomotor activity after repeated administration was due to development of tolerance to this effect of the peptide or to habituation of the animals to the behavioral test situation. Therefore the present study was designed to investigate whether development of tolerance occurs to the behavioral effects of neurotensin. METHOD Male rats of an inbred Wistar strain, weighing 140-160 g, were used. A polyethylene cannula was placed in the right ventricle of the brain for intracerebroventricular (ICV) administration of neurotensin (R 4322, Bachem, Torrance, CA) or of artificial cerebrospinal fluid (CSF) [artificial CSF contains (mM): NaCI 154, KCI 2.8, CaC12 1.1, MgSO4 0.8 and
Responsiveness to electric footshock
inulin (80 mg/100 ml)] (see [1]), which was used as placebo. The cannulation was performed 3-4 days prior to behavioral experimentation. All injection volumes were 2/xl. Locomotor activity of the animals was studied in a circular open field (diameter 75 cm) [8] during 3 min sessions. The behavioral parameters used during the sessions were ambulation (frequency of crossings of lines drawn on the floor of the open field), rearing, grooming and defecation. Two series of experiments were performed: one consisted of a daily observation session of locomotor activity in the open field for 3 consecutive days. On days 1 and 2 rats were ICV injected with 2/zl CSF one hr prior to observation. On day 3 animals received either placebo or 10 p.g neurotensin 1 hr prior to observation of the rats in the open field. In addition one group of animals received 10/zg neurotensin prior to each open field session for 3 consecutive days. In a second series of experiments animals received each day either placebo or 10/zg neurotensin during 3 consecutive days. A third group of animals received CSF for 2 days and 10/xg neurotensin on the third day. On days 1 and 2 animals were not subjected to the open field. One hr after the injection on the third day animals were tested for the various parameters in the open field. A dose of 10.0 p.g was chosen since this dose has been found in previous studies [10] to reduce locomotor activity in the open field effectively. Responsiveness to electric footshock (EFS) was observed in a 40×40×40 cm perspex box equipped with a grid floor. The responsiveness was determined by scoring the percentages of jerk/run/jump reactions, flinches and no responses to various shock levels, ranging from 0.031 mA to 0.187 mA [4]. Twelve different shock levels were used and each shock level was presented twice. The 24 E F S ' s were presented in two series of twelve shock levels. During each series the
~Requests for reprints should be addressed to TJ. B. van Wimersma Greidanus.
Copyright © 1983 Pergamon Press Ltd.--0031-9384/83/100467-04503.00
4~8
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~,
IABLE 1 THE EFFECT OF NEUROTENSIN (N'IL l0/,tg, ICV) ON OPEN F1ELD BEHAVIOR OF RATS AFTER PRETREATMENT WITH EITHER NEUROTENSIN (NT) OR PLACEBO (CSFI DURING 2 CONSECUTIVE DAYS WITHOUT PRIOR TESTING Pretreatment
Treatment
CSF CSF NT
CSF NT NT
(12) (10) (12)
Ambulation
Rearing
Grooming
Defecation
79.1 z 4.8+ 52.7 + 4.7* 84.9 ± 5.5
12.1 ~_ 1.8 5.6 ± 0.9* 10.8 + 1.3
1.5 ::~ 0.8 0.9 ± 0.4 0.6 ± 0.2
2.7 ~ 0.8 1.9 ± 0.8 2.7 ~ 0.8
( ) Number of animals/group. *p <0.05 (versus group pretreatment: CSF/treatment:CSF as well as versus group pretreatment: neurotensin/treatment: neurotensin). tmean ± S.E.M. TABLE 2 THE EFFECT OF NEUROTENSIN (NT, 10/~g, ICV) ON OPEN FIELD BEHAVIOR DURING 3 CONSECUTIVE DAYS Treatment
Day
Ambulation
Rearing
Grooming
Defecation
CSF (14) CSF (14) CSF (14)
I II III
86.6 ± 5.2t 64.7 _+ 3.5 51.2 _+ 4.3
9.3 ± 1.3 5.0 ± 1.0 3.5 ± 0.8
1.6 ± 0.4 2.9 ± 0.4 1.5 ± 0.3
3.9 + 0.6 3.4 _+ 0.7 3.4 ± 0.6
CSF (9) CSF (9) NT (9)
I 1I Ili
79.7 ± 3.3 56.8 + 2.6 35.6 ± 1.7"
10.7 ± 1.4 4.6 ± 0.5 1.5 ± 0.4*
1.3 ± (I.2 2.5 ± 0.4 0.5 ± 0.2*
4.3 ± 0.6 3.3 ± 0.6 t.8 ± 0.5*
NT NT NT
1 II II1
64.0 ± 6.2* 38.5 ± 7.5* 42.4 ± 6.3
6.0 ± 0.7* 3.3 ± 0.7 1.9 ± 0.4
0.4 ± 0.2* 0.5 ± 0.2* 1.4 ± 0.4
1.7 ± 0.9* 0.8 ± 0.8* 0.3 ± 0.3*
(7) (7) (7)
( ) Number of animals/group. *p<0.05 (vs. CSF on the same day). tmean _+ S.E.M. t w e l v e s h o c k levels w e r e p r e s e n t e d in a r a n d o m l y fixed order. T h e d u r a t i o n o f e a c h E F S p r e s e n t a t i o n w a s 1 sec a n d t h e i n t e r v a l b e t w e e n p r e s e n t a t i o n s w a s 15 sec. S c o r i n g o f m o t o r r e s p o n s i v e n e s s o f rats to E F S in a situation in w h i c h t h e v a r i o u s s h o c k levels are p r e s e n t e d in an a s c e n d i n g o r d e s c e n d i n g o r d e r h a s b e e n f o u n d to give r e s u l t s w h i c h are i n f l u e n c e d b y t h e e x p e c t a n c y o f t h e a n i m a l s to r e c e i v e a n E F S . G e n e r a l l y a n i m a l s r e s p o n d less i n t e n s e l y to a c e r t a i n s h o c k level w h e n a n a s c e n d i n g o r d e r o f s h o c k levels is u s e d a n d m o r e i n t e n s e l y w h e n a d e s c e n d i n g o r d e r is used. T h i s m a k e s a p r e c i s e d e t e r m i n a t i o n o f t h r e s h o l d levels o f E F S at w h i c h e i t h e r f l i n c h e s o r j e r k s / j u m p s / r u n s o c c u r difficult. F o r t h e s e r e a s o n s a r a n d o m l y fixed o r d e r o f E F S l e v e l s h a s b e e n c h o s e n . F o r similar r e a s o n s c a l c u l a t i o n o f t h e percentages of "no responses," flinches and jerks/jumps / r u n s is s u p p o s e d to b e m o r e a p p r o p r i a t e t h a n c a l c u l a t i o n o f t h r e s h o l d levels for f l i n c h e s a n d / o r i n t e n s e m o t o r r e s p o n s e s as j e r k s / j u m p s / r u n s [4,9]. D e v e l o p m e n t o f t o l e r a n c e to the effect o f n e u r o t e n s i n o n E F S r e s p o n s i v e n e s s was s t u d i e d b y I C V i n j e c t i o n ' o f n e u r o t e n s i n (3.0 p,g) 1 h r b e f o r e e a c h daily s e s s i o n o f 24 E F S p r e s e n t a t i o n s for 3 c o n s e c u t i v e d a y s . O t h e r g r o u p s o f a n i m a l s r e c e i v e d e i t h e r C S F for 3 c o n s e c u t i v e d a y s o r C S F d u r i n g t w o d a y s f o l l o w e d b y n e u r o t e n s i n (3.0/xg) o n t h e t h i r d day. All i n j e c t i o n s w e r e p e r f o r m e d 1 h r p r i o r to b e h a v i o r a l observations. I n a d d i t i o n , a n e x p e r i m e n t w a s p e r f o r m e d in w h i c h t h e
rats w e r e p r e t r e a t e d w i t h e i t h e r C S F or n e u r o t e n s i n (3.0 k~g I C V p e r day) d u r i n g 2 c o n s e c u t i v e d a y s w i t h o u t t e s t i n g in the E F S situation. T r e a t m e n t c o n s i s t e d o f e i t h e r n e u r o t e n s i n o," C S F o n t h e t h i r d d a y a n d was p e r f o r m e d 1 h r p r i o r to t e s t i n g o f t h e r e s p o n s i v e n e s s to E F S . T h e d o s e o f 3.0 ~ g w a s c h o s e n s i n c e this d o s e h a s b e e n f o u n d to i n d u c e a m a r k e d r e d u c t i o n o f l o c o m o t o r r e s p o n s i v e n e s s to E F S [10]. A l t h o u g h a tenfold l o w e r d o s e (300 n g ) i n d u c e s this r e d u c t i o n as welt [10], t h e 3.0 /~g d o s e w a s c h o s e n in o r d e r to m a k e a fair c o m p a r i s o n w i l h t h e d o s e o f 10.0 ~ g u s e d in the o p e n field e x p e r i m e n t . T h e d o s e s c a n b e r e g a r d e d as equally s u b m a x i m a l for the two b e h a v i o r a l effects studied. Statistical e v a l u a t i o n o f the d a t a w a s p e r f o r m e d b y a n a l y s i s o f v a r i a n c e ( A N O V A ) a n d Stud e n t ' s t-test. RESULTS R e p e a t e d I C V i n j e c t i o n o f n e u r o t e n s i n (10 ~g) w i t h o u t o b s e r v a t i o n in the o p e n field followed b y a d m i n i s t r a t i o n of this p e p t i d e p r i o r to o b s e r v a t i o n o f l o c o m o t o r a c t i v i t y rev e a l e d t h a t in this s c h e d u l e n e u r o t e n s i n did n o t i n d u c e a reduction of locomotor activity, whereas neurotensin admini s t r a t i o n p r i o r to o p e n field t e s t i n g o f a n i m a l s p r e t r e a t e d w i t h C S F r e s u l t e d in r e d u c e d l o c o m o t o r activity ( T a b l e 1). Also, r e p e a t e d t e s t i n g o f l o c o m o t o r a c t i v i t y in t h e o p e n field o f rats u n d e r p l a c e b o ( C S F ) a d m i n i s t r a t i o n for t w o d a y s , followed b y a n i n j e c t i o n o f n e u r o t e n s i n 1 h r p r i o r to t h e o p e n field o b s e r v a t i o n o n the t h i r d day r e s u l t e d in a significant
TOLERANCE
TO NEUROTENSIN
469 TABLE 3
THE EFFECT OF NEUROTENSIN (NT; 3 p,g, ICV) ON RESPONSIVENESS TO ELECTRIC FOOTSHOCK (EFS) OF RATS PRETREATED WITH EITHER PLACEBO (CSF) OR NT FOR 2 CONSECUTIVE DAYS WITHOUT PRIOR TESTING FOR EFS RESPONSIVENESS
% No responses
% Flinches
% Jerks/ runs/jumps
% Vocalization
Pretreatment Treatment
CSF CSF (5)
20.9 ± 2.9¢
18.8 ± 2.7
60.4 ± 1.2
43.8 ± 4.0
Pretreatment Treatment
CSF NT (8)
43.1 ± 1.0t
11.6 ± 1.5"
45.4 ± 1.5t
20.8 ± 3.8*
Pretreatment Treatment
NT NT (8)
37.0 ± 2.3*
15.1 ± 2.4
47.9 ± 1.4t
22.4 ± 5.7*
( ) Number of animals/group. *p<0.05 (vs. CSF/CSF). tp<0.01 (vs. CSF/CSF). Smean ± S.E.M. TABLE 4 THE EFFECT OF NEUROTENSIN (NT; 3.0/~,g)ON RESPONSIVENESS TO ELECTRIC FOOTSHOCK DURING 3 CONSECUTIVE DAYS
Treatment
Day
% No responses
% Flinches
< Jerks/ runs/jumps
% Vocalization
CSF (9) CSF (9) CSF (9)
I 1I III
26.4 _+ 3.2~t 22.7 ± 3.7 23.1 ± 3.0
19.5 _+ 2.9 22.2 --- 2.5 24.5 _ 2.2
54.2 _ 2.2 55.1 ± 2.2 52.3 ± 1.6
42.1 ± 2.9 48.2 ± 3.0 45.8 ± 3.0
CSF (10) CSF (10) NT (10)
1 II III
28.8 ± 2.4 22.5 ÷ 2.2 48.7 ± 2.5§
20.0 ± 2.2 21.7 ± 2.0 14.2 ± 2.4*
51.3 ± 1.9 55.8 ± 1.9 37.1 ± 1.8"
33.0 ± 4.5 41.7 ± 3.5 41.3 _+ 4.3
NT NT NT
I II III
47.2 ± 2.9§ 54.6 ± 4.1§ 43.5 ± 4.5*
20.8 ± 3.0 13.4 ± 2.2* 14.2 ± 2.5*
33.3 ± 2.5§ 31.9 ± 2.7§ 42.6 ± 3.0*
15.9 ± 3.2§ 20.8 ± 3.1§ 29.9 ± 3.8"t
(9) (9) (9)
( ) Number ol animals/group. *p<0.05 (vs. CSF on same day). tp<0.05 (vs. NT on day 1). ~mean ± S.E.M. §p<0.01 (vs. CSF on same day).
n e u r o t e n s i n i n d u c e d r e d u c t i o n o f a m b u l a t i o n , rearing a n d g r o o m i n g b e h a v i o r , as c o m p a r e d to C S F t r e a t e d rats (Table 2). In c o n t r a s t , t r e a t m e n t w i t h n e u r o t e n s i n 1 h r p r i o r to e a c h o p e n field s e s s i o n d u r i n g 3 c o n s e c u t i v e d a y s r e s u l t e d in red u c e d l o c o m o t o r a c t i v i t y o n t h e first a n d the s e c o n d d a y , b u t n o t o n t h e third d a y , as c o m p a r e d to t h e C S F / C S F / C S F g r o u p ( T a b l e 2). R e s p o n s i v e n e s s to e l e c t r i c f o o t s h o c k was m a r k e d l y red u c e d a f t e r n e u r o t e n s i n a d m i n i s t r a t i o n (3 /xg), as c a n b e c o n c l u d e d f r o m the i n c r e a s e d p e r c e n t a g e o f n o r e s p o n s e s and the decreased percentages of flinches and jerks/runs / j u m p s (Tables 3, 4). T h i s r e d u c e d r e s p o n s i v e n e s s to E F S d u e to n e u r o t e n s i n a d m i n i s t r a t i o n w a s p r e s e n t in a n i m a l s p r e t r e a t e d with e i t h e r C S F o r n e u r o t e n s i n ( T a b l e 3). Repeated injection of the peptide and repeated testing r e s u l t e d in a similar r e d u c t i o n o f t h e m o t o r i c r e s p o n s i v e n e s s to E F S o n t h e first, s e c o n d a n d t h i r d d a y ( T a b l e 4). A l s o a d e c r e a s e in v o c a l i z a t i o n w a s o b s e r v e d a f t e r n e u r o t e n s i n a d m i n i s t r a t i o n , w h i c h w a s still p r e s e n t at the t h i r d d a y , albeit
t h a t o n this last d a y o f t e s t i n g this r e d u c t i o n o f v o c a l i z a t i o n was less p r o n o u n c e d t h a n o n t h e first day ( T a b l e 4). H o w e v e r , i n j e c t i o n o f n e u r o t e n s i n into rats w h i c h r e c e i v e d C S F p r i o r to p r e v i o u s t e s t s e s s i o n s did n o t r e s u l t in a r e d u c t i o n o f v o c a l i z a t i o n (Table 4). In c o n t r a s t , n e u r o t e n s i n r e d u c e d m o t o r r e s p o n s i v e n e s s to E F S as well as v o c a l i z a t i o n in a n i m a l s p r e t r e a t e d w i t h e i t h e r C S F or n e u r o t e n s i n w i t h o u t p r i o r t e s t i n g (Table 3). DISCUSSION T h e p r e v i o u s finding [10] that n e u r o t e n s i n a d m i n i s t r a t i o n p r i o r to e a c h s e s s i o n in the o p e n field d u r i n g 3 c o n s e c u t i v e d a y s r e d u c e s l o c o m o t o r activity o n d a y s 1 a n d 2, b u t does n o t r e d u c e l o c o m o t o r a c t i v i t y o n d a y 3, is c o n f i r m e d in t h e p r e s e n t study. T h e o b s e r v a t i o n t h a t n e u r o t e n s i n is effective in r e d u c i n g l o c o m o t o r a c t i v i t y in a n i m a l s w h i c h r e c e i v e d p l a c e b o p r i o r to t w o p r e v i o u s s e s s i o n s in t h e o p e n field, m a y b e e x p l a i n e d by d e v e l o p m e n t o f t o l e r a n c e to this effect o f n e u r o t e n s i n o n o p e n field b e h a v i o r . H o w e v e r , n o significant
470
RINKi:]
difference exists between the groups of rats treated with neurotensin on day 3 and pretreated during previous sessions with either C S F or neurotensin (Table 2). Since this difference has to be present in case of tolerance development, the results of this experiment do not prove that development of tolerance to this effect of neurotensin takes place. On the other hand habituation to the open field as indicated by the gradual reduction of locomotor activity in the control rats over the 3 days of testing may mask a difference in locomotor activity in tolerant and non-tolerant rats. Repeated injections of 10/zg neurotensin without subjecting the animals to the open field after the first two injections results in ineffectiveness of the peptide on ambulation and rearing, as tested on the third day (Table 1). However, when neurotensin is administered for the first time to rats pretreated with C S F and subjected to the first observation in the open field, the peptide causes a significant reduction in locomotor activity (ambulation, rearing; Table I), as compared to rats pretreated with either CSF or neurotensin. This result makes development of tolerance to this behavioral effect of neurotensin likely. As can be concluded from the effect of neurotensin on the percentages of " n o responses," flinches and jerks/runs /jumps in reaction to E F S presentation, the peptide markedly decreases the responsiveness to EFS. Since this effect is still present on the third day of administration and observation, no development of tolerance occurs during this time period. These results are in agreement with previous findings with a lower dose (0.3/zg) of the peptide [10]. However, the dose of 3 /zg as used in the present study results also in a reduction of vocalization, which was not found previously [101. Interestingly, the neurotensin-induced reduction of vocalization was less pronounced on the third day of observation (Table 3), suggesting a slight development of tolerance of neurotensin to this effect of the peptide. However, neurotensin did not reduce vocalization in animals subjected
t t ~,~
previously to the EFS situation and treated wit h (?SF ( Iablc 4). Thus, it may be that these animals experienced the aver siveness to the EFS during the previous session in such a way that they vocalize as soon as they experience the f:.FS again, although at the same time their motor responsiveness is markedly reduced by neurotensin. Consequently, the observation that the effect of neurotensin on vocalization is less pronounced on the third day than on the first day (Table 4) is rather due to experience of the animals to the experimental situation than to tolerance 1o neurotensin. This is also supported by the finding that pretreatment with nettrotensin without prior testing does not affect the influence of neurotensin on vocalization. In fact, thus treated rats respond to EFS in a similar way as animals pretrealed with CS F (Table 3). Preliminary experiments in the hot plate situation reveal that neurotensin (0.3/xg) induced an increase in the latency for paw lifting when the peptide was administered for the first time. This neurotensin-induced increase was observed in naive animals as well as in rats which received CSF prior to previous test sessions on the hot plate. However, after pretreatment with neurotensin an injection of neurotensin did not cause prolonged reaction time for this response, suggesting that also to this effect of neurotensin tolerance is developing. In conclusion we observe a development of tolerance to some, but certainly not all effects of neurotensin. This development of tolerance to neurotensin is observed on locomotor activity in the open field following ICV administration of a high dose (10/zg) of the peptide, but not on responsiveness to EFS after administration of 3/xg of neurotensin.
ACKNOWLEDGEMENTS The authors thank Dr. Ch. B. Nemeroff for his generous supply with neurotensin and Dr. J. M. van Ree for his stimulating comments during the preparation of this manuscript.
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7. Nemeroff, Ch. B., G. Bissette, AI J. Prange, Jr., P. T. Loosen, T. S. Barlow and M. A. Lipton. Neurotensin: Central nervous system effects of a hypothalamic peptide. Brain Res 128: 485496, 1977. 8. Weijnen, J. A. W. M. and J. L. Slangen. Effects of ACTHanalogues on extinction of conditioned behavior. Pro~ Brain Res 32: 221-235, 1970. 9. Wimersma Greidanus, Tj. B. van, J. M. van Ree and D. H. G. Versteeg. Neurohypophyseal peptides and avoidance behavior: The involvement of vasopressin and oxytocin in memory processes. In: Neuropeptides and Neural Transmission, edited by C. A. Marsan and W. Z. Traczyk. New York: Raven Press, 1980, pp. 293-300. 10. Wimersma Greidanus, Tj. B. van, M. C. G. van Praag, R. Kalmann, G. J. E. Rinkel, G. Croiset, E. C. Hoeke, M. A. H. van Egmond and M. Fekete. Behavioral effects of neurotensin. Ann N Y Acad Sci 41111:31%329, 1982.