Psychopharmacology(1992) t09:373-376

Psychopharmacology (© Springer-Verlag 1992

Effects of intra-hippocampal scopolamine injections in a repeated spatial acquisition task in the rat Arian Blokland, Wiel Honig, and Wijnand G. M. Raaijmakers Department of Neuropsychologyand Psychobiology,University of Limburg, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands Received September 17, 1991 / Final version May 14, 1992

Abstract. The involvement of hippocampal cholinergic synapses in spatial discrimination learning was evaluated by locally administering scopolamine into the hippocampus. Sixteen 16-month-old male Lewis rats received bilaterally implanted cannulae aimed at the dorsal part of the hippocampus. The rats were trained on a repeated acquisition test in the Morris water-escape task. In this procedure the invisible platform is randomly moved from day to day to one of four possible locations. Thus, the rat has to learn to localize the platform from day to day. On each day the rats received four pairs of trials. Scopolamine injections (35 gg in 1 gl per hippocampus) were given to one group (n = 8) on days 5 and 7. On days 6 and 8 all rats received saline injections. Place learning was retarded in the scopolamine-treated rats during the first swims of pairs of trials. During second swims the scopolaminetreated rats showed a general performance deficit, indicating that first and second swims were differentially affected. The data support the hypothesis that cholinergic neurotransmission in the dorsal hippocampus is involved in spatial learning processes. Key words: Cholinergic system ........Scopolamine Hippocampus - Spatial discrimination - Learning - Rat

Behavioral deficits after peripheral administration of anticholinergic drugs have predominantly been found in short-term memory tasks (Beninger et al. 1986; Yamamoto et al. 1990) and in spatial discrimination tasks (Sutherland et al. 1982; Willner et al. 1986). Hippocampal lesions also affect short-term memory (Olton et al. 1979) and spatial discrimination learning (Morris et al. 1990). Cholinergic denervation of the hippocampus by fimbria lesions results in spatial working and reference memory per-

Correspondence to." A. Blokland

formance deficits (Van der Staay et al. 1989). These findings may suggest that the hippocampal cholinergic system could be involved in short-term memory and spatial discrimination learning. However, the above mentioned studies give no insight into the functions of the hippocampal cholinergic system. The involvement of the hippocampal cholinergic system in learning and memory processes can be studied by injecting cholinergic drugs directly into the hippocampus. Moreover, as long as there is no selective toxin for cholinergic neurons, this remains at present one of the most suitable strategies to study the functions of the hippocampal cholinergic system (Hagan and Morris 1988; Fibiger 1991). It has been reported that intra-hippocampal injection of a muscarinic antagonist disrupts working memory performance in a T-maze alternation task (Brito et al. 1983; Messer et al. 1987) and, more recently, a deficit in short-term memory has been found in a non-matching-toposition task (Dunnett et al. 1990). This suggests that the hippocampal cholinergic system is critically involved in short-term memory processes. However, to our knowledge, there are no studies which evaluated the effects of intra-hippocampal injections in an allocentric spatial discrimination paradigm (see Hagan and Morris 1988). In the Morris spatial navigation task, in which the position of a submerged escape platform is changed from day to day, rats with hippocampal lesions have an impaired performance compared to controls (Whishaw 1987). In the same test procedure, similar results have been found after peripheral administration of muscarinic antagonists (Whishaw 1985b). Although hippocampal lesions and systemic injections with cholinergic drugs are not comparable, these findings may suggest that the hippocampal cholinergic system is involved in spatial discrimination learning processes. We evaluated the role of the hippocampal cholinergic system in allocentric spatial discrimination performance by injecting a muscarinic antagonist directly into the hippocampus, Because the test procedure of Whishaw was sensitive to both a hippocampal lesion (Whishaw 1987) and systemic injection of a cholinergic drug (Whishaw 1985b), we used the same behavioral test in the present study,

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Materials and methods

Results

Animals. Sixteen male Lewis rats (16-month-old) were randomly assigned to either the control group (CON, n = 8) or the scopolamine group (SCOP, n = 8). The rats were housed individually in standard Makrolon TM cages on sawdust bedding in an air-conditioned room (20°C) with ad libitum access to standard food and tap water, and maintained on a reversed light/dark cycle (12/12, lights on at 9:00 A.M.).

Pre-training

Surgery. The rats were anesthetized with sodium pentobarbital (60 mg/kg IP) and bilateral stainless steel cannulae were stereotaxically implanted in the dorsal part of the hippocampus. The coordinates (A 3.8, L 2.5, H 3.0 mm) for the injection site were chosen according to the atlas of Paxinos and Watson (1986). The cannulae (outer and inner diameter 0.65 mm and 0.30 ram, respectively) were fixed to the skull with acrylic dental cement (PaladurTM). Behavioral procedures. One week after surgery the rats were trained on the standard Morris spatial navigation task (Morris 1981) in a black water tank with a diameter of 1.22 m. The rats were started from four different and randomly chosen start positions and trained to find an invisible platform (diameter 11 cm) that was at a fixed position in the water tank, 1 cm below the surface of the water. After the rats' performance had reached asymptotic levels, they were trained on a procedure as described by Whishaw (1985a). In this procedure the platform was moved to a different location in the water tank from day to day. The rats were given four pairs of trials per day; each trial pair consisted of a first and second swim, with an inter-swim time of 5 s. The time between pairs of trials was about 4 min. The rats were started from the same start position for both swims of a pair, but the start position of the four pairs of trials varied randomly between the four possible locations. After having reached an asymptotic level of performance, the rats were subjected to the drug sessions.

Drug effects. The SCOP group was given a bolus injection of scopolamine (35 p,g in 1 gl saline per injection site) on days 5 and 7 of training on the repeated acquisition test; the CON group was given a saline injection (1 ~tl per injection site). Injections were given 20 min prior to testing. Test sessions took about 15 min. On days 6 and 8 the rats of the CON and the SCOP groups were given saline injections (1 gl per injection site).

Histology. Immediately preceding decapitation, the rats received an intra-hippocampal injection of 1 ~tl of a methylene blue solution via the injection cannula to determine the location of the injection sites. Statistics. Group effects in the standard test were analyzed by comparing the mean escape latency per block of four trials with a t-test. For the four pre-training sessions in the repeated acquisition test, group effects were analyzed by comparing the mean escape latencies of the first swims and the mean escape latencies of the second swims separately, using t-statistics. Drug effects were analyzed by comparing the mean escape latencies of the first swims and the mean escape latencies of the second swims for the two scopolamine/saline (days 5 and 7) and saline sessions (days 6 and 8) by using t-statistics. Within session analysis compared the average escape latencies of the groups for the scopolamine/saline sessions during first and second swims separately using t-statistics. In addition, group effects on polynomial trend coefficients were analyzed for the first and second swims of the scopolamine/saline sessions seperately in order to evaluate the characteristics of the learning curves of first and second swims. Finally, it was evaluated whether the rats improved their performance from first to second swims during a test session by comparing the individual first and second swims in pairs of trials (cf Whishaw 1985b).

T h e r e were no differences between the escape latencies of the C O N a n d S C O P g r o u p s during acquisition of the s t a n d a r d task (all ts < 1.51, Ps > 0.10; d a t a n o t shown). Also, d u r i n g p r e - t r a i n i n g on the r e p e a t e d acquisition t a s k (data n o t shown), there were no differences between the g r o u p s for the m e a n escape latencies of the first swims (all ts < 1.48, Ps > 0.10) a n d second swims (all ts < 1.06, Ps > 0.10). The escape latencies of sessions three a n d four were a p p r o x i m a t e l y the same as the escape latencies of the s t a n d a r d test ( + 15 s), indicating t h a t the rats h a d reached an a s y m p t o t i c level of performance.

Drug effects Analysis of the s c o p o l a m i n e / s a l i n e sessions showed that s c o p o l a m i n e - i n j e c t e d rats n e e d e d m o r e time to locate the p l a t f o r m in b o t h the first a n d second swims It ( 1 4 ) = - 2.23, P < 0.05 a n d t (14) = - 2.99, P < 0.01, respectively; see Fig. 1A]. In the saline sessions, there was no difference between the C O N a n d S C O P g r o u p s for the m e a n latency to find the p l a t f o r m in either the first or the second swims (both ts < 1.20, Ps > 0.10; d a t a n o t shown). W i t h i n - s e s s i o n analysis of the first swims of pairs of trials revealed t h a t the S C O P g r o u p h a d a g r e m e r escape latency d u r i n g the second a n d third first swims It (14) = 4.09, P < 0.01 a n d t (14) = 3.11, P < 0.01, respectively; see Fig. 1B] whereas there were no differences between the g r o u p s for the first a n d fourth first swims (both ts < 0.89, P s > 0 . 1 0 ) . Analysis of p o l y n o m i a l trend coefficients showed that the g r o u p s differed on the q u a d r a t i c trend c o m p o n e n t I F ( l , 14) = 9.36, P < 0.01] for the escape latencies of the first swims, i n d i c a t i n g that acquisition of place learning was r e t a r d e d in the S C O P group. W i t h r e g a r d to the second swims of pairs of trials, it was f o u n d t h a t the S C O P g r o u p h a d greater escape latencies d u r i n g the second a n d fourth second swims than the C O N g r o u p [t (14) = 2.71, P < 0.05 a n d t (14) = 2.61, P < 0.05, respectively; see Fig. 1C] but there were no differences between the first a n d third second swims (both ts < 0.88, Ps > 0.39). T r e n d analysis revealed that there was a g r o u p effect on the third t r e n d c o m p o n e n t [ F (1, 14) = 4.18, P < 0.05], which was interpreted as a general p e r f o r m a n c e deficit of the S C O P g r o u p d u r i n g second swims. Analysis of the first to second swim i m p r o v e m e n t revealed t h a t the escape latency d u r i n g first a n d second swims was similar for the C O N g r o u p It ( 7 ) = 1.24, P > 0.10]. After s c o p o l a m i n e injections the escape latency of the S C O P - t r e a t e d rats decreased from first to second swims by a p p r o x i m a t e l y 5.5 s (t (7) = 3.56, P > 0.01].

Histological data I n s p e c t i o n of the injection sites s h o w e d that the methylene blue was found in the CA1 region of the d o r s a l h i p p o c a m pus in all animals.

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Fig. 1 A-C. Performance of the scopolamine-treated (SCOP) and control (CON) rats in a repeated acquisition test in a spatial navigation task. A Mean escape latency (s _+ SEM) of the first and second swims of the two scopolamine/saline sessions. B Mean escape latency of the first swims, and C mean escape latency of the second swims of pairs of trials in the scopolamine/saline sessions

Discussion

The results of this study showed that intra-hippocampal scopolamine injections increased the latency to find a new platform position, indicating that the hippocampal cholinergic system is involved in allocentric spatial discrimination learning. The scopolamine-injected rats, however, did show place learning during first swims, albeit at a slower rate than the control rats. Control rats learned a new platform position within one swim and showed a maximal performance on subsequent swims whereas the scopolamine-injected rats reached a maximal performance at the end of the session. There was a general performance deficit during second swims and the scopolamine-treated rats were able to improve their performance from first to second swims. This differential effect of scopolamine on first and second swims was probably due to differences in task demands for first and second swims (see below).

The procedure used in this study was originally developed by Whishaw (1985a) to evaluate the formation of a spatial learning set in rats: a first to second swim improvement indicates formation of a learning set. Zeldin and Olton (1986), however, have argued that first and second swims are different with regard to task demands. If a rat finds the platform during the first swim of a pair it might use a taxon or some cue-based strategy during the second swim, whereby mean escape latencies for second swims can be lower than for first swims where no taxon or cue-based strategy is used. Similar findings have been reported by Whishaw (1987)., who found a decrease in escape latencies but no reduction in heading errors between first and second swims in rats with hippocampal damage. Qualitative observations of the present study indicated that rats seemed to use a taxon strategy during second swims by swimming the same route as in the first swim. This explanation could account for the finding that there was a first to second swim improvement after intrahippocampal scopolamine injections: scopolamine-treated rats were able to make use of a strategy other than a spatial orientation strategy during second swims. It should be noted that if a rat does not find the platform during the first swim it will not be able to use a taxon strategy during the second swim. This may explain the general performance deficit during second swims. The present results are consistent with findings after peripheral administration of muscarinic antagonists (e.g. Whishaw and Tomie 1987). However, in the same procedure as was used in the present study, atropine-treated rats did not reach the performance of control rats on either the first or the second swims, even after eight pairs of trials (Whishaw 1985b). The scopolamine-treated rats of our study did reach performance levels as good as those of controls within four pairs of trials. The difference between these findings may be caused by the dose of atropine (50 mg/kg) used, which may, at least partly, be related to "stereotypies" in swimming Performance in the Morris task (Lindner and Schallert 1988). Furthermore, it is dimcult to differentiate between peripheral and central effects that may interfere with behavioral performance after peripheral administration of a cholinergic drug. Apparently, intra-hippocampal scopolamine injections affect the rate of spatial discrimination learning whereas systemic scopolamine injections cause a general performance deficit. It could be argued that the intra-cerebral injections with scopolamine could have affected other processes that interfered with the escape performance. The injection volume used in the present study (1 gl) will affect a region of maximal 1 mm which would be in the range of the hippocampus (Myers et al. 1971). It is known that almost all drugs find their way into the ventricle or the blood incidentally. However, this may not explain the majority of the behavioral effects (Myers et al. 1971; Van Dongen 1980; Brito et al. 1983). Another possibility could be that electrographic abnormalities seen after intra-cerebral injections may have attributed to the behavioral deficit (Rowntree and Bland 1986). We tried to circumvent this by waiting 20 min after injection before subjecting the rats to behavioral testing (cf Brito et al. 1983; Messer et al. 1987; Dunnet et al. 1990).

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References Beninger RJ, Jhamandas K, Boegman RJ, E1-Defrawy SR (1986) Effects of scopolamine and unilateral lesions of the basal forebrain on T-maze spatial discrimination and alternation in rats. Pharmacol Biochem Behav 24:1353-1360 Brito GNO, Davis BJ, Stopp LC, Stanton ME (1983) Memory and the septo-hippocampal cholinergic system in the rat. Psychopharmacology 81:315-320 Dunnett SB, Wareham AT, Torres EM (1990) Cholinergic blockade in prefrontal cortex and hippocampus disrupts short-term memory in rats. Neuro Report 1 : 61-64 FiNger HC (1991) Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence. TINS 14:220-223 Hagan JJ, Morris RGM (1988) The cholinergic hypothesis of memory: a review of animal experiments. In: Iversen LL, Iversen SD, Snyder SH (eds) Handbook of psychopharmacology, vol 20. Plenum Press, pp 237-323 Lindner MD, Schattert T (1988) Aging and atropine effects on spatial navigation in the Morris water task. Behav Neurosci 102 : 621-634 Messer WS, Thomas G J, Hoss W (1987) Selectivity of pirenzipine in the central nervous system. II. Differential effects of pirenzipine and scopolamine on performance of a representational memory task. Brain Res 407 : 37-45 Morris RGM (1981) Spatial localization does not require the presence of local cues. Learn Motiv 12:239 261 Morris RGM, Schenk F, Tweedie F, Jarrard LE (1990) Ibotenate lesions of hippocampus and/or subiculum: dissociating components of allocentric spatial learning. Eur J Neurosci 2:1016-1028 Myers RD, Tytell M, Kawa A, Rudy T (1971) Micro-injection of 3Hacetylcholine, 14C-serotonin and 3H-norepinephrine into the hypothalamus of the rat: diffusion into tissue and ventricles. Physiol Behav 7 : 743-751 Olton DS, Becker JT, Handelmann GE (1979) Hippocampus, space and memory. Behav Brain Sci 2:313-365 Paxinos G, Watson C (t986) The rat brain in stereotaxic co-

ordinates, 2nd edn. Academic Press, Sidney Rowntree CI, Bland BH (1986~ An analysis of chotinoceptive neurons in the hippocampal formation by direct microinfusion. Brain Res 362 : 98-113 Sutherland RJ, Whishaw IQ, Regehr JC (1982) Cholinergic receptor blockade impairs spatial localization by use of distal cues in the rat. J Comp Physiol Psychol 96:563-573 Van der Staay F J, Raaijmakers WGM, Lammers AJJC, Tonnaer JADM (1989) Selective fimbria lesions impair acquisition of working and reference memory of rats in a complex spatial discrimination task. Behav Brain Res 32:151--161 Van Dongen PAM (1980) The noradrenergic locus coeruleus: behavioral effects of intra-cerebral injections, and a survey of its structure, function and pathology. Doctoral dissertation, University of Nijmegen, The Netherlands Whishaw IQ (1985a) Formation of a place learning-set by the rat: a new paradigm for neurobehavioral studies. Physiol Behav 35 : 139-143 Whishaw IQ (1985b) Cholinergic receptor blockade in the rat impairs locale but not taxon strategies for place navigation in a swimming pool. Behav Neurosci 99 : 979---1005 Whishaw IQ (1987) Hippocampal, granule cell and CA3-4 lesions impair formation of a place learning-set in the rat and induce reflex epilepsy. Behav Brain Res 24:59-72 Whishaw IQ, Tomie J-A (1987) Cholinergic receptor blockade produces impairments in a sensorimotor subsystem for place navigation in the rat: evidence from sensory, motor, and acquisition tests in a swimming pool. Behav Neurosci 101 : 603-616 Willner P, Wise D, Ellis T (1986) Specific disruption of spatial behaviour in rats by central muscarinic receptor blockade. Psychopharmacotogy 90 : 229 235 Yamamoto Tatsugi S, Ohno M, Furuya Y, Kitajima I, Ueki S (1990) Minaprine improves impairment of working memory induced by scopolamine and cerebral ischemia in rats. Psychopharmacology t00:316-322 Zeldin RK, Olton DS (1986) Rats acquire spatial learning sets. J Exp Psychol [Anim Behav Proc] 12:412-419