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The role of medial and lateral hippocampal perforant path lesions and object distinctiveness in rats' reaction to novelty
Physiology & Behavior, Vol. 42, pp. 371-377. Copyright © Pergamon Press plc, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
The Role of Medial and Lateral Hippocampal Perforant Path Lesions and Object Distinctiveness in Rats' Reaction to Novelty TROND MYHRER 1
N o r w e g i a n D e f e n c e R e s e a r c h Establishment, Division f o r Environmental Toxicology N-2007 Kjeller, N o r w a y R e c e i v e d 20 A p r i l 1987 MYHRER, T. The role of medial and lateral hippocampal perforant path lesions and object distinctiveness in rats' reaction to novelty. PHYSIOL BEHAV 42(4) 371-377, 1988.---The purpose of the present study was to investigate whether damage to the medial (MPP) or lateral hippocampal perforant path (LPP) may differentially affect rats' ability to react to novelty or environmental change. Three different types of task were used based on various sets of stimuli; visual/tactile, olfactory, or visual only. The results showed that the lesions produced different responses to different novel stimuli. In contrast to control and MPP animals, LPP rats displayed excessive exploration of the test cage. The results are discussed in terms of impoverished cognitive structure following perforant path disruptions and functional differentiation between MPP and LPP. Perforant path lesions
Reaction to novelty
Exploratory behavior
have access to polymodal sensory information from the primary and secondary association areas of the neocortex [14]. Exploration of a discrete novel object is one form of inquisitive activity frequently seen among rats. This activity appears as a strong preference for novelty, the recognition of which is probably based on polymodal sensory information [3]. In a recent study, it was found that disruption of LPP produced a marked decrease in preference for novelty, whereas MPP lesions only caused a slight deficit in this respect [11]. However, MEC lesions have been reported to result in a pronounced reduction in preference for novelty [ 10]. A possible explanation of these discrepant findings may be that the difference between neutral and novel objects was only slight in the latter study (toy cars slightly different in form) and more conspicuous in the former (closed vs. open metal rectangle). The test procedures for the two studies were otherwise similar. Thus, it may be hypothesized that whether damage to pefforant path systems affects preference for novelty is associated with object distinctiveness in terms of the degree of differences among the discriminanda. On the basis of previous findings [11] it may be expected that MPP rats are most affected in tasks with low level of distinctiveness, whereas LPP animals are vulnerable in a larger number of tasks. The present study was carried out to test these hypotheses by applying a modified version of the novelty
T H E hippocampal formation has been suggested to integrate signals from the reticulo-septal projection with highly processed neocortical information entering the hippocampus via the entorhinal area (cf. [16]). The latter projection is organized in several elements. The orbitofronal cortex and temporal cortex project to the entorhinal cortex [13-15]. The hippocampus proper and fascia dentata receive their major extrinsic fiber input from the entorhinal cortex by way of the perforant path (cf, [4]). This pathway is composed of two distinct fiber systems which both have fields of termination along the whole axial extent of the hippocampal formation. The medial perforant path (MPP) arises in the medial entorhinal cortex (MEC) and terminates in the middle of the dentate molecular layer and in the deep part of the stratum lacunosum-moleculare of subfield CA3 [7]. The lateral perforant path (LPP) originates in the lateral entorhinal cortex (LEC) and projects to a superficial zone in the dentate molecular layer and to the superficial portion of the stratum lacunosum-moleculare of CA3 [6] (Fig. 1). These differences in fields of termination among MPP and LPP coupled with findings of different neocortical sources of input to MEC and L E C [14] may constitute a morphological substrate for functional differences between MPP and LPP. The findings of neocortical projections to the entorhinal cortices have given rise to the suggestion that the hippocampal formation may
1Requests for reprints should be addressed to Trond Myhrer, Norwegian Armed Forces, Psychological and Educational Centre, Oslo Mil/Akershus, 0015 Oslo 1, Norway.
371
372
MYHRER
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line. Each cannula was inserted into the brain in a position deviating 20 ° from the vertical in the sagittal plane (tip of cannula pointing rostrally). From this position the syringe was moved ten times back and forth to make a cut of 1.0-1.5 mm. F o r medial pp lesions the movement was made in an approximate occipito-thalamic axis and for the lateral pp lesions in an approximate occipito-temporal axis. These maneuvers were carried out in three stages for medial lesions with the depths of cannula insertions being 4, 6 and 8 ram, respectively. The lateral lesions were made in two stages with insertion depths of 6 and 8 mm.
Histology
CA]
fim FIG. 1. Diagram of a horizonal section of the entorhinalhippocampal region showing intended sites of transection of medial pefforant path (MPP) and lateral perforant path (LPP). Abbreviations: LEC=lateral entorhinal cortex; MEC=medial entorhinal cortex; ab=angular bundle; fd=fascia dentata; fim=fimbra; sub =subiculum; p=parasubiculum. test of Berlyne [2]. Rats with MPP or LPP lesions and controis were run in three discrimination tasks based on different sets of stimuli; visual/tactile, olfactory, or visual only. METHOD
Subjects Twenty-four male Wistar rats from a commercial supplier (Dyrlaege M011egaards Avlslaboratorium, Denmark), weighing 290-320 g at the time of surgery, served as subjects. They were randomly assigned to three groups: 8 animals received bilateral lesions of the medial perforant path (MPP), 8 received bilateral lesions of the lateral perforant path (LPP), and 8 served as controls and had the scalp reflected only. The rats were housed individually and had free access to commercial rat pellets and water. The rats were handled individually 3 days preoperatively and 1 day postoperatively, being allowed to explore a table top (80x60 cm) for 3 min a day. The climatized (21°C) vivarium was illuminated from 0700 to 1900 hours.
Surgery The rats were anesthetized IP with diazepam (10 mg/kg) and fentanyl fluanisone (2 mg/kg) and placed in a stereotaxic head holder with their skulls horizontal. The lesions were made mechanically by means of the sharp edges of cannulas provided with collar. The cannula to be used was mounted on a syringe. The points of insertion were the following; for the medial perforant path lesions 9.1 mm posterior to bregma and 5.0 mm lateral to midline; for lateral perforant path lesions 8.5 mm posterior to bregma and 5.5 mm lateral to mid-
Upon termination of testing the brains were removed and frozen. The brains were sectioned horizontally on a CO2freezing microtome at 30/zm, every twelfth section being preserved. The sections were stained with the GrOnwald/Giemsa method. The percentage of perforant path damage was based on the location and extent of the transections in relation to the course of MPP and LPP fibers (cf. Fig. 1). The lesions were traced on diagrams of the perforant path fibers consisting of 20 lines evenly distributed in MEC or LEC, each line representing 5 percent of the fibers. The number of lines affected in the dorso-ventral levels presented in Fig. 2 were counted, and the average percentage of damage for each animal was computed.
Apparatus Behavioral testing was carried out in a Plexiglas cage (56×34×20 cm) with a metal mesh roof. The floor was divided in 18 equal squares (9× 11 cm). Three equal wooden cubes (5×5×5 cm) with a track in the under side made up the neutral objects. These were put on screweyes fixed to the floor in the middle of three squares evenly distributed in the cage. This arrangement spaced the objects in fixed positions with approximately the same distance from the walls and one another. Three other objects provided with corresponding tracks in the under side made up the novel objects. One object only differed from the neutral ones in that its top was uneven with tracks in it making up a square pattern (visual/tactile stimuli). One was identical with the neutrals and a spot of cheese was smeared on one side (olfactory stimulus). One was smaller than the neutrals (4.5x4.5x5.0 cm) and two sides were slightly uneven (visual stimulus). All objects were painted light grey. The only light was a 15 W bulb 60 cm above the apparatus. The sound attenuated testing room was provided with a fan producing background sound (52 dB).
Procedure Training started on Postoperative Day 10. The rats were allowed to explore individually the empty apparatus for 25 min. On the next day the rats were run in Session I. In Phase 1, the rats were tested for 5 min with the three neutral objects present. The following behaviors were recorded: number of seconds in contact with each object, number of squares traversed (locomotor activity), number of rearings, and duration of grooming. Then the rats spent 10 rain in the home cage. In Phase 2, the rats were tested again for 5 min, and one of the neutral objects had been replaced by the novel object with uneven top. During this period the same measures as in Phase 1 were made. In Sessions II and III (Postoperative Days 12 and 13), the same procedure was followed
PERFORANT PATH AND NOVELTY
373 in both groups. Further, degeneration was seen among the stellate ceils in layer II and to some extent the pyramidal cells in layer III in MEC or LEC.
Behavior
FIG. 2. Representative examples of medial perforant path lesion (A) and lateral perforant path lesion (B). and the novelty was represented by a spot (diameter 1.5 cm) of cheese on one side of the object on Day 12 and a smaller object on Day 13. So-called Norwegian white cheese which hardly smells anything to humans was used. In the test cage, it was not possible to detect the cheese spot visually. To exclude effects of positional preferences the novel object was for each session placed in a position never used in preceding sessions. A pre-arranged random order for the position of novel object was made for each animal. Prior to each time a rat was placed in the apparatus the cage and objects were carefully cleaned and allowed to dry. The behaviors were hand scored by the author who was unaware of the rats' group assignment.
Statistics Overall analyses were carded out with parametric oneway ANOVA or nonparametric one-way ANOVA (Kruskal-Wallis). Group comparisons were made with twotailed t-test or two-tailed Mann-Whitney U-test. RESULTS
Histology In the MPP group, the lesion of the medial perforant path appeared as a cut in the medial entorhinal area (Fig. 2A). The medial perforant path was affected bilaterally in the entire dorso-ventral extent in all animals. Additional damage appeared as a section of both MPP and LPP systems in the ventral-most tip of ~he hippocampal formation. This damage occurred bilaterally in two rats and unilaterally in one. The mean percentage of lesion efficiency was 87 (range 65-100). In the LPP group, the lateral perforant path was disrupted bilaterally in the entire dorso-ventral extent in all animals (Fig. 2B). As for some of the MPP animals, both perforant path systems in the ventral-most tip of the hippocampal formation were occasionally damaged. This damage was observed unilaterally in four rats. Furthermore, an additional small section was occasionally observed in parts of the subiculum. The mean percentage of lesion efficiency was 86 (range 75-100). The tracks made by the cannulas in neocortex overlying the entorhinal areas were very slight and difficult to identify
Both the MPP group and the LPP group displayed reduced preference for novelty compared with the control group in all three sessions. However, the reduction in preference was more marked among the LPP rats than the MPP animals (Fig. 3). Exploration of neutral object is based on the mean time of contact with the two neutral objects. Group comparisons were made in terms of time difference between exploration of novel vs. neutral objects (Table 1). Because of uneven distribution of data nonparametric statistics were applied. In Session I (uneven top of novel object), Kruskal-Wallis ANOVA revealed a significant treatment effect, H(2) = 11.81, p <0.01. Mean comparisons with U-test showed that both MPP and LPP rats displayed significantly less preference for the novel object than the control group, U=9, p<0.02 and U = I , p<0.001, respectively. The LPP group also exhibited less preference for novelty than the MPP group, U=9, p<0.02, t-Test for dependent means only revealed significantly more exploration of novel than neutral objects among the control group, t(7)---4.74, p<0.01. In Session II (novelty smell of cheese), ANOVA revealed a reliable overall effect, H(2)---10.70, p <0.01. Both the MPP and LPP groups showed a significant reduction in preference for novelty relative to the control group, U= 11, p<0.05 and U=4, p<0.005, respectively. The two experimental groups did not differ reliably from one another. The control group and the MPP group explored the novel object significantly more than the neutral ones, t(7)=6.03, p<0.001 and t(7)=2.55, p <0.05, respectively. In Session Ill (smaller object), ANOVA showed a reliable treatment effect, H(2)=8.94, p<0.02. Both the MPP and LPP groups displayed significantly less preference for novelty than the control group, U= 10, p<0.05 and U=8, p<0.02, respectively. The LPP group also showed less preference for novelty than the MPP group, U= 10, p<0.05. The only significant difference in exploration of novel vs. neutral objects was that the LPP group explored the novel object less than the neutral ones, t(7)=3.74, p<0.01. The time spent in exploring neutral objects in Phase 2 was relatively constant from one session to another for all groups; in particular the control group (Fig. 3, Table 1). ANOVA revealed no significant differences among the groups in this respect for either of the three sessions. The time spent in exploring novel objects varied between the groups. Kruskal-WaUis ANOVA revealed a significant difference among the groups in Session I, H(2)=8.84, p<0.02. Mean comparisons showed that the LPP group explored the novel object significantly less than the control group and the MPP group, U=6, p<0.005 and U=12, p<0.05, respectively, whereas the control group and the MPP group did not differ reliably. A reliable treatment effect was also revealed in Session II, H(2)=6.54, p<0.05. The LPP group explored the novel object significantly less than the control group, U= 11, p <0.05. No other significant group differences were revealed in this respect, neither any reliable treatment effect in Session III. Table 1 shows the total time spent in exploring neutral objects in Phase 1 and neutral plus novel objects in Phase 2. No reliable treatment effect was revealed among the groups in any phase in any session. However, in Session I, the LPP group displayed a significant decrease in exploration from
374
MYHRER
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TABLE 1 MEAN MEASURES OF EXPLORATORY BEHAVIORIN SECONDS Total Time Exploring
Differential Time Exploring Session I
Session II
Session I
Session III
Session II
Session III
Group
N
Neut
Nov
Diff
Neut
Nov
Diff
Neut
Nov
Diff
Ph 1
Ph2
Ph 1
Ph2
Ph 1
Ph2
MPP LPP Cont
8 8 8
10.6 6.4 9.6
13.5 5.4 23.3
2.9 -1.1 13.6
11.4 14.0 9.1
20.6 16.0 29.1
9.4 2.0 20.0
12.8 13.0 9.5
12.3 10.0 18.4
0.0 -3.0 9.1
38. I 35.0 32.5
34.4 17.5 41.6
33.1 28.4 34.8
42.6 43.6 46.8
34.6 29.4 35.1
37.3 32.8 37.0
Ph=Phase; Neut=Neutral; Nov=Novel; Diff=Difference; MPP=Medial Perforant Path; LPP=Lateral Perforant Path; Cont=Control. Phase 1 to Phase 2, t(7)=4.26, p<0.01. In Session II, both the MPP group and the LPP group showed a significant increase in exploration from Phase 1 to Phase 2, t(7)=2.39, p < 0 . 0 5 and t(7)=5.57,p<0.001, respectively. In Session III, no significant differences were observed. Occasionally, rats urinated on the objects. This was seen among all groups and appeared during Phase 1 and/or Phase 2. As seen from FigJ 4A the LPP group tended to display more locomotor activity than the other groups. In Session I, Phase 1, one-way A N O V A revealed a reliable treatment effect, F(2,21)=4.68, p<0.02. Group comparisons with t-test showed that the LPP group was significantly more active than the control group and the MPP group, t(14)=3.24, p<0.01 and t(14)=2.93, p<0.02, respectively. The control group and the MPP group did not differ reliably from one another. A N O V A revealed no significant overall effect in Phase 2. Only the LPP group displayed a significant decrease in locomotor activity from Phase 1 to Phase 2, t(7)=2.98, p<0.05. In Session II, A N O V A revealed no significant differences in either of the two phases. However, both the control group and the LPP group had a significant decrease in activity from Phase 1 to Phase 2, t(7)=3.42, p < 0 . 0 2 and t(7)=3.63, p<0.01, respectively. In Session III, A N O V A only revealed significant treatment effect in Phase 2, F(2,21)=4.03, p<0.05. Group comparisons showed that the LPP group was significantly more active than the control
group and MPP group, t(14)=2.75 and t(14)=2.93, p<0.02, respectively. The control group and the MPP group was not reliably at variance. No groups displayed significant decrease in activity from Phase 1 to Phase 2. As seen from Fig. 4B the LPP group also tended to display more rearing activity than the other groups. In Session I Phase 1, one-way A N O V A revealed a reliable treatment effect, F(2,21) =4.01, p <0.05. Group comparisons showed that the LPP group made significantly more rearings than the control group and the MPP group, t(14)=6.37, p<0.001 and t(14)=4.27, p<0.001, respectively. The two latter groups were not reliably different. In Phase 2, A N O V A also revealed significant differences, F(2,21)=5.79, p<0.01. Mean comparisons showed that the LPP group made significantly more rearings than the control group and the MPP group, t(14)=7.07 and t(14)=5.89, respectively; p<0.001. The two latter groups did not differ reliably from one another. Only the LPP group made a significant decrease in rearings from Phase 1 to Phase 2, t(7)=2.76, p<0.05. In Session II, A N O V A revealed no significant differences for either of the phases, However, both the control group and the LPP group displayed a significant decrease in rearings from Phase 1 to Phase 2, t(7)=4.39, p<0.01 and t(7)=3.24, p<0.02, respectively. In Session III, A N O V A only revealed reliable treatment effect for Phase 2, F(2,21)=6.39, p<0.01. The LPP group displayed significantly more rearing activity than the
PERFORANT PATH AND NOVELTY
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110
100
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TABLE 2
LPP
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MEAN MEASURES OF GROOMING B E H A V I O R IN SECONDS
CONT
Session I
Session II
Session III
9O
=:
Group
N
Ph 1
Ph 2
Ph 1
Ph 2
Ph 1
Ph 2
MPP LPP Cont
8 8 8
6.4 1.6 13.5
0.5 0.0 6.1
22.9 1.6 14.1
5.3 2.3 12.3
11.3 3.4 14.6
2.5 9.0 12.3
llO 70 6O
Abbreviations as for Table 1. I
I
I
1
2
1
SESSION )
B
PHASE
I
I
I
2
1
2
SESSION I [
SESSION I l l
30
z
15
10 I
I
I
1
2
1
SESSION [
PHASE SESSION 11
I
I
t
2
I
2 SESSIONTIT
FIG. 4. Mean measures of locomotor activity (A) and rearing (B).
control group and the MPP group, t(14)=7.55 and t(14)=6.25, respectively; p<0.001. Both the control group and the MPP group displayed a reliable decrease in rearings from Phase 1 to Phase 2, t(7)=2.44, and t(7)=2.93, respectively; p <0.05. As seen from Table 2, the LPP group tended to display less grooming than the control group and the MPP group. However, Kruskal-Wallis A N O V A did not reach a statistically significant level for any phase in any of the sessions. Since the tendency for less grooming among the LPP rats was rather pronounced in Phase 1 for all sessions, the average scores of these three phases were calculated. On the basis of the average scores for Phase 1 in three sessions A N O V A revealed a significant treatment effect, H(2)=8.51, p<0.02. Group comparisons showed that the LPP group groomed significantly less than the control group and the MPP group, U = 10, p < 0 . 0 2 and U = 6 , p<0.005, respectively. The latter two groups did not differ significantly. The total time grooming for each session appeared to increase steadily for the LPP group. However, A N O V A of Grooming × Time interaction was not significant in this respect. DISCUSSION
Both medial (MPP) and lateral perforant path (LPP) lesions resulted in reduced preference for novelty. However, the LPP group was even more impoverished in responding to novelty than the MPP group. Further, the LPP rats displayed
more locomotor activity and rearing behavior than the control and MPP animals. The LPP group also tended to groom less than the control and MPP groups. The present findings are in accord with the notion that the degree of difference between neutral and novel objects appears crucial in revealing potential effects of decreased preference for novelty. In a previous study, in which the difference between the discriminanda was conspicuous MPP lesions only caused a slight discrimination deficit [11], whereas MEC lesions have been reported to produce a marked deficit in reaction to novelty when the difference between the discriminanda was very small [10]. The degree of preference for novelty was differentially affected by the two types of lesions. In Session I, both the MPP and LPP group displayed less preference for the novel object than the control group. However, the LPP group also exhibited reliably less preference for novelty than the MPP group. In Session II, both MPP and LPP groups showed a reduction in preference for novelty relative to the control group. However, the MPP group like the control group explored the novel object more than the neutral ones; this was not the case for the LPP group. In Session III, both MPP and LPP groups again displayed reduced reaction to novelty. It has been suggested that a deficit in inquisitive behavior may be expected if perforant path disruptions reduce the hippocampal formation's access to sensory information of neocortical origin [11]. In the latter study, the discrimination task was based on polymodal sensory stimuli. In the present study, a corresponding deficit was also seen when the discrimination task was based on a single sensory modality. The idea of sensory information reaching the hippocampal formation by way of the perforant path also gains support from an electrophysiological study. Deadwyler et al. [5] have shown that septal and entorhinal inputs to the dentate gyrus are activated differentially by sensory stimuli as a function of their acquired behavioral significance to the rat. The decreased preference for novelty among the experimental groups may be accounted for in other ways than reduced sensory information to the hippocampal formation. Exploratory activity is generally counteracted by fear [3]. Thus, it may be suggested that the novel object was less inspected because it elicited fear among the experimental subjects. This suggestion does not appear plausible in view of the fact that no significant differences were observed among the groups in exploring objects in Phase 1 of Session I when all objects were novel to the animals (Table 1). It may be argued that perforant path lesions caused decreased curiosity. However, this argument does not gain support from the present data, since the level of exploring neutral objects in Phase 1 and Phase 2 did not differ signifi-
376 cantly among the groups for any session (Table 1; Fig. 2). F o r the same reason it is difficult to maintain that the experimental animals were less attentive or aroused than the control rats. A deficit in short-term or long-term memory may represent a potential explanation of reduced preference for novelty among the experimental animals. However, the short-term aspect of this explanation does not seem to be reconcilable with the fact that the MPP group explored the novel object significantly more than the neutral ones in Session II, indicating that these rats still were able to discriminate provided a proper task. The LPP group also demonstrated an ability to discriminate, inasmuch as this group explored the novel object less than the neutral ones in Session III. This latter finding is not readily accounted for. Further, both experimental groups did not differ from the control group in exploring neutral objects in Phase 1 or Phase 2 in any session, indicating intact long-term memory. Among the experimental groups the LPP animals differed most markedly from the control rats. In addition to being the most impoverished subjects in preference for novelty the LPP rats also displayed high level of locomotor and rearing activity (Fig. 4). This activity which reached a peak in Phase 1 in Session I may explain the relatively low level of exploring neutral objects at this event; the LPP animals were highly engaged in exploring the test cage as such. The low level of grooming activity during Phase 1 for each session among the LPP rats may be taken as supportive for this explanation. The MPP rats did not differ from the control animals in terms of locomotor, rearing, or grooming activity. Thus, even if the MPP group also displayed reduced ability to respond to novelty, there are qualitative differences between the behaviors of the LPP group and the MPP group. It appears that MPP rats may be better off concerning access to sensory information than the LPP animals are and thus exhibited a smaller need to explore the surroundings in order to achieve cognitive structure of the environment. As seen from Fig. 4 the locomotor activity and rearing behavior generally decreased from Phase 1 to Phase 2 among all groups. This decrement may be associated with the presentation of novelty or habituation to the environment (following ten minutes in the home cage) or an interaction between these two factors. In the control group, however, the total time exploring objects only increased 7.7 sec from Phase 1 to 2 on the average for all three sessions. This small increase in exploration within a total testing time of 5 min probably indicates that the decrement in locomotor and rearing activity reflects habituation to the environment rather than preoccupation of the novelty. The MPP group followed very much the same pattern as the control group, whereas the LPP group differed somewhat. Since the experimental animals were less aware of the novelty than the control rats, habituation probably represents a plausible explanation of decreased locomotor and rearing activity for these subjects as well. In a previous study dealing with perforant path systems, both MPP and LPP rats displayed significantly more locomotor activity than the control animals [11]. In this study, LPP rats groomed more as a function of time. These results are discrepant with the present ones in which MPP rats did not exhibit increased locomotor activity and LPP animals groomed less than the rats from the other groups. These
MYHRER discrepancies are most likely due to different test procedures. In the previous study [11], the testing time each day was continuous for 15 min instead of two times for 5 rain. It appears that the longer test exposure provides more opportunity to reveal potential changes in, for instance, activity after perforant path lesions. This seems to be in accordance with the extremely high level of both locomotor and rearing activity seen among LPP rats in the previous study [11]. The behavioral differences between MPP and LPP rats can hardly be attributed to lesion efficiency, since the percentage of damage was very much the same; 87 and 86, respectively. The medial entorhinal cortex is far larger than the lateral one and LPP lesions produced the most marked effects. It is likely that the behavioral differences between MPP and LPP animals are due to different sources of input to the two entorhinal cortices. LEC receives fibers from orbito-frontal cortex and large areas of the temporal cortex, whereas MEC only receives fibers from the caudal part of temporal cortex [13-15]. Further, MPP and LPP terminate differently in the hippocampal formation (cf. the Introduction section). Thus, there seems to be a morphological substrate for functional differences between MPP and LPP. Projections from the neocortical association areas to the entorhinal cortex have been found in the monkey [14]. Corresponding projections have not been revealed in the rat with the horseradish peroxidase (HRP) labeling technique, but the existence can not be precluded since certain cells fail to transport HRP in detectable quantitities [1]. Actually, Beckstead [1] refers to unpublished findings that tritiated amino acids, when injected into either medial prefrontal or anterior cingulate cortex in the rat, are transported so as to label axons in LEC in an autoradiographically detectable manner. Extra-hippocampal fiber systems which may be differently affected by MPP and LPP lesions have been reported to run in angular bundle. One system projects from both MEC and LEC to nucleus accumbens [9], and another system projects from LEC only to cortical areas and amygdala [17]. The projections to nucleus accumbens originating in MEC and LEC are disrupted by MPP or LPP lesions, respectively. However, the projection from LEC to cortical areas and amygdala appears to enter distal parts of angular bundle and thus avoids damage from LPP sections. Accordingly, the behavioral differences seen among MPP and LPP animals are likely not due to different damage to extrahippocampal fibers made by MPP or LPP lesions. In the present study, degeneration was seen clearly among the stellate cells in layer II and to some extent among the pyramidal cells in layer III in MEC or LEC after MPP or LPP lesions, respectively. These findings correspond with the notion that the perforant path fibers arise predominantly in layer II and to some degree in layer III in the entorhinal cortices [12]. There is a marked projection from LEC to temporal cortex, whereas M E C ' s contribution is very modest in this respect [8]. This relation might imply that LEC is associated with memory processes. Such an association may explain at least some of the powerful effects seen after LPP lesions. A memory component may interact with decreased sensory information to the hippocampal formation resulting in the apparently pronounced deficit to recognize environmental change.
PERFORANT
PATH AND NOVELTY
377 REFERENCES
1. Beckstead, R. M. Afferent connections of the entorhinal area in the rat as demonstrated by retrograde cell-labeling with horseradish peroxidase. Brain Res 152: 249-264, 1978. 2. Bedyne, D. E. Novelty and curiosity as determinants of exploratory behaviour. Br J Psychol 41: 68-80, 1950. 3. Berlyne, D. E. Conflict, Arousal, and Curiosity. New York: McGraw-Hill, 1960. 4. Blackstad, T. W. On the termination of some afferents to the hippocampus and fascia dentata. An experimental study in the rat. Acta Anat 35: 202-214, 1958. 5. Deadwyler, S. A., M. O. West and J. H. Robinson. Entorhinal and septal input differentially control sensory-evoked responses in the rat dentate gyrus. Science 211: 1181-1183, 1981. 6. Hjorth-Simonsen, A. Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata. J Comp Neurol 146: 219-231, 1972. 7. Hjorth-Simonsen, A. and B. Jeune. Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J Comp Neurol 144: 215-231, 1972. 8. Kosel, K. C., G. W. Van Hoesen and D. L. Rosene. Nonhippocampal cortical projections from the entorhinal cortex in the rat and rhesus monkey. Brain Res 244: 201-213, 1982. 9. Krayniak, P. F., R. C. Meibach and A. Siegel. A projection from the entorhinal cortex to the nucleus accumbens in the rat. Brain Res 209" 427-431, 1981. 10. Kohler, C. On the role of the hippocampus, the septum and the entorhinal cortex in reaction to novelty and habituation: A behavioral analysis of the effects of selective electrolytic lesions in the male albino rat. Unpublished doctoral thesis, University of Bergen, 1979.
11. Myhrer, T. Exploratory behavior and reaction to novelty in rats with hippocampal perforant path systems disrupted. Behav Neurosci, in press, 1988. 12. Segal, M. and S. Landis. Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase. Brain Res 78: 1-15, 1974. 13. Van Hoesen, G. W. and D. N. Pandya. Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the Rhesus monkey. I. Temporal lobe afferents. Brain Res 95: 1-24, 1975. 14. Van Hoesen, G. W., D. N. Pandya and N. Butters. Cortical afferents to the entorhinal cortex of the rhesus monkey. Science 175: 1471-1473, 1972. 15. Van Hoesen, G. W., D. N. Pandya and N. Butters. Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the Rhesus monkey. II. Frontal lobe afferents. Brain Res 95: 25-38, 1975. 16. Vinogradova, O. S. Functional organization of the limbic system in the process of registration of information: Facts and hypotheses. In: The Hippocampus. Volume 2: Neurophysiology and Behavior, edited by R. L. Isaacson and K. H. Pribram. New York: Plenum Press, 1975, pp. 3-69. 17. Wyss, J. M. An autoradiographic study of the efferent connections of the entorhinal cortex in the rat. J Comp Neurol 199: 495-512, 1981.
0031-9384/88 $3.00 + .00
The Role of Medial and Lateral Hippocampal Perforant Path Lesions and Object Distinctiveness in Rats' Reaction to Novelty TROND MYHRER 1
N o r w e g i a n D e f e n c e R e s e a r c h Establishment, Division f o r Environmental Toxicology N-2007 Kjeller, N o r w a y R e c e i v e d 20 A p r i l 1987 MYHRER, T. The role of medial and lateral hippocampal perforant path lesions and object distinctiveness in rats' reaction to novelty. PHYSIOL BEHAV 42(4) 371-377, 1988.---The purpose of the present study was to investigate whether damage to the medial (MPP) or lateral hippocampal perforant path (LPP) may differentially affect rats' ability to react to novelty or environmental change. Three different types of task were used based on various sets of stimuli; visual/tactile, olfactory, or visual only. The results showed that the lesions produced different responses to different novel stimuli. In contrast to control and MPP animals, LPP rats displayed excessive exploration of the test cage. The results are discussed in terms of impoverished cognitive structure following perforant path disruptions and functional differentiation between MPP and LPP. Perforant path lesions
Reaction to novelty
Exploratory behavior
have access to polymodal sensory information from the primary and secondary association areas of the neocortex [14]. Exploration of a discrete novel object is one form of inquisitive activity frequently seen among rats. This activity appears as a strong preference for novelty, the recognition of which is probably based on polymodal sensory information [3]. In a recent study, it was found that disruption of LPP produced a marked decrease in preference for novelty, whereas MPP lesions only caused a slight deficit in this respect [11]. However, MEC lesions have been reported to result in a pronounced reduction in preference for novelty [ 10]. A possible explanation of these discrepant findings may be that the difference between neutral and novel objects was only slight in the latter study (toy cars slightly different in form) and more conspicuous in the former (closed vs. open metal rectangle). The test procedures for the two studies were otherwise similar. Thus, it may be hypothesized that whether damage to pefforant path systems affects preference for novelty is associated with object distinctiveness in terms of the degree of differences among the discriminanda. On the basis of previous findings [11] it may be expected that MPP rats are most affected in tasks with low level of distinctiveness, whereas LPP animals are vulnerable in a larger number of tasks. The present study was carried out to test these hypotheses by applying a modified version of the novelty
T H E hippocampal formation has been suggested to integrate signals from the reticulo-septal projection with highly processed neocortical information entering the hippocampus via the entorhinal area (cf. [16]). The latter projection is organized in several elements. The orbitofronal cortex and temporal cortex project to the entorhinal cortex [13-15]. The hippocampus proper and fascia dentata receive their major extrinsic fiber input from the entorhinal cortex by way of the perforant path (cf, [4]). This pathway is composed of two distinct fiber systems which both have fields of termination along the whole axial extent of the hippocampal formation. The medial perforant path (MPP) arises in the medial entorhinal cortex (MEC) and terminates in the middle of the dentate molecular layer and in the deep part of the stratum lacunosum-moleculare of subfield CA3 [7]. The lateral perforant path (LPP) originates in the lateral entorhinal cortex (LEC) and projects to a superficial zone in the dentate molecular layer and to the superficial portion of the stratum lacunosum-moleculare of CA3 [6] (Fig. 1). These differences in fields of termination among MPP and LPP coupled with findings of different neocortical sources of input to MEC and L E C [14] may constitute a morphological substrate for functional differences between MPP and LPP. The findings of neocortical projections to the entorhinal cortices have given rise to the suggestion that the hippocampal formation may
1Requests for reprints should be addressed to Trond Myhrer, Norwegian Armed Forces, Psychological and Educational Centre, Oslo Mil/Akershus, 0015 Oslo 1, Norway.
371
372
MYHRER
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line. Each cannula was inserted into the brain in a position deviating 20 ° from the vertical in the sagittal plane (tip of cannula pointing rostrally). From this position the syringe was moved ten times back and forth to make a cut of 1.0-1.5 mm. F o r medial pp lesions the movement was made in an approximate occipito-thalamic axis and for the lateral pp lesions in an approximate occipito-temporal axis. These maneuvers were carried out in three stages for medial lesions with the depths of cannula insertions being 4, 6 and 8 ram, respectively. The lateral lesions were made in two stages with insertion depths of 6 and 8 mm.
Histology
CA]
fim FIG. 1. Diagram of a horizonal section of the entorhinalhippocampal region showing intended sites of transection of medial pefforant path (MPP) and lateral perforant path (LPP). Abbreviations: LEC=lateral entorhinal cortex; MEC=medial entorhinal cortex; ab=angular bundle; fd=fascia dentata; fim=fimbra; sub =subiculum; p=parasubiculum. test of Berlyne [2]. Rats with MPP or LPP lesions and controis were run in three discrimination tasks based on different sets of stimuli; visual/tactile, olfactory, or visual only. METHOD
Subjects Twenty-four male Wistar rats from a commercial supplier (Dyrlaege M011egaards Avlslaboratorium, Denmark), weighing 290-320 g at the time of surgery, served as subjects. They were randomly assigned to three groups: 8 animals received bilateral lesions of the medial perforant path (MPP), 8 received bilateral lesions of the lateral perforant path (LPP), and 8 served as controls and had the scalp reflected only. The rats were housed individually and had free access to commercial rat pellets and water. The rats were handled individually 3 days preoperatively and 1 day postoperatively, being allowed to explore a table top (80x60 cm) for 3 min a day. The climatized (21°C) vivarium was illuminated from 0700 to 1900 hours.
Surgery The rats were anesthetized IP with diazepam (10 mg/kg) and fentanyl fluanisone (2 mg/kg) and placed in a stereotaxic head holder with their skulls horizontal. The lesions were made mechanically by means of the sharp edges of cannulas provided with collar. The cannula to be used was mounted on a syringe. The points of insertion were the following; for the medial perforant path lesions 9.1 mm posterior to bregma and 5.0 mm lateral to midline; for lateral perforant path lesions 8.5 mm posterior to bregma and 5.5 mm lateral to mid-
Upon termination of testing the brains were removed and frozen. The brains were sectioned horizontally on a CO2freezing microtome at 30/zm, every twelfth section being preserved. The sections were stained with the GrOnwald/Giemsa method. The percentage of perforant path damage was based on the location and extent of the transections in relation to the course of MPP and LPP fibers (cf. Fig. 1). The lesions were traced on diagrams of the perforant path fibers consisting of 20 lines evenly distributed in MEC or LEC, each line representing 5 percent of the fibers. The number of lines affected in the dorso-ventral levels presented in Fig. 2 were counted, and the average percentage of damage for each animal was computed.
Apparatus Behavioral testing was carried out in a Plexiglas cage (56×34×20 cm) with a metal mesh roof. The floor was divided in 18 equal squares (9× 11 cm). Three equal wooden cubes (5×5×5 cm) with a track in the under side made up the neutral objects. These were put on screweyes fixed to the floor in the middle of three squares evenly distributed in the cage. This arrangement spaced the objects in fixed positions with approximately the same distance from the walls and one another. Three other objects provided with corresponding tracks in the under side made up the novel objects. One object only differed from the neutral ones in that its top was uneven with tracks in it making up a square pattern (visual/tactile stimuli). One was identical with the neutrals and a spot of cheese was smeared on one side (olfactory stimulus). One was smaller than the neutrals (4.5x4.5x5.0 cm) and two sides were slightly uneven (visual stimulus). All objects were painted light grey. The only light was a 15 W bulb 60 cm above the apparatus. The sound attenuated testing room was provided with a fan producing background sound (52 dB).
Procedure Training started on Postoperative Day 10. The rats were allowed to explore individually the empty apparatus for 25 min. On the next day the rats were run in Session I. In Phase 1, the rats were tested for 5 min with the three neutral objects present. The following behaviors were recorded: number of seconds in contact with each object, number of squares traversed (locomotor activity), number of rearings, and duration of grooming. Then the rats spent 10 rain in the home cage. In Phase 2, the rats were tested again for 5 min, and one of the neutral objects had been replaced by the novel object with uneven top. During this period the same measures as in Phase 1 were made. In Sessions II and III (Postoperative Days 12 and 13), the same procedure was followed
PERFORANT PATH AND NOVELTY
373 in both groups. Further, degeneration was seen among the stellate ceils in layer II and to some extent the pyramidal cells in layer III in MEC or LEC.
Behavior
FIG. 2. Representative examples of medial perforant path lesion (A) and lateral perforant path lesion (B). and the novelty was represented by a spot (diameter 1.5 cm) of cheese on one side of the object on Day 12 and a smaller object on Day 13. So-called Norwegian white cheese which hardly smells anything to humans was used. In the test cage, it was not possible to detect the cheese spot visually. To exclude effects of positional preferences the novel object was for each session placed in a position never used in preceding sessions. A pre-arranged random order for the position of novel object was made for each animal. Prior to each time a rat was placed in the apparatus the cage and objects were carefully cleaned and allowed to dry. The behaviors were hand scored by the author who was unaware of the rats' group assignment.
Statistics Overall analyses were carded out with parametric oneway ANOVA or nonparametric one-way ANOVA (Kruskal-Wallis). Group comparisons were made with twotailed t-test or two-tailed Mann-Whitney U-test. RESULTS
Histology In the MPP group, the lesion of the medial perforant path appeared as a cut in the medial entorhinal area (Fig. 2A). The medial perforant path was affected bilaterally in the entire dorso-ventral extent in all animals. Additional damage appeared as a section of both MPP and LPP systems in the ventral-most tip of ~he hippocampal formation. This damage occurred bilaterally in two rats and unilaterally in one. The mean percentage of lesion efficiency was 87 (range 65-100). In the LPP group, the lateral perforant path was disrupted bilaterally in the entire dorso-ventral extent in all animals (Fig. 2B). As for some of the MPP animals, both perforant path systems in the ventral-most tip of the hippocampal formation were occasionally damaged. This damage was observed unilaterally in four rats. Furthermore, an additional small section was occasionally observed in parts of the subiculum. The mean percentage of lesion efficiency was 86 (range 75-100). The tracks made by the cannulas in neocortex overlying the entorhinal areas were very slight and difficult to identify
Both the MPP group and the LPP group displayed reduced preference for novelty compared with the control group in all three sessions. However, the reduction in preference was more marked among the LPP rats than the MPP animals (Fig. 3). Exploration of neutral object is based on the mean time of contact with the two neutral objects. Group comparisons were made in terms of time difference between exploration of novel vs. neutral objects (Table 1). Because of uneven distribution of data nonparametric statistics were applied. In Session I (uneven top of novel object), Kruskal-Wallis ANOVA revealed a significant treatment effect, H(2) = 11.81, p <0.01. Mean comparisons with U-test showed that both MPP and LPP rats displayed significantly less preference for the novel object than the control group, U=9, p<0.02 and U = I , p<0.001, respectively. The LPP group also exhibited less preference for novelty than the MPP group, U=9, p<0.02, t-Test for dependent means only revealed significantly more exploration of novel than neutral objects among the control group, t(7)---4.74, p<0.01. In Session II (novelty smell of cheese), ANOVA revealed a reliable overall effect, H(2)---10.70, p <0.01. Both the MPP and LPP groups showed a significant reduction in preference for novelty relative to the control group, U= 11, p<0.05 and U=4, p<0.005, respectively. The two experimental groups did not differ reliably from one another. The control group and the MPP group explored the novel object significantly more than the neutral ones, t(7)=6.03, p<0.001 and t(7)=2.55, p <0.05, respectively. In Session Ill (smaller object), ANOVA showed a reliable treatment effect, H(2)=8.94, p<0.02. Both the MPP and LPP groups displayed significantly less preference for novelty than the control group, U= 10, p<0.05 and U=8, p<0.02, respectively. The LPP group also showed less preference for novelty than the MPP group, U= 10, p<0.05. The only significant difference in exploration of novel vs. neutral objects was that the LPP group explored the novel object less than the neutral ones, t(7)=3.74, p<0.01. The time spent in exploring neutral objects in Phase 2 was relatively constant from one session to another for all groups; in particular the control group (Fig. 3, Table 1). ANOVA revealed no significant differences among the groups in this respect for either of the three sessions. The time spent in exploring novel objects varied between the groups. Kruskal-WaUis ANOVA revealed a significant difference among the groups in Session I, H(2)=8.84, p<0.02. Mean comparisons showed that the LPP group explored the novel object significantly less than the control group and the MPP group, U=6, p<0.005 and U=12, p<0.05, respectively, whereas the control group and the MPP group did not differ reliably. A reliable treatment effect was also revealed in Session II, H(2)=6.54, p<0.05. The LPP group explored the novel object significantly less than the control group, U= 11, p <0.05. No other significant group differences were revealed in this respect, neither any reliable treatment effect in Session III. Table 1 shows the total time spent in exploring neutral objects in Phase 1 and neutral plus novel objects in Phase 2. No reliable treatment effect was revealed among the groups in any phase in any session. However, in Session I, the LPP group displayed a significant decrease in exploration from
374
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TABLE 1 MEAN MEASURES OF EXPLORATORY BEHAVIORIN SECONDS Total Time Exploring
Differential Time Exploring Session I
Session II
Session I
Session III
Session II
Session III
Group
N
Neut
Nov
Diff
Neut
Nov
Diff
Neut
Nov
Diff
Ph 1
Ph2
Ph 1
Ph2
Ph 1
Ph2
MPP LPP Cont
8 8 8
10.6 6.4 9.6
13.5 5.4 23.3
2.9 -1.1 13.6
11.4 14.0 9.1
20.6 16.0 29.1
9.4 2.0 20.0
12.8 13.0 9.5
12.3 10.0 18.4
0.0 -3.0 9.1
38. I 35.0 32.5
34.4 17.5 41.6
33.1 28.4 34.8
42.6 43.6 46.8
34.6 29.4 35.1
37.3 32.8 37.0
Ph=Phase; Neut=Neutral; Nov=Novel; Diff=Difference; MPP=Medial Perforant Path; LPP=Lateral Perforant Path; Cont=Control. Phase 1 to Phase 2, t(7)=4.26, p<0.01. In Session II, both the MPP group and the LPP group showed a significant increase in exploration from Phase 1 to Phase 2, t(7)=2.39, p < 0 . 0 5 and t(7)=5.57,p<0.001, respectively. In Session III, no significant differences were observed. Occasionally, rats urinated on the objects. This was seen among all groups and appeared during Phase 1 and/or Phase 2. As seen from FigJ 4A the LPP group tended to display more locomotor activity than the other groups. In Session I, Phase 1, one-way A N O V A revealed a reliable treatment effect, F(2,21)=4.68, p<0.02. Group comparisons with t-test showed that the LPP group was significantly more active than the control group and the MPP group, t(14)=3.24, p<0.01 and t(14)=2.93, p<0.02, respectively. The control group and the MPP group did not differ reliably from one another. A N O V A revealed no significant overall effect in Phase 2. Only the LPP group displayed a significant decrease in locomotor activity from Phase 1 to Phase 2, t(7)=2.98, p<0.05. In Session II, A N O V A revealed no significant differences in either of the two phases. However, both the control group and the LPP group had a significant decrease in activity from Phase 1 to Phase 2, t(7)=3.42, p < 0 . 0 2 and t(7)=3.63, p<0.01, respectively. In Session III, A N O V A only revealed significant treatment effect in Phase 2, F(2,21)=4.03, p<0.05. Group comparisons showed that the LPP group was significantly more active than the control
group and MPP group, t(14)=2.75 and t(14)=2.93, p<0.02, respectively. The control group and the MPP group was not reliably at variance. No groups displayed significant decrease in activity from Phase 1 to Phase 2. As seen from Fig. 4B the LPP group also tended to display more rearing activity than the other groups. In Session I Phase 1, one-way A N O V A revealed a reliable treatment effect, F(2,21) =4.01, p <0.05. Group comparisons showed that the LPP group made significantly more rearings than the control group and the MPP group, t(14)=6.37, p<0.001 and t(14)=4.27, p<0.001, respectively. The two latter groups were not reliably different. In Phase 2, A N O V A also revealed significant differences, F(2,21)=5.79, p<0.01. Mean comparisons showed that the LPP group made significantly more rearings than the control group and the MPP group, t(14)=7.07 and t(14)=5.89, respectively; p<0.001. The two latter groups did not differ reliably from one another. Only the LPP group made a significant decrease in rearings from Phase 1 to Phase 2, t(7)=2.76, p<0.05. In Session II, A N O V A revealed no significant differences for either of the phases, However, both the control group and the LPP group displayed a significant decrease in rearings from Phase 1 to Phase 2, t(7)=4.39, p<0.01 and t(7)=3.24, p<0.02, respectively. In Session III, A N O V A only revealed reliable treatment effect for Phase 2, F(2,21)=6.39, p<0.01. The LPP group displayed significantly more rearing activity than the
PERFORANT PATH AND NOVELTY
A
110
100
375
tt~
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TABLE 2
LPP
~
MEAN MEASURES OF GROOMING B E H A V I O R IN SECONDS
CONT
Session I
Session II
Session III
9O
=:
Group
N
Ph 1
Ph 2
Ph 1
Ph 2
Ph 1
Ph 2
MPP LPP Cont
8 8 8
6.4 1.6 13.5
0.5 0.0 6.1
22.9 1.6 14.1
5.3 2.3 12.3
11.3 3.4 14.6
2.5 9.0 12.3
llO 70 6O
Abbreviations as for Table 1. I
I
I
1
2
1
SESSION )
B
PHASE
I
I
I
2
1
2
SESSION I [
SESSION I l l
30
z
15
10 I
I
I
1
2
1
SESSION [
PHASE SESSION 11
I
I
t
2
I
2 SESSIONTIT
FIG. 4. Mean measures of locomotor activity (A) and rearing (B).
control group and the MPP group, t(14)=7.55 and t(14)=6.25, respectively; p<0.001. Both the control group and the MPP group displayed a reliable decrease in rearings from Phase 1 to Phase 2, t(7)=2.44, and t(7)=2.93, respectively; p <0.05. As seen from Table 2, the LPP group tended to display less grooming than the control group and the MPP group. However, Kruskal-Wallis A N O V A did not reach a statistically significant level for any phase in any of the sessions. Since the tendency for less grooming among the LPP rats was rather pronounced in Phase 1 for all sessions, the average scores of these three phases were calculated. On the basis of the average scores for Phase 1 in three sessions A N O V A revealed a significant treatment effect, H(2)=8.51, p<0.02. Group comparisons showed that the LPP group groomed significantly less than the control group and the MPP group, U = 10, p < 0 . 0 2 and U = 6 , p<0.005, respectively. The latter two groups did not differ significantly. The total time grooming for each session appeared to increase steadily for the LPP group. However, A N O V A of Grooming × Time interaction was not significant in this respect. DISCUSSION
Both medial (MPP) and lateral perforant path (LPP) lesions resulted in reduced preference for novelty. However, the LPP group was even more impoverished in responding to novelty than the MPP group. Further, the LPP rats displayed
more locomotor activity and rearing behavior than the control and MPP animals. The LPP group also tended to groom less than the control and MPP groups. The present findings are in accord with the notion that the degree of difference between neutral and novel objects appears crucial in revealing potential effects of decreased preference for novelty. In a previous study, in which the difference between the discriminanda was conspicuous MPP lesions only caused a slight discrimination deficit [11], whereas MEC lesions have been reported to produce a marked deficit in reaction to novelty when the difference between the discriminanda was very small [10]. The degree of preference for novelty was differentially affected by the two types of lesions. In Session I, both the MPP and LPP group displayed less preference for the novel object than the control group. However, the LPP group also exhibited reliably less preference for novelty than the MPP group. In Session II, both MPP and LPP groups showed a reduction in preference for novelty relative to the control group. However, the MPP group like the control group explored the novel object more than the neutral ones; this was not the case for the LPP group. In Session III, both MPP and LPP groups again displayed reduced reaction to novelty. It has been suggested that a deficit in inquisitive behavior may be expected if perforant path disruptions reduce the hippocampal formation's access to sensory information of neocortical origin [11]. In the latter study, the discrimination task was based on polymodal sensory stimuli. In the present study, a corresponding deficit was also seen when the discrimination task was based on a single sensory modality. The idea of sensory information reaching the hippocampal formation by way of the perforant path also gains support from an electrophysiological study. Deadwyler et al. [5] have shown that septal and entorhinal inputs to the dentate gyrus are activated differentially by sensory stimuli as a function of their acquired behavioral significance to the rat. The decreased preference for novelty among the experimental groups may be accounted for in other ways than reduced sensory information to the hippocampal formation. Exploratory activity is generally counteracted by fear [3]. Thus, it may be suggested that the novel object was less inspected because it elicited fear among the experimental subjects. This suggestion does not appear plausible in view of the fact that no significant differences were observed among the groups in exploring objects in Phase 1 of Session I when all objects were novel to the animals (Table 1). It may be argued that perforant path lesions caused decreased curiosity. However, this argument does not gain support from the present data, since the level of exploring neutral objects in Phase 1 and Phase 2 did not differ signifi-
376 cantly among the groups for any session (Table 1; Fig. 2). F o r the same reason it is difficult to maintain that the experimental animals were less attentive or aroused than the control rats. A deficit in short-term or long-term memory may represent a potential explanation of reduced preference for novelty among the experimental animals. However, the short-term aspect of this explanation does not seem to be reconcilable with the fact that the MPP group explored the novel object significantly more than the neutral ones in Session II, indicating that these rats still were able to discriminate provided a proper task. The LPP group also demonstrated an ability to discriminate, inasmuch as this group explored the novel object less than the neutral ones in Session III. This latter finding is not readily accounted for. Further, both experimental groups did not differ from the control group in exploring neutral objects in Phase 1 or Phase 2 in any session, indicating intact long-term memory. Among the experimental groups the LPP animals differed most markedly from the control rats. In addition to being the most impoverished subjects in preference for novelty the LPP rats also displayed high level of locomotor and rearing activity (Fig. 4). This activity which reached a peak in Phase 1 in Session I may explain the relatively low level of exploring neutral objects at this event; the LPP animals were highly engaged in exploring the test cage as such. The low level of grooming activity during Phase 1 for each session among the LPP rats may be taken as supportive for this explanation. The MPP rats did not differ from the control animals in terms of locomotor, rearing, or grooming activity. Thus, even if the MPP group also displayed reduced ability to respond to novelty, there are qualitative differences between the behaviors of the LPP group and the MPP group. It appears that MPP rats may be better off concerning access to sensory information than the LPP animals are and thus exhibited a smaller need to explore the surroundings in order to achieve cognitive structure of the environment. As seen from Fig. 4 the locomotor activity and rearing behavior generally decreased from Phase 1 to Phase 2 among all groups. This decrement may be associated with the presentation of novelty or habituation to the environment (following ten minutes in the home cage) or an interaction between these two factors. In the control group, however, the total time exploring objects only increased 7.7 sec from Phase 1 to 2 on the average for all three sessions. This small increase in exploration within a total testing time of 5 min probably indicates that the decrement in locomotor and rearing activity reflects habituation to the environment rather than preoccupation of the novelty. The MPP group followed very much the same pattern as the control group, whereas the LPP group differed somewhat. Since the experimental animals were less aware of the novelty than the control rats, habituation probably represents a plausible explanation of decreased locomotor and rearing activity for these subjects as well. In a previous study dealing with perforant path systems, both MPP and LPP rats displayed significantly more locomotor activity than the control animals [11]. In this study, LPP rats groomed more as a function of time. These results are discrepant with the present ones in which MPP rats did not exhibit increased locomotor activity and LPP animals groomed less than the rats from the other groups. These
MYHRER discrepancies are most likely due to different test procedures. In the previous study [11], the testing time each day was continuous for 15 min instead of two times for 5 rain. It appears that the longer test exposure provides more opportunity to reveal potential changes in, for instance, activity after perforant path lesions. This seems to be in accordance with the extremely high level of both locomotor and rearing activity seen among LPP rats in the previous study [11]. The behavioral differences between MPP and LPP rats can hardly be attributed to lesion efficiency, since the percentage of damage was very much the same; 87 and 86, respectively. The medial entorhinal cortex is far larger than the lateral one and LPP lesions produced the most marked effects. It is likely that the behavioral differences between MPP and LPP animals are due to different sources of input to the two entorhinal cortices. LEC receives fibers from orbito-frontal cortex and large areas of the temporal cortex, whereas MEC only receives fibers from the caudal part of temporal cortex [13-15]. Further, MPP and LPP terminate differently in the hippocampal formation (cf. the Introduction section). Thus, there seems to be a morphological substrate for functional differences between MPP and LPP. Projections from the neocortical association areas to the entorhinal cortex have been found in the monkey [14]. Corresponding projections have not been revealed in the rat with the horseradish peroxidase (HRP) labeling technique, but the existence can not be precluded since certain cells fail to transport HRP in detectable quantitities [1]. Actually, Beckstead [1] refers to unpublished findings that tritiated amino acids, when injected into either medial prefrontal or anterior cingulate cortex in the rat, are transported so as to label axons in LEC in an autoradiographically detectable manner. Extra-hippocampal fiber systems which may be differently affected by MPP and LPP lesions have been reported to run in angular bundle. One system projects from both MEC and LEC to nucleus accumbens [9], and another system projects from LEC only to cortical areas and amygdala [17]. The projections to nucleus accumbens originating in MEC and LEC are disrupted by MPP or LPP lesions, respectively. However, the projection from LEC to cortical areas and amygdala appears to enter distal parts of angular bundle and thus avoids damage from LPP sections. Accordingly, the behavioral differences seen among MPP and LPP animals are likely not due to different damage to extrahippocampal fibers made by MPP or LPP lesions. In the present study, degeneration was seen clearly among the stellate cells in layer II and to some extent among the pyramidal cells in layer III in MEC or LEC after MPP or LPP lesions, respectively. These findings correspond with the notion that the perforant path fibers arise predominantly in layer II and to some degree in layer III in the entorhinal cortices [12]. There is a marked projection from LEC to temporal cortex, whereas M E C ' s contribution is very modest in this respect [8]. This relation might imply that LEC is associated with memory processes. Such an association may explain at least some of the powerful effects seen after LPP lesions. A memory component may interact with decreased sensory information to the hippocampal formation resulting in the apparently pronounced deficit to recognize environmental change.
PERFORANT
PATH AND NOVELTY
377 REFERENCES
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