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Late post-learning effect of entorhinal cortex electrical stimulation persists despite destruction of the perforant path
174
Brain Research, 310 (1984) 174-179
Elsevier BRE20369
Late post-learning effect of entorhinal cortex electrical stimulation persists despite destruction of the perforant path MONIQUE GAUTHIER and CLAUDE DESTRADE Laboratoire de Psychophysiologie, Universit~ de Bordeaux 1, 33405 Talence Cedex (France)
(Accepted May 8th, 1984) Key words: post-training stimulation - - entorhinal cortex - - perforant path lesion - - operant conditioning - - mice
In an appetitive learning task in mice, stimulation of the lateral entorhinal cortex (LEC) 30 min after training produced an improvement in retention 24 h later, as well as faster extinction of conditioning, This effect persisted in animals with bilateral lesions of the perforant path. In addition, the threshold for hippocampal after-discharges produced by LEC stimulation was raised significantlyin perforant-path lesioned animals. The results indicate a functional dissociation between hippocampal and cortical mechanisms involved in memory consolidation.
When post-trial electrical stimulation of the brain is used, there is some evidence in the literature that the entorhinal cortex is involved in late mnemonic processes2,8,15. This suggestion received further confirmation by our recent observation, using appetitive operant conditioning tasks in mice, that a subseizure electrical stimulation of the lateral entorhinal cortex (LEC) delivered 30 min after a brief learning session produced a clear improvement of performance in a retention test 24 h later. Conversely, the stimulation was totally ineffective when delivered 30 s or 3 h after the learning session8. This facilitatory effect was quite different from the one observed after hippocampal6,14, hypothalamic3,11 or septal 7 electrical stimulation in so far as in that case the temporal gradient of the facilitation was brief (less than 10 min). As the entorhinal cortex is the major cortical structure which projects to the hippocampus and the dentate gyrus, by the way of the perforant path (pp)10,22, the mechanisms involved in the behavioral effects of delayed L E C stimulation remained unclear. Entorhinal stimulation could either involve some kind of reverberatory reactivation of the hippocampus through the perforant path or indicate a specific in-
volvement of the L E C in late information processing8. To clarify these issues, an attempt was made to dissociate entorhinal and hippocampal mechanisms through behavioral investigation. The facilitatory effect of a subseizure electrical stimulation of the L E C 30 min after training was examined on retention performance 24 h later in animals with perforant-path lesions. In addition, an electrophysiological control study was carried out in order to determine the effectiveness of the PP lesions. The subjects were 58 male B A L B / c mice, 40-50 days old at the time of surgery. Eight days prior to surgery, they were housed in individual cages with ad libitum access to food and water in a climatized room (21 °C) maintained on a light-dark cycle (12 h - 1 2
h). According to previously described procedures 8,9, the 58 animals were operated under general anesthesia (sodium penthiobarbital: 100 mg/kg). Twentytwo subjects were bilaterally implanted with bipolar electrodes in the ventral part of the lateral entorhinal cortex. Twenty-seven mice received bilateral lesions of the perforant path; additionally in 15 of these sub-
Correspondence: M. Gauthier, Laboratoire de Psychophysiologie, Universit6 de Bordeaux I, avenue des Facult6s, 33405 Talence
Cedex, France. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
175 jects, two bipolar electrodes were located in the LEC. Nine animals served as sham-operated controis; the electrodes were lowered into the PP bilaterally but no current was passed. The bipolar LEC electrodes were made from insulated platinum wire 90gm in diameter; the PP lesions were produced by passing anodal high frequency (500 kHz) current (0.05 mA) through the tip of an epoxylite-coated stainless-steel electrode (0.35 mm diameter, 0.1 mm bare tip) for 12 s. For each target structure, the stereotaxic coordinates with respect to Bregma were the following: perforant path: -3.6 mm in the antero-posterior plane (AP), laterally (L) 3.0 mm and vertically (V) 2.4 mm below the surface of the skull; lateral entorhinal cortex: (AP -3.0 mm, (L) 3.0 mm, (V) 4.9 mm at an angle of 12° to the sagittal plane. In both cases, the incisor bar was level with the interaural line. The first experiment focused on the effects of perforant path lesions on afterdischarge (AD) thresholds. Using a previously described procedure 8, 8 days after surgery, 12 mice (6 with bilateral LEC electrodes and 6 with LEC electrodes plus PP lesions) were subjected to a single daily bilateral stimulation of the LEC in order to determine their individual A D threshold. 100 Hz sine-wave stimulation was delivered to both entorhinal cortices in trains of 200 ms alternating with 200 ms of no stimulation, for a total of 4 s. Each electrode could be connected either to the stimulator or to the inputs of a two-channel polygraph. On the first day, seizure thresholds were determined by starting with a 3 0 g A (peak to peak) current delivered to both LEC electrodes connected in parallel to the stimulator (15/~A per electrode). Each day thereafter, the current intensity was increased by 30 ~ A steps until afterdischarges were elicited. The results are presented in Table I. In control animals, the AD thresholds ranged from 90 to 120 /~A. These values were significantly increased in animals with lesions of the perforant path (U = 34.5, P < 0.005). Current intensities up to 240 g A failed to induce afterdischarges in 3 of the 6 PPlesioned mice. In the second experiment, the remaining 46 subjects were trained in an operant lever-press task with food reward (5 mg pellets). Two weeks after surgery, a food deprivation schedule was initiated gradually over 4 days so that, at the time of the experiment, the
TABLE I Effect of perforant-path lesions on threshold for afterdischarges elicited by LEC stimulation. Afterdischarge threshold
Control group (LEC)
Experimental group (LEC + PP lesion)
Median values (~A)
110
200**
Interquartile range
90-120
150-240
** P < 0.005 mice weighed 82 to 84% of their ad libitum weight. As previously described 12, the learning task consisted of partial acquisition, retention, and extinction of operant conditioning in a Skinner box. The apparatus has been described in detail elsewhere 6. The Skinner box (14 × 14 × 18 cm high) had a food dispenser which differed somewhat from the standard ones in that it was equipped with a device which permitted the removal of any pellets that were not eaten. The lever and the food cup were separated by a 5-cm long partition, so the animal had to turn around to press the lever or to find food. Photocells detected the animal's presence in front of the lever or the food cup. A continuous reinforcement (CRF) schedule was used. Lever-presses which were not followed by an approach towards the food-cup within 10 s ('complete' responses) were not counted. All the animals underwent an initial acquisition session in which they were allowed 16 'complete' responses. They were fed 1 h after each learning session so as to maintain a constant body weight. To this end, stimulated, non-stimulated and PP-lesioned animals received the same amount of food so that there were no weight differences on the following training days. The 46 animals were divided into 5 groups corresponding to the treatment applied immediately after the acquisition session; there were 3 experimental and two control groups: 12 PP-lesioned subjects (PP. Les), 10 LEC stimulated subjects (LEC ST), 9 subjects with PP lesion and LEC stimulation (PP Les + LEC ST), 6 LEC non-stimulated subjects (nST), 9 sham-operated subjects (sham). The post-training treatment was administered in a waiting box, adjacent to the Skinner box, 30 rain after the end of this first session. Based on the results of the first (see Table I) and previous experiments8, animals in the stimulated groups received bilateral, discontinuous (200 ms on, 200 ms off) brain stimulation (100 Hz) de-
176 livered for an 80 s period which consisted of 10 trains of 4 s of stimulation alternating with 4 s of no stimulation. For all animals, the current intensity was half of the median value recorded in control animals in the first experiment; i.e. 55/~A delivered to both entorhinal cortices connected in parallel to the stimulator (27.5 /~A/electrode). The E E G was monitored for each animal to verify that no afterdischarges were present. Retention testing was carried out 24 h and 48 h later during a 15-min lever-pressing session. Fig. 1 summarizes the results. No significant difference appeared between sham and nST subjects during the first (F(1,13) = 1.61, n.s.) or second retention sessions (F(1,13) = 0.05, n.s.). These scores were pooled and the 15 subjects served as a single control group. During the first retention test, significant differences among the 4 groups were observed (F(3,42) = 37.94, P < 0.001) and there were significant differences among the groups in the progression of re-
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•
RETENTION 1 O-
;,
,'o
PP. L e m + L E C
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RETENTION 2
,'5 TIME
~
,'o
,~
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Fig. 1. Effects of LEC stimulation alone or combined with perforant-path lesions on retention scores measured during two sessions 24 h and 48 h after partial learning of CRF conditioning. Each session is divided into three 5-min periods (small bars indicate the standard error of the mean).
sponding over the three 5-min periods (treatment × period interaction: F(6,84) = 17.44, P < 0.001). In addition, significant treatment × period interactions were found when the scores of the control group and of each experimental group were compared. At the start of the retention test (first 5-min period), a slight impairment of performance was observed in the PP Les group (PP vs Control: F(1,25) = 11.86, P < 0.01), whereas L E C ST animals and PP Les + L E C ST animals showed an improvement (P < 0.001 in both cases). However, stimulated animals with PP lesions performed less than L E C ST animals (F(1,17) = 17.98, P < 0.001). During the second retention session, all the groups reached the same performance level (F(3,42) = 0.80, n.s.).
For all the subjects, retention testing was continued until they reached the criterion of 200 'complete' responses 4. During the next few days, the 46 mice underwent a daily 6-min session of extinction of the C R F conditioning. The LEC-implanted animals (LEC ST and PP Les + L E C ST groups) were stimulated 30 min after the end of the first 6-min extinction session, using the same parameters (discontinuous stimulation, 55 ~ A intensity) as in the first part of the experiment. No disruption was observed in the E E G records. All the subjects were fed 1 h after each extinction session so as to maintain a constant body weight (82-84% of their ad libitum weight at the start of each extinction session). The scores were calculated as the number of 'complete' responses per min. Fig. 2 summarizes the results. No difference was found between sham-operated and non-stimulated subjects over the 3 extinction sessions (1st session: F(1,13) -- 0.11, n.s.; 2nd session: F(1,13) = 0.37, n.s.; 3rd session: F(1,13) = 0.03, n.s.); thus the 15 subjects served as one control group. No differences appeared among the 4 groups during the first extinction session (F(3,42) = 0.12, n.s.). Twenty-four h later, no significant differences were found between controls and PP-lesioned animals (F(1,25) = 1.54, n.s.). Compared to the control animals, the mice that received the L E C electrical stimulation 30 min after the first extinction exhibited faster extinction of conditioning (F(1,23) = 10.67, P < 0.005 for the L E C ST group and F(1,22) = 14.73, P < 0.001 for the PP Les + L E C ST group). In both
177 7-
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Fig. 2. Effects of LEC stimulation alone or combined with perforant-path lesions on extinction scores measured during three 24 h delayed sessions.
cases; there were significant differences among the groups in the progression of responding over the 6 1-min periods (treatment x period interaction, P < 0.05). No differences were found between LEC ST and PP Les + LEC ST animals (F(1,17) = 0.31, n.s.). During the 3rd extinction session there were no significant differences among the 4 groups. The histological analysis showed that all electrode tips were located in the ventral part of the lateral entorhinal cortex. The lesioned area was measured at 3.6 mm posterior to Bregma using a planimeter for every subject. This area fell vertically between 1.9 and 2.5 mm and the average size of bilateral lesions ranged from 0.38 mm 2 to 0.45 mm 2. There was no significant difference in the distribution of the lesions between PP Les and PP Les + LEC ST groups. A reconstruction of the lesioned area in a representative animal is shown in Fig. 3. The present results show that the effect of LEC stimulation that is delayed 30 min persisted in spite of bilateral lesion of the perforant path. Compared to
control animals, the PP Les + LEC ST subjects showed, as did the non-lesioned LEC ST animals, a clear improvement in retention and exhibited a faster extinction of conditioning. Several issues must be considered before the implications of our data can be discussed. First of all, the finding that animals with PP lesions show poorer retention of operant conditioning does not agree with the results obtained after lesions of the LEC 9. The deficit seen at the beginning of the retention session (Fig. 1) may be due to the method used; in the present procedure, all animals received the same amount of limited information (16 reinforced responses) 30 min before the stimulation of the LEC. In particular, the reminiscence phenomenon (timedependent improvement in retention performance6,12) observed in control animals did not occur in the animals that had undergone lesion of the perforant path. In other words, during an appetitive test, the PP lesion may interact with the consolidation of a response which is not fully learned. Under these conditions the significant difference observed between
178
1.900
Fig. 3. Drawings of horizontal sections of the mouse brain showing the placement and the extent of the lesion (dotted area). 1.900, 2.200 and 2.500 = distances (in mm) of the section from the surface of the skull.
for afterdischarges triggered in the hippocampus b y stimulation of the lateral entorhinal cortex in PP-lesioned animals. We thus have here a good index for the rapid verification of perforant-path lesions before behavioral testing. In summary, our results show up a functional dissociation between hippocampal and cortical mechanisms involved in memory facilitation produced by stimulation of the lateral entorhinal cortex; they argue in favor of a specific involvement of this cortical region in delayed information processing. It is difficult to see, however, from a functional-anatomical point of view, how electrical stimulation of the entorhinal cortex could bring into play mechanisms that are strictly limited to the stimulated area. It is more likely that the electrical stimulation, which can no longer reach the hippocampus after perforant-path lesion, reveals the recruitment of cortical or subcortical regions that are directly or indirectly connected with the entorhinal cortex. For example, it is known that there are reciprocal connections between the entorhinal cortex and the (cingulate) retrosplenial area 5,16,18. We have recently shown that the cingulate cortex, whose posterior part is apparently a special region participating in the organization of attention and memory 1.21, is involved in memory consolidation in mice 4. However, it should be pointed out that there are numerous other projections of the entorhinal cortex, besides the cingulate cortex, to cortical areas other than the hippocampus 13 or to subcortical regions 19,20 that are indirectly connected by the Papez circuit 2~. In conclusion, our results support the hypothesis of a delayed recruitment of one or several cortical loops in information processing mechanisms. This recruitment apparently does not involve a direct reactivation of the hippocampus.
L E C ST and PP Les + L E C ST groups could result from the disruptive effect of the PP lesion. This difference is no longer present when one looks at the performance in extinction for these two groups of animals. Similarly, the PP-lesioned animals behave like the control animals. This result comes close to a finding by Myrher 17 that PP lesion did not disrupt passive avoidance conditioning. Once again, however, this result does not agree with the data obtained after L E C lesion9,17. According to Myrher's hypothesis, the entorhinal lesion, but not PP lesion, can interact with the extinction of a well-learned response. Besides the behavioral results, the effectiveness of the lesion is indicated by the anatomical (Fig. 3) and electrophysiological data we collected. The first experiment showed a significant rise in the thresholds
We thank Mrs. Ducout and Mrs. Perret for technical assistance and Dr. M. Caudarella for help in the preparation of the manuscript. This research was supported by the C.N.R.S. (L.A. no 339).
1 Baleydier, C. and Mauguiere, F., The duality of the cingulate gyrus in monkey, Brain, 103 (1980) 525-554. 2 Collier, T. J. and Routtenberg, A., Entorhinal cortex electrical stimulation disrupts retention performance when applied after but not during learning, Brain Research, 152
(1978) 411-417. 3 Destrade, C., Two types of diencephalically driven RSA (theta) as a means of studying memory formation in mice, Brain Research, 234 (1982) 486-493. 4 Destrade, C. and Gauthier, M., Facilitation de la r6tention
179
5
6
7
8
9
10
11
12
et accrlrration de l'extinction d'un conditionnement oprrant apr~s 16sion du cortex cingulaire chez la souris BALB/c, C.R. Acad. Sci. Paris, 293 (1981) Srrie III, 843-846. Destrade, C. and Ott, T., Is a retrosplenial (cingulate) pathway involved in the mediation of high frequency hippocampal rhythmical slow activity (theta)?, Brain Research, 252 (1982) 29-37. Destrade, C., Soumireu-Mourat, B. and Cardo, B., Effects of post-trial hippocampal stimulation on acquisition of operant behavior in the mouse, Behav. Biol., 8 (1973) 713-724. Galey, D., Jeantet, Y., Destrade, C. and Jaffard, R., Facilitation of memory consolidation by post-training electrical stimulation of the medial septal nucleus: is it mediated by changes in rhythmic slow activity?, Behav. Neural Biol., 38 (1983) 240-250. Gauthier, M., Destrade, C. and Soumireu-Mourat, B., Late post-learning participation of entorhinal cortex in memory processes, Brain Research, 233 (1982) 255-264. Gauthier, M. and Soumireu-Mourat, B., Behavioral effect of bilateral entorhinal cortex lesions in the BALB/c mouse, Behav. Neural Biol., 33 (1981) 419-436. Hjorth-Simonsen, A. and Jeune, B., Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation, J. cornp. Neurol., 144 (1972) 215-232. Huston, J. P., Mueller, C. and Mondadori, C., Memory facilitation by post-trial hypothalamic stimulation and other reinforcers: a central theory of reinforcement, Biobehav. Rev., 1 (1977) 143-150. Jaffard, R., Destrade, C., Soumireu-Mourat, B. and Cardo, B., Time-dependent improvement of performance on
appetitive tasks in mice, Behav. Biol., 11 (1974) 89-100. 13 Kosel, K. C., Van Hoesen, G. W. and Rosene, D. L., Nonhippocampal cortical projections from the entorhinal cortex in the rat and rhesus monkey, Brain Research, 244 (1982) 201-214. 14 Landfield, P. W., Tusa, R. J. and McGaugh, J. L., Effects of post-trial hippocampal stimulation on memory storage and EEG activity, Behav. Biol., 8 (1973) 485-505. 15 Martinez, J. L., Jr., McGaugh, J. L., Hanes, C. L. and Lacob, J. S., Modulation of memory processes induced by stimulation of the entorhinal cortex, Physiol. Behav., 19 (1977) 139-144. 16 Meibach, R. C. and Siegel, A., Subicular projections to the posterior cingulate cortex in rats, Exp. Neurol., 57 (1977) 264-274. 17 Myrher, T., Locomotor and avoidance behavior in rats with partial or total hippocampal perforant paths sections, Physiol. Behav., 15 (1977) 217-224. 18 Sorensen, K. E., Ipsilateral projection from the subiculum to the retrosplenial cortex in the guinea pig, J. comp. Neurol., 113 (1980) 893-911. 19 Sorensen, K. E. and Turner, B., Entorhinal efferents to widespread cortical and subcortical structures in guinea pig and rat, Neurosci. Lett., Suppl. 7 (1981) S 45. 20 Sorensen, K. E. and Witter, M. P., Entorhinal efferents reach the caudato-putamen, Neurosci. Lett., 35 (1983) 259-264. 21 Stafekhina, V. S., Effect of electrical stimulation of the hippocampus and neocortex on cingulate cortical neurons in rabbits, Neirofiziologiya, 14 (1982) 270-277. 22 Steward, O., Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. comp. Neurol., 167 (1976) 285-314.
Brain Research, 310 (1984) 174-179
Elsevier BRE20369
Late post-learning effect of entorhinal cortex electrical stimulation persists despite destruction of the perforant path MONIQUE GAUTHIER and CLAUDE DESTRADE Laboratoire de Psychophysiologie, Universit~ de Bordeaux 1, 33405 Talence Cedex (France)
(Accepted May 8th, 1984) Key words: post-training stimulation - - entorhinal cortex - - perforant path lesion - - operant conditioning - - mice
In an appetitive learning task in mice, stimulation of the lateral entorhinal cortex (LEC) 30 min after training produced an improvement in retention 24 h later, as well as faster extinction of conditioning, This effect persisted in animals with bilateral lesions of the perforant path. In addition, the threshold for hippocampal after-discharges produced by LEC stimulation was raised significantlyin perforant-path lesioned animals. The results indicate a functional dissociation between hippocampal and cortical mechanisms involved in memory consolidation.
When post-trial electrical stimulation of the brain is used, there is some evidence in the literature that the entorhinal cortex is involved in late mnemonic processes2,8,15. This suggestion received further confirmation by our recent observation, using appetitive operant conditioning tasks in mice, that a subseizure electrical stimulation of the lateral entorhinal cortex (LEC) delivered 30 min after a brief learning session produced a clear improvement of performance in a retention test 24 h later. Conversely, the stimulation was totally ineffective when delivered 30 s or 3 h after the learning session8. This facilitatory effect was quite different from the one observed after hippocampal6,14, hypothalamic3,11 or septal 7 electrical stimulation in so far as in that case the temporal gradient of the facilitation was brief (less than 10 min). As the entorhinal cortex is the major cortical structure which projects to the hippocampus and the dentate gyrus, by the way of the perforant path (pp)10,22, the mechanisms involved in the behavioral effects of delayed L E C stimulation remained unclear. Entorhinal stimulation could either involve some kind of reverberatory reactivation of the hippocampus through the perforant path or indicate a specific in-
volvement of the L E C in late information processing8. To clarify these issues, an attempt was made to dissociate entorhinal and hippocampal mechanisms through behavioral investigation. The facilitatory effect of a subseizure electrical stimulation of the L E C 30 min after training was examined on retention performance 24 h later in animals with perforant-path lesions. In addition, an electrophysiological control study was carried out in order to determine the effectiveness of the PP lesions. The subjects were 58 male B A L B / c mice, 40-50 days old at the time of surgery. Eight days prior to surgery, they were housed in individual cages with ad libitum access to food and water in a climatized room (21 °C) maintained on a light-dark cycle (12 h - 1 2
h). According to previously described procedures 8,9, the 58 animals were operated under general anesthesia (sodium penthiobarbital: 100 mg/kg). Twentytwo subjects were bilaterally implanted with bipolar electrodes in the ventral part of the lateral entorhinal cortex. Twenty-seven mice received bilateral lesions of the perforant path; additionally in 15 of these sub-
Correspondence: M. Gauthier, Laboratoire de Psychophysiologie, Universit6 de Bordeaux I, avenue des Facult6s, 33405 Talence
Cedex, France. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
175 jects, two bipolar electrodes were located in the LEC. Nine animals served as sham-operated controis; the electrodes were lowered into the PP bilaterally but no current was passed. The bipolar LEC electrodes were made from insulated platinum wire 90gm in diameter; the PP lesions were produced by passing anodal high frequency (500 kHz) current (0.05 mA) through the tip of an epoxylite-coated stainless-steel electrode (0.35 mm diameter, 0.1 mm bare tip) for 12 s. For each target structure, the stereotaxic coordinates with respect to Bregma were the following: perforant path: -3.6 mm in the antero-posterior plane (AP), laterally (L) 3.0 mm and vertically (V) 2.4 mm below the surface of the skull; lateral entorhinal cortex: (AP -3.0 mm, (L) 3.0 mm, (V) 4.9 mm at an angle of 12° to the sagittal plane. In both cases, the incisor bar was level with the interaural line. The first experiment focused on the effects of perforant path lesions on afterdischarge (AD) thresholds. Using a previously described procedure 8, 8 days after surgery, 12 mice (6 with bilateral LEC electrodes and 6 with LEC electrodes plus PP lesions) were subjected to a single daily bilateral stimulation of the LEC in order to determine their individual A D threshold. 100 Hz sine-wave stimulation was delivered to both entorhinal cortices in trains of 200 ms alternating with 200 ms of no stimulation, for a total of 4 s. Each electrode could be connected either to the stimulator or to the inputs of a two-channel polygraph. On the first day, seizure thresholds were determined by starting with a 3 0 g A (peak to peak) current delivered to both LEC electrodes connected in parallel to the stimulator (15/~A per electrode). Each day thereafter, the current intensity was increased by 30 ~ A steps until afterdischarges were elicited. The results are presented in Table I. In control animals, the AD thresholds ranged from 90 to 120 /~A. These values were significantly increased in animals with lesions of the perforant path (U = 34.5, P < 0.005). Current intensities up to 240 g A failed to induce afterdischarges in 3 of the 6 PPlesioned mice. In the second experiment, the remaining 46 subjects were trained in an operant lever-press task with food reward (5 mg pellets). Two weeks after surgery, a food deprivation schedule was initiated gradually over 4 days so that, at the time of the experiment, the
TABLE I Effect of perforant-path lesions on threshold for afterdischarges elicited by LEC stimulation. Afterdischarge threshold
Control group (LEC)
Experimental group (LEC + PP lesion)
Median values (~A)
110
200**
Interquartile range
90-120
150-240
** P < 0.005 mice weighed 82 to 84% of their ad libitum weight. As previously described 12, the learning task consisted of partial acquisition, retention, and extinction of operant conditioning in a Skinner box. The apparatus has been described in detail elsewhere 6. The Skinner box (14 × 14 × 18 cm high) had a food dispenser which differed somewhat from the standard ones in that it was equipped with a device which permitted the removal of any pellets that were not eaten. The lever and the food cup were separated by a 5-cm long partition, so the animal had to turn around to press the lever or to find food. Photocells detected the animal's presence in front of the lever or the food cup. A continuous reinforcement (CRF) schedule was used. Lever-presses which were not followed by an approach towards the food-cup within 10 s ('complete' responses) were not counted. All the animals underwent an initial acquisition session in which they were allowed 16 'complete' responses. They were fed 1 h after each learning session so as to maintain a constant body weight. To this end, stimulated, non-stimulated and PP-lesioned animals received the same amount of food so that there were no weight differences on the following training days. The 46 animals were divided into 5 groups corresponding to the treatment applied immediately after the acquisition session; there were 3 experimental and two control groups: 12 PP-lesioned subjects (PP. Les), 10 LEC stimulated subjects (LEC ST), 9 subjects with PP lesion and LEC stimulation (PP Les + LEC ST), 6 LEC non-stimulated subjects (nST), 9 sham-operated subjects (sham). The post-training treatment was administered in a waiting box, adjacent to the Skinner box, 30 rain after the end of this first session. Based on the results of the first (see Table I) and previous experiments8, animals in the stimulated groups received bilateral, discontinuous (200 ms on, 200 ms off) brain stimulation (100 Hz) de-
176 livered for an 80 s period which consisted of 10 trains of 4 s of stimulation alternating with 4 s of no stimulation. For all animals, the current intensity was half of the median value recorded in control animals in the first experiment; i.e. 55/~A delivered to both entorhinal cortices connected in parallel to the stimulator (27.5 /~A/electrode). The E E G was monitored for each animal to verify that no afterdischarges were present. Retention testing was carried out 24 h and 48 h later during a 15-min lever-pressing session. Fig. 1 summarizes the results. No significant difference appeared between sham and nST subjects during the first (F(1,13) = 1.61, n.s.) or second retention sessions (F(1,13) = 0.05, n.s.). These scores were pooled and the 15 subjects served as a single control group. During the first retention test, significant differences among the 4 groups were observed (F(3,42) = 37.94, P < 0.001) and there were significant differences among the groups in the progression of re-
30-
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,
T/
,
,
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/
•
/
,
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•
RETENTION 1 O-
;,
,'o
PP. L e m + L E C
ST
RETENTION 2
,'5 TIME
~
,'o
,~
(min)
Fig. 1. Effects of LEC stimulation alone or combined with perforant-path lesions on retention scores measured during two sessions 24 h and 48 h after partial learning of CRF conditioning. Each session is divided into three 5-min periods (small bars indicate the standard error of the mean).
sponding over the three 5-min periods (treatment × period interaction: F(6,84) = 17.44, P < 0.001). In addition, significant treatment × period interactions were found when the scores of the control group and of each experimental group were compared. At the start of the retention test (first 5-min period), a slight impairment of performance was observed in the PP Les group (PP vs Control: F(1,25) = 11.86, P < 0.01), whereas L E C ST animals and PP Les + L E C ST animals showed an improvement (P < 0.001 in both cases). However, stimulated animals with PP lesions performed less than L E C ST animals (F(1,17) = 17.98, P < 0.001). During the second retention session, all the groups reached the same performance level (F(3,42) = 0.80, n.s.).
For all the subjects, retention testing was continued until they reached the criterion of 200 'complete' responses 4. During the next few days, the 46 mice underwent a daily 6-min session of extinction of the C R F conditioning. The LEC-implanted animals (LEC ST and PP Les + L E C ST groups) were stimulated 30 min after the end of the first 6-min extinction session, using the same parameters (discontinuous stimulation, 55 ~ A intensity) as in the first part of the experiment. No disruption was observed in the E E G records. All the subjects were fed 1 h after each extinction session so as to maintain a constant body weight (82-84% of their ad libitum weight at the start of each extinction session). The scores were calculated as the number of 'complete' responses per min. Fig. 2 summarizes the results. No difference was found between sham-operated and non-stimulated subjects over the 3 extinction sessions (1st session: F(1,13) -- 0.11, n.s.; 2nd session: F(1,13) = 0.37, n.s.; 3rd session: F(1,13) = 0.03, n.s.); thus the 15 subjects served as one control group. No differences appeared among the 4 groups during the first extinction session (F(3,42) = 0.12, n.s.). Twenty-four h later, no significant differences were found between controls and PP-lesioned animals (F(1,25) = 1.54, n.s.). Compared to the control animals, the mice that received the L E C electrical stimulation 30 min after the first extinction exhibited faster extinction of conditioning (F(1,23) = 10.67, P < 0.005 for the L E C ST group and F(1,22) = 14.73, P < 0.001 for the PP Les + L E C ST group). In both
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cases; there were significant differences among the groups in the progression of responding over the 6 1-min periods (treatment x period interaction, P < 0.05). No differences were found between LEC ST and PP Les + LEC ST animals (F(1,17) = 0.31, n.s.). During the 3rd extinction session there were no significant differences among the 4 groups. The histological analysis showed that all electrode tips were located in the ventral part of the lateral entorhinal cortex. The lesioned area was measured at 3.6 mm posterior to Bregma using a planimeter for every subject. This area fell vertically between 1.9 and 2.5 mm and the average size of bilateral lesions ranged from 0.38 mm 2 to 0.45 mm 2. There was no significant difference in the distribution of the lesions between PP Les and PP Les + LEC ST groups. A reconstruction of the lesioned area in a representative animal is shown in Fig. 3. The present results show that the effect of LEC stimulation that is delayed 30 min persisted in spite of bilateral lesion of the perforant path. Compared to
control animals, the PP Les + LEC ST subjects showed, as did the non-lesioned LEC ST animals, a clear improvement in retention and exhibited a faster extinction of conditioning. Several issues must be considered before the implications of our data can be discussed. First of all, the finding that animals with PP lesions show poorer retention of operant conditioning does not agree with the results obtained after lesions of the LEC 9. The deficit seen at the beginning of the retention session (Fig. 1) may be due to the method used; in the present procedure, all animals received the same amount of limited information (16 reinforced responses) 30 min before the stimulation of the LEC. In particular, the reminiscence phenomenon (timedependent improvement in retention performance6,12) observed in control animals did not occur in the animals that had undergone lesion of the perforant path. In other words, during an appetitive test, the PP lesion may interact with the consolidation of a response which is not fully learned. Under these conditions the significant difference observed between
178
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Fig. 3. Drawings of horizontal sections of the mouse brain showing the placement and the extent of the lesion (dotted area). 1.900, 2.200 and 2.500 = distances (in mm) of the section from the surface of the skull.
for afterdischarges triggered in the hippocampus b y stimulation of the lateral entorhinal cortex in PP-lesioned animals. We thus have here a good index for the rapid verification of perforant-path lesions before behavioral testing. In summary, our results show up a functional dissociation between hippocampal and cortical mechanisms involved in memory facilitation produced by stimulation of the lateral entorhinal cortex; they argue in favor of a specific involvement of this cortical region in delayed information processing. It is difficult to see, however, from a functional-anatomical point of view, how electrical stimulation of the entorhinal cortex could bring into play mechanisms that are strictly limited to the stimulated area. It is more likely that the electrical stimulation, which can no longer reach the hippocampus after perforant-path lesion, reveals the recruitment of cortical or subcortical regions that are directly or indirectly connected with the entorhinal cortex. For example, it is known that there are reciprocal connections between the entorhinal cortex and the (cingulate) retrosplenial area 5,16,18. We have recently shown that the cingulate cortex, whose posterior part is apparently a special region participating in the organization of attention and memory 1.21, is involved in memory consolidation in mice 4. However, it should be pointed out that there are numerous other projections of the entorhinal cortex, besides the cingulate cortex, to cortical areas other than the hippocampus 13 or to subcortical regions 19,20 that are indirectly connected by the Papez circuit 2~. In conclusion, our results support the hypothesis of a delayed recruitment of one or several cortical loops in information processing mechanisms. This recruitment apparently does not involve a direct reactivation of the hippocampus.
L E C ST and PP Les + L E C ST groups could result from the disruptive effect of the PP lesion. This difference is no longer present when one looks at the performance in extinction for these two groups of animals. Similarly, the PP-lesioned animals behave like the control animals. This result comes close to a finding by Myrher 17 that PP lesion did not disrupt passive avoidance conditioning. Once again, however, this result does not agree with the data obtained after L E C lesion9,17. According to Myrher's hypothesis, the entorhinal lesion, but not PP lesion, can interact with the extinction of a well-learned response. Besides the behavioral results, the effectiveness of the lesion is indicated by the anatomical (Fig. 3) and electrophysiological data we collected. The first experiment showed a significant rise in the thresholds
We thank Mrs. Ducout and Mrs. Perret for technical assistance and Dr. M. Caudarella for help in the preparation of the manuscript. This research was supported by the C.N.R.S. (L.A. no 339).
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(1978) 411-417. 3 Destrade, C., Two types of diencephalically driven RSA (theta) as a means of studying memory formation in mice, Brain Research, 234 (1982) 486-493. 4 Destrade, C. and Gauthier, M., Facilitation de la r6tention
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