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LTP of mossy fiber-stimulated potentials in CA3 during learning in rats
Physiology & Behavior, Vol. 55, No. 4, pp. 633-638, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0031-9384/94 $6.00 + .00
Pergamon
LTP of Mossy Fiber-Stimulated Potentials in CA3 During Learning in Rats KAMEYOSHI
M I T S U N O , *l M A S A S H I S A S A , t K U M A T O S H I I S H I H A R A , : ~ M A S A T S U N E AND HARUHIKO KIKUCHI*
ISHIKAWA*
Departments of *Neurosurgery and ##Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan and tDepartment of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734, Japan R e c e i v e d 20 M a y 1993 MITSUNO, K., M. SASA, K. ISHIHARA, M. ISHIKAWA AND H. KIKUCHI. LTP of mossyfiber-stimulatedpotentials in CA3 during learningin rats. PHYSIOL BEHAV 55(4) 633-638, 1994.--The study was done to determine whether long-term potentiation (LTP) of the potentials occurs at mossy fiber (MF)-CA3 synapses with the advance of learning in unrestrained and unanesthetized rats. The rats were divided into two groups, the test and control groups. The test group was given daily learning tasks in the radial ann maze, whereas the control group was similarly handled without learning tasks. Complete acquisition of learning was observed in the test group on day 5, and the learning was maintained over 3 days. Under freely moving conditions, a significant increase in population spikes (PS) elicited by MF stimulation with the progress in learning was observed in the test group, and the PS potentiation remained stable after day 4. Furthermore, on day 7, when MF stimulation-induced PS in the test group were compared with that in the control group in pentobarbital-anesthetized rats, the responses were comparatively higher in the former. As the training-induced PS potentiation in CA3 occurred with the advance of learning, these findings suggest that LTP in CA3 induced by learning may be related to memory storage. LTP
CA3
Mossy fiber
Hippocampus
Learning
THERE are three major fiber connections in the hippocampus (1,23): perforant path-dentate gyrus, mossy fiber ( M F ) - C A 3 field, and Schaffer collateral-CA1 field. Because population spikes (PS) in the dentate gyrus induced by perforant path stimulation are enhanced on completion of learning (12,22,31,35,36), such long-term potentiation (LTP) of PS in the hippocampus is thought to be involved in memory and learning. However, such PS enhancements in the perforant path-dentate gyrus system have been denied in recent findings with the radial arm maze (14). Ischemic lesions in the CA1 region following transient fourvessel occlusion reportedly impair working memory, whereas learning per se is not disturbed at all (38,39). In contrast, lesions in the CA3 region have been reported to impair learning via spatial memory disturbance (13). A striking difference is found in performance of rats with lesions in the CA1 and CA3 regions. Furthermore, significant changes in distribution of protein kinase C following learning are found only in CA3 (26). Nootrophic drugs affect LTP in the M F - C A 3 system but not in the Schaffer collateral-CA 1 system (17,33,34). It is suggested, therefore, that the CA3 region is more involved in memory formation. Since the discovery of LTP (8,9), which is possibly related to memory formation (11,25,37), the phenomenon has been well examined with slice preparations and in vivo mainly in the perforant path-dentate gyrus and Schaffer collateral-CA1 systems
Rats
(1-7,10,12,22,27,28,31,35,36,40-42). In the M F - C A 3 system, however, there are only a few studies examining LTP (1,1517,32-34,43), and the relation between LTP and learning remains to be established. Thus, it is significant to investigate electrophysiologically the M F - C A 3 system in connection with behavior. This is the first in vivo investigation conducted on the LTP/learning relationship in the M F - C A 3 system of rats using a radial arm maze. METHOD
Male Wistar rats weighing 2 5 0 - 3 5 0 g at the start of testing were used. They were maintained in individual cages under a 12h day and night cycle, and were given ad lib food and water. Before a radial arm maze task commenced, the rats were handled and fed once a day for a week. Their body weights were reduced to approximately 80% of their free-feeding weight, supplemented for normal growth.
Experiment 1 The rats were divided into two groups of 10 each: test and control groups. The former was given tasks in a radial arm maze, whereas the latter was handled in a similar way without any tasks. Behavioral studies. The radial arm maze used had eight arms radiating from a center platform 30 cm in diameter. Each arm
Requests for reprints should be addressed to Kameyoshi Mitsuno, Department of Neurosurgery, Kyoto University Hospital, 54 Shogoin KawaharaCho, Sakyo-ku, Kyoto 606, Japan. 633
634
MITSUNO ET AL,
A
13
FIG. 1. Location of tips of the stimulating (A) and recording (B) electrodes, as indicated by arrows. DG: hilus of dentate gyms, CA3: pyramidal cell layer of CA3 field. was 60 cm long and had a 5-cm-high rail on both sides. The testing room was well illuminated by fluorescent ceiling lights and there were numerous potential visual cues present; these included the experimenter and conspicuous objects such as tables and chairs. Rats were adapted to the maze for 15 rain to go to the end of the arms, where 50-mg pellets were scattered, on 3 consecutive days. After the adaptation, the rats were given one trial each day. One pellet was placed at the end of each arm, and each rat was allowed to choose freely among all arms until all pellets in eight arms had been taken, or until 10 rain had elapsed from the start of the task. The numbers of correct responses in the first eight choices, as well as erroneous responses, were counted. Acute electrophysiological study. On day 7 of the trials, all 20 rats in both groups were anesthetized with sodium pentobarbital (60 mg/kg, IP) and fixed in a stereotaxic instrument. A monopolar stainless steel recording electrode (o.d., 50 #m) was inserted in the left CA3 field (4.5 mm posterior to bregma, 4.5
mm lateral to the midline, and 4.5 mm from the cortical surface) (29). A bipolar stimulating electrode with a tip diameter of 0.1 mm and tip separation of 0.2 mm was placed in the hilus of the left dentate gyrus (6.3 mm posterior to bregma, 4.5 mm lateral to the midline, and 6.5 mm from the skull surface) (29) to stimulate the MF. A square pulse stimulus, 0.1-ms duration and 0.5mA intensity, was applied to the MF every 5 s to induce the PS. The resulting PS were used to guide the final placement of both electrodes. The amplitude of the PS induced by stimulation with nine different current intensities (from 0.05 to 0.75 mA) was measured in each rat. Three successive PS induced by stimuli of each intensity were averaged, using a computer (Nihon Kohden, ATAC-450), and were recorded on a recticorder (Nihon Kohden, WT-625G). The amplitude of the PS was measured according to the method of others as indicated in Fig. 4 (24). Input/output curves (IOC) were obtained for each rat by plotting the nine PS amplitudes against the nine current intensities, and a stimulus intensity that produced 80% of the maximum response for each
LTP IN M O S S Y F I B E R - C A 3 AND L E A R N I N G
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FIG. 2. Correct (open circles, n = 7) and erroneous (closed circles, n = 7) responses to the task in the radial arm maze. Each point and vertical
bar indicates mean and SEM. rat was determined as an intensity for tetanic stimuli. After establishing the basal IOC, tetanic stimuli with 0.1-ms pulse width and 0.05-0.75-mA intensity were delivered to the MF at 100 Hz for 1 s. Posttetanic IOC were achieved by inducing PS with the respective current intensity for each rat 5, 10, 15, 30, 60, and 120 min after the tetanic stimulation (TS). Experiment 2
To investigate the change of the PS following a task in a radial arm maze, responses from 18 rats with chronically implanted electrodes were analyzed. They were anesthetized with sodium pentobarbital (60 mg/kg, IP), and recording and stimulating electrodes were implanted in the same sites as in Experiment 1. All electrodes were soldered to connector pins and the entire assembly was fixed to the skull with dental acrylic. After a 1-week recovery period, 14 rats that showed a reproducibly stable PS pattern elicited by a test stimulus (0.l-ms duration and 0.1-0.5mA intensity) were selected for further test. These rats were then divided into test and control groups of seven rats each. Rats in the former were given a daily task in the radial arm maze after handling, food deprivation, and acclimatization (see Experiment 1), whereas those in the latter were similarly treated without any
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FIG. 4. Input/output curves (IOC) when the intensity of the stimulus was increased from 0.05 to 0.75 mA in the control (open circles, n = 7) and test (closed circles, n = 7) groups. Each point and vertical bar indicates mean and SEM. The IOC was obtained after acclimatization in the control group, and on day 7 in the test group.
learning tasks. In the electrophysiological studies, the rats, which were allowed to move freely, were placed in a shielded box (30 x 24 x 42 cm3). At a 23-h lapse after every task, MF stimulationelicited PS were induced in the CA3 field with coordination and stimulus parameters similar to those employed in Experiment 1. Daily IOC were constructed for each rat. The postlearning area under the IOC curve (AUC~oc) was then calculated and compared with prelearning values. After completion of the study, the stimulating and recording sites were marked by the passing of a direct current of 20 #A for 5 s, and they were histologically verified with cresyl violet stain. The data were analyzed through a series of analysis of variance repeated-measure designs (ANOVA), and the statistical significance of the data was determined using Student's t-test. RESULTS Histological examination showed that both recording and stimulation electrodes were located in CA3 field and hilus in 14/ 20 and 14/18 rats in Experiments 1 and 2, respectively (Fig. 1). Only results from 28 rats, where electrodes were precisely appropriated in both the CA3 field and hilus, were used in this study.
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FIG. 5. Pretetanic (solid line) and posttetanic (dotted line) IOC in the test group (triangles, n = 7) and control group (circles, n = 7). Each point and vertical bar indicates mean and SEM.
636
MITSUN{)
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FIG. 6. Correct (open circles, n = 7) and erroneous (closed circles, n = 7) responses to the task in the radial arm maze in rats chronically implanted with electrodes. Each point and vertical bar indicates mean and SEM.
Experiment 1 On day 1 of the trial, 4.4 __ 0.3 correct and 16.0 + 1.4 erroneous responses were registered (n = 7). The number of correct responses gradually increased and the number of erroneous responses gradually decreased during training, respectively (Fig. 2). Performance measures from the radial arm maze were found to be significant both for the number of correct responses, F(6, 42) = 11.74, p < 0.01, and for the number of erroneous responses, F(6, 42) = 42.18, p < 0.01. The MF-stimulated PS were recorded on day 7 of the trials in both the test and control groups. An increase in stimulus intensity resulted in an enhancement of the amplitude in both groups (Fig. 3). Comparison revealed that the PS amplitudes at any intensity were consistently higher in the test group (Fig. 4). Statistical analysis of the PS amplitudes confirmed the overall difference between the test and control groups, F(I, 12) = 42.72, p < 0.01, and the difference between the groups in the change over stimulation intensities, F(8, 96) = 29.31, p < 0.01. The stimulus intensity at which the PS plateaued was approximately 0.375 m A in both groups, and the PS induced by that stimulus intensity were significantly larger (p < 0.001) in the test group than in the control group. Residual ability to induce LTP was found in both groups, but as to absolute increase in the PS amplitude after tetanic stimuli, no significant difference was found between two groups (Fig. 5).
I:{'1 , M .
previous findings by others (13,14). Gradual potenltauon of MFstimulated PS in the hippocampal CA3 field was accompanied by advances in learning. The PS potentiation following learning has been called behavioral LTP /37). Investigations of this potentiation or excitability of neurons have been focused on the perforant path--dentate gyrus system (4 7,12,22,31,35,36, 40,41 ). A recent study, however, demonstrated that such behavioral LTP could not be found in this system (14) because PS amplitude varied depending upon the ongoing behavior at the time of the recording. Because our results were obtained in the resting state of the rats and under anesthesia, the behavioral LTP is considered to be actually induced in the mossy tiber- CA3 system. However, it remains unknown what this LTP reflects, because many parameters are involved in behavioral per|brmante. Because successful learning cannot be achieved unless rats are allowed to move in the maze, behavioral [.TP may reflect both the memory and motional aspects. However, as the PS remained stable after day 4 of the trials, behavioral LTP may well be regarded as an index of memory. Given that neurons are activated all-or-none, reproducible PS amplitudes would be consistently achieved if the same afferent fiber was stimulated and the PS were obtained from a designated population of neurons via securely positioned electrodes. Although postlearning PS potentiation (22) may be related to coactivation of the neuronal population (19,20,23,37,44), the underlying mechanisms remains unknown. The results of Experiment 1 demonstrated a significant difference in the rate of increase (gradient) of the PS amplitude/stimulation intensity plot. As a difference in the threshold level required to activate a population of neurons exists between any two groups, training probably has lowered this threshold level accompanied by an increase in the number of activated neurons to subsequently produce PS enhancement. The IOC changes accompanied by learning in Experiment 2 converged with this postulation. In Experiment 2, PS potentiation peaked on day 4 of the trail, and thereafter, further enhancement was not detected until day 7. Defining the interval from day 1 to 4 and day 5 to 7 as the acquisition and retention periods, respectively, IOC changes in CA3 field were found in the former. Furthermore, PS potentiation
300
250
200'
Experiment 2 The time course for learning in the rats with chronically implanted electrodes was similar to that observed in Experiment 1 (Fig. 6). The MF-stimulated PS were recorded only when the rats were in the resting condition, i.e., when they were not moving about, grooming, or rearing up. The AUC~oc increased with the advances of learning in the maze until day 4 of the trials, whereas changes were not registered in the control group throughout the 7-day observation period (Fig. 7). Statistical analysis of values for A U Q o c before and after the trials confirmed the overall differences between the test and control groups, F(I, 12) = 15.71, p < 0.01, and the differences between the groups in the change over days, F(7, 84) = 7.06, p < 0.01.
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1
2
3
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DISCUSSION The time course for learning in which the rats showed correct and erroneous responses in the radial arm maze coincided with
of
trials
FIG. 7. Percentage areas under input/output curves obtained every day in the test group (closed circles, n = 7) and control group (open circles, n = 7). Each point and vertical bar indicates mean and SEM.
LTP IN M O S S Y F I B E R - C A 3 AND L E A R N I N G
637
manifested in the acquisition period persisted for at least 24 h, because recording of the parameter was performed at 24-h intervals. The 24-h repetitive training may have facilitated and enhanced the memory storage because of the presence of residual PS. Although PS potentiation was not found during the retention period, preceding potentiation was maintained, suggesting retention of the learned behavior. Such a suggestion corresponds to a previous finding by others (22). Present findings suggest that the CA3 field plays an important role in acquiring new information. However, this field is not the only site responsible for learning in rats because PS induced by perforant path stimulation in the dentate gyrus are enhanced on completion of learning (12,22,31,35,36). Thus, neuronal pathway from entorhinal cortex to CA3 field would have to remain intact for rats to learn. A previous study (26), which advocates our postulation, has demonstrated distributional changes of protein kinase C (PKC) only in CA3 field after discrimination learning. Such changes may be associated with training-induced LTP in CA3 field in our present study because a shift in the phosphorylation of B50, a PKC substrate, has been found to be associated with maintenance of LTP (21). This suggestion is especially reinforced by the impairment of spatial memory as a result of neurotoxic lesions in the CA3 (13). Furthermore, rats subjected to
transient forebrain ischemic injury with irreversible damage in the CA1 by four-vessel occlusion did not show any negative effects on their learning performance (38,39). The fact that CA3 insults (but not CA1 lesions) affect rodent performance in learning strongly advocates that the LTP development in the CA3 and previously documented dentate gyrus (22) may be responsible for the memory formation. This conclusion is also supported by the recent electrophysiological studies using hippocampal slice preparations that LTP of the PS induced by M F stimulation in the CA3 were augmented by nootrophic drugs such as aniracetam, idebenone, and vinpocetine, but those LTP responses in the Schaffer collateral-CA1 system were not affected (17,33,34). There is no evidence, however, as to whether synaptic transmission in the M F - C A 3 system would remain totally intact or be damaged to some degree after transient ischemic injury, although CA3 cells have been demonstrated to recover morphologically (30). Prominent among theories of hippocampal function is the suggestion of a transient memory storage. The present evidence supports the view that CA3 field plays a crucial role to acquire new information, and that LTP induced in synapses on neuronal pathway from entorhinal cortex to CA3 field may participate in memory formation and/or storage.
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14. Hargreaves, E. L.; Cain, D. P.; Vanderwolf, C. H. Learning and behavioral long-term potentiation: Importance of controlling for motor activity. J. Neurosci. 10:1472-1478; 1990. 15. Harris, E. W.; Cotman, C. W. Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl D-aspartate antagonists. Neurosci. Lett. 70:132-137; 1986. 16. Ishihara, K.; Katsuki, H.; Kawabata, A.; Sasa, M.; Satoh, M.; Takaori, S. Effects of thyrotropin-releasing hormone and a related analog, CNK-602A, on long-term potentiation in the mossy fiber-CA3 pathway of guinea pig hippocampal slices. Brain Res. 554:203-208; 1991. 17. Ishihara, K.; Katsuki, H.; Sugimura, M.; Satoh, M. Idebenone and vinpocetine augment long-term potentiation in hippocampal slices in the guinea pig. Neuropharmacology 28:569-573; 1989. 18. Jaffard, R.; Jeantet, Y. Posttraining changes in excitability of the commissural path-CA 1 pyramidal cell synapse in the hippocampus of mice. Brain Res. 220:167-172; 1981. 19. Levy, W. B.; Steward, O. Synapses as associative memory elements in the hippocampal formation. Brain Res. 175:233-245; 1979. 20. Levy, W. B.; Steward, O. Temporal contiguity requirement for longterm associative potentiation/depression in the hippocampus. Neuroscience 8:791-797; 1983. 21. Lovinger, D. M.; Routtenberg, A. Synapse-specific protein kinase C activation enhances maintenance of long-term potentiation in rat hippocampus. J. Physiol. (Lond.) 400:321-333; 1988. 22. Matthies, H.; Ruethrich, H.; Ott, T.; Matthies, H. K.; Matthies, R. Low frequency perforant path stimulation as a conditioned stimulus demonstrates correlations between long-term synaptic potentiation and learning. Physiol. Behav. 36:811-821; 1986. 23. McNaughton, B. L.; Douglas, R. M.; Goddard, G. V. Synaptic enhancement in fascia dentata: Cooperativity among coactive afferents. Brain Res. 157:277-293; 1978. 24. Morimoto, K.; Goddard, G. V.; Sirett, N. E. Effects of a new thyrotropin-releasing hormone analogue (DN-1417) on evoked responses and long-term potentiation in the dentate gyrus of rat. Neurosci. Lett. 63:195-199; 1986. 25. Morris, R.; Baker, M. Does long-term potentiation/synaptic enhancement have anything to do with learning or memory? In: Squire, L. R.; Butters, N., eds. Neuropsychology of memory. New York: Guilford Press; 1984:521-535. 26. Olds, J. L.; Golski, S.; McPhie, D. L.; Olton, D.; Mishkin, M.; A1kon, D. L. Discrimination learning alters the distribution of protein
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35. Sharp, P. E.; McNaughton, B. L.; Barnes, C. A. Enhancement of hippocampal field potentials in rats exposed to a novel, complex environment. Brain Res. 339:361 -365; 1985. 36. Skelton, R. W.; Scarth, A. S.; Wilkie, D. M.; Miller, 1. J.; Phillips, A. G. Long-term increases in dentate granule cell responsivity accompany operant conditioning. J. Neurosci. 7:3081-3087; 1987. 37. Teyler, T. J.; Discenna, P, Long-term potentiation as a candidate mnemonic device. Brain Res. Rev. 7:15 28; 1984. 38. Volpe, B. T.; Pulsinelli, W. A.; Tribuna, J.; Davis, H. P. Behavioral performance of rats following transient forebrain ischemia. Stroke 15:558-562; 1984. 39. Volpe, B. T.; Davis, H. P.; Colombo, P. J. Preoperative training modifies radial maze performance in rats with ischemic hippocampal injury. Stroke 20:1700-1706; 1989. 40. Weisz, D. G.; Clark, G. A.; Thompson, R. F. Increased responsivity of dentate granule cells during nictitating membrane response conditioning in rabbit. Behav. Brain Res. 12:145-154; 1984. 41. West, M. O.; Christian, E.; Robinson, J. H.; Deadwyler, S. A. Dentate granule cell discharge during conditioning. Exp. Brain Res 44:287-294; 1981. 42. Wigstrom, H.; McNaughton, B. L.; Barnes, C. A. Long-term syn-aptic enhancement in hippocampus is not regulated by postsynaptic membrane potential. Brain Res. 233:195-199; 1982. 43. Yamamoto, C.; Chujo, T. Long-term potentiation in thin hippocampal sections studied by intracellular and extracellular recordings. Exp. Neurol. 58:242-250; 1978. 44. Yamamoto, C.; Sawada, S. Important factors in induction of longterm potentiation in thin hippocampal sections. Exp. Neurol. 74:122-130; 1981.
Pergamon
LTP of Mossy Fiber-Stimulated Potentials in CA3 During Learning in Rats KAMEYOSHI
M I T S U N O , *l M A S A S H I S A S A , t K U M A T O S H I I S H I H A R A , : ~ M A S A T S U N E AND HARUHIKO KIKUCHI*
ISHIKAWA*
Departments of *Neurosurgery and ##Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan and tDepartment of Pharmacology, Hiroshima University School of Medicine, Hiroshima 734, Japan R e c e i v e d 20 M a y 1993 MITSUNO, K., M. SASA, K. ISHIHARA, M. ISHIKAWA AND H. KIKUCHI. LTP of mossyfiber-stimulatedpotentials in CA3 during learningin rats. PHYSIOL BEHAV 55(4) 633-638, 1994.--The study was done to determine whether long-term potentiation (LTP) of the potentials occurs at mossy fiber (MF)-CA3 synapses with the advance of learning in unrestrained and unanesthetized rats. The rats were divided into two groups, the test and control groups. The test group was given daily learning tasks in the radial ann maze, whereas the control group was similarly handled without learning tasks. Complete acquisition of learning was observed in the test group on day 5, and the learning was maintained over 3 days. Under freely moving conditions, a significant increase in population spikes (PS) elicited by MF stimulation with the progress in learning was observed in the test group, and the PS potentiation remained stable after day 4. Furthermore, on day 7, when MF stimulation-induced PS in the test group were compared with that in the control group in pentobarbital-anesthetized rats, the responses were comparatively higher in the former. As the training-induced PS potentiation in CA3 occurred with the advance of learning, these findings suggest that LTP in CA3 induced by learning may be related to memory storage. LTP
CA3
Mossy fiber
Hippocampus
Learning
THERE are three major fiber connections in the hippocampus (1,23): perforant path-dentate gyrus, mossy fiber ( M F ) - C A 3 field, and Schaffer collateral-CA1 field. Because population spikes (PS) in the dentate gyrus induced by perforant path stimulation are enhanced on completion of learning (12,22,31,35,36), such long-term potentiation (LTP) of PS in the hippocampus is thought to be involved in memory and learning. However, such PS enhancements in the perforant path-dentate gyrus system have been denied in recent findings with the radial arm maze (14). Ischemic lesions in the CA1 region following transient fourvessel occlusion reportedly impair working memory, whereas learning per se is not disturbed at all (38,39). In contrast, lesions in the CA3 region have been reported to impair learning via spatial memory disturbance (13). A striking difference is found in performance of rats with lesions in the CA1 and CA3 regions. Furthermore, significant changes in distribution of protein kinase C following learning are found only in CA3 (26). Nootrophic drugs affect LTP in the M F - C A 3 system but not in the Schaffer collateral-CA 1 system (17,33,34). It is suggested, therefore, that the CA3 region is more involved in memory formation. Since the discovery of LTP (8,9), which is possibly related to memory formation (11,25,37), the phenomenon has been well examined with slice preparations and in vivo mainly in the perforant path-dentate gyrus and Schaffer collateral-CA1 systems
Rats
(1-7,10,12,22,27,28,31,35,36,40-42). In the M F - C A 3 system, however, there are only a few studies examining LTP (1,1517,32-34,43), and the relation between LTP and learning remains to be established. Thus, it is significant to investigate electrophysiologically the M F - C A 3 system in connection with behavior. This is the first in vivo investigation conducted on the LTP/learning relationship in the M F - C A 3 system of rats using a radial arm maze. METHOD
Male Wistar rats weighing 2 5 0 - 3 5 0 g at the start of testing were used. They were maintained in individual cages under a 12h day and night cycle, and were given ad lib food and water. Before a radial arm maze task commenced, the rats were handled and fed once a day for a week. Their body weights were reduced to approximately 80% of their free-feeding weight, supplemented for normal growth.
Experiment 1 The rats were divided into two groups of 10 each: test and control groups. The former was given tasks in a radial arm maze, whereas the latter was handled in a similar way without any tasks. Behavioral studies. The radial arm maze used had eight arms radiating from a center platform 30 cm in diameter. Each arm
Requests for reprints should be addressed to Kameyoshi Mitsuno, Department of Neurosurgery, Kyoto University Hospital, 54 Shogoin KawaharaCho, Sakyo-ku, Kyoto 606, Japan. 633
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MITSUNO ET AL,
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FIG. 1. Location of tips of the stimulating (A) and recording (B) electrodes, as indicated by arrows. DG: hilus of dentate gyms, CA3: pyramidal cell layer of CA3 field. was 60 cm long and had a 5-cm-high rail on both sides. The testing room was well illuminated by fluorescent ceiling lights and there were numerous potential visual cues present; these included the experimenter and conspicuous objects such as tables and chairs. Rats were adapted to the maze for 15 rain to go to the end of the arms, where 50-mg pellets were scattered, on 3 consecutive days. After the adaptation, the rats were given one trial each day. One pellet was placed at the end of each arm, and each rat was allowed to choose freely among all arms until all pellets in eight arms had been taken, or until 10 rain had elapsed from the start of the task. The numbers of correct responses in the first eight choices, as well as erroneous responses, were counted. Acute electrophysiological study. On day 7 of the trials, all 20 rats in both groups were anesthetized with sodium pentobarbital (60 mg/kg, IP) and fixed in a stereotaxic instrument. A monopolar stainless steel recording electrode (o.d., 50 #m) was inserted in the left CA3 field (4.5 mm posterior to bregma, 4.5
mm lateral to the midline, and 4.5 mm from the cortical surface) (29). A bipolar stimulating electrode with a tip diameter of 0.1 mm and tip separation of 0.2 mm was placed in the hilus of the left dentate gyrus (6.3 mm posterior to bregma, 4.5 mm lateral to the midline, and 6.5 mm from the skull surface) (29) to stimulate the MF. A square pulse stimulus, 0.1-ms duration and 0.5mA intensity, was applied to the MF every 5 s to induce the PS. The resulting PS were used to guide the final placement of both electrodes. The amplitude of the PS induced by stimulation with nine different current intensities (from 0.05 to 0.75 mA) was measured in each rat. Three successive PS induced by stimuli of each intensity were averaged, using a computer (Nihon Kohden, ATAC-450), and were recorded on a recticorder (Nihon Kohden, WT-625G). The amplitude of the PS was measured according to the method of others as indicated in Fig. 4 (24). Input/output curves (IOC) were obtained for each rat by plotting the nine PS amplitudes against the nine current intensities, and a stimulus intensity that produced 80% of the maximum response for each
LTP IN M O S S Y F I B E R - C A 3 AND L E A R N I N G
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bar indicates mean and SEM. rat was determined as an intensity for tetanic stimuli. After establishing the basal IOC, tetanic stimuli with 0.1-ms pulse width and 0.05-0.75-mA intensity were delivered to the MF at 100 Hz for 1 s. Posttetanic IOC were achieved by inducing PS with the respective current intensity for each rat 5, 10, 15, 30, 60, and 120 min after the tetanic stimulation (TS). Experiment 2
To investigate the change of the PS following a task in a radial arm maze, responses from 18 rats with chronically implanted electrodes were analyzed. They were anesthetized with sodium pentobarbital (60 mg/kg, IP), and recording and stimulating electrodes were implanted in the same sites as in Experiment 1. All electrodes were soldered to connector pins and the entire assembly was fixed to the skull with dental acrylic. After a 1-week recovery period, 14 rats that showed a reproducibly stable PS pattern elicited by a test stimulus (0.l-ms duration and 0.1-0.5mA intensity) were selected for further test. These rats were then divided into test and control groups of seven rats each. Rats in the former were given a daily task in the radial arm maze after handling, food deprivation, and acclimatization (see Experiment 1), whereas those in the latter were similarly treated without any
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learning tasks. In the electrophysiological studies, the rats, which were allowed to move freely, were placed in a shielded box (30 x 24 x 42 cm3). At a 23-h lapse after every task, MF stimulationelicited PS were induced in the CA3 field with coordination and stimulus parameters similar to those employed in Experiment 1. Daily IOC were constructed for each rat. The postlearning area under the IOC curve (AUC~oc) was then calculated and compared with prelearning values. After completion of the study, the stimulating and recording sites were marked by the passing of a direct current of 20 #A for 5 s, and they were histologically verified with cresyl violet stain. The data were analyzed through a series of analysis of variance repeated-measure designs (ANOVA), and the statistical significance of the data was determined using Student's t-test. RESULTS Histological examination showed that both recording and stimulation electrodes were located in CA3 field and hilus in 14/ 20 and 14/18 rats in Experiments 1 and 2, respectively (Fig. 1). Only results from 28 rats, where electrodes were precisely appropriated in both the CA3 field and hilus, were used in this study.
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FIG. 5. Pretetanic (solid line) and posttetanic (dotted line) IOC in the test group (triangles, n = 7) and control group (circles, n = 7). Each point and vertical bar indicates mean and SEM.
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Experiment 1 On day 1 of the trial, 4.4 __ 0.3 correct and 16.0 + 1.4 erroneous responses were registered (n = 7). The number of correct responses gradually increased and the number of erroneous responses gradually decreased during training, respectively (Fig. 2). Performance measures from the radial arm maze were found to be significant both for the number of correct responses, F(6, 42) = 11.74, p < 0.01, and for the number of erroneous responses, F(6, 42) = 42.18, p < 0.01. The MF-stimulated PS were recorded on day 7 of the trials in both the test and control groups. An increase in stimulus intensity resulted in an enhancement of the amplitude in both groups (Fig. 3). Comparison revealed that the PS amplitudes at any intensity were consistently higher in the test group (Fig. 4). Statistical analysis of the PS amplitudes confirmed the overall difference between the test and control groups, F(I, 12) = 42.72, p < 0.01, and the difference between the groups in the change over stimulation intensities, F(8, 96) = 29.31, p < 0.01. The stimulus intensity at which the PS plateaued was approximately 0.375 m A in both groups, and the PS induced by that stimulus intensity were significantly larger (p < 0.001) in the test group than in the control group. Residual ability to induce LTP was found in both groups, but as to absolute increase in the PS amplitude after tetanic stimuli, no significant difference was found between two groups (Fig. 5).
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previous findings by others (13,14). Gradual potenltauon of MFstimulated PS in the hippocampal CA3 field was accompanied by advances in learning. The PS potentiation following learning has been called behavioral LTP /37). Investigations of this potentiation or excitability of neurons have been focused on the perforant path--dentate gyrus system (4 7,12,22,31,35,36, 40,41 ). A recent study, however, demonstrated that such behavioral LTP could not be found in this system (14) because PS amplitude varied depending upon the ongoing behavior at the time of the recording. Because our results were obtained in the resting state of the rats and under anesthesia, the behavioral LTP is considered to be actually induced in the mossy tiber- CA3 system. However, it remains unknown what this LTP reflects, because many parameters are involved in behavioral per|brmante. Because successful learning cannot be achieved unless rats are allowed to move in the maze, behavioral [.TP may reflect both the memory and motional aspects. However, as the PS remained stable after day 4 of the trials, behavioral LTP may well be regarded as an index of memory. Given that neurons are activated all-or-none, reproducible PS amplitudes would be consistently achieved if the same afferent fiber was stimulated and the PS were obtained from a designated population of neurons via securely positioned electrodes. Although postlearning PS potentiation (22) may be related to coactivation of the neuronal population (19,20,23,37,44), the underlying mechanisms remains unknown. The results of Experiment 1 demonstrated a significant difference in the rate of increase (gradient) of the PS amplitude/stimulation intensity plot. As a difference in the threshold level required to activate a population of neurons exists between any two groups, training probably has lowered this threshold level accompanied by an increase in the number of activated neurons to subsequently produce PS enhancement. The IOC changes accompanied by learning in Experiment 2 converged with this postulation. In Experiment 2, PS potentiation peaked on day 4 of the trail, and thereafter, further enhancement was not detected until day 7. Defining the interval from day 1 to 4 and day 5 to 7 as the acquisition and retention periods, respectively, IOC changes in CA3 field were found in the former. Furthermore, PS potentiation
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Experiment 2 The time course for learning in the rats with chronically implanted electrodes was similar to that observed in Experiment 1 (Fig. 6). The MF-stimulated PS were recorded only when the rats were in the resting condition, i.e., when they were not moving about, grooming, or rearing up. The AUC~oc increased with the advances of learning in the maze until day 4 of the trials, whereas changes were not registered in the control group throughout the 7-day observation period (Fig. 7). Statistical analysis of values for A U Q o c before and after the trials confirmed the overall differences between the test and control groups, F(I, 12) = 15.71, p < 0.01, and the differences between the groups in the change over days, F(7, 84) = 7.06, p < 0.01.
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DISCUSSION The time course for learning in which the rats showed correct and erroneous responses in the radial arm maze coincided with
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FIG. 7. Percentage areas under input/output curves obtained every day in the test group (closed circles, n = 7) and control group (open circles, n = 7). Each point and vertical bar indicates mean and SEM.
LTP IN M O S S Y F I B E R - C A 3 AND L E A R N I N G
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manifested in the acquisition period persisted for at least 24 h, because recording of the parameter was performed at 24-h intervals. The 24-h repetitive training may have facilitated and enhanced the memory storage because of the presence of residual PS. Although PS potentiation was not found during the retention period, preceding potentiation was maintained, suggesting retention of the learned behavior. Such a suggestion corresponds to a previous finding by others (22). Present findings suggest that the CA3 field plays an important role in acquiring new information. However, this field is not the only site responsible for learning in rats because PS induced by perforant path stimulation in the dentate gyrus are enhanced on completion of learning (12,22,31,35,36). Thus, neuronal pathway from entorhinal cortex to CA3 field would have to remain intact for rats to learn. A previous study (26), which advocates our postulation, has demonstrated distributional changes of protein kinase C (PKC) only in CA3 field after discrimination learning. Such changes may be associated with training-induced LTP in CA3 field in our present study because a shift in the phosphorylation of B50, a PKC substrate, has been found to be associated with maintenance of LTP (21). This suggestion is especially reinforced by the impairment of spatial memory as a result of neurotoxic lesions in the CA3 (13). Furthermore, rats subjected to
transient forebrain ischemic injury with irreversible damage in the CA1 by four-vessel occlusion did not show any negative effects on their learning performance (38,39). The fact that CA3 insults (but not CA1 lesions) affect rodent performance in learning strongly advocates that the LTP development in the CA3 and previously documented dentate gyrus (22) may be responsible for the memory formation. This conclusion is also supported by the recent electrophysiological studies using hippocampal slice preparations that LTP of the PS induced by M F stimulation in the CA3 were augmented by nootrophic drugs such as aniracetam, idebenone, and vinpocetine, but those LTP responses in the Schaffer collateral-CA1 system were not affected (17,33,34). There is no evidence, however, as to whether synaptic transmission in the M F - C A 3 system would remain totally intact or be damaged to some degree after transient ischemic injury, although CA3 cells have been demonstrated to recover morphologically (30). Prominent among theories of hippocampal function is the suggestion of a transient memory storage. The present evidence supports the view that CA3 field plays a crucial role to acquire new information, and that LTP induced in synapses on neuronal pathway from entorhinal cortex to CA3 field may participate in memory formation and/or storage.
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