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Acidic fibroblast growth factor facilitates generation of long-term potentiation in rat hippocampal slices
Brain Research Bulletin, Vol. 33, No. 5, pp. 505-511, 1994 Copyright 8 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + .OO
Pergamon 0361.9230(93)EOOO4-6
Acidic Fibroblast Growth Factor Facilitates Generation of Long-term Potentiation in Rat Hippocampal Slices KAZUO
SASAKI,*’
YUTAKA
OOMURA,?
ALEXANDER
FIGUROV*’
AND
HIROSHI
YAGI*
*Department of Electronics and Computer Science, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930, Japan TInstitute of Bio-Active Science, Nippon Zoki Pharmaceutical Co. Ltd., Hyogo 673-14, Japan Received
30 August
1993; Accepted
4 October
1993
SASAKl, K., Y. OOMURA, A. FIGUROV AND H. YAGI. Acidic fibroblasi growth factor facilitates generation of long-term potentiation in rat hippocampal slices. BRAIN RES BULL 33(S) 505-511, 1994. In the present study, effects of acidic fibroblast growth factor (aFGF, 0.5-2.5 @ml) on synaptic transmission were investigated in rat hippocampal slices. Stimulation was applied to Schaffer collateral/commissural afferents and evoked spikes were recorded in CA1 pyramidal cell layer. Continuous perfusion of slices with aFGF slightly decreased the basal amplitude of the spikes and significantly increased the paired-pulse facilitation. When brief tetanic stimulation (7 impulses at 100 Hz) was applied 30 min after the perfusion of aFGF, aFGF-treated slices enhanced the magnitude of short-term potentiation after the tetanus and facilitated the generation of long-term potentiation. aFGF also enhanced post-tetanic potentiation directly after the tetanus. These effects of aFGF were dose-dependent. The enhancement of short-term potentiation and facilitation of the generation of long-term potentiation were not evident when aFGF was applied with or 10 min after the tetanus. The results suggest that aFGF is implicated in modulation of synaptic efficacy and can activate some mechanisms related to the generation of long-term potentiation. Paired-pulse facilitation Acidic fibroblast growth factor Rat Long-term potentiation Hippocampus Slice
ACIDIC fibroblast growth factor (aFGF), which has been purified from brain and pituitary extracts, is a member of a large family of heparin-binding polypeptide growth factors that include basic FGF @FGF), int-2, hstlks, FGF-5, FGF-6, and keratinocyte growth factor (KGF) (6,7,42). aFGF exhibits mitogenic activity for glial cells, endothelial cells, vascular smooth muscle cells, and others, in vitro (6,7,42). It also promotes the survival and neurite outgrowth of various cultured brain neurons such as cortical, hippocampal, striatal, and thalamic neurons (41). Administration of aFGF to the severed stump of the optic nerve in viva augmented survival of retinal ganglion cells in rats (35). Continuous infusion of aFGF into the cerebral lateral ventricles before or after transient ischemia protected CA1 pyramidal cells in the hippocampus of gerbil from death (33). Taken together, these tindings indicate that aFGF acts as a neurotrophic factor in various neurons in the central nervous system (CNS). However, the physiological role(s) of aFGF in the CNS other than as the neurotrophic factor is largely unknown. In previous studies, we demonstrated that aFGF, which is produced by ependymal cells in the walls of cerebral ventricles (30), increases 1000 times in cerebrospinal fluid of rats 2 h after food
Post-tetanic
potentiation
Short-term
potentiation
or after intraperitoneal (IP) injection of glucose (13,34), and that neurons in some brain regions such as the hippocampus and the hypothalamus immunoreact to anti-aFGF antibody when examined 2 h after IP glucose injection (30). These neurons also immunoreact to anti-FGF-receptor antibody (39). In addition, we reported that in a step-through passive avoidance task of mice IP injection of glucose 2 h before an acquisition trial significantly increased latency in a retention trial of avoidance tested 24 h later (30,32). In the Morris water maze task, IP glucose injection 2 h before a first trial block significantly reduced latency to find and climb onto a platform hidden just below the water surface (32). The effects of IP glucose on the passive avoidance and the Morris water maze tasks were abolished by pretreatment with anti-aFGF antibody applied into the cerebral ventricles (30,32). Continuous infusion of aFGF into the lateral cerebral ventricles by osmotic minipumps in rats also increased latency in retention trials of passive avoidance tests throughout the infusion time (30). The results suggest that aFGF may be implicated in some neural substrates related to learning and memory. Brief high frequency stimulation of a population of presynaptic fibers in the hippocampus results in a substantial increase in intake,
’ TO whom requests for reprints should be addressed. ’ Present address: Department de Pharmacologic, Centre MCdical Universitaire,
GenBve, Switzerland.
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FIG. 1. (A) The time course of potentiation induced by tetanic stimulation composed of 7 (0, n = 9), 11 (N, n = 9), 31 (V, n = 9), 51 (A, n = 10) or 100 (0, n = 10) impulses at 100 Hz. Abscissa indicates the time before and after tetanic stimulation in min. Ordinate indicates spike amplitude expressed as a percent change of mean basal spike amplitude before tetanic stimulation, defined as 100%. The tetanic stimulation was applied at 0 min. Data represented as the mean 2 SEM. (B) Frequency of LTP induction. The number of slices in which LTP was induced by tetanic stimulation was expressed as percent of the total number of slices tested. LTP frequency induced by tetanic stimulation of 7 impulses at 100 Hz was 0%.
synaptic effectiveness. This basically consists of three sequential events: (a) post-tetanic potentiation (PTP); (b) decremental shortterm potentiation (SIP); and (c) persistent long-term potentiation (LTP) (28). Several studies have shown that STP and LTP have similar inductive mechanisms (3,4,5,22), so it is possible that some drugs that enhance SIP also facilitate the generation of LTP. In fact, it has been shown that phorbol ester and unsaturated fatty acids transform SIP to LTP and facilitate the generation of LTP (10,18,22,43). Interestingly, it was reported recently that some polypeptide growth factors such as epidermal growth factor (EGF) and bFGF enhance the magnitude of LTP in the CA1 region of rat hippocampal slices (1,37,38). In another experiment, Abe et al. (2) demonstrated that EGF enhances the magnitude of SIP, transforms SIP to LTP, and facilitates the generation of
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LTP. These findings are of great interest in view of the novel physiological functions of growth factors and suggest that other growth factors such as aFGF also may have some effects on LTP. Thus, the present study was undertaken to clarify relations between aFGF and LTP by investigating possible effects of aFGF on SIP in the CA1 region of rat hippocampal slices. MATERIALS AND METHODS
Slice Preparation
Animals were 5-8 week old Wistar rats (weighing 140-250 g). Following decapitation, the brain was quickly isolated from the skull and transverse hippocampal slices (400 pm) were cut with a microslicer in oxygenated, ice cooled Ringer’s solution. The slices were then placed into an incubation chamber containing Ringer’s solution, which was maintained at room temperature (27°C) and continuously oxygenated with 95% 02-5% COz. The composition of Ringer’s solution was as follows (mM): NaCl, 124.0; KCl, 5.0; CaCl*, 2.4; MgS04, 1.3; KH2P04, 1.24; NaHC03, 26.0; glucose, 10.0. Neurophysiological
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Time ( min ) FIG. 2. The time course of potentiation induced by the first and second tetanic stimulation composed of 7 impulses at 100 Hz. There was no significant difference between the time courses of potentiation induced by the first (0) and second (0) tetanus. The number of slices tested was 8. Other descriptions as in Fig. 1A.
Procedures
After preincubation for more than 1.5 h, each slice was transferred into a recording chamber which was maintained at 32.5 +. 0.5”C, constantly aerated with 95% 02-5% CO1, and perfused continuously with Ringer’s solution at a rate of 1 ml/mm A bipolar stainless steel stimulating electrode was placed in the stratum radiatum of the CA3 region of the slice to stimulate Schaffer collateral/commissural afferents. The extracellular evoked potential was recorded from the pyramidal cell layer of the CA1 region with a glass microelectrode filled with 0.9% NaCl solution (electrode resistance, 3-8 MO), and fed into an amplifier. The output of the amplifier was then monitored on digital oscilloscopes and charted on an ink recorder. Single test stimulation, which consisted of a 0.05 ms constant current 200-600
ACIDIC FIBROBLAST
GROWTH FACTOR
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“‘I’
was always adjusted so that the amplitude of the spike was 45% to 55% of the maximum and the paired-pulse facilitation (ratio of second to first response) was 150% to 160%. Any slice that could not meet this criterion was not used in the experiments. In addition, experiments were only started after stable evoked potentials were established, Spike amplitude was expressed as the percent change from the average spike amplitude that was measured during the control period and defined as 100%. LTP was considered to have occurred when the potentiated spike amplitude was maintained at a level more than 10% above the baseline level 30 min after tetanic stimulation. The ratio of slices in which LTP was induced to the total number of slices tested was expressed as percentage and referred as the frequency of LTP generation.
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Acidic Fibroblast Growth Factor
Bovine aFGF, which was purchased from R & D Systems Inc. (Minneapolis, MN), was first diluted to a concentration of 25 &ml in distilled water and small aliquots was stored at -20°C. One aliquot was taken in each experiment and further diluted to the desired concentration by adding Ringer’s solution just before use.
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Statistics
Results were analyzed using analysis of variance (ANOVA) followed by Student’s t-test or the Cochran-Cox test. The criterion of significance was p < 0.05. I -30
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RESULTS
The relation between the number of impulses of high frequency tetanic stimulation and the generation of LTP was first investigated. Figure 1A shows the time course of potentiation following tetanic stimulation composed of 7, 11, 31, 51 or 100 impulses delivered at 100 Hz. Increase of the number of impulses of tetanic stimulation increased the magnitude of potentiation, and the frequency of LTP generation (Fig. 1B). In any slices tested, the tetanic stimulation of 7 impulses at 100 Hz only produced potentiation which returned to baseline about 30 min after the tetanus, and was regarded as STP, but not LTP. Therefore, the frequency of LTP generation was O%, and we selected the stimulation of 7 impulses at 100 Hz as a subthreshold tetanic stimulation required for the generation of LTP. Figure 2 shows the time course of STP when 7 impulses of tetanic stimulation was applied at 100 Hz twice with an interval
FIG. 3. Effects of aFGF on potentiation induced by 7 impulses, 100 Hz tetanus. After the control ~tentiation (0) induced by the first tetanus was observed for 30 min in Ringer’s sofution, aFGF was perfused. The second tetanic stimulation was applied 30 min after beginning of aFGF perfusion. (A, B and C) Effects of 0.5 (n = 9), 1.0 (n = 9) and 2.5 (n = 9) ng/ml aFGF on potentiation (e) induced by the second tetanic stimulation, respectively. *p < 0.05; **p < 0.01; ***p < 0.001. Other descriptions as in Fig. 1A.
PA square-wave pulse, was applied at intervals of 10 s except during high frequency tetanic stimulation and paired-pulse stimulation. High frequency tetanic stimulation (7-100 impulses at 100 Hz) to produce potentiation and paired-pulse stimulation (interval: 51 ms) to induce paired-pulse facilitation were delivered at the same intensity through the stimulating electrode used for single test stimulation. In the present study, spike amplitudes were defined as the amplitude from the negative peak to the late positive peak. Before the recording session, the stimulus intensity
0.5 ng/ml
10 ng/mf
2.5 ng/mi
FIG. 4. Effects of aFGF on the frequency of LTf’ generation. The number of slices in which LTP was induced is expressed as percentage of the total number of slices treated by 0.5, 1.0 and 2.5 ng/ml aFGF. LTF’ frequency increased dose-dependently.
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Time ( min ) FIG. 5. Effects of aFGF on paired-pulse facilitation. After the basal spike amplitude (open marks) and paired-pulse facilitation (filled marks) were observed for 30 min in Ringer’s solution, 1.0 (A, A, n = 8) or 2.5 (0, D, n = 9) &ml aFGF was perfused. In controls, Ringer’s solution was continuously perfused (0, lt n = 6). Abscissa indicates the time before and after the application of aFGF in min. aFGF was applied at 0 min. For basal spike amplitude, ordinate indicates spike amplitude expressed as a percent change of mean basal amplitude before tetanic stimulation that was defined as 100%. For paired-pulse facilitation, ordinate indicates spike amplitude by the second pulse expressed as a percent of that by the first pulse at each time point. *p < 0.05; **p < 0.01; ***p < 0.001.
of 1 h in the same slice. In the magnitude of initial potentiation and the decay time course, STP induced by a second tetanus was identical to that induced by a first tetanus, and there was no statistical significance between them. In later experiments, therefore, the control STP and the STP in the presence of drugs were observed and compared in the same slice. After a basal spike amplitude was observed for 20 min (control period), the first tetanic stimulation was applied and the control STP was observed for 30 min. Perfusion of a slice with aFGF was then started. The second tetanic stimulation was delivered 30 min after the beginning of perfusion. The concentrations of aFGF used in the present study were 0.5 (0.32), 1.0 (0.65) and 2.5 (1.61) n&&/ml(lo-l*M). After 0.5 ng/mi aFGF was perfused, the basal spike amplitude did not change significantly compared to controls (Fig. 3A). However, perfusion of the slices with 1.0 and 2.5 @ml aFGF significantly and dose-dependently decreased the basal amplitude of spikes (Fig. 3B and C). The magnitude of the basal spike amplitude by perfusion of 1.0 and 2.5 @ml aFGF was 96.9 + 0.6% (mean ? SEM) and 92.8 f 0.6%, respectively, when measured 1 min before the second tetanus. In the presence of 0.5 @ml aFGF, the tetanic stimulation had no effect on the magnitude of initial potentiation observed within 2 min after the tetanus, but it significantly increased the magnitude of the following potentiation (Fig. 3A). In the presence of 1.0 and 2.5 ng/ml aFGF, the magnitude of the initial potentiation as well as the following potentiation was significantly increased (Fig. 3B and C). The degree of initial potentiation directly after the tetanus by 0.5, 1.0 and 2.5 r&ml aFGF was 156.3 2 1.2%, 160.4 + 1.7% and 165.4 + 1.4%, respectively, and that of the following ~tentiation 30 min after the tetanus was 106.1 t 1.2%, 109.1 t 0.9% and 114.0 2 1.4%, respectively. Thus, the enhancing effects of aFGF on the magnitude of STP was dosedependent. The frequency of LTP generation also increased in
dose-dependently: 11.1% for 0.5 &ml aFGF, 33.3% for 1.0 ng/ ml aFGF and 77.8% for 2.5 ng/ml aFGF (Fig. 4). Figure 5 shows the effects of aFGF on paired-pulse facilitation induced by paired-pulse stimulation, After basal spike amplitude in first response and paired-pulse facilitation were observed for 30 min (control period), perfusion of aFGF was started. Perfusion of 1.0 and 2.5 r&ml aFGF significantly and dose-dependently decreased the basal spike amplitude in a way and to a degree similar to those observed in Fig. 3. In contrast, aFGF significantly and dose-dependently increased the pairedpulse facilitation. The increase of paired-pulse facilitation was from 158.8 2 1.5% 5 min before the beginning of perfusion to 182.6 t 1.3% 1 h after the beginning of perfusion for 1.0 @ml aFGF, and from 157.6 -t 0.9% to 190.8 t 0.6% for 2.5 ng/ml aFGF. Since paired-pulse facilitation is defined as the ratio of the second response amplitude to the first response amplitude, it increases relatively when the magnitude of first response decreases and the second response does not change. Such a relative increase in paired-pulse facilitation due to the decrease of basal spike amplitude is estimated as a change from about 158% in control periods to about 165% for 1.0 ng/ml aFGF and to about 172% for 2.5 @ml aFGF. In the present study, the degree of increase of the paired-pulse facilitation was rather greater than those estimated values. Therefore, it seems to be reasonable to state that aFGF enhances paired-pulse facilitation. In the above experiments, we started to apply aFGF 30 min before the second tetanic stimulation. We also investigated the effects of aFGF on STP when it was administered at times different than 30 min before the second tetanus. The results are shown in Fig. 6. aFGF applied 20 min before the second tetanw significantly increased the magnitude of initial potentiation within 2 min after the tetanus and the magnitude of following potentiation, except 3 min after the tetanus. The degree of in-
ACIDIC FIBROBLAST
GROWTH FACTOR
Time ( min )
Time ( min )
FIG. 6. Effects on potentiation of aFGF applied before, with and after tetanic stimulation. The control potentiation (0) was observed for 30 min in Ringer’s solution after the first tetanic stimulation. The second tetanic stimulation (0) was applied 60 min after the first tetanus. (A, B, C and D) 2.5 @ml aFGF was applied 20 min before, 10 min before, at the time of, and 10 min after the second tetanic stimulation of 7 impulses at 100 Hz, respectively. *p < 0.05; **p < 0.01; ***p < 0.001. Other descriptions as in Fig. 1A.
crease in following potentiation 30 min after tetanus was 112.1 ? 1.9% and the LTP was induced in 5 of 8 slices (62.5%, Pig. 7). When aFGF was applied 10 min before the second tetanus, the magnitude of the potentiation within 2 min after the tetanus did not change si~ificantly, but the magni~de of following potentiation increased significantly. The degree of following potentiation 30 min after the tetanus was 106.8 t 1.0% and LTP was induced in 1 of 8 slices (12.5%). aFGF applied with, and 10 min after, the second tetanus had almost no effect on the initial potentiation and the following potentiation and did not generate LTP, DISCUSSION In the present study, perfusion of slices with aFGF enhanced the magnitude of SIP, which leads to the generation of LTP, and increased paired-pulse facilitation in the CA1 region of the hippocampus. In contrast to this, aFGF reduced the basal amplitude of spikes. A recent study in another laboratory has demonstrated that bFGF increases the magnitude of LTP and paired-pulse facilitation and decreases the basal spike amplitude in the CA1 region of rat hippocampal slices (1). From in vivo studies, it was also reported that aFGF and bFGF enhance the potentiation induced by weak tetanic stimulation in the dentate gyms of anaesthetized rats (15,17). Therefore, it seems that aFGF and bFGF have identical biological effects in the hippocampus. In support of this, it has been reported that aFGF and bFGF are 55% ho-
mologous in amino acid sequence (6,7,42) and interact with the same surface receptors (27). STP and LTP have similar inductive mechanisms and are considered to be caused by postsynaptic mechanisms including opening of N-methyl-D-asp~ate (NMDA) receptor channels and subsequent postsynaptic entry of Ca’+ ions (3,4,22). The rise in intracellular Ca” is thought to stimulate kinase activity. Ca*+phospholipid dependent kinase (PKC) (8) tyrosine kinase (29) and Ca’+-calmodulin kinase II (24) have important functions in LTP. Previous studies demons~ated that activation of PKC participates in the maintenance phase but not in the early phase of LTP (12). However, Asztely et al. (5) recently reported that activation of PKC by phorbol-12,13-diacetate reduces early decay of LTP in the CA1 region of the hippocampus, suggesting that PKC activation may be involved in biochemical events underlying the early phase, as well as those underlying the later phase of LTP. So far, several studies have shown that FGFs activate PKC. In our previous study, intraventricular infusion of aFGF significantly suppressed feeding in rats. This suppression of feeding was concluded to be due to suppression of glucose-sensitive neurons in the LHA, which in turn was caused by activation of PKC induced by aFGF (30). Prester et al. (31) found that bFGF induces PKC-dependent mitogenic response of transformed fetal bovine aortic endothelial GM 7373 cells. Tsuda et al. (40) and Takeyama et al. (36) also reported that bFGF stimulated the generation of diacylglycerol with subsequent activation of protein
510
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Time (min) FIG. 7. Effects of aFGF applied before, with and after tetanic stimulation
on the frequency of LTF’generation. The number of slices in which LTP was induced is expressedas a percent of the total number of slices treated by 2.5 @ml aFGF applied 20 min before (-20), 10 min before (-lo), at the time of (0), and 10 min after (10) the second tetanic stim~ation by 7 impulses at 100 Hz. LTP frequency was 0% when aFGF was applied with or 10 min after the second tetanus.
kinase C in Swiss 3T3 cells. Other than PKC, a recent study shows that aFGF activates an FGF receptor-associated tyrosine kinase (16). However, tyrosine kinase inhibitors such as Lavendustin A and Genistein had no significant effect on ST%’in the hippocampus, although they blocked LTP (29). Therefore, it might be possible that FGFs, in the present and other experiments (l&5,17), promote the generation of LTP by enhancing the magnitude of STP throu~ activation of protein ~n~e(s} such as PKC. In addition, it should be noted that FGFs rapidly induce the expression of the fos and myc protooncogenes in some ceils (26,44), since there is a possibility that protooncogenes are involved in the mechanisms of LTP (9). Initial potentiation, which lasts for a few minutes after tetanic stim~ation, consists mainly of the component of PTP. In addition to the increase of paired-pulse facilitation, initial potentiation after tetanus in the present study was found to be increased by the presence of aFGF. PTP as well as paired-pulse facilitation is considered to be caused through increase of transmitter release from presynaptic terminals (12,14,20,21,25). Therefore, the present study showing that aFGF enhances paired-pulse facilitation and PTP suggests a possibility that, at least, aFGF transiently modulates presynaptic mechanisms after high frequency stimulation such as tetanic stimulation and paired-pulse stimulation. In the present study, the magnitude of the basal spike amplitude decreased significantly and dose-dependently in the presence of aFGF. In previous studies, effects of PKC activation on basal response were inconsistent. It has been reported that activation of PKC by phorbol esters can induce lasting increases in
fcr AI,.
basal responses in the hippocampus (11,23). In contrast, Lovinger and ~outtenberg (19) demonstrated that application of PKC stimulators such as lZ-O-tetradecanoyl-phorbol- 13-acetate and c&unsaturated fatty acid did not alter basal responses but did enhance the maintenance of LTP. Application of tyrosine kinase inhibitors also had no effect on the basal responses in the hippocampus (29). Further investigations are required to investigate relations between the reduction of basal responses and aFGF, considering some other mechanisms such as the expression of protooncogenes mentioned above in addition to activation of protein kinase(s). Terlau and Seifelt (38) reported that bFGF does not lead to changes in the evoked potentials in the CA1 region of the hippocampus when it is added 20 min after tetanic stimulation, although it enhances LTP when added 20 min before tetanic stimulation. They discussed that processes which are activated during or in the first 20 min after tetanic stimulation are involved in the modulatory effect of bFGF on LTP. In the present study, aFGF produced almost no changes on the magnitude of potentiation when it was added at the time of and 10 min after tetanic stimulation. increase of potentiation, except the initial part, was observed when aFGF was added 10 min before tetanus, whereas increase of initial potentiation and following potentiation was observed when it was added 20 min before tetanus. Therefore, it appears that the enhancement of STP by aFGF, which leads to the generation of LTP, requires some biochemical processes activated by aFGF before tetanic stimulation. In conclusion, we demonstrated that aFGF enhances STP, transforms STP to LTP, and promotes the generation of LTP. The results suggest that aFGF may be involved in the modulatory mechanisms of synaptic plasticity in the adult CNS and that aFGF may activate some mechanisms related to the generation of LTP. In a previous study, we demonstrated that 1P glucose, which increases aFGF in the cerebrospinal fluid (13,34), facilitates behavioral performance in passive avoidance and Morris water maze tasks (30,32). Since these glucose effects are abolished by pretreatment with anti-aFGF antibody applied into cerebral ventricles (30,32), it might be possible that these behavioral effects by glucose are attributed to endogenous aFGF. And it might be expected that endogenous aFGF may contribute to these effects by having the same effect in the modulation of synaptic plasticity as exogenous aFGF. Further studies are required
to clarify the detailed mechanisms involved in the LTP-promoting effects of aFGF and the relation between enhancement of behavioral performance in learning tasks and aFGF. ACKNOWLEDGEMENTS
This work was partly supported by the Japanese Ministry of Education, Science and Culture Grant-in-Aid for Scientific Research, 02454127 (KS.), and by the Science and Technology Agency. using the Special Coordination Funds for Promoting Science and Technology of Japan (Y.O., KS).
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7. Baird, A.; Walicke, P. A. Fibroblast growth factors. Br. Med. Bull. 45:438-452; 1989. 8. Ben-Ari, Y.; Aniksztejn, L.; Bregestovski, P. Protein kinase C modulation of NMDA currents: An important link for LTP induction. Trends Neurosci. 15:333-339; 1992. 9. Cole, A. J.; Saffen, D. W.; Baraban, J. M.; Worley, P. F. Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340:474476; 1989. 10. Colley, P. A.; Sheu, F. S.; Routtenberg, A. Dose-dependent phorbol ester facilitation or blockade of hippocampal long-term potentiation: Relation to membrane/cytosol distribution of protein kinase C activity. Brain Res. 495:205-216; 1989. 11. Gustafsson, B.; Huang, Y.; Wigstrom, H. Photbol ester-induced potentiation differs from long-term potentiation in the guinea pig hippoeampus in vitro. Neurosci. Lett. 85:77-81; 1988. 12. Gustafsson, B.; Wigstrom, H. Physiological mechanisms underlying long-term potentia;on. Trends Neurosci. 11:156-162; 1988. 13. Hanai, K.; Oomura, Y.; Kai, Y.; Nishikawa, K.; Shim& N.; Morita, H.; Plata-Salaman, C. R. Central action of acidic fibroblast growth factor in feeding regulation. Am. J. Physiol. 256:R217-R223; 1989. 14 Hess, G.; Kuhnt, U.; Voronin, L. L. Quantai analysis of paired-pulse facilitation in guinea pig hippocampal slices. Neurosci. Lett. 77:187-192; 1987. 15 Hisajima, H.; Saito, H.; Abe, K.; Nishiyama, N. Effects of acidic fibroblast growth factor on hippocampal long-term potentiation in fasted rats. J. Neurosci. Res. 31549-553; 1992. 16 Huang, S. S.; Huang, 1. S. Association of bovine bran-derived growth factor receptor with protein tyrosine kinase activity. J. Biol. Chem. 261:9568-9571; 1986. 17. Ishiyama, J.; Saito, H.; Abe, K. Epidennal growth factor and basic fibroblast growth factor promote the generation of long-term potentiation in the dentate gyrus of anaesthetized rats. Neurosci. Res. 12403-411; 1991. 18 Linden, D. J.; Murakami, K.; Routtenberg, A. A newly discovered protein kinase C activator (oleic acid) enhances long-term potentiation in the intact hippocampus. Brain Res. 379:358-363; 1986. 19. Lovinger, D. M.; Routtenberg, A. Synapse-specific protein kinase C activation enhances maintenance of long-term potentiation in rat hippocampus. J. Physioi. 400:321-333; 1988. 20. Magleby, R. L.; Zengel, J. E. A dual effect of repetitive stimuiation on post-tetanic potentiation of transmitter release at the frog neuromuscular junction. J. Physiol. 245:163-182; 1975. 21. Magleby, K. L.; Zengel, J. E. A quantitative description of tetanic and post-tetanic potentiation of transmitter release at the frog neuromuscular junction. J. Physiot. 245:183-208; 1975. 22. Malenka, R. C. Postsynaptic factors control the duration of synaptic enhancement in area CA1 of the hippocampus. Neuron 653-60; 1991. 23. Malenka, R. C.; Madison, D. V.; Nicoll, R. A. Potentiation of synaptic transmission in the hippocampus by phorbol esters, Nature 321:175-177; 1986. 24. Malinow, R.; Schulman, H.; Tsien, R. W. Inhibition of postsynaptic PKC or CaMKB blocks induction but not expression of LTP. Science 245862-866; 1989. 25. McNaughton, B. L. Long-term synaptic enhancement and short-term potentiaiion in rat fascia dentate act through different mechanisms. J. Phvsiol. 324:249-262: 1982. 26. M&r, R.; Bravo, R.; Burckhardt, J. Induction of c-fos gene and protein by growth factors precedes activation of c-my; Nature 312:716-720; 1984.
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27. Neufeld, G.; Gospodarowicz, D. Basic and acidic fibroblast growth factors interact with the same surface receptors. J. Biol. Chem. 261:5631-5637; 1986. 28. Nicoll, R. A.; Kauer, J. A.; Malenka, R. C. The current excitement in long-term potentiation. Neuron 1:97-103; 1988. 29. O’Dell, T. J.; Kandel, E. R.; Grant, S. G. N. Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors. Nature 353558-560; 1991. 30. Oomura, Y.; Sasaki, K.; Suzuki, K.; Muto, T.; Li, A.; Ogita, Z.; Hanai, K.; Tooyama, I.; Kimura, H.; Yanaihara, N. A new brain glucosensor and its physiological significance. Am. J. Clin. Nutr. 55:278S-2823; 1992. 31 Presta, M.; Tiberio, L.; Rusnati, M.; Dell’Era, P.; Ragnotti, G. Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endotheliacells. Cell Regul. 2719-726; _ 1991. _.. __ 32. Sasaki, K.; Oomura, Y.; Li, A.; Hanai, K.; Tooyama, I.; Kimma, H.; Yagi, H. Effects of acidic fibroblast growth factor on memory in rodents. Neurosci. Abstr. 17:llOO; 1991. 33. Sasaki, K.; Oomura, Y.; Suzuki, K.; Hanai, K.; Yagi, H. Acidic fibroblast growth factor prevents death of hippocampal CA1 pyramidal cells following ischemia. Neurochem. Int. 21:397-402: 1992. 34. Sasaki, K.; Oomur\, Y.; Suzuki, K.; Muto, T.; Hanai, K.; Tooyama, I.; Kimura, H.; Yanaihara, N. Effects of fibroblast growth factors and platelet-derived growth factor on food intake in rats, Brain Res. Bull. 27:327-332; 1991. 35. Sievers, J.; Hausmann, B.; Unsicker, K.; Berry, M. Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neurosci. Lett. 76:157-162; 1987. 36. Takeyama, Y.; Tanimoto, T.; Hoshijima, M.; Kaibuchi, K.; Ohyanagi, H.; Saitoh, Y.; Takai, Y. Enhancement of fibroblast growth factor-induced diacylglycerol formation and protein kinase C activation by colon tumor-promoting bile acid in Swiss 3T3 cells. Different modes of action between bile acid and phorbol ester. FEBS Len. 197:339-343; 1986. 37. Terlau, H.; Seifeit, W. Influence of epidermal growth factor on longterm potentiation in the hippocampal slice. Brain Res. 484:352-356; 1989. 38. Terlau, H.; Seifelt, W. Fibroblast growth factor enhances long-term potentiation in the hippocampal slice. Eur. J. Neurosci. 2:973-977; 1990. 39. Tooyama, 1.; Yasuhara, 0.; Matsuo, A.; Hanai, K.; Isobe, M.; Oomura, Y.; McGeer, P. L.; Kamo, H.; Kimura, H. immunohistochemical study of fibroblast growth factor receptor in brains of the rat and human, Neurosci. Abstr. 17:196; 1991. 40. Tsuda, T.; Kaibuchi, K.; Kawahara, Y.; Fukuzaki, H.; Takai, Y. Induction of protein kinase C activation and Ca” mobilization by tibroblast growth factor in Swiss 3T3 cells. FEBS Lett. 191:205210; 1985. 41. Walicke, P. A. Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J. Neurosci. 826X-2627; 1988. 42. Walicke, P. A. Novel neurotrophic factors, receptors, and oncogenes. Ann. Rev. Neurosci. 12:103-126; 2989. 43. Williams, J. H.; Errington, M. L.; Lynch, M. A.; Bliss, T. V. P. Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus. Nature 341:739742: 1989. 44. Zeytin, F. M.; Rusk, S. F.; Baird A.; Raymond, V.; Leff, S. E.; Mandell, A. J. Induction of c-fos, calcitonin gene expression, and acidic FGF production in a multipeptide-se~eting neuroend~ine cell line. EndocrinoIogy 122:1114-1120; 1988.
Pergamon 0361.9230(93)EOOO4-6
Acidic Fibroblast Growth Factor Facilitates Generation of Long-term Potentiation in Rat Hippocampal Slices KAZUO
SASAKI,*’
YUTAKA
OOMURA,?
ALEXANDER
FIGUROV*’
AND
HIROSHI
YAGI*
*Department of Electronics and Computer Science, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930, Japan TInstitute of Bio-Active Science, Nippon Zoki Pharmaceutical Co. Ltd., Hyogo 673-14, Japan Received
30 August
1993; Accepted
4 October
1993
SASAKl, K., Y. OOMURA, A. FIGUROV AND H. YAGI. Acidic fibroblasi growth factor facilitates generation of long-term potentiation in rat hippocampal slices. BRAIN RES BULL 33(S) 505-511, 1994. In the present study, effects of acidic fibroblast growth factor (aFGF, 0.5-2.5 @ml) on synaptic transmission were investigated in rat hippocampal slices. Stimulation was applied to Schaffer collateral/commissural afferents and evoked spikes were recorded in CA1 pyramidal cell layer. Continuous perfusion of slices with aFGF slightly decreased the basal amplitude of the spikes and significantly increased the paired-pulse facilitation. When brief tetanic stimulation (7 impulses at 100 Hz) was applied 30 min after the perfusion of aFGF, aFGF-treated slices enhanced the magnitude of short-term potentiation after the tetanus and facilitated the generation of long-term potentiation. aFGF also enhanced post-tetanic potentiation directly after the tetanus. These effects of aFGF were dose-dependent. The enhancement of short-term potentiation and facilitation of the generation of long-term potentiation were not evident when aFGF was applied with or 10 min after the tetanus. The results suggest that aFGF is implicated in modulation of synaptic efficacy and can activate some mechanisms related to the generation of long-term potentiation. Paired-pulse facilitation Acidic fibroblast growth factor Rat Long-term potentiation Hippocampus Slice
ACIDIC fibroblast growth factor (aFGF), which has been purified from brain and pituitary extracts, is a member of a large family of heparin-binding polypeptide growth factors that include basic FGF @FGF), int-2, hstlks, FGF-5, FGF-6, and keratinocyte growth factor (KGF) (6,7,42). aFGF exhibits mitogenic activity for glial cells, endothelial cells, vascular smooth muscle cells, and others, in vitro (6,7,42). It also promotes the survival and neurite outgrowth of various cultured brain neurons such as cortical, hippocampal, striatal, and thalamic neurons (41). Administration of aFGF to the severed stump of the optic nerve in viva augmented survival of retinal ganglion cells in rats (35). Continuous infusion of aFGF into the cerebral lateral ventricles before or after transient ischemia protected CA1 pyramidal cells in the hippocampus of gerbil from death (33). Taken together, these tindings indicate that aFGF acts as a neurotrophic factor in various neurons in the central nervous system (CNS). However, the physiological role(s) of aFGF in the CNS other than as the neurotrophic factor is largely unknown. In previous studies, we demonstrated that aFGF, which is produced by ependymal cells in the walls of cerebral ventricles (30), increases 1000 times in cerebrospinal fluid of rats 2 h after food
Post-tetanic
potentiation
Short-term
potentiation
or after intraperitoneal (IP) injection of glucose (13,34), and that neurons in some brain regions such as the hippocampus and the hypothalamus immunoreact to anti-aFGF antibody when examined 2 h after IP glucose injection (30). These neurons also immunoreact to anti-FGF-receptor antibody (39). In addition, we reported that in a step-through passive avoidance task of mice IP injection of glucose 2 h before an acquisition trial significantly increased latency in a retention trial of avoidance tested 24 h later (30,32). In the Morris water maze task, IP glucose injection 2 h before a first trial block significantly reduced latency to find and climb onto a platform hidden just below the water surface (32). The effects of IP glucose on the passive avoidance and the Morris water maze tasks were abolished by pretreatment with anti-aFGF antibody applied into the cerebral ventricles (30,32). Continuous infusion of aFGF into the lateral cerebral ventricles by osmotic minipumps in rats also increased latency in retention trials of passive avoidance tests throughout the infusion time (30). The results suggest that aFGF may be implicated in some neural substrates related to learning and memory. Brief high frequency stimulation of a population of presynaptic fibers in the hippocampus results in a substantial increase in intake,
’ TO whom requests for reprints should be addressed. ’ Present address: Department de Pharmacologic, Centre MCdical Universitaire,
GenBve, Switzerland.
506
0
20
lo
Time
30
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7
11
3l
51
loo
impulses, lOOH
FIG. 1. (A) The time course of potentiation induced by tetanic stimulation composed of 7 (0, n = 9), 11 (N, n = 9), 31 (V, n = 9), 51 (A, n = 10) or 100 (0, n = 10) impulses at 100 Hz. Abscissa indicates the time before and after tetanic stimulation in min. Ordinate indicates spike amplitude expressed as a percent change of mean basal spike amplitude before tetanic stimulation, defined as 100%. The tetanic stimulation was applied at 0 min. Data represented as the mean 2 SEM. (B) Frequency of LTP induction. The number of slices in which LTP was induced by tetanic stimulation was expressed as percent of the total number of slices tested. LTP frequency induced by tetanic stimulation of 7 impulses at 100 Hz was 0%.
synaptic effectiveness. This basically consists of three sequential events: (a) post-tetanic potentiation (PTP); (b) decremental shortterm potentiation (SIP); and (c) persistent long-term potentiation (LTP) (28). Several studies have shown that STP and LTP have similar inductive mechanisms (3,4,5,22), so it is possible that some drugs that enhance SIP also facilitate the generation of LTP. In fact, it has been shown that phorbol ester and unsaturated fatty acids transform SIP to LTP and facilitate the generation of LTP (10,18,22,43). Interestingly, it was reported recently that some polypeptide growth factors such as epidermal growth factor (EGF) and bFGF enhance the magnitude of LTP in the CA1 region of rat hippocampal slices (1,37,38). In another experiment, Abe et al. (2) demonstrated that EGF enhances the magnitude of SIP, transforms SIP to LTP, and facilitates the generation of
150
1
LTP. These findings are of great interest in view of the novel physiological functions of growth factors and suggest that other growth factors such as aFGF also may have some effects on LTP. Thus, the present study was undertaken to clarify relations between aFGF and LTP by investigating possible effects of aFGF on SIP in the CA1 region of rat hippocampal slices. MATERIALS AND METHODS
Slice Preparation
Animals were 5-8 week old Wistar rats (weighing 140-250 g). Following decapitation, the brain was quickly isolated from the skull and transverse hippocampal slices (400 pm) were cut with a microslicer in oxygenated, ice cooled Ringer’s solution. The slices were then placed into an incubation chamber containing Ringer’s solution, which was maintained at room temperature (27°C) and continuously oxygenated with 95% 02-5% COz. The composition of Ringer’s solution was as follows (mM): NaCl, 124.0; KCl, 5.0; CaCl*, 2.4; MgS04, 1.3; KH2P04, 1.24; NaHC03, 26.0; glucose, 10.0. Neurophysiological
II
a
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Time ( min ) FIG. 2. The time course of potentiation induced by the first and second tetanic stimulation composed of 7 impulses at 100 Hz. There was no significant difference between the time courses of potentiation induced by the first (0) and second (0) tetanus. The number of slices tested was 8. Other descriptions as in Fig. 1A.
Procedures
After preincubation for more than 1.5 h, each slice was transferred into a recording chamber which was maintained at 32.5 +. 0.5”C, constantly aerated with 95% 02-5% CO1, and perfused continuously with Ringer’s solution at a rate of 1 ml/mm A bipolar stainless steel stimulating electrode was placed in the stratum radiatum of the CA3 region of the slice to stimulate Schaffer collateral/commissural afferents. The extracellular evoked potential was recorded from the pyramidal cell layer of the CA1 region with a glass microelectrode filled with 0.9% NaCl solution (electrode resistance, 3-8 MO), and fed into an amplifier. The output of the amplifier was then monitored on digital oscilloscopes and charted on an ink recorder. Single test stimulation, which consisted of a 0.05 ms constant current 200-600
ACIDIC FIBROBLAST
GROWTH FACTOR
507
“‘I’
was always adjusted so that the amplitude of the spike was 45% to 55% of the maximum and the paired-pulse facilitation (ratio of second to first response) was 150% to 160%. Any slice that could not meet this criterion was not used in the experiments. In addition, experiments were only started after stable evoked potentials were established, Spike amplitude was expressed as the percent change from the average spike amplitude that was measured during the control period and defined as 100%. LTP was considered to have occurred when the potentiated spike amplitude was maintained at a level more than 10% above the baseline level 30 min after tetanic stimulation. The ratio of slices in which LTP was induced to the total number of slices tested was expressed as percentage and referred as the frequency of LTP generation.
I f
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11
1
0
10
20
30
B
s
Acidic Fibroblast Growth Factor
Bovine aFGF, which was purchased from R & D Systems Inc. (Minneapolis, MN), was first diluted to a concentration of 25 &ml in distilled water and small aliquots was stored at -20°C. One aliquot was taken in each experiment and further diluted to the desired concentration by adding Ringer’s solution just before use.
150
Statistics
Results were analyzed using analysis of variance (ANOVA) followed by Student’s t-test or the Cochran-Cox test. The criterion of significance was p < 0.05. I -30
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lime
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Ill
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( min )
RESULTS
The relation between the number of impulses of high frequency tetanic stimulation and the generation of LTP was first investigated. Figure 1A shows the time course of potentiation following tetanic stimulation composed of 7, 11, 31, 51 or 100 impulses delivered at 100 Hz. Increase of the number of impulses of tetanic stimulation increased the magnitude of potentiation, and the frequency of LTP generation (Fig. 1B). In any slices tested, the tetanic stimulation of 7 impulses at 100 Hz only produced potentiation which returned to baseline about 30 min after the tetanus, and was regarded as STP, but not LTP. Therefore, the frequency of LTP generation was O%, and we selected the stimulation of 7 impulses at 100 Hz as a subthreshold tetanic stimulation required for the generation of LTP. Figure 2 shows the time course of STP when 7 impulses of tetanic stimulation was applied at 100 Hz twice with an interval
FIG. 3. Effects of aFGF on potentiation induced by 7 impulses, 100 Hz tetanus. After the control ~tentiation (0) induced by the first tetanus was observed for 30 min in Ringer’s sofution, aFGF was perfused. The second tetanic stimulation was applied 30 min after beginning of aFGF perfusion. (A, B and C) Effects of 0.5 (n = 9), 1.0 (n = 9) and 2.5 (n = 9) ng/ml aFGF on potentiation (e) induced by the second tetanic stimulation, respectively. *p < 0.05; **p < 0.01; ***p < 0.001. Other descriptions as in Fig. 1A.
PA square-wave pulse, was applied at intervals of 10 s except during high frequency tetanic stimulation and paired-pulse stimulation. High frequency tetanic stimulation (7-100 impulses at 100 Hz) to produce potentiation and paired-pulse stimulation (interval: 51 ms) to induce paired-pulse facilitation were delivered at the same intensity through the stimulating electrode used for single test stimulation. In the present study, spike amplitudes were defined as the amplitude from the negative peak to the late positive peak. Before the recording session, the stimulus intensity
0.5 ng/ml
10 ng/mf
2.5 ng/mi
FIG. 4. Effects of aFGF on the frequency of LTf’ generation. The number of slices in which LTP was induced is expressed as percentage of the total number of slices treated by 0.5, 1.0 and 2.5 ng/ml aFGF. LTF’ frequency increased dose-dependently.
508
SASAKl
_&
*
_&
.
_._
~
a
lo
&
ET Al..
&
40
50
60
Time ( min ) FIG. 5. Effects of aFGF on paired-pulse facilitation. After the basal spike amplitude (open marks) and paired-pulse facilitation (filled marks) were observed for 30 min in Ringer’s solution, 1.0 (A, A, n = 8) or 2.5 (0, D, n = 9) &ml aFGF was perfused. In controls, Ringer’s solution was continuously perfused (0, lt n = 6). Abscissa indicates the time before and after the application of aFGF in min. aFGF was applied at 0 min. For basal spike amplitude, ordinate indicates spike amplitude expressed as a percent change of mean basal amplitude before tetanic stimulation that was defined as 100%. For paired-pulse facilitation, ordinate indicates spike amplitude by the second pulse expressed as a percent of that by the first pulse at each time point. *p < 0.05; **p < 0.01; ***p < 0.001.
of 1 h in the same slice. In the magnitude of initial potentiation and the decay time course, STP induced by a second tetanus was identical to that induced by a first tetanus, and there was no statistical significance between them. In later experiments, therefore, the control STP and the STP in the presence of drugs were observed and compared in the same slice. After a basal spike amplitude was observed for 20 min (control period), the first tetanic stimulation was applied and the control STP was observed for 30 min. Perfusion of a slice with aFGF was then started. The second tetanic stimulation was delivered 30 min after the beginning of perfusion. The concentrations of aFGF used in the present study were 0.5 (0.32), 1.0 (0.65) and 2.5 (1.61) n&&/ml(lo-l*M). After 0.5 ng/mi aFGF was perfused, the basal spike amplitude did not change significantly compared to controls (Fig. 3A). However, perfusion of the slices with 1.0 and 2.5 @ml aFGF significantly and dose-dependently decreased the basal amplitude of spikes (Fig. 3B and C). The magnitude of the basal spike amplitude by perfusion of 1.0 and 2.5 @ml aFGF was 96.9 + 0.6% (mean ? SEM) and 92.8 f 0.6%, respectively, when measured 1 min before the second tetanus. In the presence of 0.5 @ml aFGF, the tetanic stimulation had no effect on the magnitude of initial potentiation observed within 2 min after the tetanus, but it significantly increased the magnitude of the following potentiation (Fig. 3A). In the presence of 1.0 and 2.5 ng/ml aFGF, the magnitude of the initial potentiation as well as the following potentiation was significantly increased (Fig. 3B and C). The degree of initial potentiation directly after the tetanus by 0.5, 1.0 and 2.5 r&ml aFGF was 156.3 2 1.2%, 160.4 + 1.7% and 165.4 + 1.4%, respectively, and that of the following ~tentiation 30 min after the tetanus was 106.1 t 1.2%, 109.1 t 0.9% and 114.0 2 1.4%, respectively. Thus, the enhancing effects of aFGF on the magnitude of STP was dosedependent. The frequency of LTP generation also increased in
dose-dependently: 11.1% for 0.5 &ml aFGF, 33.3% for 1.0 ng/ ml aFGF and 77.8% for 2.5 ng/ml aFGF (Fig. 4). Figure 5 shows the effects of aFGF on paired-pulse facilitation induced by paired-pulse stimulation, After basal spike amplitude in first response and paired-pulse facilitation were observed for 30 min (control period), perfusion of aFGF was started. Perfusion of 1.0 and 2.5 r&ml aFGF significantly and dose-dependently decreased the basal spike amplitude in a way and to a degree similar to those observed in Fig. 3. In contrast, aFGF significantly and dose-dependently increased the pairedpulse facilitation. The increase of paired-pulse facilitation was from 158.8 2 1.5% 5 min before the beginning of perfusion to 182.6 t 1.3% 1 h after the beginning of perfusion for 1.0 @ml aFGF, and from 157.6 -t 0.9% to 190.8 t 0.6% for 2.5 ng/ml aFGF. Since paired-pulse facilitation is defined as the ratio of the second response amplitude to the first response amplitude, it increases relatively when the magnitude of first response decreases and the second response does not change. Such a relative increase in paired-pulse facilitation due to the decrease of basal spike amplitude is estimated as a change from about 158% in control periods to about 165% for 1.0 ng/ml aFGF and to about 172% for 2.5 @ml aFGF. In the present study, the degree of increase of the paired-pulse facilitation was rather greater than those estimated values. Therefore, it seems to be reasonable to state that aFGF enhances paired-pulse facilitation. In the above experiments, we started to apply aFGF 30 min before the second tetanic stimulation. We also investigated the effects of aFGF on STP when it was administered at times different than 30 min before the second tetanus. The results are shown in Fig. 6. aFGF applied 20 min before the second tetanw significantly increased the magnitude of initial potentiation within 2 min after the tetanus and the magnitude of following potentiation, except 3 min after the tetanus. The degree of in-
ACIDIC FIBROBLAST
GROWTH FACTOR
Time ( min )
Time ( min )
FIG. 6. Effects on potentiation of aFGF applied before, with and after tetanic stimulation. The control potentiation (0) was observed for 30 min in Ringer’s solution after the first tetanic stimulation. The second tetanic stimulation (0) was applied 60 min after the first tetanus. (A, B, C and D) 2.5 @ml aFGF was applied 20 min before, 10 min before, at the time of, and 10 min after the second tetanic stimulation of 7 impulses at 100 Hz, respectively. *p < 0.05; **p < 0.01; ***p < 0.001. Other descriptions as in Fig. 1A.
crease in following potentiation 30 min after tetanus was 112.1 ? 1.9% and the LTP was induced in 5 of 8 slices (62.5%, Pig. 7). When aFGF was applied 10 min before the second tetanus, the magnitude of the potentiation within 2 min after the tetanus did not change si~ificantly, but the magni~de of following potentiation increased significantly. The degree of following potentiation 30 min after the tetanus was 106.8 t 1.0% and LTP was induced in 1 of 8 slices (12.5%). aFGF applied with, and 10 min after, the second tetanus had almost no effect on the initial potentiation and the following potentiation and did not generate LTP, DISCUSSION In the present study, perfusion of slices with aFGF enhanced the magnitude of SIP, which leads to the generation of LTP, and increased paired-pulse facilitation in the CA1 region of the hippocampus. In contrast to this, aFGF reduced the basal amplitude of spikes. A recent study in another laboratory has demonstrated that bFGF increases the magnitude of LTP and paired-pulse facilitation and decreases the basal spike amplitude in the CA1 region of rat hippocampal slices (1). From in vivo studies, it was also reported that aFGF and bFGF enhance the potentiation induced by weak tetanic stimulation in the dentate gyms of anaesthetized rats (15,17). Therefore, it seems that aFGF and bFGF have identical biological effects in the hippocampus. In support of this, it has been reported that aFGF and bFGF are 55% ho-
mologous in amino acid sequence (6,7,42) and interact with the same surface receptors (27). STP and LTP have similar inductive mechanisms and are considered to be caused by postsynaptic mechanisms including opening of N-methyl-D-asp~ate (NMDA) receptor channels and subsequent postsynaptic entry of Ca’+ ions (3,4,22). The rise in intracellular Ca” is thought to stimulate kinase activity. Ca*+phospholipid dependent kinase (PKC) (8) tyrosine kinase (29) and Ca’+-calmodulin kinase II (24) have important functions in LTP. Previous studies demons~ated that activation of PKC participates in the maintenance phase but not in the early phase of LTP (12). However, Asztely et al. (5) recently reported that activation of PKC by phorbol-12,13-diacetate reduces early decay of LTP in the CA1 region of the hippocampus, suggesting that PKC activation may be involved in biochemical events underlying the early phase, as well as those underlying the later phase of LTP. So far, several studies have shown that FGFs activate PKC. In our previous study, intraventricular infusion of aFGF significantly suppressed feeding in rats. This suppression of feeding was concluded to be due to suppression of glucose-sensitive neurons in the LHA, which in turn was caused by activation of PKC induced by aFGF (30). Prester et al. (31) found that bFGF induces PKC-dependent mitogenic response of transformed fetal bovine aortic endothelial GM 7373 cells. Tsuda et al. (40) and Takeyama et al. (36) also reported that bFGF stimulated the generation of diacylglycerol with subsequent activation of protein
510
SASAKI
1
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0
’
-20
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0
10
Time (min) FIG. 7. Effects of aFGF applied before, with and after tetanic stimulation
on the frequency of LTF’generation. The number of slices in which LTP was induced is expressedas a percent of the total number of slices treated by 2.5 @ml aFGF applied 20 min before (-20), 10 min before (-lo), at the time of (0), and 10 min after (10) the second tetanic stim~ation by 7 impulses at 100 Hz. LTP frequency was 0% when aFGF was applied with or 10 min after the second tetanus.
kinase C in Swiss 3T3 cells. Other than PKC, a recent study shows that aFGF activates an FGF receptor-associated tyrosine kinase (16). However, tyrosine kinase inhibitors such as Lavendustin A and Genistein had no significant effect on ST%’in the hippocampus, although they blocked LTP (29). Therefore, it might be possible that FGFs, in the present and other experiments (l&5,17), promote the generation of LTP by enhancing the magnitude of STP throu~ activation of protein ~n~e(s} such as PKC. In addition, it should be noted that FGFs rapidly induce the expression of the fos and myc protooncogenes in some ceils (26,44), since there is a possibility that protooncogenes are involved in the mechanisms of LTP (9). Initial potentiation, which lasts for a few minutes after tetanic stim~ation, consists mainly of the component of PTP. In addition to the increase of paired-pulse facilitation, initial potentiation after tetanus in the present study was found to be increased by the presence of aFGF. PTP as well as paired-pulse facilitation is considered to be caused through increase of transmitter release from presynaptic terminals (12,14,20,21,25). Therefore, the present study showing that aFGF enhances paired-pulse facilitation and PTP suggests a possibility that, at least, aFGF transiently modulates presynaptic mechanisms after high frequency stimulation such as tetanic stimulation and paired-pulse stimulation. In the present study, the magnitude of the basal spike amplitude decreased significantly and dose-dependently in the presence of aFGF. In previous studies, effects of PKC activation on basal response were inconsistent. It has been reported that activation of PKC by phorbol esters can induce lasting increases in
fcr AI,.
basal responses in the hippocampus (11,23). In contrast, Lovinger and ~outtenberg (19) demonstrated that application of PKC stimulators such as lZ-O-tetradecanoyl-phorbol- 13-acetate and c&unsaturated fatty acid did not alter basal responses but did enhance the maintenance of LTP. Application of tyrosine kinase inhibitors also had no effect on the basal responses in the hippocampus (29). Further investigations are required to investigate relations between the reduction of basal responses and aFGF, considering some other mechanisms such as the expression of protooncogenes mentioned above in addition to activation of protein kinase(s). Terlau and Seifelt (38) reported that bFGF does not lead to changes in the evoked potentials in the CA1 region of the hippocampus when it is added 20 min after tetanic stimulation, although it enhances LTP when added 20 min before tetanic stimulation. They discussed that processes which are activated during or in the first 20 min after tetanic stimulation are involved in the modulatory effect of bFGF on LTP. In the present study, aFGF produced almost no changes on the magnitude of potentiation when it was added at the time of and 10 min after tetanic stimulation. increase of potentiation, except the initial part, was observed when aFGF was added 10 min before tetanus, whereas increase of initial potentiation and following potentiation was observed when it was added 20 min before tetanus. Therefore, it appears that the enhancement of STP by aFGF, which leads to the generation of LTP, requires some biochemical processes activated by aFGF before tetanic stimulation. In conclusion, we demonstrated that aFGF enhances STP, transforms STP to LTP, and promotes the generation of LTP. The results suggest that aFGF may be involved in the modulatory mechanisms of synaptic plasticity in the adult CNS and that aFGF may activate some mechanisms related to the generation of LTP. In a previous study, we demonstrated that 1P glucose, which increases aFGF in the cerebrospinal fluid (13,34), facilitates behavioral performance in passive avoidance and Morris water maze tasks (30,32). Since these glucose effects are abolished by pretreatment with anti-aFGF antibody applied into cerebral ventricles (30,32), it might be possible that these behavioral effects by glucose are attributed to endogenous aFGF. And it might be expected that endogenous aFGF may contribute to these effects by having the same effect in the modulation of synaptic plasticity as exogenous aFGF. Further studies are required
to clarify the detailed mechanisms involved in the LTP-promoting effects of aFGF and the relation between enhancement of behavioral performance in learning tasks and aFGF. ACKNOWLEDGEMENTS
This work was partly supported by the Japanese Ministry of Education, Science and Culture Grant-in-Aid for Scientific Research, 02454127 (KS.), and by the Science and Technology Agency. using the Special Coordination Funds for Promoting Science and Technology of Japan (Y.O., KS).
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