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Involvement of postsynaptic G proteins in hippocampal long-term potentiation
Brain Researt:/~. 581 (1992) ltJ~; ~14 © 1992 Elsevier Science Publishers B.V. All rights reserved, !)006-8993/02/$(J5~i~(i
108 BRES 17752
Involvement of postsynaptic G proteins in hippocampal long-term potentiation Hiroshi Katsuki, Shuji Kaneko and Masamichi Satoh Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto (Japan) (Accepted 7 January 1992)
Key words: Long-term potentiation; Hippocampal slice; CA3 region; Guanosine 5-triphosphate-binding protein; Guanosine 5-diphosphate flS; GTPvS; Whole-cell clamp
The possible involvement of postsynapic guanosine 5-triphosphate (GTP)-binding proteins (G proteins) in long-term potentiation (LTP) was studied in rat hippocampal slices, using whole-cell recording techniques. The inclusion of guanosine 5'-O-(2-thiodiphosphate) (GDPflS) or guanosine 5'-O-(3-thiotriphosphate) (GTPyS) in the recording pipette significantly reduced or abolished the baclofen-induced hyperpolarization of pyramidal neurons, which indicates uncoupling of the signal transduction from G protein-coupled receptors by these compounds. Both GDPflS and GTPTS significantly attenuated the magnitude of LTP in the fimbria-CA3 synapses, but not in the mossy fiber-CA3 synapses. GTP?S did not attenuate LTP in the Schaffer-CA1 synapses. The effects of guanine nucleotide analogs on fimbrial LTP were reversed by postsynaptic depolarization during high frequency stimulation. These results suggest that postsynaptic G proteins may be involved in the generation of LTP in the fimbrial synapses, possibly by affecting membrane depolarization during high frequency afferent activation. INTRODUCTION It is now well known that many central synapses show activity-dependent changes in transmission efficacy, which is considered to be the cellular basis of learning and memory. In the hippocampus, all the major excitatory pathways exhibit a persistent increase in synaptic efficacy; this is called long-term potentiation 2 (LTP). Hippocampal CA3 pyramidal neurons receive 3 types of excitatory inputs; mossy fiber, commissural/associational, and fimbrial input. Zalutsky and Nicol122 have reported that the induction of commissural/associational LTP requires N-methyl-D-aspartate ( N M D A ) receptor activation, m e m b r a n e depolarization, and a rise in Ca 2÷ in the postsynaptic cell, while mossy fiber LTP does not. We have also previously shown that the activation of N M D A receptors, increase in intracellular Ca 2÷ in the postsynaptic cell, and postsynaptic membrane depolarization are required for the induction of fimbrial, but not of mossy fiber LTP 14. Thus the mechanisms underlying LTP may be different for these inputs, but the precise mechanisms, particularly the signal transduction pathways involved in LTP, are not yet clear. Guanosine 5-triphosphate (GTP)-binding proteins (G proteins) are involved in many cellular signal transduction processes. The involvement of G proteins in LTP has been suggested by the observation that pretreatment
of animals with pertussis toxin suppresses LTP in the mossy fiber-CA313 and in the Schaffer-CA19 synapses. However, these pretreatment experiments have not clarified the location of G proteins (pre- or postsynaptic, or in the interneurons) responsible for LTP. Goh and Pennefather 9 have reported that postsynaptic injection of guanosine 5'-0-(3-thiotriphosphate) (GTPyS) does not occlude LTP in the Schaffer-CA1 synapses, thus indicating that postsynaptic G proteins are not responsible for LTP in those synapses. However, there is no direct evidence regarding whether postsynaptic G proteins are involved in LTP in the CA3 region. In the present study, we have addressed this issue; we found that, in contrast to findings in the Schaffer-CA1 synapses, modification of the activity of postsynaptic G proteins can affect the induction of LTP in the fimbria-CA3 synapses. MATERIALS AND METHODS Tight seal (>1 Gf~), whole-cell recordings were carried out in thin hippocampal slices as described previously6"14. Hippocampi of 20-28-day-old rats (both sexes) were cut in 200-/~mthick slices and maintained at 30-33°C in the following medium (in mM): NaCt 124, KC1 2, KH2PO4 1.24, MgSO4 5, NaHCO 3 26, CaC12 2, and glucose 10, bubbled with 95% 02/5% CO 2. Picrotoxin (10 ~M) was routinely added to the perfusion medium to block 7-aminobotyric acid (GABA)ergic inhibition. Pyramidal neurons were viewed using an upright microscope with Nomarski optics, and the surface of the cell membrane was cleaned with a jet stream of the perfusion medium, following which a patch electrode (tip resistance 3-5
Correspondence: M. Satoh, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Fax: (81) (75) 753-4586.
109
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Fig. 1. Whole-cell recording from hippocampal pyramidal neurons. A: schematic representation of a transverse hippocampal slice showing stimulating (stim.) and recording (rec.) electrodes. DG, dentate gyrus; MF, mossy fibers; Sch, Schaffer collateral/commissural fibers. B: effects of baclofen on CA3 pyramidal neuron dialyzed with 300/IM GTP (upper) and 100 ~M GTPTS (lower). The cell was held in currentclamp, and baclofen (10 ~M) was applied by bath application at the time indicated by the bars. The resting membrane potential is indicated to the left of each trace. Downward deflections are responses to 500 ms, 50 pA hyperpolarizing current pulses applied at 0.2 Hz to monitor input resistance of the cell. Application of baclofen caused membrane hyperpolarization and concomitant decrease in input resistance in the cell dialyzed with GTP. With intracellular application of GTPTS, the membrane was hyperpolarized and input resistance was reduced, and application of baclofen caused no further hyperpolarization.
Mf~) was accessed to the middle part of the visualized cell. The pipette solution for whole-cell recordings contained (in mM) K-gluconate 128, KCI 7, MgCI2 1, CaC12 0.1, EGTA 1, ATP 2, and HEPES 10: the pH was adjusted to 7.3 with KOH. In several experiments, K-gluconate and KC1 were replaced by equimolar Csgluconate and CsCI. Liquid-junction potentials between external and internal solutions were measured with a 3 M-KCl-agar electrode l° to calibrate the actual membrane potential. Rectangular stimulating pulses (300-1000 ~A, 10-120/~s in duration) were delivered at 0.2 Hz through a bipolar electrode (tip spacing 200 ~m) positioned in the upper blade of the dentate granule cell layer, or ventral fimbria. Excitatory postsynaptic currents (EPSC) were recorded with a patch-clamp amplifier (EPC7, List) from CA3 pyramidal neurons voltage-clamped at -80 mV. In several experiments, whole-cell recordings from CA1 neurons were also performed, and in this case, Schaffer collateral/commissural input was stimulated by an electrode positioned in the stratum radiatum (Fig. 1A). The intensity of the test stimulation was set to evoke EPSP of 5-10 mV in amplitude in the current-clamp mode. High frequency tetanic stimulation (TS; 3 trains of 100 Hz 1 s at 0.2 Hz) was delivered with the same intensity as the test stimulation, while the cell was held in the current-clamp unless otherwise indicated. In all the experiments with mossy fiber-CA3 synapses, TS was delivered in the presence of 25 /~M ~-2-amino-5phosphonovalerate to avoid contributing potentiation of the nonmossy fiber input. Current and voltage outputs were filtered at 10 kHz and stored for later off-line analysis using an analysis program (QPlllJ; Nihon Kohden) on a personal computer (PC9801; NEC). Averaged 10 consecutive responses were sampled at 5-min intervals. Percent changes in EPSC amplitudes were calculated taking the values immediately before TS as 100%. All results were expressed as mean _+ S.E.M. GTP (sodium or lithium salt, Sigma), guanosine 5'-O-(2-thiodiphosphate) (GDPflS) and GTPTS (lithium salt, Boehringer) were dissolved in the pipette solution at the indicated concentrations. Since the GTP sodium and lithium salts gave similar results, their
data were pooled. RESULTS
Internal dialyzation with guanine nucleotide analogs Modification of the activity of G proteins in pyramidal neurons was achieved by intracellular perfusion with pipette solution containing the non-hydrolysable guanine nucleotide analogs, GDPflS and GTPTS. To assess the effectiveness of the loading of these compounds, we tested the effect of the G A B A B agonist baclofen. Baclofen hyperpolarizes hippocampal pyramidal n e u r o n s through the activation of K + channels which are coupled to G A B A ~ receptors via pertussis toxin-sensitive G proteins ~'7'21. Thus, modification of the activity of G proteins affects the hyperpolarizing action of baclofen t. Baclofen was bath applied for 1 min at 23-28 rain after whole-cell access to the CA3 pyramidal neurons, and the m e m b r a n e potential was monitored while the cell was held in the current-clamp mode. Sample records are shown in Fig. lB. W h e n recordings were made with pipette solution containing 300/~M GTP, the responses of the CA3 pyramidal n e u r o n s to 10/~M baclofen averaged -16.7 + 1.6 m V (8 cells). Even when the internal solution contained no added GTP, a similar magnitude of hyperpolarization was observed (-18.9 _+ 0.5 mV, 4 cells). This may indicate that the local concentration of endogenous G T P remaining near the m e m b r a n e is suf-
110 TABLE I
Effects of guanine nucleotide analogs on passive membrane properties and responses of hippocampal pyramidal neuron~ lo bacl&~n Whole-cell recording was made with pipette solutions in which each guanine nucleotide analog was dissolved. Resting membrane potentml and input resistance were measured at 15-30 rain after whole-cell access. Responses to 10/~M baclofen were measured at 23-28 mm (in CA3) and at 22-24 rain (in CAt) after whole-cell access.
Resting membrane potential: mV
CA3
GTP 300/~M GDP/~S 300 ~M GTPyS 100 #M GTP 100/~M GTP?S 100/~M
CA1
-62.3 -66.1 -79.5 -57.8 -68.6
-+ 1.7 (17) + 2.1 (16) + 1.4"* (16) + 1.9 (12) + 1.5"* (11)
Input resistance: Mg2
Responses ~o 10 #M baclo/'en: mV
145 128 72 189 94
-16.7 -10.4 -0.8 -7.9 -0.5
+ + + + +
[1 (15) 7 (12) 6** (13) 18 (12) 3** (11)
+_ 1.0 ('8) + 1.4" (6) _+ 0.3 ** (6) + 0.6 (6) _+ 0.1"* (6)
*P < 0.05, **P < 0.01 vs. GTP group (one-way analysis of variance, followed by the Bonferroni method). The numbers in parentheses indicate numbers of neurons observed.
K + channels normally opened by baclofen. These results suggest that significant modification of the activity of G
ficient for G proteins to function H'2°. W h e n the pipette solution contained 3 0 0 / , M GDPflS, the resting potential and input resistance of the cell was not significantly changed; however, the response to baclofen was significantly reduced (Table I). GTPyS was applied to the CA3 neurons, was gradually hyperpolarized and input reduced, and a steady state was reached
proteins by guanine nucleotide analogs occurred at 23L28 min after whole-cell access.
W h e n 100/,M the m e m b r a n e resistance was within 15 min
Effects o f guanine nucleotide analogs on fimbrial L T P Fig. 2 shows the effect of guanine nucleotide analogs on LTP in the fimbria-CA3 synapses. In this set of experiments, TS was delivered at the same time window used in the previous section, that is, at 2 3 - 2 8 rain after whole-cell access. W h e n recordings were made with
after whole-cell access (Table I). Application of baclofen induced almost no further hyperpolarization (Table I, Fig. 1B), which suggests that GTPyS fully activated the
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Fig. 2. Effects of the modification of the activity of postsynaptic G proteins on fimbrial long-term potentiation (LTP). A: intracellular application of GDPflS attenuates LTP. o, GTP 300/*M (n = 6); o, GDPflS 100/tM (n = 5); A, GDPflS 300 #M (n = 6). B: GTPyS inhibits LTP. o, GTP 100 gM (n = 5); e, GTPyS 100 gM (n = 5). Statistical significance was evaluated at each time point with one-way analysis of variance, followed by the Bonferroni method. *P < 0.05, '~ P < 0.01, vs. GTP (data of 100 and 300 #M were pooled). C: sample traces of LTP recorded with pipettes containing 100/~M GTP, 300/*M GDPffS and 100 #M GTPyS. Traces before and 20 min after TS are superimposed.
111
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200 pA 10 ms Fig. 3. Irreversible inactivation or activation of postsynaptic G proteins does not inhibit LTP in mossy fiber-CA3 synapses. A: plot of potentiation time course, o, GTP (100 ktM n = 2, 300/~M n = 3); • GDPflS (100 BM n = 2, 300/~M n = 4); A, GTPTS 100 ~M (n = 5). B: sample records of mossy fiber LTP with pipettes containing 300 BM GTP and 100 ~M GTPyS. G T P - c o n t a i n i n g solution, EPSC was greatly enhanced immediately (within 5-10 min) after TS, and then stable potentiation was observed for up to 90 rain. A n averaged potentiation of 109.8 + 18.2% was obtained at 20 min after TS ( G T P 100 /~M n = 5, 300 /zM, n = 6). This value was comparable to one we had obtained previously, using a pipette solution with no added G T P (129.3 _+ 33.3%, n = 6) 14. As shown in Fig. 2A, substitution of G T P with GDP/3S significantly attenuated LTP. The effect was apparent at a concentration of 100 /~M (49.9 _+ 12.1% at 20 min after TS, n = 5), and when 300 # M GDPflS was applied, EPSC gave an averaged potentiation of only 29.6 + 21.5% (n = 6) at 20 min after TS. Next, we tested the effects of GTPTS on LTP in the fimbrial synapses. LTP in the fimbrial synapses was negligible after internal dialyzation with GTPTS (Fig. 2B). The potentiation of EPSC at 20 min after TS was only 6.1 + 14.7% (n = 5).
~/'"'after
200 pA 10 ms
Fig. 4. Intracellular GTPTS attenuates short-term potentiation but not LTP in the Schaffer-CA1 synapses. A: potentiation time course. o, GTP 100/~M (n = 6); e, GTPTS 100 BM (n = 5). *P < 0.05 vs. GTP 1013~M. B: sample records from CA1 neurons with pipettes containing 100 gM GTP and 100/tM GTPyS.
Effects of guanine nucleotide analogs on mossy fiber LTP We also carried out analogous experiments in the mossy fiber-CA3 synapses. The same TS condition resulted in a similar time course but much lower degree of potentiation than in the fimbrial synapses, which averaged a 28.5 + 15.5% increase at 20 min after TS in neurons recorded when G T P solution was added (100 ktM n = 2 , 3 0 0 / ~ M n = 3). This value was not significantly different from that recorded when there was no added G T F (42.4 _+ 20.5%, n = 5) 14. In contrast to that in the timbrial synapses, LTP in the mossy fiber synapses was not inhibited by 100-300 ~M GDP/3S or 100 ktM GTPTS (Fig. 3).
Effects of GTPTS on LTP in the CA1 region The above results on the fimbria-CA3 synapses contrast with the results of a previous report 9 in which it was
112 shown that postsynaptic injection of GTPvS did not affect LTP in the Schaffer-CA1 synapses, although these two synapses share common features, in that the induction of LTP is dependent on the activation of N M D A receptors 4"14. However, it is possible that the apparent discrepancy is due to some differences between the experimental conditions of the present study and those in the previous report. To test this possibility, we also performed whole-cell recording from CA1 pyramidal neurons. In the CA1 region, a pairing procedure > 30 min after whole-cell access fails to induce LTP 17. To avoid extensive washout of cytoplasmic factors, and at the same time to ensure the loading of the guanine nucleotide analogs into the CA1 neurons, we applied TS at 22-24 min after whole-cell access in all cases in this set of experiments. The results are shown in Fig. 4. When recordings were made with internal solution containing 100 ,uM GTP, EPSC was greatly enhanced immediately (within 5 min) after TS, and then the potentiation remained stable at levels of 30-40% (32.0 + 12.3% increase at 20 min after TS) for up to 60 min. As was the case with the CA3 neurons, hyperpolarization and significant reduction of input resistance resuited in the CA1 pyramidal neurons when GTPvS was included in the pipette solution (Table I). The responses to baclofen were also largely abolished, which indicates the effectiveness of the loading of GTPTS in the blockade of signal transduction from the receptor to the G proteins. However, even under these conditions, TS induced LTP in the Schaffer-CA1 synapses. Although initial potentiation was significantly reduced, as reported previously 9, the magnitude of subsequent potentiation was not attenuated or occluded (37.3 + 9.8% increase at 20 min after TS) by the irreversible activation of postsynaptic G proteins with GTPTS. These results confirmed the qualitative difference between the fimbria-CA3 and the Schaffer-CA1 synapses regarding the involvement of G proteins in LTP.
Reversal of the effects of guanine nucleotide analogs on fimbrial LTP by postsynaptic depolarization As the inhibition of fimbrial LTP by GDPflS and GTPTS was apparent immediately after TS, it is likely that factor(s) responsible for the induction of LTP may be altered. As reported previously, induction of LTP in the fimbrial synapses requires postsynaptic membrane depolarization 14. Thus, one possible mechanism underlying the effects of GDPflS and GTPyS is that the inhibition of depolarization during TS is responsible for the attenuation of LTP. To assess this possibility, we attempted to control the membrane potential during TS. To ensure more reliable control of this potential, K + in the pipette solution was
A Fim stim.
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Fig. 5. Reversal of the effects of guanine nucleotide analogs on LTP in the fimbrial synapses by postsynaptic depolarization during TS.
A: stimulation paradigm. Fimbrial fibers were stimulated 3 times at 100 Hz for 1 s (Fire stim.), while the CA3 neuron was depolarized from -80 mV to -20 mV (Vm). B: potentiation time course. o, GTP 100/zM (n = 6); o, GDPflS 300/xM (n = 4); A, GTP%S 100/~M (n = 5). replaced by Cs + in this set of experiments. TS was delivered while the membrane was depolarized: from -80 to -20 mV by voltage command pulses (Fig. 5A). Fig. 5B shows the results of this set of experiments. When the membrane was sufficiently depolarized during TS, LTP in the fimbrial synapses was observed to the same extent in neurons dialyzed with 100 ~M GTP as in neurons dialyzed with 100/~M GTPTS or 300/~M GDPflS. In other words, the effects of non-hydrolysable guanine nucleotide analogs were reversed by postsynaptic membrane depolarization during TS. These results suggest that the inhibition of depolarization during TS may be responsible for the inhibition of LTP in the fimbrial synapses by guanine nueleotide analogs. DISCUSSION
The present study showed that postsynaptic G proteins could affect the induction of LTP in firnbria-CA3 synapses. To assess the involvement of G proteins, we used two kinds of non-hydrolysable guanine nucIeotide analogs, GDPflS and GTPyS. GDPflS inhibits G proteins, presumably by preventing the reaction of G pro-
113 teins and GTP 5. On the other hand, GTPvS binds to the G proteins and induces them to become continuously active 8. Both compounds have now been shown to inhibit LTP in the fimbrial synapses. The inhibitory effects of guanine nucleotide analogs on fimbrial LTP are probably due to the inhibition of depolarization in the CA3 pyramidal neurons during TS, since the effect was reversed by depolarization during TS by delivery of voltage command pulses. Reduction of depolarization by GTP~,S may be explained by the fact that GTPyS opens G protein-linked K + channels, thus causing significant hyperpolarization of the resting membrane potential. However, GDPflS did not significantly affect the resting membrane potential (Table I). GDPflS may specifically reduce the depolarization induced by TS. One explanation of the mechanisms underlying the effect of GDPflS is as follows: in physiological conditions, repetitive stimulation of afferents and extensive release of neurotransmitter(s) activates postsynaptic receptors coupling to G proteins, and the activated G proteins may then activate certain factors that help to depolarize the CA3 pyramidal neurons; GDPflS could inhibit these processes, thereby reducing membrane depolarization. In this context, G proteincoupled, metabotropic glutamate (Qp) receptors might be involved in these processes. Activation of Qp receptors has been shown to block K ÷ conductances and to depolarize CA3 neurons 3. Therefore, it is possible that during TS, a substantial amount of glutamate is released, and that postsynaptic Qp receptors are activated; this activation then blocks some types of K + currents and accelerates the depolarization of postsynaptic neurons. Greater depolarization opens N M D A receptor channels more effectively, and this permits a large influx of Ca 2+ which is necessary for the induction of LTP. The CA3 pyramidal neurons also have many other types of receptors, including muscarinic and fl-adrenergic receptors, which couple to G proteins. There is also a possibility that neurotransmitters other than glutamate are co-released during high frequency afferent activation, and that these are responsible for the membrane depolarization of the CA3 neurons ~5. Other factors should also be considered in explaining the reversal of the effects of guanine nucleotide analogs by membrane depolarization: a blockade of G proteins could reduce the rise in intracellular Ca 2+ by means other than a reduction of membrane depolarization. Depolarizing the cells could counterbalance this action by increasing the Ca 2+ influx through N M D A receptor-mediated conductances or through voltage-gated Ca z+ channels. One possibility is the occurrence of a blockade of the processes that follow activation of Qp and/or muscarinic receptors, since both of these receptors enhance the release of Ca 2+ from intracellular stores via G pro-
tein-mediated processes 12'x9. As reported previously, LTP in the Schaffer-CA1 synapses was not inhibited or occluded by GTPTS. This may indicate that the activation of postsynaptic G proteins during TS, if it occurs, contributes little to the induction of LTP in the CA1 region. It is interesting that fewer Qp receptors are expressed in the CA1 pyramidal neurons than in the CA3 neurons, as shown by in situ hybridization histochemistry TM. During whole-cell recording, cytoplasmic factors are gradually lost, and this 'wash-out' had been reported to inhibit the induction of LTP in the CA1 region 17. Therefore, we must take special care when using this technique to compare LTP in different cells. It is possible that rates of dialysis in CA1 and CA3 cells are different, and this difference might contribute to the observed difference in LTP. Indeed, we observed robust LTP on delivering TS even 40 min after whole-cell access to CA3 cells, as reported previously ~4, which suggested that CA3 neurons may be more resistant to dialysis than CA1 neurons. In the present study, we applied TS at 23-28 min after whole-cell access in the case of CA3, and at 22-24 min after whole-cell access in the case of CA1. By setting these time windows, we were able to achieve effective loading of guanine nucleotide analogs into the pyramidal neurons, while retaining the ability to induce LTP in both CA3 and CA1. Thus, although this procedure could have minimized the artificial effects of wash-out on LTP, we cannot exclude the possibility that intracellular dialysis affected the magnitude of LTP to some extent. LTP in the mossy fiber-CA3 synapses, in contrast to that in the fimbrial synapses, was not inhibited by modification of the activity of postsynaptic G proteins. These results suggest that, in the mossy fiber-CA3 synapses, postsynaptic G proteins are not involved in LTP in any way. However, in vivo pretreatment experiments have suggested that pertussis toxin-sensitive G proteins are involved in mossy fiber LTP 13. Therefore, the location of G proteins responsible for mossy fiber LTP is probably in the presynaptic terminals. The extent of postsynaptic depolarization and, probably, the subsequent increase in intracellular Ca 2+ in the postsynaptic neurons may be important in determining the magnitude of the N M D A receptor-dependent form of LTP. In fact, it was recently demonstrated that the magnitude of postsynaptic Ca 2+ increase is a critical factor in controlling the duration of synaptic enhancement in the CA1 region 16. However, as demonstrated by the present study, the physiological factors controlling the intracellular C a 2+ dynamics of pyramidal neurons may differ in CA3 and CA1. Investigation of the mechanisms modulating intracellular C a 2+ will provide further understanding of plastic changes in various synaptic connections.
114 REFERENCES 1 Andrade, R., Malenka, R.C. and Nicoll, R.A., A G protein couples serotonin and GABAn receptors to the same channels in hippoeampus, Science, 234 (1986) 1261-1265. 2 Bliss, T.V.R and Lynch, M.A., Long-term potentiation of synaptic transmission in the hippocampus: properties and mechanisms. In P.W. Landfield and S.A. Deadwyler (Eds.), LongTerm Potentiation: From Biophysiscs to Behavior, Alan R. Liss, New York, 1988, pp. 3-72. 3 Charpak, S., G~ihwiler, B.H., Do, K.Q. and KnOpfel, T., Potassium conductances in hippocampal neurons blocked by excitatory amino acid transmitters, Nature, 347 (1990) 765-767. 4 Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateralcommissural pathway of the rat hippocampus, J. Physiol.. 334 (1983) 33-46. 5 Eckstein, F., Cassel, D., Levkovitz, H., Lowe, M. and Selinger, Z., Guanosine 5'-O-(2-thiodiphosphate). An inhibitor of adenylate cyclase stimulation by guanine nucleotides and fluoride ions, J. Biol. Chem., 254 (1979) 9829-9834. 6 Edwards, E A . , Konnerth, A., Sakmann, B. and Takahashi, T., A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system, Pfliigers Arch., 414 (1989) 600-612. 7 G~ihwiler, B.H. and Brown, D.A., GABAB-receptor-activated K + current in voltage-clamped CA3 pyramidal cells in hippocampal cultures, Proc. Natl. Acad. Sci. USA, 82 (1985) 15581562. 8 Gilman, A.G., G-proteins and dual control of adenylate cyclase, Cell, 36 (1984) 577-579. 9 Goh, J.W. and Pennefather, ES., A pertussis toxin-sensitive G protein in hippocampal long-term potentiation, Science, 244 (1988) 980-983. 10 Hagiwara, S. and Ohmori, H., Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp, J. Physiol., 331 (1982) 231-252. 11 Hescheler, J., Rosenthal, W., Trautwein, W. and Schultz, G., The GTP-binding protein, Go, regulates neuronal calcium channels, Nature, 325 (1987) 445-447. 12 Iijima, T., Kudo, Y., Ogura, A., Akita, K. and Matsumoto, G.,
13
14
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Variation in the pattern of [Ca z~]l change induced by acetylcholine in cultured hippocampat neurons. Brain Res., 521 (199~J) 273-280. Ito, I., Okada, D and Sugiyama, H., Pertussis toxin suppresses long-term potentiation of hippocampal mossy fiber synapses, Neurosci. Lett., 90 (1988) 181-185. Katsuki, H., Kaneko, S., Tajima, A. and Satoh, M., Separate mechanisms of long-term potentiation in two input systems to CA3 pyramidal neurons of rat hippocampal slices as revealed by whole-cell patch-clamp technique, Neurosci. Res., 12 (I991) 393-402. Kn6pfel, T., Vranesic, 1., G~hwiler, B.H. and Brown, D.A., Muscarinic and fl-adrenergic depression of the slow CaZ+-acti vated potassium conductance in hippocampal CA3 pyramidal cells is not mediated by a reduction of depolarization-induced cytosolic Ca 2+ transients, Proc. Natl. Acad. Sci. USA, 87 (1990) 4083-4087. Malenka, R.C., Postsynaptic factors control the duration of synaptic enhancement in area CA1 of the hippocampus, Neuron, 6 (1991) 53-60. Malinow, R. and Tsien, R.W., Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices, Nature, 346 (1990) 177-180. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R. and Nakanishi, S., Sequence and expression of a metabotropic glutamate receptor, Nature, 349 (1991) 760-765. Murphy, S.N. and Miller, R.J., A glutamate receptor regulates Ca 2+ mobilization in hippocampal neurons, Proc. Natl. Acad. Sci. USA, 85 (1988) 8737-8741. Nakajima, Y., Nakajima, S. and Inoue, M., Pertussis toxin-insensitive G protein mediates substance P-induced inhibition of potassium channels in brain neurons, Proe. Natl. Acad. Sei. USA, 85 (1988) 3643-3647. Thalmann, R.H., Evidence that guanosine triphosphate (GTP)binding proteins control a synapic response in brain: effect of pertussis toxin and GTP7S on the late inhibitory postsynaptic potential of hippocampal CA3 neurons, J. Neurosei., 8 (1988) 4589-4602. Zalutsky, R.A. and Nicoll, R.A., Comparison of two forms of long-term potentiation in single hippocampat neurons, Science, 248 (1990) 1619-1624.
108 BRES 17752
Involvement of postsynaptic G proteins in hippocampal long-term potentiation Hiroshi Katsuki, Shuji Kaneko and Masamichi Satoh Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto (Japan) (Accepted 7 January 1992)
Key words: Long-term potentiation; Hippocampal slice; CA3 region; Guanosine 5-triphosphate-binding protein; Guanosine 5-diphosphate flS; GTPvS; Whole-cell clamp
The possible involvement of postsynapic guanosine 5-triphosphate (GTP)-binding proteins (G proteins) in long-term potentiation (LTP) was studied in rat hippocampal slices, using whole-cell recording techniques. The inclusion of guanosine 5'-O-(2-thiodiphosphate) (GDPflS) or guanosine 5'-O-(3-thiotriphosphate) (GTPyS) in the recording pipette significantly reduced or abolished the baclofen-induced hyperpolarization of pyramidal neurons, which indicates uncoupling of the signal transduction from G protein-coupled receptors by these compounds. Both GDPflS and GTPTS significantly attenuated the magnitude of LTP in the fimbria-CA3 synapses, but not in the mossy fiber-CA3 synapses. GTP?S did not attenuate LTP in the Schaffer-CA1 synapses. The effects of guanine nucleotide analogs on fimbrial LTP were reversed by postsynaptic depolarization during high frequency stimulation. These results suggest that postsynaptic G proteins may be involved in the generation of LTP in the fimbrial synapses, possibly by affecting membrane depolarization during high frequency afferent activation. INTRODUCTION It is now well known that many central synapses show activity-dependent changes in transmission efficacy, which is considered to be the cellular basis of learning and memory. In the hippocampus, all the major excitatory pathways exhibit a persistent increase in synaptic efficacy; this is called long-term potentiation 2 (LTP). Hippocampal CA3 pyramidal neurons receive 3 types of excitatory inputs; mossy fiber, commissural/associational, and fimbrial input. Zalutsky and Nicol122 have reported that the induction of commissural/associational LTP requires N-methyl-D-aspartate ( N M D A ) receptor activation, m e m b r a n e depolarization, and a rise in Ca 2÷ in the postsynaptic cell, while mossy fiber LTP does not. We have also previously shown that the activation of N M D A receptors, increase in intracellular Ca 2÷ in the postsynaptic cell, and postsynaptic membrane depolarization are required for the induction of fimbrial, but not of mossy fiber LTP 14. Thus the mechanisms underlying LTP may be different for these inputs, but the precise mechanisms, particularly the signal transduction pathways involved in LTP, are not yet clear. Guanosine 5-triphosphate (GTP)-binding proteins (G proteins) are involved in many cellular signal transduction processes. The involvement of G proteins in LTP has been suggested by the observation that pretreatment
of animals with pertussis toxin suppresses LTP in the mossy fiber-CA313 and in the Schaffer-CA19 synapses. However, these pretreatment experiments have not clarified the location of G proteins (pre- or postsynaptic, or in the interneurons) responsible for LTP. Goh and Pennefather 9 have reported that postsynaptic injection of guanosine 5'-0-(3-thiotriphosphate) (GTPyS) does not occlude LTP in the Schaffer-CA1 synapses, thus indicating that postsynaptic G proteins are not responsible for LTP in those synapses. However, there is no direct evidence regarding whether postsynaptic G proteins are involved in LTP in the CA3 region. In the present study, we have addressed this issue; we found that, in contrast to findings in the Schaffer-CA1 synapses, modification of the activity of postsynaptic G proteins can affect the induction of LTP in the fimbria-CA3 synapses. MATERIALS AND METHODS Tight seal (>1 Gf~), whole-cell recordings were carried out in thin hippocampal slices as described previously6"14. Hippocampi of 20-28-day-old rats (both sexes) were cut in 200-/~mthick slices and maintained at 30-33°C in the following medium (in mM): NaCt 124, KC1 2, KH2PO4 1.24, MgSO4 5, NaHCO 3 26, CaC12 2, and glucose 10, bubbled with 95% 02/5% CO 2. Picrotoxin (10 ~M) was routinely added to the perfusion medium to block 7-aminobotyric acid (GABA)ergic inhibition. Pyramidal neurons were viewed using an upright microscope with Nomarski optics, and the surface of the cell membrane was cleaned with a jet stream of the perfusion medium, following which a patch electrode (tip resistance 3-5
Correspondence: M. Satoh, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Fax: (81) (75) 753-4586.
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Fig. 1. Whole-cell recording from hippocampal pyramidal neurons. A: schematic representation of a transverse hippocampal slice showing stimulating (stim.) and recording (rec.) electrodes. DG, dentate gyrus; MF, mossy fibers; Sch, Schaffer collateral/commissural fibers. B: effects of baclofen on CA3 pyramidal neuron dialyzed with 300/IM GTP (upper) and 100 ~M GTPTS (lower). The cell was held in currentclamp, and baclofen (10 ~M) was applied by bath application at the time indicated by the bars. The resting membrane potential is indicated to the left of each trace. Downward deflections are responses to 500 ms, 50 pA hyperpolarizing current pulses applied at 0.2 Hz to monitor input resistance of the cell. Application of baclofen caused membrane hyperpolarization and concomitant decrease in input resistance in the cell dialyzed with GTP. With intracellular application of GTPTS, the membrane was hyperpolarized and input resistance was reduced, and application of baclofen caused no further hyperpolarization.
Mf~) was accessed to the middle part of the visualized cell. The pipette solution for whole-cell recordings contained (in mM) K-gluconate 128, KCI 7, MgCI2 1, CaC12 0.1, EGTA 1, ATP 2, and HEPES 10: the pH was adjusted to 7.3 with KOH. In several experiments, K-gluconate and KC1 were replaced by equimolar Csgluconate and CsCI. Liquid-junction potentials between external and internal solutions were measured with a 3 M-KCl-agar electrode l° to calibrate the actual membrane potential. Rectangular stimulating pulses (300-1000 ~A, 10-120/~s in duration) were delivered at 0.2 Hz through a bipolar electrode (tip spacing 200 ~m) positioned in the upper blade of the dentate granule cell layer, or ventral fimbria. Excitatory postsynaptic currents (EPSC) were recorded with a patch-clamp amplifier (EPC7, List) from CA3 pyramidal neurons voltage-clamped at -80 mV. In several experiments, whole-cell recordings from CA1 neurons were also performed, and in this case, Schaffer collateral/commissural input was stimulated by an electrode positioned in the stratum radiatum (Fig. 1A). The intensity of the test stimulation was set to evoke EPSP of 5-10 mV in amplitude in the current-clamp mode. High frequency tetanic stimulation (TS; 3 trains of 100 Hz 1 s at 0.2 Hz) was delivered with the same intensity as the test stimulation, while the cell was held in the current-clamp unless otherwise indicated. In all the experiments with mossy fiber-CA3 synapses, TS was delivered in the presence of 25 /~M ~-2-amino-5phosphonovalerate to avoid contributing potentiation of the nonmossy fiber input. Current and voltage outputs were filtered at 10 kHz and stored for later off-line analysis using an analysis program (QPlllJ; Nihon Kohden) on a personal computer (PC9801; NEC). Averaged 10 consecutive responses were sampled at 5-min intervals. Percent changes in EPSC amplitudes were calculated taking the values immediately before TS as 100%. All results were expressed as mean _+ S.E.M. GTP (sodium or lithium salt, Sigma), guanosine 5'-O-(2-thiodiphosphate) (GDPflS) and GTPTS (lithium salt, Boehringer) were dissolved in the pipette solution at the indicated concentrations. Since the GTP sodium and lithium salts gave similar results, their
data were pooled. RESULTS
Internal dialyzation with guanine nucleotide analogs Modification of the activity of G proteins in pyramidal neurons was achieved by intracellular perfusion with pipette solution containing the non-hydrolysable guanine nucleotide analogs, GDPflS and GTPTS. To assess the effectiveness of the loading of these compounds, we tested the effect of the G A B A B agonist baclofen. Baclofen hyperpolarizes hippocampal pyramidal n e u r o n s through the activation of K + channels which are coupled to G A B A ~ receptors via pertussis toxin-sensitive G proteins ~'7'21. Thus, modification of the activity of G proteins affects the hyperpolarizing action of baclofen t. Baclofen was bath applied for 1 min at 23-28 rain after whole-cell access to the CA3 pyramidal neurons, and the m e m b r a n e potential was monitored while the cell was held in the current-clamp mode. Sample records are shown in Fig. lB. W h e n recordings were made with pipette solution containing 300/~M GTP, the responses of the CA3 pyramidal n e u r o n s to 10/~M baclofen averaged -16.7 + 1.6 m V (8 cells). Even when the internal solution contained no added GTP, a similar magnitude of hyperpolarization was observed (-18.9 _+ 0.5 mV, 4 cells). This may indicate that the local concentration of endogenous G T P remaining near the m e m b r a n e is suf-
110 TABLE I
Effects of guanine nucleotide analogs on passive membrane properties and responses of hippocampal pyramidal neuron~ lo bacl&~n Whole-cell recording was made with pipette solutions in which each guanine nucleotide analog was dissolved. Resting membrane potentml and input resistance were measured at 15-30 rain after whole-cell access. Responses to 10/~M baclofen were measured at 23-28 mm (in CA3) and at 22-24 rain (in CAt) after whole-cell access.
Resting membrane potential: mV
CA3
GTP 300/~M GDP/~S 300 ~M GTPyS 100 #M GTP 100/~M GTP?S 100/~M
CA1
-62.3 -66.1 -79.5 -57.8 -68.6
-+ 1.7 (17) + 2.1 (16) + 1.4"* (16) + 1.9 (12) + 1.5"* (11)
Input resistance: Mg2
Responses ~o 10 #M baclo/'en: mV
145 128 72 189 94
-16.7 -10.4 -0.8 -7.9 -0.5
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[1 (15) 7 (12) 6** (13) 18 (12) 3** (11)
+_ 1.0 ('8) + 1.4" (6) _+ 0.3 ** (6) + 0.6 (6) _+ 0.1"* (6)
*P < 0.05, **P < 0.01 vs. GTP group (one-way analysis of variance, followed by the Bonferroni method). The numbers in parentheses indicate numbers of neurons observed.
K + channels normally opened by baclofen. These results suggest that significant modification of the activity of G
ficient for G proteins to function H'2°. W h e n the pipette solution contained 3 0 0 / , M GDPflS, the resting potential and input resistance of the cell was not significantly changed; however, the response to baclofen was significantly reduced (Table I). GTPyS was applied to the CA3 neurons, was gradually hyperpolarized and input reduced, and a steady state was reached
proteins by guanine nucleotide analogs occurred at 23L28 min after whole-cell access.
W h e n 100/,M the m e m b r a n e resistance was within 15 min
Effects o f guanine nucleotide analogs on fimbrial L T P Fig. 2 shows the effect of guanine nucleotide analogs on LTP in the fimbria-CA3 synapses. In this set of experiments, TS was delivered at the same time window used in the previous section, that is, at 2 3 - 2 8 rain after whole-cell access. W h e n recordings were made with
after whole-cell access (Table I). Application of baclofen induced almost no further hyperpolarization (Table I, Fig. 1B), which suggests that GTPyS fully activated the
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Fig. 2. Effects of the modification of the activity of postsynaptic G proteins on fimbrial long-term potentiation (LTP). A: intracellular application of GDPflS attenuates LTP. o, GTP 300/*M (n = 6); o, GDPflS 100/tM (n = 5); A, GDPflS 300 #M (n = 6). B: GTPyS inhibits LTP. o, GTP 100 gM (n = 5); e, GTPyS 100 gM (n = 5). Statistical significance was evaluated at each time point with one-way analysis of variance, followed by the Bonferroni method. *P < 0.05, '~ P < 0.01, vs. GTP (data of 100 and 300 #M were pooled). C: sample traces of LTP recorded with pipettes containing 100/~M GTP, 300/*M GDPffS and 100 #M GTPyS. Traces before and 20 min after TS are superimposed.
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200 pA 10 ms Fig. 3. Irreversible inactivation or activation of postsynaptic G proteins does not inhibit LTP in mossy fiber-CA3 synapses. A: plot of potentiation time course, o, GTP (100 ktM n = 2, 300/~M n = 3); • GDPflS (100 BM n = 2, 300/~M n = 4); A, GTPTS 100 ~M (n = 5). B: sample records of mossy fiber LTP with pipettes containing 300 BM GTP and 100 ~M GTPyS. G T P - c o n t a i n i n g solution, EPSC was greatly enhanced immediately (within 5-10 min) after TS, and then stable potentiation was observed for up to 90 rain. A n averaged potentiation of 109.8 + 18.2% was obtained at 20 min after TS ( G T P 100 /~M n = 5, 300 /zM, n = 6). This value was comparable to one we had obtained previously, using a pipette solution with no added G T P (129.3 _+ 33.3%, n = 6) 14. As shown in Fig. 2A, substitution of G T P with GDP/3S significantly attenuated LTP. The effect was apparent at a concentration of 100 /~M (49.9 _+ 12.1% at 20 min after TS, n = 5), and when 300 # M GDPflS was applied, EPSC gave an averaged potentiation of only 29.6 + 21.5% (n = 6) at 20 min after TS. Next, we tested the effects of GTPTS on LTP in the fimbrial synapses. LTP in the fimbrial synapses was negligible after internal dialyzation with GTPTS (Fig. 2B). The potentiation of EPSC at 20 min after TS was only 6.1 + 14.7% (n = 5).
~/'"'after
200 pA 10 ms
Fig. 4. Intracellular GTPTS attenuates short-term potentiation but not LTP in the Schaffer-CA1 synapses. A: potentiation time course. o, GTP 100/~M (n = 6); e, GTPTS 100 BM (n = 5). *P < 0.05 vs. GTP 1013~M. B: sample records from CA1 neurons with pipettes containing 100 gM GTP and 100/tM GTPyS.
Effects of guanine nucleotide analogs on mossy fiber LTP We also carried out analogous experiments in the mossy fiber-CA3 synapses. The same TS condition resulted in a similar time course but much lower degree of potentiation than in the fimbrial synapses, which averaged a 28.5 + 15.5% increase at 20 min after TS in neurons recorded when G T P solution was added (100 ktM n = 2 , 3 0 0 / ~ M n = 3). This value was not significantly different from that recorded when there was no added G T F (42.4 _+ 20.5%, n = 5) 14. In contrast to that in the timbrial synapses, LTP in the mossy fiber synapses was not inhibited by 100-300 ~M GDP/3S or 100 ktM GTPTS (Fig. 3).
Effects of GTPTS on LTP in the CA1 region The above results on the fimbria-CA3 synapses contrast with the results of a previous report 9 in which it was
112 shown that postsynaptic injection of GTPvS did not affect LTP in the Schaffer-CA1 synapses, although these two synapses share common features, in that the induction of LTP is dependent on the activation of N M D A receptors 4"14. However, it is possible that the apparent discrepancy is due to some differences between the experimental conditions of the present study and those in the previous report. To test this possibility, we also performed whole-cell recording from CA1 pyramidal neurons. In the CA1 region, a pairing procedure > 30 min after whole-cell access fails to induce LTP 17. To avoid extensive washout of cytoplasmic factors, and at the same time to ensure the loading of the guanine nucleotide analogs into the CA1 neurons, we applied TS at 22-24 min after whole-cell access in all cases in this set of experiments. The results are shown in Fig. 4. When recordings were made with internal solution containing 100 ,uM GTP, EPSC was greatly enhanced immediately (within 5 min) after TS, and then the potentiation remained stable at levels of 30-40% (32.0 + 12.3% increase at 20 min after TS) for up to 60 min. As was the case with the CA3 neurons, hyperpolarization and significant reduction of input resistance resuited in the CA1 pyramidal neurons when GTPvS was included in the pipette solution (Table I). The responses to baclofen were also largely abolished, which indicates the effectiveness of the loading of GTPTS in the blockade of signal transduction from the receptor to the G proteins. However, even under these conditions, TS induced LTP in the Schaffer-CA1 synapses. Although initial potentiation was significantly reduced, as reported previously 9, the magnitude of subsequent potentiation was not attenuated or occluded (37.3 + 9.8% increase at 20 min after TS) by the irreversible activation of postsynaptic G proteins with GTPTS. These results confirmed the qualitative difference between the fimbria-CA3 and the Schaffer-CA1 synapses regarding the involvement of G proteins in LTP.
Reversal of the effects of guanine nucleotide analogs on fimbrial LTP by postsynaptic depolarization As the inhibition of fimbrial LTP by GDPflS and GTPTS was apparent immediately after TS, it is likely that factor(s) responsible for the induction of LTP may be altered. As reported previously, induction of LTP in the fimbrial synapses requires postsynaptic membrane depolarization 14. Thus, one possible mechanism underlying the effects of GDPflS and GTPyS is that the inhibition of depolarization during TS is responsible for the attenuation of LTP. To assess this possibility, we attempted to control the membrane potential during TS. To ensure more reliable control of this potential, K + in the pipette solution was
A Fim stim.
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Fig. 5. Reversal of the effects of guanine nucleotide analogs on LTP in the fimbrial synapses by postsynaptic depolarization during TS.
A: stimulation paradigm. Fimbrial fibers were stimulated 3 times at 100 Hz for 1 s (Fire stim.), while the CA3 neuron was depolarized from -80 mV to -20 mV (Vm). B: potentiation time course. o, GTP 100/zM (n = 6); o, GDPflS 300/xM (n = 4); A, GTP%S 100/~M (n = 5). replaced by Cs + in this set of experiments. TS was delivered while the membrane was depolarized: from -80 to -20 mV by voltage command pulses (Fig. 5A). Fig. 5B shows the results of this set of experiments. When the membrane was sufficiently depolarized during TS, LTP in the fimbrial synapses was observed to the same extent in neurons dialyzed with 100 ~M GTP as in neurons dialyzed with 100/~M GTPTS or 300/~M GDPflS. In other words, the effects of non-hydrolysable guanine nucleotide analogs were reversed by postsynaptic membrane depolarization during TS. These results suggest that the inhibition of depolarization during TS may be responsible for the inhibition of LTP in the fimbrial synapses by guanine nueleotide analogs. DISCUSSION
The present study showed that postsynaptic G proteins could affect the induction of LTP in firnbria-CA3 synapses. To assess the involvement of G proteins, we used two kinds of non-hydrolysable guanine nucIeotide analogs, GDPflS and GTPyS. GDPflS inhibits G proteins, presumably by preventing the reaction of G pro-
113 teins and GTP 5. On the other hand, GTPvS binds to the G proteins and induces them to become continuously active 8. Both compounds have now been shown to inhibit LTP in the fimbrial synapses. The inhibitory effects of guanine nucleotide analogs on fimbrial LTP are probably due to the inhibition of depolarization in the CA3 pyramidal neurons during TS, since the effect was reversed by depolarization during TS by delivery of voltage command pulses. Reduction of depolarization by GTP~,S may be explained by the fact that GTPyS opens G protein-linked K + channels, thus causing significant hyperpolarization of the resting membrane potential. However, GDPflS did not significantly affect the resting membrane potential (Table I). GDPflS may specifically reduce the depolarization induced by TS. One explanation of the mechanisms underlying the effect of GDPflS is as follows: in physiological conditions, repetitive stimulation of afferents and extensive release of neurotransmitter(s) activates postsynaptic receptors coupling to G proteins, and the activated G proteins may then activate certain factors that help to depolarize the CA3 pyramidal neurons; GDPflS could inhibit these processes, thereby reducing membrane depolarization. In this context, G proteincoupled, metabotropic glutamate (Qp) receptors might be involved in these processes. Activation of Qp receptors has been shown to block K ÷ conductances and to depolarize CA3 neurons 3. Therefore, it is possible that during TS, a substantial amount of glutamate is released, and that postsynaptic Qp receptors are activated; this activation then blocks some types of K + currents and accelerates the depolarization of postsynaptic neurons. Greater depolarization opens N M D A receptor channels more effectively, and this permits a large influx of Ca 2+ which is necessary for the induction of LTP. The CA3 pyramidal neurons also have many other types of receptors, including muscarinic and fl-adrenergic receptors, which couple to G proteins. There is also a possibility that neurotransmitters other than glutamate are co-released during high frequency afferent activation, and that these are responsible for the membrane depolarization of the CA3 neurons ~5. Other factors should also be considered in explaining the reversal of the effects of guanine nucleotide analogs by membrane depolarization: a blockade of G proteins could reduce the rise in intracellular Ca 2+ by means other than a reduction of membrane depolarization. Depolarizing the cells could counterbalance this action by increasing the Ca 2+ influx through N M D A receptor-mediated conductances or through voltage-gated Ca z+ channels. One possibility is the occurrence of a blockade of the processes that follow activation of Qp and/or muscarinic receptors, since both of these receptors enhance the release of Ca 2+ from intracellular stores via G pro-
tein-mediated processes 12'x9. As reported previously, LTP in the Schaffer-CA1 synapses was not inhibited or occluded by GTPTS. This may indicate that the activation of postsynaptic G proteins during TS, if it occurs, contributes little to the induction of LTP in the CA1 region. It is interesting that fewer Qp receptors are expressed in the CA1 pyramidal neurons than in the CA3 neurons, as shown by in situ hybridization histochemistry TM. During whole-cell recording, cytoplasmic factors are gradually lost, and this 'wash-out' had been reported to inhibit the induction of LTP in the CA1 region 17. Therefore, we must take special care when using this technique to compare LTP in different cells. It is possible that rates of dialysis in CA1 and CA3 cells are different, and this difference might contribute to the observed difference in LTP. Indeed, we observed robust LTP on delivering TS even 40 min after whole-cell access to CA3 cells, as reported previously ~4, which suggested that CA3 neurons may be more resistant to dialysis than CA1 neurons. In the present study, we applied TS at 23-28 min after whole-cell access in the case of CA3, and at 22-24 min after whole-cell access in the case of CA1. By setting these time windows, we were able to achieve effective loading of guanine nucleotide analogs into the pyramidal neurons, while retaining the ability to induce LTP in both CA3 and CA1. Thus, although this procedure could have minimized the artificial effects of wash-out on LTP, we cannot exclude the possibility that intracellular dialysis affected the magnitude of LTP to some extent. LTP in the mossy fiber-CA3 synapses, in contrast to that in the fimbrial synapses, was not inhibited by modification of the activity of postsynaptic G proteins. These results suggest that, in the mossy fiber-CA3 synapses, postsynaptic G proteins are not involved in LTP in any way. However, in vivo pretreatment experiments have suggested that pertussis toxin-sensitive G proteins are involved in mossy fiber LTP 13. Therefore, the location of G proteins responsible for mossy fiber LTP is probably in the presynaptic terminals. The extent of postsynaptic depolarization and, probably, the subsequent increase in intracellular Ca 2+ in the postsynaptic neurons may be important in determining the magnitude of the N M D A receptor-dependent form of LTP. In fact, it was recently demonstrated that the magnitude of postsynaptic Ca 2+ increase is a critical factor in controlling the duration of synaptic enhancement in the CA1 region 16. However, as demonstrated by the present study, the physiological factors controlling the intracellular C a 2+ dynamics of pyramidal neurons may differ in CA3 and CA1. Investigation of the mechanisms modulating intracellular C a 2+ will provide further understanding of plastic changes in various synaptic connections.
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13
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