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Pertussis toxin prevents induction of hippocampal long-term potentiation in the stratum radiatum and stratum oriens inputs to CA1 neurons
Brain Research, 511 (1990)345-348 Elsevier
345
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Pertussis toxin prevents induction of hippocampal long-term potentiation in the stratum radiatum and stratum oriens inputs to CA1 neurons Joanne W. Goh* and Peter S. Pennefather Faculty of Pharmacy, University of Toronto, Toronto, Ont. (Canada) (Accepted 28 November 1989) Key words: G-protein; Long-term potentiation; Pertussis toxin; Hippocampus; Stratum radiatum; Stratum oriens; Adenosine; Excitatory postsynaptic potential
Stereotaxic injections of pertussis toxin (3-4/~g) over the right hippocampus resulted in blockade of long-term potentation (LTP) induction in the ipsilateral stratum radiatum-CA1 and stratum oriens-CA1 synaptic systems. LTP of intracellularly recorded excitatory postsynaptic potentials was prevented in slices obtained from the hippocampus at 3 and 4 but not 6 days post-toxin injection. Slices taken from the left (contralateral) hippocampus on the same days as above exhibited LTP which was similar to that obtained in control slices from uninjected rats. The post- but not presynaptic actions of adenosine were antagonized at 3, 4 and 6 days post-toxin injection. The observations suggest that the guanosine triphosphate binding proteins involved in LTP induction (GLTp)and those coupled to the postsynaptic adenosine receptors exhibit different turnover times.
Long-term potentiation (LTP) observed in the mammalian hippocampus is a prolonged enhancement of synaptic transmission that is thought to be a physiological correlate of certain types of learning and memory. LTP can be induced by delivering a transient high frequency tetanic stimulation to the presynaptic fibers. While it has been established that the induction phase of LTP requires concurrent postsynaptic depolarization and presynaptic activation 14'16, the cellular mechanisms responsible for both the induction as well as the maintenance phases of LTP are still controversial. Previous work from this laboratory demonstrated the participation of a pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)binding protein (GLTP) in the initiation of LTP of the stratum radiatum-CA1 population spike 7. The present study was undertaken to examine whether PT could inhibit LTP of the intraceUularly recorded stratum radiatum as well as stratum oriens evoked excitatory postsynaptic potentials (EPSPs) in CA1 neurons. Stratum oriens contains axons of the commissural pathway originating from the CA3 region of the contralateral hippocampus. We were curious, therefore, to examine whether incorporation of pertussis toxin in the cells of origin of the commissural pathway would affect GLTp involved in LTP of the stratum oriens pathway. Thalmann 15 has reported that unilateral injection of pertussis toxin into the CA3 region of the hippocampus resulted in a decrease
of pertussis substrate in the contralateral hippocampus. Male Wistar rats (250-300 g) were stereotaxically injected directly above the right hippocampus with PT (List Biochemicals) dissolved in 0.01 M sodium phosphate buffer and 0.05 M NaCI (pH 7). The coordinates are (1) posterior to Bregma 3 mm; (2) lateral 4 mm and (3) ventral to dural surface 2.5 mm. The concentration of the PT solution was 2 pg/IA and the dose used for each rat was 3-4 pg so that the final volume administered would be 1.5-2 pl. The rate of injection through a Hamilton microsyringe was 0.2 pl/min to limit dispersion of the toxin to adjacent brain structures. Experiments were conducted on transversely sectioned rat hippocampal slices prepared and maintained as described previously 7. Briefly, they were submerged and perfused with a bathing medium containing (in mM): NaCI 120, KCI 3.1, N a H C O 3 26, dextrose 10, CaCI 2 4, MgCI 2 4, picrotoxin 0.1. Slices were obtained from ipsilateral (right, treated) and contralateral (left, untreated) hippocampi of the injected rats at 3, 4 and 6 days postinjection as well as from uninjected control rats. CA1 neurons were impaled with 'Omega Dot' glass microelectrodes (resistance 30-50 Mg2) filled with 3 M KC1. Concentric bipolar stimulating electrodes (SNEX 100, Rhodes Electronics) were placed in stratum oriens and stratum radiatum on either side of the CA1 cell body layer. Stimulus frequency for each input was once every
Correspondence: J.W. Goh, Department of Pharmacology and Toxicology,Faculty of Medicine, Queen's University, Kingston, Ont., Canada K7L 3N6. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
346 10 s and the stimuli were alternated so that there was a 5-s interval b e t w e e n activation of each input. To induce, LTP, a 400-Hz, 200-pulse tetanus at twice control stimulus intensity was delivered to the a p p r o p r i a t e input. A d e n osine (Sigma Chemical Co.) was dissolved in normal bathing m e d i u m and perfused over the slices at concentrations of 20 and 100 MM. With PT p r e t r e a t m e n t , LTP of the stratum r a d i a t u m CA1 population spike could be induced at 2 days but not 3 and 4 days post-toxin injection 7. Similarly, the inhibition of the population spike by baclofen was p r o n o u n c e d in control, u n t r e a t e d slices and in slices o b t a i n e d at 2 days postinjection 7, but was reduced in slices obtained at 3 and 4 days after toxin injection. These observations suggest that PT requires at least 2 days to exert its effects and, therefore, in the present study, slices were obtained from injected animals after 3, 4 and 6 days. It was not clear from the study of G o h and Pennefather 7 whether the inhibition of the population spike by the G A B A B r e c e p t o r agonist, baclofen, which was used as an index of G T P - b i n d i n g protein ( G - p r o t e i n ) activity, was due to a pre- or postsynaptic action of the drug. According to D u t a r and Nicoll 5, baclofen has both pre- and postsynaptic actions which can be distinguished from each other pharmacologically, in that postsynaptic G A B A B receptors are sensitive to PT whereas presynaptic ones are not
(see also Colmers and Williams/). This being the case, the antagonism of the reduction of the population spike by baclofen after PT p r e t r e a t m e n t p r o b a b l y reflects an inactivation of G - p r o t e i n s coupled to postsynaptic G A B A B receptors. It a p p e a r s that the intracellular EPSP is much less affected than the extracellular population spike by changes in postsynaptic m e m b r a n e potential and resistance 5'7. This may be expected if the excitability of CA1 neurons and, therefore, threshold for spike discharge are m o r e sensitive to alterations in postsynaptic m e m b r a n e properties than the EPSP. Since it was d e t e r m i n e d that GETP is not located in the postsynaptic CA1 neuron 7, we decided to utilize an agent which elicits a pharmacological response due to a relatively selective action on presynaptic G - p r o t e i n s as a possible correlate of GETP activity. A d e n o s i n e has been r e p o r t e d to inhibit synaptic transmission primarily through a G - p r o t e i n - d e p e n d e n t presynaptic action on transmitter release 3'4't3. It was for this reason that the response to adenosine was utilized in the present study as a possible indication of G - p r o t e i n activity in the presynaptic terminal. The results o b t a i n e d , however, suggest that the G-proteins coupled to presynaptic adenosine receptors are not sensitive to PT because adenosine inhibits both the stratum o r i e n s - C A 1 and stratum radia t u m - C A 1 EPSPs equally well after toxin t r e a t m e n t (Figs. 1 and 2). F r e d h o l m et al. 6 have r e p o r t e d similar findings.
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Fig. 1. Effect of pertussis toxin (PT) on adenosine-induced suppression and LTP of the intracellularly recorded CA1 neuronal EPSP evoked by stratum radiatum stimulation. The PT-treated slices were obtained from the ipsilateral, right hippocampus 3-4 days after stereotaxic PT injections. Control slices were from uninjected rats. Records taken during adenosine application were at 2 min during perfusion of each concentration. At least 15 min was allowed for recovery from adenosine effects before the LTPinducing stimulus was delivered (400 Hz, 200 pulses, 2× control stimulus intensity). Each data point on graph represents mean + S.E.M. Asterisks indicate P < 0.05 (significantly different from control).
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Fig. 2. Effect of pertussis toxin (PT) on adenosine-induced suppression and LTP of the intracellularly recorded CA1 neuronal EPSP evoked by stratum oriens stimulation. Experimental protocols were similar to those for Fig. 1. Each data point on graph represents mean + S.E.M. Asterisks indicate P < 0.05 (significantly different from control). This level of significance was attained only upon exclusion of the one potentiated cell out of 7 experiments (filled diamonds). Inclusion of this cell in calculating means (open circles) markedly increased the error and reduced the significance of the difference between control and pertussis-treated groups.
347 TABLE I Effect o f adenosine (lOOpM) on CA1 neuronal membrane potential
Control (not injected) 3-4 days post-pertussistoxin (contralateral hippocampus) 3-4 days post-pertussistoxin (ipsilateral hippocampus) 6 days post-pertussistoxin (ipsilateral hippocampus)
-3.1±0.7mY(n=8) -3.7±0.3mV(n=3) 0.1±0.4mV*(n=9) -0.5±0.5mV(n=2).
*P < 0.01. Significantlydifferent from uninjectedcontrols.
Adenosine produces a minor hyperpolarization of CA1 neurons in control situations but shows no response following PT administration (Table I). Therefore, it appears that postsynaptic adenosine receptors are uncoupled from their G-proteins by PT, whereas their presynaptic counterparts are not affected. Nevertheless, adenosine provides an indication of the bioavailability of PT in the hippocampal CA1 region following injection. The observation that adenosine effects on EPSPs evoked by both inputs show no significant difference between slices obtained from PT-injected and control animals (Figs. 1 and 2) in spite of the blockade of postsynaptic hyperpolarization by PT indicates that the postsynaptic effect of adenosine contributes minimally to modulation of EPSP amplitude. Tetanus-induced LTP of both inputs is prevented in slices obtained from PT-treated hippocampi up to 4 days post-toxin injection (Figs. 1 and 2, Table II). When slices are taken at 6 days following injection, the effect of the toxin is no longer present and LTP can be produced (Table II). The time course of inactivation of GETP and the G-protein coupled to adenosine receptors appears to be dissimilar, pointing to different turnover rates for these proteins. Although G-proteins associated with presynaptic adenosine receptors are not sensitive to PT in the hippocampus, this is not true for all presynaptic G-proteins. For instance, presynaptic adenosine receptors in the cerebellum 3 and presynaptic serotonin re-
TABLE II Effect o f pertussis toxin on LTP in stratum oriens and stratum radiatum inputs to CA1 neurons Number o f cells exhibiting LTP
Control (not injected) 3-4 days post-pertussis toxin (contralateralhippocampus) 3-4 days post-pertussis toxin (ipsilaterai hippocampus) 6 days post-pertussis toxin (ipsilaterai hippocampus)
Stratum radiatum
Stratum oriens
3 of 4
3of6
3of3
3of3
I of 7
1 of 7
2 of 2
2of2 •
ceptors 2'12 are uncoupled from their G-proteins by the toxin. It is obvious that, if GETP is located in the presynaptic terminal, it is affected differently than the G-protein linked to presynaptic adenosine receptors. PT, when injected over the right, ipsilateral hippocampus does not travel to the left, contralateral hippocampus because LTP induction and the responses to adenosine application are virtually indistinguishable between slices obtained from uninjected control and contralateral hippocampi (Tables I and II). In some cases, PT-injected rats exhibited seizure activity. One concern was that the epileptic behavior, which is caused by massive neuronal discharges, could induce LTP in PT-treated animals. If this were the case, slices obtained from pre-potentiated hippocampi should exhibit little or no further LTP. Since the seizures were of the generalized type and LTP could be induced in the contralateral but not ipsilateral hippocampus, one can conclude that increased cellular firing during epileptic activity did not induce LTP in the animals. Furthermore, even when PT effects on GLTP had worn off in the ipsilateral hippocampus, as evidenced by success of LTP development at 6 days postinjection (Table II), the seizures had not subsided. Perhaps, the epileptic activity can be directly correlated with a suppression of the adenosine and other (e.g. GABAB) inhibitory systems. The prediction in this instance would be that the time course for recovery of the postsynaptic adenosine (and baclofen?) response following PT-treatment should closely parallel a reduction in epileptic seizures. Evidence in the literature suggests that an activation of the enzyme protein kinase C (PKC) may be involved in LTP 1"9'11. As suggested previously7, activation of GETP may be linked to stimulation of PKC. It is of note that Malinow et al. 11 have presented evidence suggesting that, although postsynaptic protein kinases are involved in the induction of LTP, a non-postsynaptic protein kinase, inhibited by H-7, is involved in expression of LTP. We have demonstrated that GETP is not located postsynaptically. We showed that occlusion of postsynaptic Gprotein-dependent pathways by loading the neuron with GTPyS had no effect on LTP induction 7. Other second messenger pathways have been implicated in induction or maintenance of LTP. These include production of arachidonic acid 17 and activation of Ca2+/ calmodulin-dependent protein kinase II (CaMKII) 1°. The exact position of GLTP in the complex web of interacting second messenger cascades remains to be determined. Reports indicate that LTP of population responses in both the stratum radiatum-CA1 and mossy fibre-CA3 synapses are sensitive to PT 7"s. The results presented in this paper provide evidence for the involvement of a G-protein in the induction of LTP in yet another synaptic
348 system, the stratum o r i e n s - C A 1 system. The observation that LTP in 3 different hippocampal inputs is blocked by PT treatment suggests a universal mechanism involving
extracellular population responses is confirmed in this study using intracellular recordings of EPSPs evoked by stimulation of two different synaptic systems terminating
GETP which may ultimately be linked to the chain of events leading to stimulation of PKC, C a M K I I or the
on CA1 neurons. F u r t h e r m o r e , it has b e e n established that G-proteins involved in LTP and those coupled to
arachidonic acid cascade. Previous studies which examined the effects of PT on LTP utilized extracellular population recordings 7's. Pop-
postsynaptic adenosine receptors exhibit different turnover times (presynaptic adenosine receptors appear not to be affected by PT). The inhibitory effect of PT on LTP is lost by 6 days post-toxin administration, whereas postsynaptic adenosine responses are still suppressed.
ulation responses may not be an accurate measure of synaptic transmission because of the possible recruitment or inhibition of action potentials due to changes in CA1 n e u r o n a l m e m b r a n e properties. Therefore, recording EPSPs intracellularly give a better reflection of events occurring at synapses. The effect of PT on LTP of
This work was supported by the Canadian Medical Research Council. P.S.P. is a Career Scientist of the Ontario Ministry of Health and J.W.G. is an MRC Fellow.
1 Akers, R.E, Lovinger, D.M., Colley, P.A., Linden, D.J. and Routtenberg, A., Translocation of protein kinase C activity may mediate hippocampal long-term potentiation, Science, 231 (1986) 587-589. 2 Colmers, W.E and Williams, J.T., Pertussis toxin pretreatment discriminates between pre- and postsynaptic actions of baclofen in rat dorsal raphe nucleus in vitro, Neurosci. Lett., 93 (1988) 300-306. 3 Dolphin, A.C. and Prestwich, S.A., Pertussis toxin reverses adenosine inhibition of neuronal glutamate release, Nature (Lond.), 316 (1985) 148-150. 4 Dunwiddie, T.V. and Haas, H., Adenosine increases synaptic facilitation in the in vitro rat hippocampus: evidence for a presynaptic site of action, J. Physiol. (Lond.), 369 (1985) 365-377. 5 Dutar, P. and Nicoll, R.A., Pre- and postsynaptic GABAB receptors in the hippocampus have different pharmacological properties, Neuron, 1 (1988) 585-591. 6 Fredholm, B.B., Proctor, W., Van der Ploeg, I. and Dunwiddie, T.V., In vivo pertussis toxin treatment attenuates some, but not all, adenosine A 1 effects in slices of the rat hippocampus, Eur. J. Pharmacol., 172 (1989) 249-262. 7 Goh, J.W. and Pennefather, P.S., A pertussis toxin-sensitive G protein in hippocampal long-term potentiation, Science, 244 (1989) 980-983. 8 Ito, I., Okada, D. and Sugiyama, H., Pertussis toxin suppresses long-term potentiation in the rat hippocampal mossy fiber synapses, Neurosci. Lett., 90 (1988) 181-185. 9 Malenka, R.C., Madison, D.V. and Nicoll, R.A., Potentiation of synaptic transmission in the hippocampus by phorbol esters,
Nature (Lond.), 321 (1986) 175-177. 10 Malenka, R.C., Kauer, J.A., Perkel, D.J., Mauk, M.D., Kelly, P.T., Nicoll, R.A. and Waxham, M.N., An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation, Nature (Lond.), 340 (1989) 554-557. 11 Malinow, R., Schuiman, H. and Tsien, R.W., Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP, Science, 245 (1989) 862-866. 12 Passarelli, E, Costa, T. and Almeida, O.EX., Pertussis toxin inactivates the presynaptic serotonin autoreceptor in the hippocampus, Eur. J. Pharrnacol., 155 (1988) 297-299. 13 Proctor, W.R. and Dunwiddie, T.V., Pre- and postsynaptic actions of adenosine in the in vitro rat hippocampus, Brain Research, 426 (1987) 187-190. 14 Sastry, B.R., Goh, J.W. and Auyeung, A., Associative induction of posttetanic and long-term potentiation in CA1 neurons of rat hippocampus, Science, 232 (1986) 988-990. 15 Thalmann, R.H., Evidence that guanosine triphosphate (GTP)binding proteins control a synaptic response in brain: effects of pertussis toxin and GTP),S on the late inhibitory postsynaptic potential of hippocampus CA3 neurons, J. Neurosci., 8 (1988) 4589-4602. 16 Wigstr6m, H., Gustafsson, B., Huang, Y.-Y. and Abraham, W.C., Hippocampal long-term potentiation is induced by pairing single afferent volleys with intracellularly injected depolarizing current pulses, Acta Physiol. Scand., 126 (1986) 317-319. 17 Williams, J.H., Errington, M.L., Lynch, M.A. and Bliss, T.V.P., Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus, Nature (Lond.), 341 (1989) 739-742.
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Pertussis toxin prevents induction of hippocampal long-term potentiation in the stratum radiatum and stratum oriens inputs to CA1 neurons Joanne W. Goh* and Peter S. Pennefather Faculty of Pharmacy, University of Toronto, Toronto, Ont. (Canada) (Accepted 28 November 1989) Key words: G-protein; Long-term potentiation; Pertussis toxin; Hippocampus; Stratum radiatum; Stratum oriens; Adenosine; Excitatory postsynaptic potential
Stereotaxic injections of pertussis toxin (3-4/~g) over the right hippocampus resulted in blockade of long-term potentation (LTP) induction in the ipsilateral stratum radiatum-CA1 and stratum oriens-CA1 synaptic systems. LTP of intracellularly recorded excitatory postsynaptic potentials was prevented in slices obtained from the hippocampus at 3 and 4 but not 6 days post-toxin injection. Slices taken from the left (contralateral) hippocampus on the same days as above exhibited LTP which was similar to that obtained in control slices from uninjected rats. The post- but not presynaptic actions of adenosine were antagonized at 3, 4 and 6 days post-toxin injection. The observations suggest that the guanosine triphosphate binding proteins involved in LTP induction (GLTp)and those coupled to the postsynaptic adenosine receptors exhibit different turnover times.
Long-term potentiation (LTP) observed in the mammalian hippocampus is a prolonged enhancement of synaptic transmission that is thought to be a physiological correlate of certain types of learning and memory. LTP can be induced by delivering a transient high frequency tetanic stimulation to the presynaptic fibers. While it has been established that the induction phase of LTP requires concurrent postsynaptic depolarization and presynaptic activation 14'16, the cellular mechanisms responsible for both the induction as well as the maintenance phases of LTP are still controversial. Previous work from this laboratory demonstrated the participation of a pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)binding protein (GLTP) in the initiation of LTP of the stratum radiatum-CA1 population spike 7. The present study was undertaken to examine whether PT could inhibit LTP of the intraceUularly recorded stratum radiatum as well as stratum oriens evoked excitatory postsynaptic potentials (EPSPs) in CA1 neurons. Stratum oriens contains axons of the commissural pathway originating from the CA3 region of the contralateral hippocampus. We were curious, therefore, to examine whether incorporation of pertussis toxin in the cells of origin of the commissural pathway would affect GLTp involved in LTP of the stratum oriens pathway. Thalmann 15 has reported that unilateral injection of pertussis toxin into the CA3 region of the hippocampus resulted in a decrease
of pertussis substrate in the contralateral hippocampus. Male Wistar rats (250-300 g) were stereotaxically injected directly above the right hippocampus with PT (List Biochemicals) dissolved in 0.01 M sodium phosphate buffer and 0.05 M NaCI (pH 7). The coordinates are (1) posterior to Bregma 3 mm; (2) lateral 4 mm and (3) ventral to dural surface 2.5 mm. The concentration of the PT solution was 2 pg/IA and the dose used for each rat was 3-4 pg so that the final volume administered would be 1.5-2 pl. The rate of injection through a Hamilton microsyringe was 0.2 pl/min to limit dispersion of the toxin to adjacent brain structures. Experiments were conducted on transversely sectioned rat hippocampal slices prepared and maintained as described previously 7. Briefly, they were submerged and perfused with a bathing medium containing (in mM): NaCI 120, KCI 3.1, N a H C O 3 26, dextrose 10, CaCI 2 4, MgCI 2 4, picrotoxin 0.1. Slices were obtained from ipsilateral (right, treated) and contralateral (left, untreated) hippocampi of the injected rats at 3, 4 and 6 days postinjection as well as from uninjected control rats. CA1 neurons were impaled with 'Omega Dot' glass microelectrodes (resistance 30-50 Mg2) filled with 3 M KC1. Concentric bipolar stimulating electrodes (SNEX 100, Rhodes Electronics) were placed in stratum oriens and stratum radiatum on either side of the CA1 cell body layer. Stimulus frequency for each input was once every
Correspondence: J.W. Goh, Department of Pharmacology and Toxicology,Faculty of Medicine, Queen's University, Kingston, Ont., Canada K7L 3N6. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
346 10 s and the stimuli were alternated so that there was a 5-s interval b e t w e e n activation of each input. To induce, LTP, a 400-Hz, 200-pulse tetanus at twice control stimulus intensity was delivered to the a p p r o p r i a t e input. A d e n osine (Sigma Chemical Co.) was dissolved in normal bathing m e d i u m and perfused over the slices at concentrations of 20 and 100 MM. With PT p r e t r e a t m e n t , LTP of the stratum r a d i a t u m CA1 population spike could be induced at 2 days but not 3 and 4 days post-toxin injection 7. Similarly, the inhibition of the population spike by baclofen was p r o n o u n c e d in control, u n t r e a t e d slices and in slices o b t a i n e d at 2 days postinjection 7, but was reduced in slices obtained at 3 and 4 days after toxin injection. These observations suggest that PT requires at least 2 days to exert its effects and, therefore, in the present study, slices were obtained from injected animals after 3, 4 and 6 days. It was not clear from the study of G o h and Pennefather 7 whether the inhibition of the population spike by the G A B A B r e c e p t o r agonist, baclofen, which was used as an index of G T P - b i n d i n g protein ( G - p r o t e i n ) activity, was due to a pre- or postsynaptic action of the drug. According to D u t a r and Nicoll 5, baclofen has both pre- and postsynaptic actions which can be distinguished from each other pharmacologically, in that postsynaptic G A B A B receptors are sensitive to PT whereas presynaptic ones are not
(see also Colmers and Williams/). This being the case, the antagonism of the reduction of the population spike by baclofen after PT p r e t r e a t m e n t p r o b a b l y reflects an inactivation of G - p r o t e i n s coupled to postsynaptic G A B A B receptors. It a p p e a r s that the intracellular EPSP is much less affected than the extracellular population spike by changes in postsynaptic m e m b r a n e potential and resistance 5'7. This may be expected if the excitability of CA1 neurons and, therefore, threshold for spike discharge are m o r e sensitive to alterations in postsynaptic m e m b r a n e properties than the EPSP. Since it was d e t e r m i n e d that GETP is not located in the postsynaptic CA1 neuron 7, we decided to utilize an agent which elicits a pharmacological response due to a relatively selective action on presynaptic G - p r o t e i n s as a possible correlate of GETP activity. A d e n o s i n e has been r e p o r t e d to inhibit synaptic transmission primarily through a G - p r o t e i n - d e p e n d e n t presynaptic action on transmitter release 3'4't3. It was for this reason that the response to adenosine was utilized in the present study as a possible indication of G - p r o t e i n activity in the presynaptic terminal. The results o b t a i n e d , however, suggest that the G-proteins coupled to presynaptic adenosine receptors are not sensitive to PT because adenosine inhibits both the stratum o r i e n s - C A 1 and stratum radia t u m - C A 1 EPSPs equally well after toxin t r e a t m e n t (Figs. 1 and 2). F r e d h o l m et al. 6 have r e p o r t e d similar findings.
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Fig. 1. Effect of pertussis toxin (PT) on adenosine-induced suppression and LTP of the intracellularly recorded CA1 neuronal EPSP evoked by stratum radiatum stimulation. The PT-treated slices were obtained from the ipsilateral, right hippocampus 3-4 days after stereotaxic PT injections. Control slices were from uninjected rats. Records taken during adenosine application were at 2 min during perfusion of each concentration. At least 15 min was allowed for recovery from adenosine effects before the LTPinducing stimulus was delivered (400 Hz, 200 pulses, 2× control stimulus intensity). Each data point on graph represents mean + S.E.M. Asterisks indicate P < 0.05 (significantly different from control).
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Fig. 2. Effect of pertussis toxin (PT) on adenosine-induced suppression and LTP of the intracellularly recorded CA1 neuronal EPSP evoked by stratum oriens stimulation. Experimental protocols were similar to those for Fig. 1. Each data point on graph represents mean + S.E.M. Asterisks indicate P < 0.05 (significantly different from control). This level of significance was attained only upon exclusion of the one potentiated cell out of 7 experiments (filled diamonds). Inclusion of this cell in calculating means (open circles) markedly increased the error and reduced the significance of the difference between control and pertussis-treated groups.
347 TABLE I Effect o f adenosine (lOOpM) on CA1 neuronal membrane potential
Control (not injected) 3-4 days post-pertussistoxin (contralateral hippocampus) 3-4 days post-pertussistoxin (ipsilateral hippocampus) 6 days post-pertussistoxin (ipsilateral hippocampus)
-3.1±0.7mY(n=8) -3.7±0.3mV(n=3) 0.1±0.4mV*(n=9) -0.5±0.5mV(n=2).
*P < 0.01. Significantlydifferent from uninjectedcontrols.
Adenosine produces a minor hyperpolarization of CA1 neurons in control situations but shows no response following PT administration (Table I). Therefore, it appears that postsynaptic adenosine receptors are uncoupled from their G-proteins by PT, whereas their presynaptic counterparts are not affected. Nevertheless, adenosine provides an indication of the bioavailability of PT in the hippocampal CA1 region following injection. The observation that adenosine effects on EPSPs evoked by both inputs show no significant difference between slices obtained from PT-injected and control animals (Figs. 1 and 2) in spite of the blockade of postsynaptic hyperpolarization by PT indicates that the postsynaptic effect of adenosine contributes minimally to modulation of EPSP amplitude. Tetanus-induced LTP of both inputs is prevented in slices obtained from PT-treated hippocampi up to 4 days post-toxin injection (Figs. 1 and 2, Table II). When slices are taken at 6 days following injection, the effect of the toxin is no longer present and LTP can be produced (Table II). The time course of inactivation of GETP and the G-protein coupled to adenosine receptors appears to be dissimilar, pointing to different turnover rates for these proteins. Although G-proteins associated with presynaptic adenosine receptors are not sensitive to PT in the hippocampus, this is not true for all presynaptic G-proteins. For instance, presynaptic adenosine receptors in the cerebellum 3 and presynaptic serotonin re-
TABLE II Effect o f pertussis toxin on LTP in stratum oriens and stratum radiatum inputs to CA1 neurons Number o f cells exhibiting LTP
Control (not injected) 3-4 days post-pertussis toxin (contralateralhippocampus) 3-4 days post-pertussis toxin (ipsilaterai hippocampus) 6 days post-pertussis toxin (ipsilaterai hippocampus)
Stratum radiatum
Stratum oriens
3 of 4
3of6
3of3
3of3
I of 7
1 of 7
2 of 2
2of2 •
ceptors 2'12 are uncoupled from their G-proteins by the toxin. It is obvious that, if GETP is located in the presynaptic terminal, it is affected differently than the G-protein linked to presynaptic adenosine receptors. PT, when injected over the right, ipsilateral hippocampus does not travel to the left, contralateral hippocampus because LTP induction and the responses to adenosine application are virtually indistinguishable between slices obtained from uninjected control and contralateral hippocampi (Tables I and II). In some cases, PT-injected rats exhibited seizure activity. One concern was that the epileptic behavior, which is caused by massive neuronal discharges, could induce LTP in PT-treated animals. If this were the case, slices obtained from pre-potentiated hippocampi should exhibit little or no further LTP. Since the seizures were of the generalized type and LTP could be induced in the contralateral but not ipsilateral hippocampus, one can conclude that increased cellular firing during epileptic activity did not induce LTP in the animals. Furthermore, even when PT effects on GLTP had worn off in the ipsilateral hippocampus, as evidenced by success of LTP development at 6 days postinjection (Table II), the seizures had not subsided. Perhaps, the epileptic activity can be directly correlated with a suppression of the adenosine and other (e.g. GABAB) inhibitory systems. The prediction in this instance would be that the time course for recovery of the postsynaptic adenosine (and baclofen?) response following PT-treatment should closely parallel a reduction in epileptic seizures. Evidence in the literature suggests that an activation of the enzyme protein kinase C (PKC) may be involved in LTP 1"9'11. As suggested previously7, activation of GETP may be linked to stimulation of PKC. It is of note that Malinow et al. 11 have presented evidence suggesting that, although postsynaptic protein kinases are involved in the induction of LTP, a non-postsynaptic protein kinase, inhibited by H-7, is involved in expression of LTP. We have demonstrated that GETP is not located postsynaptically. We showed that occlusion of postsynaptic Gprotein-dependent pathways by loading the neuron with GTPyS had no effect on LTP induction 7. Other second messenger pathways have been implicated in induction or maintenance of LTP. These include production of arachidonic acid 17 and activation of Ca2+/ calmodulin-dependent protein kinase II (CaMKII) 1°. The exact position of GLTP in the complex web of interacting second messenger cascades remains to be determined. Reports indicate that LTP of population responses in both the stratum radiatum-CA1 and mossy fibre-CA3 synapses are sensitive to PT 7"s. The results presented in this paper provide evidence for the involvement of a G-protein in the induction of LTP in yet another synaptic
348 system, the stratum o r i e n s - C A 1 system. The observation that LTP in 3 different hippocampal inputs is blocked by PT treatment suggests a universal mechanism involving
extracellular population responses is confirmed in this study using intracellular recordings of EPSPs evoked by stimulation of two different synaptic systems terminating
GETP which may ultimately be linked to the chain of events leading to stimulation of PKC, C a M K I I or the
on CA1 neurons. F u r t h e r m o r e , it has b e e n established that G-proteins involved in LTP and those coupled to
arachidonic acid cascade. Previous studies which examined the effects of PT on LTP utilized extracellular population recordings 7's. Pop-
postsynaptic adenosine receptors exhibit different turnover times (presynaptic adenosine receptors appear not to be affected by PT). The inhibitory effect of PT on LTP is lost by 6 days post-toxin administration, whereas postsynaptic adenosine responses are still suppressed.
ulation responses may not be an accurate measure of synaptic transmission because of the possible recruitment or inhibition of action potentials due to changes in CA1 n e u r o n a l m e m b r a n e properties. Therefore, recording EPSPs intracellularly give a better reflection of events occurring at synapses. The effect of PT on LTP of
This work was supported by the Canadian Medical Research Council. P.S.P. is a Career Scientist of the Ontario Ministry of Health and J.W.G. is an MRC Fellow.
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