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A postsynaptic G-protein in hippocampal long-term potentiation
Brain Research, 611 (1993) 81-86
81
Elsevier Science Publishers B.V.
BRES 18794
A postsynaptic G-protein in hippocampal long-term potentiation B a r b a r a A n n Ballyk a n d J o a n n e W. G o h Departma~t of Pharmacology and Toxicology, Queen's Unicersity, Kingston, ON (Canada) (Accepted 8 December 1992)
Key words: Lithium; G-protein; Hippocampus; CA I region; Postsynaptic; Metabotropic glutamate receptor
The effects of guanosine triphosphate (GTP)-binding protein (G-protein) blockade on hippocampal LTP at stratum radiatum-CA i synapses was studied. Bath application of 20 mM lithium chloride (LiCI) inhibited long-term potentiation (LTP) of extracellularly-recorded excitatory postsynaptic potentials (EPSPs). Inclusion of 100 mM LiCi in intracellular recording electrodes was shown to block postsynaptic G-proteins by bath-application of baclofen, an agonist at the G-protein linked y-aminobutyric acid (GABA n) receptor. Under normal conditions, GABA B receptor activation causes a hyperpolarization postsynapticaUy, and a decrease in neurotransmitter release presynaptically. With LiCi in the recording electrodes, the postsynaptically-mediated hyperpolarization was blocked, while the presynaptically-mediated depression of EPSPs was unaffected. With postsynaptic G-proteins blocked in this manner, LTP at these synapses was inhibited. These studies provide evidence for the involvement of a postsynaptic G-protein in LTP of stratum radiatum-CA i synapses.
INTRODUCTION LTP is a prolonged form of activity-dependent synaptic plasticity thought to be related to learning and memory s. The involvement of G-proteins in this phenomenon has been thc subject of recent studies 12'It'. With the discovery of the metabotropic glutamate receptor (mGluR) 35'3s, interest in this question of G-protein involvement in LTP has gained new relevance. Glutamate is the excitatory neurotransmitter at stratum radiatum-CA~ synapses, where it acts on multiple receptor subtypes, both ionotropic and metabotropic 24'a2. The ionotropic receptors include the Nmethyl-D-aspartate (NMDA) and non-NMDA subtypes 7,s, while the G-protein-linked mGluR subtypes continue to be identified 2'39. While the involvement of the NMDA receptor in the induction of stratum radiatum-CA~ LTP has been firmly established 7,s, recent studies in this lab suggest that coactivation of both mGluR and NMDA receptors is required 26. A pertussis toxin (PTX)-sensitive G-protein is involved in LTP in the CA~ region ~2. It is possible that this PTX-sensitive G-protein may in fact mediate a response to mGluR activation. It has also been shown
that maximal activation of CA i neuronal G-proteins by intracellular injection of guanosine-5'-O-(3-thiotriphosphate) (GTPyS), a nonhydrolyzable GTP analogue, does not occlude LTP 12'16. Although this result has been interpreted to negate the involvement of a postsynaptie G-protein in CA~ LTP, recent findings in this lab provide for a second interpretation. If, as we have found, the induction of stratum radiatum-CA~ LTP requires coactivation of both NMDA and mGluRs 2~', it appears that G-protein activation (as with GTPyS) at these synapses is in itself insufficient to induce LTP. The second requirement, NMDA receptor activation, can be fulfilled by tetanization of afferents leading to neurotransmitter release. The location(s) of Gprotein(s) involved in LTP remains to be determined. Li + is known to inhibit both adrenergic and cholinergic agonist-stimulated increases in [3H]GTP binding to rat cortical membranes a, therefore blocking the activation of G s and G i or Go. Li + also blocks Gs-mediated increases in cAMP accumulation ~°'2°, G~-mediated decrcases in cAMP 2~, and Go-mediated increases in phosphoinositol (PI) turnover ~3. Since mGluR activation can trigger a variety of second messenger cascades 2'39, and the identity of the G-protein(s) in-
Correspondence: J.W. Goh, Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, C~mada KL7 3N6. Fax: (1) (613) 545 6412.
82 volved in LTP has not been established, we used Li + as a ubiquitous blocker to probe the subceilular localization (ie. pre- vs. postsynaptic) of G-proteins important for LTP induction.
A 160
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MATERIALS AND METHODS 120
O
Care of animals was in accordance with Canadian Council on Animal Care guidelines. Male Wistar rats were anesthetized with halothane (2%), and their brains removed. The hippocampi were dissected out and 41}{} ttm transverse slices cut using a Vibroslice tissue slicer (Campden Instruments Ltd.). For one hour prior to recording, control slices were incubated at room temperature in artificial cerebrospinal fluid (ACSF)containing in raM: 120 NaCI, 3. l KCI, 1.3 NaH,PO4, 26 NaHCO 3, 10 dextrose, 2 CaCI, and 1.5 MgCI,: pH 7.4, bubbled with 95% O2/5r~ CO.,. Lie-incubated slices were maintained at room temperature for at least two hours prior to recording in oxygenated ACSF containing 20 mM LiCI, osmotically balanced by a reduction in NaCI content. For recording, slices were transferred to a superfusion recording chamber, to which the same (Li "-containing or control) oxygenated ACSF was delivered at a rate of 2 ml per rain, maintained at 30+0.2°C. Recording electrodes were fashioned from 1.2 mm O.D., 0.68 mm I.D. glass, using a Flaming-Brown model P-87 microelectrode puller (Sutter Instrument Co.). For extracellular recording, electrodes were pulled to a resistance of 3-5 MI2 and filled with 4 M NaCI. For intracellular recording, electrodes were pulled to a resistance of 30-50 MO and filled with 3 M potassium acetate (KAc), with or without 100 mM LiCI added. EPSPs were elicited at a control frequency of {}.2 Hz by concentric bipolar stimulating electrodes (SNEX Illl), Rhodes Electronics) placed in the stratum radiatum. EPSPs were recorded extracellularly from the stratum radiatum or intraccUularly from the stratum pyramidale of the CA I region, in the intracellular experiments, G-protein flmction was monitored using a 2 rain bath-application of 5ll/~ M baclofen. Signals were amplified by an Axoclamp model 2A amplil'ier (Axon Instruments), digitized and stored on videotape, and analyzed by pClamp software (Axon Instruments). LTP was defined as a greater than 2()~,: increase in EPSP slope, seen 30 rain folk+wing the delivery of a one second, I(10 l+lz tetanus to afferent fibres.
RESULTS
Bath-applied fithium Slices were incubated at room temperature for at least 2 h prior to recording in oxygenated ACSF containing 20 mM LiCi. During recording, the same ACSF containing Li + was used to perfuse the bath. Normal ACSF (lacking Li ~ ) was used to incubate control slices prior to recording, and to perfuse the bath during recording. Expe:iments commenced once dendriticallyrecorded EPSPs remained stable for at least 15 min. Following a 100 Hz, Is tetanus delivered to the stratum radiatum, short-term potentiation (STP) was seen in both control and experimental slices (Fig. 1). However, at 30 min post-tetanus, only the control slices exhibited LTP, the slopes of the EPSPs being 136.0 + 9.7% of pretetanus values (n = 10), while EPSPs in the experimental slices were unchanged (98.6_+ 4.1% of pretetanus values, n = 11). The slopes of control EPSPs were significantly greater than experimental at 10, 20, and 30 min post-tetanus (Fig. 1).
t~
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80
5
i
I
0
15
•
30
Time (rain) Fig. I. Extracellularly-applied Li+ blocks the induction of stratum radiatum-CA I LTP. EPSPs were recorded extracellularly in the stratum radiatum of CA I in response to test stimuli delivered at 0.2 Hz. At 0 time, a 100 Hz, Is tetanus was delivered to the afferent fibres, as indicated by the open arrow. • EPSPs recorded from slices incubated for 2 h in 20 mM Li+ (n = 11). • EPSPs recorded from control slices ( n = 10). Data plotted as mean =l:S.E.M. Statistical analysis consisted of two way analysis of variance, followed by Tukey's post-hoe test. * P < 0.05. ** P < 0.01. A.
2,-
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B.
'
CONTROL
,
.
100~ Lz' ZR ELECTEODE
.I Fig. 2. lntracellular Li + blocks the postsynaptic G-protein-mediated response to baclofen, while having no effect on ionotropic glutamate responses. A: continuous record of membrane potential with EPSPs (deflections above baseline) elicited by 0.2 Hz test stimuli and voltage responses to -0.2hA, 50 m~ pulses (deflections below baseline) recorded from a CA I pyramidal neuron. Expanded EPSP • records are taken from the trace as indicated by the arrows. At the bars, 50 ~ M baclofen was bath-applied for 2 rain. The initial exposure to baclofen (20 rain post-impalement) elicited both a postsynaptically-mediated hyperpolarization, and a presynaptically-mediated suppressio!a of EPSPs. The second exposure to baclofen (32 rain post-impalement) dicited only the presynaptically-mediated effect, indicating blockade of the postsynaptic G-protein-mediated hyperpolarization by the progressive introduction of Li + into the cell. Following washout of each exposure to baclofen, it can be seen that EPSPs elicited by low-frequency activation of afferents are unchanged by intraceilular LiCI. The scale bar represents 40 ms, 8 mV for expanded EPSPs (top) and 2 rain, 10 mV for continuous trace (bottom). Resting membrane potential was - 7 1 inV. B: the response of a CA l pyramidal neuron to tetanic artivation of afferents are unchanged by intraceilular Li +. Response of the control cell was recorded using a 3 M K + acetate-filled electrode, while that in the experimental cell was recorded with 100 mM LiCI/3 M K + acetate in the electrode. The bars indicate delivery of the 100 Hz, ls tetanus. in the latter case, the affercnts were tetanized after the postsynaptic G-protein-mediated response to baclofen was blocked. The scale bar represents 250 ms, 2 mV. Resting membrane potential of the control cell was - 78 mV and of the Li +-loaded cell was - 75 mV.
83 A. CONTROL
i m m
B.
I00~4 Lx+
ZN ELECTRODE
I
Fig. 3. Introduction of Li + into a CA I pyramidal neuron blocks both the G-protein-mediated response to the G A B A n agonist baciofen and the induction of LTP. Top traces show continuous record of membrane potential with EPSPs elicited by 0.2 Hz test stimuli and voltage responses to - 0 . 2 nA, 50 ms pulses recorded from CA r pyramidal neurons. Expanded EPSP records are taken from the upper traces as indicated by the filled arrows. At the open arrow, a 1 s 100 Hz tetanus was delivered to afferent fibres. The scale bar represents 2 min, 10 mV for the upper records and 40 ms, 6 mV for the EPSP records. A: record from a control neuron. At ~ae bar, 50/zM baclofen was applied for 2 min. The cell exhibited both postsynaptically mediated hyperpolarization and presynaptically mediated depression of EPSPs with activation of GABA a receptors. Tetanus resulted in LTP. B: 32 min after impalement with an intracellular recording electrode containing 100 mM LiCI, a 2 rain exposure to 50 p.M baclofen elicited no hyperpolarizing response, indicating blockade of postsynaptic G-proteins, while the presynaptically mediated depression of EPSPs is unaffected. Under these conditions, the inducton of LTP was inhibited. Records in (A) and (B) are from 2 different cells. Membrane potentials were - 6 7 and - 7 1 mV, respectively.
lntracellular lithium Li + was introduced to the post-synaptic neuron by the inclusion of 100 mM LiC! with the 3 M KAc solution Used to fill intracellular recording electrodes. Control cells were impaled with electrodes containing 3 M KAc, with no LiCI. In these experiments, prior to tetanization of afferents, the status of post-synaptic G-proteins was assessed using the GABA a receptor agonist baclofen. GABA B receptors in hippocampal pyramidale neurons are linked via a G-protein to a K + channel TM. Activation of these receptors by baclofen results in neuronal hyperpolarization. GABAa receptors are also located presynaptically, their activation decreasing the release of excitatory neurotransmitter, resulting in depression of EPSPs 42. Thus, G-protein function w~s monitored by the neuronal response to a 2 min bath-application of 50/zM baclofen (Fig. 2; Fig. 3; Table I). In control cells, baclofen elicited a - 4 . 8 + 1.0 mV hyperpolarization (n = 4). We found that with Li+-containing electrodes, it took from 30 to 60 min following neuronal impalement for post-synaptic GABA B receptors to be blocked (Fig. 2), resulting in no hyperpolarization in response to the bath-applied baclofen ( - 0 . 5 + 0.3 mV, n - 8). In both control and Li +-containing cells, the presynaptic effects of baclofen
were the same, both demonstrating a comparable depression of EPSP slope (Fig. 3, Table !). A second indication of the blockade of postsynaptic G-proteins TABLE I
Effects of intracellular lithium on IPSPs, baclofen-induced hyperpolarization, tetanus-induced depolarization and L TP Control
100 mM Li + hz recording electrode
Slow IPSP amplitude (mV)
- 1.8 + 0.4 (n = 8)
+ 0.2 5:0.1 * (n = 16)
Maximum baclofeninduced hyperpolarization (mV)
- 4.8 + 1.0 (n = 4)
- 0.5 + 0.3 * (n = 8)
EPSP slope during baciofen (% control)
28 + 4 (n = 8)
34 +5 (n = 8)
Depolarization during 100 Hz, 1 s tetanus (mV)
2.4 + 0.3 01 = 10)
2.7 + 0.1 (n = 16)
EPSP slope 30 min post tetanus (% control)
154 + 10 (n = 7)
100 + 6 * (n = 8)
Data expressed as mean + SEM. * Significantly different from control ( P < 0.005, Mann-Whitney two-sample test).
84 was the amplitude of the late inhibitory, postsynaptic potential (IPSP). The late IPSP in pyramidal neurons is also mediated by post-synaptic GABA a receptors, and was seen to decrease progressively following neuronal impalement, until it was entirely abolished (Table I). While these G-protein-mediated effects were blocked by intracellular Li +, ionotropic events were unaffected (Fig. 2). The amplitude and wavef~l'm of EPSPs recorded using a LiCI filled electrode were unchanged by the progressive introduction of Li + to the neuron (Fig. 2A). Once these G-protein-mediated effects were blocked in neurons impaled with Li+-containing electrodes, a Is 100 Hz tetanus was delivered to stratum radiatum afferents, and the EPSP monitored for LTP. The neuronal response to the tetanus was the same in control cells and in cells recorded with Li+-filled electrodes (Fig. 2B, Table 1). Again, STP was elicited in both control and experimental slices. Howevel', at 30 min post-tetanus, only control cells exhibited LTP (Figs. 3 and 4; Table I), the EPSP slope being 154.0 + 10.1% of pretetanus value (n = 7), as compared to Li+-contain ing cells, which were unchanged (100.4 + 5.9% of pretctanus value, n = 8). The slopes of control EPSPs were significantly greater than Li+-treated at 5, 10, 20 and 30 rain post-tetanus (Fig. 4).
180
-
I
!
Q ,_.
140
120
-IS
0
15
30
Time (rain) Fig. 4. Introduction of Li ~ inlo CA u pyramidal neurons inhibits the induction of LTP at stratum radiatum-CA t s~napses. Li ÷ was introduced to the post-synaptic neuron only by the inclusion of 100 mM LiCI in the intracellular recording electrode. EPSPs were recorded from CA I pyramidal neurons in response to activation of stratum radiatum afferents by test stimuli delivered at 0.2 Hz. At 0 time, a 100 Hz ! s tetanus was delivered to the afferent fibres, as indicated by the open arrow. • EPSPs recorded from cells using Li*.containing electrodes (n = 8). II EPSPs recorded from control cells in = 7). Data plotted as mean + S.E.M. Statistical analysis consisted of two way analysis of variance, followed by Tukey's post-hoe test. * * P < 0.01
DISCUSSION An index of G-protein function is agonist-induced increases in [3H]GTP binding 3. In vivo and in vitro exposure of rat cortical membranes to Li + blocks both adrenergic and cholinergic agonist-induced increases in [3H]GTP binding, indicating that Li + acts to block the activation of multiple G-proteins, in this case 'G~ and G i or Go '3. Electrophysiological responses mediated by PI turnover are blocked by in vitro incubation of slices in Li+-containing ACSF 45. Although the mechanism underlying this blockade was not investigated, these authors suggest that the effects of Li + on PI metabolism may account for the effects seen. Li + is known to inhibit the activity of inositol-l-phosphatase 4.3t, thus blocking the normal recycling of membrane phosphoinositides, and depleting stores of precursors essential for normal PI turnover. However, inhibition of Pl turnover due to Li ÷ treatment in rat cerebral cortex is not reversed by addition of inositol, an affected precursor, and is therefore not due to depleted phosphoinositides 17. Further, flouride-stimulated increases in PI turnover, mediated at the level of the G-protein, are blocked by Li +nn. These results and others 28 suggest that Li + decreases PI turnover by an action at the G-protein level. Li + blocks Gi-mediated decreases in cAMP by activation of receptors coupled negatively to adenylyl cyclase (AC), such as the muscarinic M 2~'43 2 and serotonergic 5-HT~,, receptors 27. it has been suggested that Li ÷ acts to stabilize Gi, preventing its dissociation into active components 2~. G:mediated /3adrenoceptor stimulation of AC is inhibited by chronic Li + treatment 2~J. In vitro, Li + blocks flouride-stimulated increases in cAMP when applied at concentrations comparable to those used in this study, demonstrating an effect at the G-protein level 2s. Thus the stimulation or inhibition of second messenger systems mediated by a variety of G-proteins is inhibited by Li +. It is clear from our results that incubation of slices in Li+-containing ACSF blocks the induction of LTP. However, the mechanism underlying this blockade could not be determined using extracellular recording electrodes and bath-application of Li +, We therefore used intracellular recording electrodes containing Li + in an attempt to determine where and how Li + was exerting this effect. We found that following an appropriate period post-impalement to allow for the Li + to enter the cell, the postsynaptic responses to baclofen (membrane hyperpolarization and late IPSP)were inhibited, while the presynaptic action (suppression of EPSPs) was unaffected. Thus, the effects of Li + on these G-protein-mediated processes were confined to the neuron from which we were recording. Under
85 these conditions of blockade of postsynaptic G-proteins, the induction of LTP was blocked. The demonstration that intracellular Li + did not change EPSPs or the neuronal response to tetanus is important in this study, as it shows that Li + does not alter the basic ionotropic responses to afferent activation. Single EPSPs elicited by low-frequency activation of afferents are mediated predominantly by nonNMDA ionotropic glutamate receptors, while neuronal responses to high-frequency activation of afferents are mediated predominantly by NMDA receptors 14. Thus, Li + did not alter non-G-protein mediated synaptic transmission, including the NMDA receptor activation required for the induction of LTP. The metabotropic actions of glutamate were first demonstrated by Sladeczek et al. 35, who reported the ability of this neurotransmitter to stimulate phosphoinositide hydrolysis in cultured striatal neurons. Since then, metabotropic glutamatergic activity has been described in many brain areas 9, including the hippocampus 33'44. Electrophysiologically, mGluR activation using the selective agonist 1-aminocyclopentane-trans-l,3-di. carboxylic acid (trans-ACPD) has been shown to cause neuronal depolarization, inhibit after-hyperpolarization and spike accommodation, and decrease neurotransmission in the CA~ region 3°'36. Antagonists of mGluR stimulated PI turnover have no effects on these electrophysiological parameters aT. Conversely, an antagonist of mGluR-mediated depolarization has been shown to have so effect on mGluR-mediated PI turnover ~s. Thus, it is possible that the electrophysiological effects of mGluR activation are mediated by different second messenger systems. Recently, multiplicity of the mGluR has been described 24, and four mGluR subtypes (mGluRl-mGluR4) have been cloned 2'4°. Tanabe, et al. 4°, have shown that mGluR2 activation leads to an inhibition of the cAMP cascade, and although the specific second messenger systems linked to mGluR3 and mGluR4 have not been elucidated, mGluR activation in the hippocampus has been shown to increase cAMP accumulationTM, decrease forskolin stimulated c A M P 6't9'34'39, depress calcium currents ts, and stimulate arachidonic acid release 2. It has been shown that bath-application of transACPD facilitates the induction of LTP in the CA~ region 22'23'29and even induces a slowly developing form of LTP without the need for tetanic activation 5. Our work suggests induction of LTP requires activation of both NMDA and metabotropic glutamate receptors 26. Thus, it is possible that the G-protein-linked glutamate receptor is involved in LTP in the CA~ area of the hippocampus. While the present study demonstrates the involvement of a postsynaptic G-protein in the
induction of LTP, it does not rule out the possibility that a presynaptic G-protein plays a role in the expression phase of LTP 12. It is possible that the postsynaptic G-protein links a metabotropic glutamate receptor to its second messenger system. However, whether this is the case, and which second messenger system is involved will require further investigation. Acknowledgements. This study was supported by the Medical Research Council of Canada. BAB is the recipient of an Ontario Graduate Scholarship and a Queen's Graduate Fellowship. JWG is a Scholar of the MRC (Canada). REFERENCES 1 Andrade, R., Malenka, R.C. and Nicoll, R.A., A G protein couples serotonin and GABA B receptors to the same channels in hippocampus, Science, 234 (1986) 1261-1265. 2 Aramori, I. and Nakanishi, S., Signal transduction and pharmacological characteristics of a metabotropic glutamate receptor, mGluR1, in transfected CHO cells, Neuron, 8 (1992) 757-765. 3 Avissar, S., Schreiber, G., Danon, A. and Belmaker, R.H., Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex, Nature, 331 (1988) 440-442. 4 Berridge, M.J. and Irvine, R.F., Inositol phosphates and cell signalling, Nature, 341 (1989) 197-205. 5 Bortolotto, Z.A. and Collingridge, G.L., Activation of glutamate metabotropic receptors induces long-term potentiation, Eur. J. Pharmacol., 214 (1992) 297-298. 6 Cartmell, J., Kemp, J.A., Alexander, S.P.H., Hill, S.J. and Kendall, D.A., Inhibition of forskolin-stimulated cyclic AMP formation by 1-aminocyclopentane-trans-l,3-dicarboxylate in guinea-pig cerebral cortical slices, J. Neurochem., 58 (1992) 1964-1966. 7 Coilingridge, G.L. and Bliss, T.V.P., NMDA receptors - their role in long-term potentiation, Trends Neurosci., 10 (1987) 288293. 8 Collingridge, G.L. and Singer, W., Excitatory amino acid receptors and synaptic plasticity, Trends Pharmacol. Sci., 11 (1990) 290-296. 9 Conn, P.J. and Desai, M.A., Pharmacology and physiology of metabotropic glutamate receptors in mammalian central nervous system, Drug Dec. Res., 24 (1991) 207-229. 10 Ebstein, R.P., Hermoni, M. and Belmaker, R.H., The effect of lithium on noradrenaline-induced cyclic AMP accumulation in rat brain: inhibition after chronic treatment and absence of supersensitivity, J. Pharmacol. Exp. Ther., 213 (1980) 161-167. 11 Godfrey, P.P., McClue, S.J., White, A.M., Wood, A.J. and Grahame-Smith, D.G., Subacute and chronic in vivo lithium treatment inhibits agonist- and sodium fluoride-stimulated inositoi phosphate production in rat cortex, J. Neurochem., 52 (1989) 498-506. 12 Goh, J.W. and Pennefather, P.S., A pertussis toxin-sensitive G protein in hippocampal long-term potentiation, Science, 244 (1,989) 980-983. 13 Hallacher, L.M. and Sherman, W.R., The effects of lithium ion and other agents on the activity of myo.inositol-l-phosphatase from bovine brain, J. Biol. Chem., 255 (1980) 10896-10901. 14 Herron, C.E., Lester, R.A.J., Coan, E.J. and Collingridge, G.L., Frequency-dependent involvement of NMDA receptors in the hippocampus: a novel synaptic mechanism, Nature, 322 (1986) 265 -268. 15 Jones, P.L.St.J., Porter, R.H.P., Birse, E.F., Pook, P.C.-K., Sunter, D.C., Udvarhelyi, P.M., Wharton, B., Roberts, P.J. and Watkins, J.C., Characterization of a new excitatory amino acid (EAA) receptor type activated by (1S,3R)-ACPD and selectively blocked by (S)-4C3H-PG, Br. J. Pharmacol., 106 (1992) 47P. 16 Katsuki, H., Kaneko, S. and Satoh, M., Involvement of postsynaptic G proteins in hippocampal long-term potentiation, Brain Res., 581 (1992) 108-114.
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32 Schoepp, D., Bockaert, J. and Sladeczek, F., Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors, Trends Pharmacol. Sci., ! 1 (1990) 508-515. 33 Schoepp, D.D. and Johnson, B.G., Comparison of excitatory amino acid-stimulated phosphoinositide hydrolysis and N[JH]acetylaspartylglutamate binding in rat brain: selective inhibition of phosphoinositide hydrolysis by 2-amino-3-phosphonopropionate, J. Neurochem., 53 (1989) 273-278. 34 Schoepp, D.D., Johnson, B.G. and Monn, J.A., Inhibition of cyclic AMP formation by a selective metabotropic glutamate receptor agonist,/. Neurochem., 58 (1992) 1184-1186. 35 Sladeczek, F., Pin, J.-P., Recasens, M., Bockaert, J. and Weiss, S., Glutamate stimulates inositol phosphate formation in striatal neurons, Nature, 317 (1985) 717-719. 36 Stratton, K.R., Worley, P.F. and Baraban, J.M., Excitation of hippocampal neurons by stimulation of glutamate Qp receptors, Eur. J. Pharmacol., 173 (1989) 235-237. 37 Stratton, K.R., Worley, P.F. and Baraban, J.M., Pharmacological characterization of phosphoinositide-linked glutamate receptor excitation of hippocampal neurons, Fur..l. Pharmacol., 186 (1990) 357-361. 38 Sugiyama, H., lto, I. and Hirono, C., A new type of glutamate receptor linked to inositol phospholipid metabolism, Nature, 325 (1987) 531-533. 39 Tanabe, S., lto, I. and Sugiyama, H., Possible heterogeneity of metabotropic glutamate receptors induced in Xenopus oocytes by rat brain mRNA, Neurosci. Res., 10 (1991) 71-77. 40 Tanabe, Y., Masu, M., Ishii, T., Shigemoto, R. and Nakanishi, S., A family of metabotropic glutamate receptors, Neuron, 8 (1992) 169-179. 41 Thalmann, R.H., Evidence that guanosine triphosphate (GTP)binding proteins control a synaptic response in brain: effect of pertussis toxin and GTPyS on the late inhibitory postsynaptie potential of hippocampal CA 3 neurons, J. Neurosci., 8 (1988) 4589-4602. 42 Waldmeier, P.C. and Baumann, P.A., Presynaptic GABA receptors, Ann. NYAcad. Sci., 604 (1990) 136-151. 43 Whitworth, P. and Dendall, D,A., Effects of lithium on inositol phospholipid hydrolysis and inhibition of dopamine D i receptormediated cyclic AMP formation by carbachol in rat brain slices, J. Neurochem., 53 (1989) 536-541. 44 Winder, D.G. and Corm, P.J., Activation of metabotropic glutamate receptors in the hippocampus increases cyclic AMP accumulation, J. Neurochem., 59 (1992)375-378. 45 Worley, P.F., Heller, W.A., Snyder, S.H. and Baraban, J.M., Lithium blocks a phosphoinositide-mediated cholinergic response in hippocampal slices, Science, 239 (1988) 1428-1429.
81
Elsevier Science Publishers B.V.
BRES 18794
A postsynaptic G-protein in hippocampal long-term potentiation B a r b a r a A n n Ballyk a n d J o a n n e W. G o h Departma~t of Pharmacology and Toxicology, Queen's Unicersity, Kingston, ON (Canada) (Accepted 8 December 1992)
Key words: Lithium; G-protein; Hippocampus; CA I region; Postsynaptic; Metabotropic glutamate receptor
The effects of guanosine triphosphate (GTP)-binding protein (G-protein) blockade on hippocampal LTP at stratum radiatum-CA i synapses was studied. Bath application of 20 mM lithium chloride (LiCI) inhibited long-term potentiation (LTP) of extracellularly-recorded excitatory postsynaptic potentials (EPSPs). Inclusion of 100 mM LiCi in intracellular recording electrodes was shown to block postsynaptic G-proteins by bath-application of baclofen, an agonist at the G-protein linked y-aminobutyric acid (GABA n) receptor. Under normal conditions, GABA B receptor activation causes a hyperpolarization postsynapticaUy, and a decrease in neurotransmitter release presynaptically. With LiCi in the recording electrodes, the postsynaptically-mediated hyperpolarization was blocked, while the presynaptically-mediated depression of EPSPs was unaffected. With postsynaptic G-proteins blocked in this manner, LTP at these synapses was inhibited. These studies provide evidence for the involvement of a postsynaptic G-protein in LTP of stratum radiatum-CA i synapses.
INTRODUCTION LTP is a prolonged form of activity-dependent synaptic plasticity thought to be related to learning and memory s. The involvement of G-proteins in this phenomenon has been thc subject of recent studies 12'It'. With the discovery of the metabotropic glutamate receptor (mGluR) 35'3s, interest in this question of G-protein involvement in LTP has gained new relevance. Glutamate is the excitatory neurotransmitter at stratum radiatum-CA~ synapses, where it acts on multiple receptor subtypes, both ionotropic and metabotropic 24'a2. The ionotropic receptors include the Nmethyl-D-aspartate (NMDA) and non-NMDA subtypes 7,s, while the G-protein-linked mGluR subtypes continue to be identified 2'39. While the involvement of the NMDA receptor in the induction of stratum radiatum-CA~ LTP has been firmly established 7,s, recent studies in this lab suggest that coactivation of both mGluR and NMDA receptors is required 26. A pertussis toxin (PTX)-sensitive G-protein is involved in LTP in the CA~ region ~2. It is possible that this PTX-sensitive G-protein may in fact mediate a response to mGluR activation. It has also been shown
that maximal activation of CA i neuronal G-proteins by intracellular injection of guanosine-5'-O-(3-thiotriphosphate) (GTPyS), a nonhydrolyzable GTP analogue, does not occlude LTP 12'16. Although this result has been interpreted to negate the involvement of a postsynaptie G-protein in CA~ LTP, recent findings in this lab provide for a second interpretation. If, as we have found, the induction of stratum radiatum-CA~ LTP requires coactivation of both NMDA and mGluRs 2~', it appears that G-protein activation (as with GTPyS) at these synapses is in itself insufficient to induce LTP. The second requirement, NMDA receptor activation, can be fulfilled by tetanization of afferents leading to neurotransmitter release. The location(s) of Gprotein(s) involved in LTP remains to be determined. Li + is known to inhibit both adrenergic and cholinergic agonist-stimulated increases in [3H]GTP binding to rat cortical membranes a, therefore blocking the activation of G s and G i or Go. Li + also blocks Gs-mediated increases in cAMP accumulation ~°'2°, G~-mediated decrcases in cAMP 2~, and Go-mediated increases in phosphoinositol (PI) turnover ~3. Since mGluR activation can trigger a variety of second messenger cascades 2'39, and the identity of the G-protein(s) in-
Correspondence: J.W. Goh, Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, C~mada KL7 3N6. Fax: (1) (613) 545 6412.
82 volved in LTP has not been established, we used Li + as a ubiquitous blocker to probe the subceilular localization (ie. pre- vs. postsynaptic) of G-proteins important for LTP induction.
A 160
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MATERIALS AND METHODS 120
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Care of animals was in accordance with Canadian Council on Animal Care guidelines. Male Wistar rats were anesthetized with halothane (2%), and their brains removed. The hippocampi were dissected out and 41}{} ttm transverse slices cut using a Vibroslice tissue slicer (Campden Instruments Ltd.). For one hour prior to recording, control slices were incubated at room temperature in artificial cerebrospinal fluid (ACSF)containing in raM: 120 NaCI, 3. l KCI, 1.3 NaH,PO4, 26 NaHCO 3, 10 dextrose, 2 CaCI, and 1.5 MgCI,: pH 7.4, bubbled with 95% O2/5r~ CO.,. Lie-incubated slices were maintained at room temperature for at least two hours prior to recording in oxygenated ACSF containing 20 mM LiCI, osmotically balanced by a reduction in NaCI content. For recording, slices were transferred to a superfusion recording chamber, to which the same (Li "-containing or control) oxygenated ACSF was delivered at a rate of 2 ml per rain, maintained at 30+0.2°C. Recording electrodes were fashioned from 1.2 mm O.D., 0.68 mm I.D. glass, using a Flaming-Brown model P-87 microelectrode puller (Sutter Instrument Co.). For extracellular recording, electrodes were pulled to a resistance of 3-5 MI2 and filled with 4 M NaCI. For intracellular recording, electrodes were pulled to a resistance of 30-50 MO and filled with 3 M potassium acetate (KAc), with or without 100 mM LiCI added. EPSPs were elicited at a control frequency of {}.2 Hz by concentric bipolar stimulating electrodes (SNEX Illl), Rhodes Electronics) placed in the stratum radiatum. EPSPs were recorded extracellularly from the stratum radiatum or intraccUularly from the stratum pyramidale of the CA I region, in the intracellular experiments, G-protein flmction was monitored using a 2 rain bath-application of 5ll/~ M baclofen. Signals were amplified by an Axoclamp model 2A amplil'ier (Axon Instruments), digitized and stored on videotape, and analyzed by pClamp software (Axon Instruments). LTP was defined as a greater than 2()~,: increase in EPSP slope, seen 30 rain folk+wing the delivery of a one second, I(10 l+lz tetanus to afferent fibres.
RESULTS
Bath-applied fithium Slices were incubated at room temperature for at least 2 h prior to recording in oxygenated ACSF containing 20 mM LiCi. During recording, the same ACSF containing Li + was used to perfuse the bath. Normal ACSF (lacking Li ~ ) was used to incubate control slices prior to recording, and to perfuse the bath during recording. Expe:iments commenced once dendriticallyrecorded EPSPs remained stable for at least 15 min. Following a 100 Hz, Is tetanus delivered to the stratum radiatum, short-term potentiation (STP) was seen in both control and experimental slices (Fig. 1). However, at 30 min post-tetanus, only the control slices exhibited LTP, the slopes of the EPSPs being 136.0 + 9.7% of pretetanus values (n = 10), while EPSPs in the experimental slices were unchanged (98.6_+ 4.1% of pretetanus values, n = 11). The slopes of control EPSPs were significantly greater than experimental at 10, 20, and 30 min post-tetanus (Fig. 1).
t~
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80
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Time (rain) Fig. I. Extracellularly-applied Li+ blocks the induction of stratum radiatum-CA I LTP. EPSPs were recorded extracellularly in the stratum radiatum of CA I in response to test stimuli delivered at 0.2 Hz. At 0 time, a 100 Hz, Is tetanus was delivered to the afferent fibres, as indicated by the open arrow. • EPSPs recorded from slices incubated for 2 h in 20 mM Li+ (n = 11). • EPSPs recorded from control slices ( n = 10). Data plotted as mean =l:S.E.M. Statistical analysis consisted of two way analysis of variance, followed by Tukey's post-hoe test. * P < 0.05. ** P < 0.01. A.
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CONTROL
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.I Fig. 2. lntracellular Li + blocks the postsynaptic G-protein-mediated response to baclofen, while having no effect on ionotropic glutamate responses. A: continuous record of membrane potential with EPSPs (deflections above baseline) elicited by 0.2 Hz test stimuli and voltage responses to -0.2hA, 50 m~ pulses (deflections below baseline) recorded from a CA I pyramidal neuron. Expanded EPSP • records are taken from the trace as indicated by the arrows. At the bars, 50 ~ M baclofen was bath-applied for 2 rain. The initial exposure to baclofen (20 rain post-impalement) elicited both a postsynaptically-mediated hyperpolarization, and a presynaptically-mediated suppressio!a of EPSPs. The second exposure to baclofen (32 rain post-impalement) dicited only the presynaptically-mediated effect, indicating blockade of the postsynaptic G-protein-mediated hyperpolarization by the progressive introduction of Li + into the cell. Following washout of each exposure to baclofen, it can be seen that EPSPs elicited by low-frequency activation of afferents are unchanged by intraceilular LiCI. The scale bar represents 40 ms, 8 mV for expanded EPSPs (top) and 2 rain, 10 mV for continuous trace (bottom). Resting membrane potential was - 7 1 inV. B: the response of a CA l pyramidal neuron to tetanic artivation of afferents are unchanged by intraceilular Li +. Response of the control cell was recorded using a 3 M K + acetate-filled electrode, while that in the experimental cell was recorded with 100 mM LiCI/3 M K + acetate in the electrode. The bars indicate delivery of the 100 Hz, ls tetanus. in the latter case, the affercnts were tetanized after the postsynaptic G-protein-mediated response to baclofen was blocked. The scale bar represents 250 ms, 2 mV. Resting membrane potential of the control cell was - 78 mV and of the Li +-loaded cell was - 75 mV.
83 A. CONTROL
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Fig. 3. Introduction of Li + into a CA I pyramidal neuron blocks both the G-protein-mediated response to the G A B A n agonist baciofen and the induction of LTP. Top traces show continuous record of membrane potential with EPSPs elicited by 0.2 Hz test stimuli and voltage responses to - 0 . 2 nA, 50 ms pulses recorded from CA r pyramidal neurons. Expanded EPSP records are taken from the upper traces as indicated by the filled arrows. At the open arrow, a 1 s 100 Hz tetanus was delivered to afferent fibres. The scale bar represents 2 min, 10 mV for the upper records and 40 ms, 6 mV for the EPSP records. A: record from a control neuron. At ~ae bar, 50/zM baclofen was applied for 2 min. The cell exhibited both postsynaptically mediated hyperpolarization and presynaptically mediated depression of EPSPs with activation of GABA a receptors. Tetanus resulted in LTP. B: 32 min after impalement with an intracellular recording electrode containing 100 mM LiCI, a 2 rain exposure to 50 p.M baclofen elicited no hyperpolarizing response, indicating blockade of postsynaptic G-proteins, while the presynaptically mediated depression of EPSPs is unaffected. Under these conditions, the inducton of LTP was inhibited. Records in (A) and (B) are from 2 different cells. Membrane potentials were - 6 7 and - 7 1 mV, respectively.
lntracellular lithium Li + was introduced to the post-synaptic neuron by the inclusion of 100 mM LiC! with the 3 M KAc solution Used to fill intracellular recording electrodes. Control cells were impaled with electrodes containing 3 M KAc, with no LiCI. In these experiments, prior to tetanization of afferents, the status of post-synaptic G-proteins was assessed using the GABA a receptor agonist baclofen. GABA B receptors in hippocampal pyramidale neurons are linked via a G-protein to a K + channel TM. Activation of these receptors by baclofen results in neuronal hyperpolarization. GABAa receptors are also located presynaptically, their activation decreasing the release of excitatory neurotransmitter, resulting in depression of EPSPs 42. Thus, G-protein function w~s monitored by the neuronal response to a 2 min bath-application of 50/zM baclofen (Fig. 2; Fig. 3; Table I). In control cells, baclofen elicited a - 4 . 8 + 1.0 mV hyperpolarization (n = 4). We found that with Li+-containing electrodes, it took from 30 to 60 min following neuronal impalement for post-synaptic GABA B receptors to be blocked (Fig. 2), resulting in no hyperpolarization in response to the bath-applied baclofen ( - 0 . 5 + 0.3 mV, n - 8). In both control and Li +-containing cells, the presynaptic effects of baclofen
were the same, both demonstrating a comparable depression of EPSP slope (Fig. 3, Table !). A second indication of the blockade of postsynaptic G-proteins TABLE I
Effects of intracellular lithium on IPSPs, baclofen-induced hyperpolarization, tetanus-induced depolarization and L TP Control
100 mM Li + hz recording electrode
Slow IPSP amplitude (mV)
- 1.8 + 0.4 (n = 8)
+ 0.2 5:0.1 * (n = 16)
Maximum baclofeninduced hyperpolarization (mV)
- 4.8 + 1.0 (n = 4)
- 0.5 + 0.3 * (n = 8)
EPSP slope during baciofen (% control)
28 + 4 (n = 8)
34 +5 (n = 8)
Depolarization during 100 Hz, 1 s tetanus (mV)
2.4 + 0.3 01 = 10)
2.7 + 0.1 (n = 16)
EPSP slope 30 min post tetanus (% control)
154 + 10 (n = 7)
100 + 6 * (n = 8)
Data expressed as mean + SEM. * Significantly different from control ( P < 0.005, Mann-Whitney two-sample test).
84 was the amplitude of the late inhibitory, postsynaptic potential (IPSP). The late IPSP in pyramidal neurons is also mediated by post-synaptic GABA a receptors, and was seen to decrease progressively following neuronal impalement, until it was entirely abolished (Table I). While these G-protein-mediated effects were blocked by intracellular Li +, ionotropic events were unaffected (Fig. 2). The amplitude and wavef~l'm of EPSPs recorded using a LiCI filled electrode were unchanged by the progressive introduction of Li + to the neuron (Fig. 2A). Once these G-protein-mediated effects were blocked in neurons impaled with Li+-containing electrodes, a Is 100 Hz tetanus was delivered to stratum radiatum afferents, and the EPSP monitored for LTP. The neuronal response to the tetanus was the same in control cells and in cells recorded with Li+-filled electrodes (Fig. 2B, Table 1). Again, STP was elicited in both control and experimental slices. Howevel', at 30 min post-tetanus, only control cells exhibited LTP (Figs. 3 and 4; Table I), the EPSP slope being 154.0 + 10.1% of pretetanus value (n = 7), as compared to Li+-contain ing cells, which were unchanged (100.4 + 5.9% of pretctanus value, n = 8). The slopes of control EPSPs were significantly greater than Li+-treated at 5, 10, 20 and 30 rain post-tetanus (Fig. 4).
180
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120
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15
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Time (rain) Fig. 4. Introduction of Li ~ inlo CA u pyramidal neurons inhibits the induction of LTP at stratum radiatum-CA t s~napses. Li ÷ was introduced to the post-synaptic neuron only by the inclusion of 100 mM LiCI in the intracellular recording electrode. EPSPs were recorded from CA I pyramidal neurons in response to activation of stratum radiatum afferents by test stimuli delivered at 0.2 Hz. At 0 time, a 100 Hz ! s tetanus was delivered to the afferent fibres, as indicated by the open arrow. • EPSPs recorded from cells using Li*.containing electrodes (n = 8). II EPSPs recorded from control cells in = 7). Data plotted as mean + S.E.M. Statistical analysis consisted of two way analysis of variance, followed by Tukey's post-hoe test. * * P < 0.01
DISCUSSION An index of G-protein function is agonist-induced increases in [3H]GTP binding 3. In vivo and in vitro exposure of rat cortical membranes to Li + blocks both adrenergic and cholinergic agonist-induced increases in [3H]GTP binding, indicating that Li + acts to block the activation of multiple G-proteins, in this case 'G~ and G i or Go '3. Electrophysiological responses mediated by PI turnover are blocked by in vitro incubation of slices in Li+-containing ACSF 45. Although the mechanism underlying this blockade was not investigated, these authors suggest that the effects of Li + on PI metabolism may account for the effects seen. Li + is known to inhibit the activity of inositol-l-phosphatase 4.3t, thus blocking the normal recycling of membrane phosphoinositides, and depleting stores of precursors essential for normal PI turnover. However, inhibition of Pl turnover due to Li ÷ treatment in rat cerebral cortex is not reversed by addition of inositol, an affected precursor, and is therefore not due to depleted phosphoinositides 17. Further, flouride-stimulated increases in PI turnover, mediated at the level of the G-protein, are blocked by Li +nn. These results and others 28 suggest that Li + decreases PI turnover by an action at the G-protein level. Li + blocks Gi-mediated decreases in cAMP by activation of receptors coupled negatively to adenylyl cyclase (AC), such as the muscarinic M 2~'43 2 and serotonergic 5-HT~,, receptors 27. it has been suggested that Li ÷ acts to stabilize Gi, preventing its dissociation into active components 2~. G:mediated /3adrenoceptor stimulation of AC is inhibited by chronic Li + treatment 2~J. In vitro, Li + blocks flouride-stimulated increases in cAMP when applied at concentrations comparable to those used in this study, demonstrating an effect at the G-protein level 2s. Thus the stimulation or inhibition of second messenger systems mediated by a variety of G-proteins is inhibited by Li +. It is clear from our results that incubation of slices in Li+-containing ACSF blocks the induction of LTP. However, the mechanism underlying this blockade could not be determined using extracellular recording electrodes and bath-application of Li +, We therefore used intracellular recording electrodes containing Li + in an attempt to determine where and how Li + was exerting this effect. We found that following an appropriate period post-impalement to allow for the Li + to enter the cell, the postsynaptic responses to baclofen (membrane hyperpolarization and late IPSP)were inhibited, while the presynaptic action (suppression of EPSPs) was unaffected. Thus, the effects of Li + on these G-protein-mediated processes were confined to the neuron from which we were recording. Under
85 these conditions of blockade of postsynaptic G-proteins, the induction of LTP was blocked. The demonstration that intracellular Li + did not change EPSPs or the neuronal response to tetanus is important in this study, as it shows that Li + does not alter the basic ionotropic responses to afferent activation. Single EPSPs elicited by low-frequency activation of afferents are mediated predominantly by nonNMDA ionotropic glutamate receptors, while neuronal responses to high-frequency activation of afferents are mediated predominantly by NMDA receptors 14. Thus, Li + did not alter non-G-protein mediated synaptic transmission, including the NMDA receptor activation required for the induction of LTP. The metabotropic actions of glutamate were first demonstrated by Sladeczek et al. 35, who reported the ability of this neurotransmitter to stimulate phosphoinositide hydrolysis in cultured striatal neurons. Since then, metabotropic glutamatergic activity has been described in many brain areas 9, including the hippocampus 33'44. Electrophysiologically, mGluR activation using the selective agonist 1-aminocyclopentane-trans-l,3-di. carboxylic acid (trans-ACPD) has been shown to cause neuronal depolarization, inhibit after-hyperpolarization and spike accommodation, and decrease neurotransmission in the CA~ region 3°'36. Antagonists of mGluR stimulated PI turnover have no effects on these electrophysiological parameters aT. Conversely, an antagonist of mGluR-mediated depolarization has been shown to have so effect on mGluR-mediated PI turnover ~s. Thus, it is possible that the electrophysiological effects of mGluR activation are mediated by different second messenger systems. Recently, multiplicity of the mGluR has been described 24, and four mGluR subtypes (mGluRl-mGluR4) have been cloned 2'4°. Tanabe, et al. 4°, have shown that mGluR2 activation leads to an inhibition of the cAMP cascade, and although the specific second messenger systems linked to mGluR3 and mGluR4 have not been elucidated, mGluR activation in the hippocampus has been shown to increase cAMP accumulationTM, decrease forskolin stimulated c A M P 6't9'34'39, depress calcium currents ts, and stimulate arachidonic acid release 2. It has been shown that bath-application of transACPD facilitates the induction of LTP in the CA~ region 22'23'29and even induces a slowly developing form of LTP without the need for tetanic activation 5. Our work suggests induction of LTP requires activation of both NMDA and metabotropic glutamate receptors 26. Thus, it is possible that the G-protein-linked glutamate receptor is involved in LTP in the CA~ area of the hippocampus. While the present study demonstrates the involvement of a postsynaptic G-protein in the
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