Different effects of ω-conotoxin GVIA at excitatory and inhibitory synapses in rat CA1 hippocampal neurons

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Brain Research, 616 (1993) 236-241 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19033

Different effects of w-conotoxin GVIA at excitatory and inhibitory synapses in rat CA1 hippocampal neurons Brigitte Potier

a

Patrick Dutar a and Y v o n L a m o u r a,b

Laboratoire de Physiopharmacologie du Syst~me NerL,eux, INSERM U161, Paris (France) and b Sercice d'Explorations Fonctionnelles du Syst~me Nerueux, H~pital Lariboisi~re, Paris (France) (Accepted 16 February 1993)

Key words: Calcium channel; Hippocampus; w-Conotoxin; y-Aminobutyric acid; Glutamate; G-protein; Neurotransmitter release

The nature of the coupling mechanism of presynaptic calcium channels involved in the release of neurotransmitters in the mammalian central nervous system is unknown. Using intracellular recordings from CA1 neurons in the rat hippocampal slice preparation, we show that the N-type calcium channels antagonist omega-conotoxin GVIA (w-CgTx) blocks partially the excitatory (EPSP) and totally the inhibitory (IPSP) synaptic transmission in CA1 hippocampal pyramidal neurons. In addition, the inhibitory effect of oJ-CgTx on IPSPs is strongly depressed by intrahippocampal injection of PTX, while the effect on EPSP is not. The results suggest that the nature or the regulation of calcium channels might be different, depending on the location of these channels on excitatory or inhibitory terminals.

INTRODUCTION A family of guanine nucleotide binding proteins (or G-proteins) has been identified as coupling some neurotransmitter receptors to a variety of effectors in different tissues, especially in the central nervous system (see refs. 4, 27). Some of these G-proteins are inactivated by toxins such as pertussis toxin (PTX) 17. Inhibition of calcium channel activity by various neurotransmitters involves a PTX-sensitive G-protein in sensory neurons (see ref. 5 for references), and also in hippocampal neurons 29. The direct effect of calcium channel agonists or antagonists (especially the dihydropyridines) on neuronal calcium channels can also involve a PTX-sensitive G-protein 2'24. However, the studies of calcium currents using voltage-clamp or patch-clamp methods have been done exclusively on postsynaptic membranes. For technical reasons, presynaptic terminals in the central nervous system cannot be studied by these approaches. As a consequence, the nature of the membrane mechanisms responsible for the release of neurotransmitters in vertebrate presynaptic nerve terminals remains unknown. Recently, w-conotoxin (w-CgTx), a peptide isolated from the

venom of a marine mollusc, has been demonstrated to be an inhibitor of N- and L-type Ca 2÷ channels in dorsal root ganglion neurons 14. In contrast, w-CgTx does not seem to affect L-type Ca 2+ channels in hippocampus 22'311. w-CgTx blocks the synaPtic transmission in CA3 13 and CA1 8,12 fields of the rat hippocampus. It was suggested that to-CgTx acts through N-type calcium channels to block calcium entry into the nerve terminal and thereby inhibits neurotransmitter release 7'15'25. An action through L-type Ca 2+ channels is unlikely since L-type Ca 2÷ channels antagonists such as dihydropyridines do not depress the synaptic events in the hippocampus t8'21. The inhibitory action of ~o-CgTx has been suggested to differentially affect excitatory and inhibitory transmission, the inhibitory events being more sensitive to the toxin t2"21. However, no quantitative measurement have been done. In addition, the cellular mechanisms underlying these effects in hippocampal neurons are unknown. In the present study, we analysed the effect of o~-CgTx on hippocampal synaptic transmission at Schaffer collaterals/CA1 pyramidal synapses. We directly injected PTX into the stratum radiatum of the

Correspondence: P. Dutar, INSERM U161, 2, rue d'Alesia, 75014 Paris, France. Fax: (33) 45.88.1304.

237 hippocampus to block the presynaptic mechanisms coupled with PTX-sensitive G-proteins. We report here that the inhibitory effect of o~-CgTx on the excitatory postsynaptic potentials (EPSPs) is not affected by a pretreatment with PTX, while the blockade of inhibitory potentials (IPSPs) by ~o-CgTx is strongly reduced by PTX. Our results suggest that the mechanisms governing the release of excitatory and inhibitory neurotransmitters in hippocampus are different.

EPSP

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CONTROL

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MATERIALS AND METHODS Male Sprague-Dawley rats (200-300 g) were anesthetized with pentobarbital (50 mg/kg) and placed in a stereotaxic frame. Using a stereotaxic approach, we injected toxin of Bordetella pertussis, pertussis toxin (PTX, Sigma) unilaterally into the third cerebral ventricule (2/zg diluted in 15-20 izl saline, at coordinates A - 1, L 1.5, H - 4 (Paxinos and Watson 19) and into the stratum radiatum of the hippocampus (1 /zg in 2 /zl saline at coordinates A -4.3, L 2.5, H - 3 . 2 (Paxinos and Watson19). Control rats were injected with saline at the same coordinates (sham). Rats were allowed to live for 3 days, whereafter they were anesthetized with halothane and decapitated. The hippocampus was quickly removed and placed in a cold oxygenated medium. Slices (400 /zm thick) were cut from the injected side and placed in a holding chamber at least 1 h before recording. A single slice was then transferred to the recording chamber, where it was held between two nylon nets and submerged beneath a continuously superfusion medium (pH 7.4) warmed to 30-33°C and pregassed with 95% 0 2 and 5% CO 2. The composition of the medium was (in mM): NaCI 119, KCI 2.5, MgSO4 1.3, NaH2PO 4 1.0, CaCI 2 2.5, NaHCO 3 26.2, Glucose 11. The drugs were applied in the superfusion medium. They included: baclofen (30 /IM, Ciba-Geigy), bicuculline (2 ~M, Sigma), phaclofen (0.5 mM, Tocris), D(-)-2-amino-5-phosphonovaleric acid (APV, 30/~M, CRB), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 ~M, CRB), oJ-conotoxin GVIA (0.1-1 /zM; Sigma). Conventional intracellular recordings from CA1 pyramidal neurons were obtained by using glass micropipettes filled with 3 M potassium acetate and having resistance of 60-120 MO. These recordings were made using an Axoclamp-2A amplifier (Axon Instrument). A bridge circuit was used to apply current pulses for the measurement of membrane resistance and for triggering afterhyperpolarizations (AHP) which follow a train of spikes. Intracellular voltage and current recordings were stored on a rectilinear strip chart recorder. Fast events such as excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs respectively) were stored on a Nicolet 4094B digital oscilloscope and plotted on a Hewlett Packard 7470A digital plotter. EPSPs and IPSPs were induced by activation of Schaffer collateral/commissural fibers (20 to 60 V, 0.1 ms pulse width) with bipolar electrodes positioned in the stratum radiatum. Stimulation pulses were applied between the two poles of the bipolar electrode, one pole inserted into the slice and the other pole just above the slice. Amplitude of IPSPs were measured at the pic of hyperpolarizing response.

RESULTS The sensitivity of each neuron of the two groups (sham and PTX) was assessed using 3 different concentrations of oJ-CgTx: 0.1 /zM, 0.5 tzM and 1 tzM. The effect of the toxin on the amplitude of the synaptic events was measured 15 min after the beginning of the

~ - C.,gTx 05uM

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Fig. 1. Effect of different concentrations of oJ-CgTx on synaptic events in sham-operated animals. EPSPs (20 V, 0.1 ms, '-90 mV) are induced in the presence of bicuculline (2 IxM) and phaclofen (0.3 mM). Increasing concentrations of ~o-CgTxdepress the EPSP but did not block it. On this example, 1 /zM co-CgTx decreases the EPSP by 56%. IPSPs (right column) are induced in another cell (60 V, 0.1 ms, - 55 mV). Control IPSPs are biphasic, reflecting the effect of GABA on GABA A (fast IPSP) and GABA B (slow IPSP) receptors. The slow IPSP disappears first. Notice on this record the increase in EPSP size (at 0.5 /~M) due to the loss of the inhibitory control. 1 /~M ~o-CgTx leads to the full blockade of IPSPs.

superfusion, time to get the maximal effect of the toxin.

Effect of oo-CgTx on synaptic events in slices from sham-operated rats EPSPs. Excitatory synaptic events were induced at - 90 mV to avoid the contamination by action potentials. We used bicuculline and phaclofen to eliminate contaminating hyperpolarizing potentials. The inhibition of EPSPs by ~o-CgTx applied at 0.1 /zM, was 23% (n = 7), 49% (n = 11) at 0.5 /zM, and 66% at 1 /zM (n = 4). oJ-CgTx decreased but never fully abolished the EPSPs (Figs. 1, 2, 3A). IPSPs. Inhibitory synaptic events were induced at - 5 5 mV. Control IPSPs were biphasic. The fast phase of the IPSP was due to the action of GABA on GABA A receptors, and the slow phase was due to the action of GABA on GABA B receptors, o~-CgTx inhibited in a few minutes the two phases of the IPSPs. The slow phase disappeared before the fast one and for lower concentrations of ~o-CgTx (Fig. 1). Our results show that IPSPs were more sensitive to a given concentration of o~-CgTx than EPSPs in slices

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from s h a m - o p e r a t e d rats. T h e m e a n p e r c e n t a g e inhibition of I P S P a m p l i t u d e was 48% at 0.1 / z M (n = 6), 78% at 0 . 5 / x M (n = 11), a n d 92% at 1 /zM of to-CgTx (n = 3). A s seen in Fig. 2, the d i f f e r e n c e o f sensitivity b e t w e e n E P S P s a n d I P S P s was statistically significant at e a c h c o n c e n t r a t i o n o f ~o-CgTx ( * * P < 0.01, c o m p a r i s o n o f the m e a n s , S t u d e n t ' s t-test). In s o m e e x p e r i m e n t s , we r e c o r d e d i s o l a t e d I P S P s in the p r e s e n c e o f A P V (30 /zM) a n d C N Q X (10 /zM), which respectively b l o c k e d N M D A a n d n o n - N M D A g l u t a m a t e r e c e p t o r s subtypes. W e d i d n ' t a d d A P V a n d C N Q X in all e x p e r i m e n t s since it is impossible to study the slow I P S P which is p a r t i a l l y lost in the p r e s e n c e o f t h e s e drugs. T h e s t i m u l a t i n g e l e c t r o d e was l o c a t e d n e a r the s o m a of t h e r e c o r d e d cell, to directly activate i n t e r n e u r o n s . In t h e s e conditions, m o n o p h a s i c I P S P s a r e r e c o r d e d , mainly d u e to t h e activation o f G A B A A r e c e p t o r s l o c a t e d on the soma. to-CgTx (0.5 /xM) strongly d e p r e s s e d t h e m o n o p h a s i c I P S P s (n = 3; Fig.

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Fig. 4. Comparison of the inhibitory effect of o~-CgTx on EPSPs in control and PTX pretreated rats as a function of to-CgTx concentration. The depression of EPSPs are comparable in slices from control and PTX-pretreated rats. Vertical bars indicate S.E.M. The differences between the two groups are not statistically significant.

5A). T h e d e p r e s s i n g effect o f synaptic events by to-CgTx was quasi i r r e v e r s i b l e in o u r e x p e r i m e n t a l c o n d i t i o n s (maximal time o f washing: 2 h).

Effect of to-CgTx on synaptic ecents in slices from PTXtreated rats T h e efficacy of P T X injection was c h e c k e d by testing the effect o f b a c l o f e n o n h i p p o c a m p a l p y r a m i d a l n e u r o n s , since p o s t s y n a p t i c G A B A n r e c e p t o r s are b l o c k e d by p r e t r e a t m e n t with P T X 1. R a t s whose neurons p r e s e n t e d h y p e r p o l a r i z i n g r e s p o n s e s to b a c l o f e n and biphasic IPSPs were eliminated. Effect of to-CgTx on EPSPs. E P S P s w e r e i n d u c e d as previously d e s c r i b e d . T h e m e a n p e r c e n t a g e inhibition o f E P S P s by to-CgTx a p p l i e d at 0.1 /zM was 24% ( n = 5), 40% at 0.5 /zM (n = 10), a n d 57% at 1 /xM ( n = 5). T h e r e was no statistically significant d i f f e r e n c e b e t w e e n t h e inhibition o f E P S P s r e c o r d e d f r o m P T X p r e t r e a t e d rats a n d E P S P s r e c o r d e d from s h a m - o p e r a t e d rats ( S t u d e n t ' s t-test; see Figs. 3, 4). In a d d i t i o n , we have t e s t e d the action o f b a c l o f e n (30 /~M) in the

A Control

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Fig. 2. Percentage inhibition of EPSPs and IPSPs as a function of oJ-CgTx concentration in sham-operated rats. Notice the lowest sensitivity of EPSPs as compared to IPSPs. Vertical bars indicate S.E.M. • * P < 0.01, Student t-test.

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Fig. 3. Effect of ~o-CgTx on EPSPs in a control (A) and a PTX-pretreated rat (B). A I : an EPSP is induced by electrical stimulation of C A I afferents (20 V) in a slice from a control rat. A2: ~o-CgTx (0.5 /zM) depresses (partially) the EPSP. A3: traces in 1 and 2 are superimposed. B: the same experiment in a PTX-pretreated rat. ~o-CgTx is still able to depress the EPSP. In this example, the

inhibitory effect of to-CgTx is less dramatic in the PTX-pretreated rat than in the control, but the difference is not statistically significant in the whole population of neurons (see Fig. 4).

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Fig. 5. Effect of to-CgTx on IPSPs in a control (A) and a PTX-pretreated rat (B). A: a synaptic response is induced by stimulation of CA1 afferents (60 V). Addition of APV and CNQX reveals a pure IPSP which is abolished by addition of to-CgTx (0.5 /~M) in the superfusion medium. B: same experiment in a slice from a PTX-pretreated rat. Notice the absence of the slow component of the IPSP in the control trace and the lack of effect of to-CgTx in this cell.

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Fig. 6. Comparison of the inhibitory effect of ~o-CgTx on IPSPs in control and PTX-pretreatcd rats as a function of w-CgTx concentrations. The differences between the 2 groups of rats are statistically significant. * P < 0.05, ** * P < 0.001, (Student t-test). Vertical bars indicate S.E.M.

presence of to-CgTx 0.5 ~M. The remaining EPSPs (insensitive to to-CgTx) were blocked by baclofen. Effect of to-CgTx on IPSPs. The inhibitory action of to-CgTx on IPSPs was strongly prevented by PTX pretreatment. Percentages of inhibition of IPSPs in PTXpretreated rats were 20% (n = 4) at 0.1 /~M, 31% (n = 9) at 0.5 tzM, and 58% (n = 4) at 1 /zM (Fig. 6). Differences between inhibition of IPSPs recorded in slices from PTX-pretreated rats and IPSPs recorded in slices from sham-operated rats were highly significant at each concentration of to-CgTx (* P < 0.05; *** P < 0.001; Student's t-test). Monophasic IPSPs were induced in 2 cells in the presence of APV and CNQX. In those cells, w-CgTx was almost ineffective in blocking the IPSP as illustrated in Fig. 5B. DISCUSSION

Effect of to-CgTx on EPSP and IPSP The biphasic IPSP following the EPSP is due to the action of GABA on GABA A (fast IPSP) and GABA B (slow IPSP) postsynaptic receptors. We and others have shown previously that these inhibitory events are very sensitive to w-CgTx 8'~2'21. We showed that this effect is a presynaptic effect since the postsynaptic hyperpolarizing effect of baclofen is not affected by to-CgTx8. In the present study, we compared the respective sensitivity to to-CgTx of IPSPs and EPSPs. We report that IPSPs are completely and easily abolished by to-CgTx while the depression of the EPSP is incomplete. This result suggests that another calcium channel, insensitive to to-CgTx, could be involved in the control of the release of excitatory amino-acids. Dihydropyridines antagonists do not depress EPSPs in the

CA1 hippocampal neurons 18'2~, ruling out the implication of an L-type calcium channel. It has been recently demonstrated that a novel toxin isolated from the venom of a funnel web spider, to-Aga-IVA, a specific blocker of P-type calcium channels, strongly inhibits Ca 2+ entry into rat brain synaptosomes much more efficiently than to-CgYx 16. This toxin blocks the Ca 2+dependent and w-CgTx-resistant release of glutamate from rat synaptosomes 31. Thus, an to-Agatoxin-sensitive calcium channel could be involved in the to-CgTxresistant EPSP. The lack of a complete inhibition of EPSPs by to-CgTx could also be explained by a calcium-independent release of excitatory amino acids. This point is extensively discussed in a recent review 3.

Implication of a PTX-sensitive G-protein in the effects of o~-CgTx Postsynaptic receptors to neurotransmitters or neuropeptides are often linked to calcium channels through a PTX-sensitive G-protein. For instance, this is the case for GABA, noradrenalin and baclofen in dorsal root ganglion (DRG) neurons 6'11, acetylchofine in rat sympathetic neurons 26, and for peptides such as NPY and bradykinin in DRG neurons 9 or enkephalins in neuroblastoma/glioma hybrid cells l°. G-protein activation or inactivation can also change the response to calcium channels ligands in PC12 cells 2 and in sensory neurons (see ref. 5 for a review). In the central nervous system (CNS), it was shown in the hippocampus that the postsynaptic inhibitory action of acetylcholine on voltage-dependent calcium channels is mediated by a G-protein zg. In contrast, because of technical limitations, the link between receptors and intracellular effectors at the level of central presynaptic terminals remains virtually unknown. Our results show that ~o-CgTx blocks the excitatory synaptic transmission by a mechanism independent of a PTX-sensitive G-protein. The EPSP, reflecting the release of glutamate, is depressed to the same extent in slices from sham-operated and PTXpretreated rats. In contrast, to-CgTx is not able to block IPSPs in slices from PTX-pretreated rats. These results suggest that the blockade of calcium channels linked to the release of neurotransmitters may be mediated in a different way at excitatory and inhibitory terminals. How can we explain the effect of PTX on the inhibitory action of oJ-CgTx? If a G-protein mediates the effect of to-CgTx on the release of GABA, we can hypothesize that the G-protein is located between the binding site of to-CgTx and the calcium channel closed by to-CgTx. No information are currently available on the structure of the presynaptic binding site of to-CgTx which could confirm this hypothesis, which,

240 however, seems unlikely. An alternative explanation would be that PTX acts on a G-protein into the terminals which would interfere indirectly with some reactions linked to the function of calcium channels. Gprotein could regulate indirectly calcium channels through activation of cytoplasmic kinases, leading to changes in the mechanisms of phosphorylation or dephosphorylation. We can also hypothesize that PTX pretreatment leads to a conformational change of the binding site and modifies w-CgTx binding. A change in the gating kinetics of calcium channels after G-protein activation has also been proposed in sensory neurons for the binding of dihydropyridines 23'24. The explanation of the differential susceptibility of EPSPs and IPSPs could be that PTX reaches more easily the inhibitory than the excitatory terminals. However, experiments measuring ADP-ribosylation have demonstrated that PTX reaches as effectively synaptic and non-synaptic sites 32. In addition, it has also been demonstrated that the GABA B agonist baclofen is able to depress EPSPs and IPSPs in these ceils by respectively PTX-insensitive and PTX-sensitive mechanisms when PTX is applied into the hippocampus 2° or after incubation of neuronal cultures in the presence of PTX28. In this latter case, the problem of interpretation linked to the location of the injection site is ruled out. These results of the effect of baclofen as well as the present result on the effect of w-CgTx show that the presynaptic mechanisms on excitatory terminals are not associated with a PTX-sensitive Gprotein. The parallel between the effect of ~o-CgTx and baclofen is interesting and suggests that baclofen depresses the synaptic events by acting, at least in part, through o~-CgTx-sensitive calcium channels. In conclusion, our results have shown that in spite of the relative universal action of ~0-CgTx as a blocker of neurotransmitter release in the CNS, is has different effects in the hippocampus, depending on the nature of the terminal. The release of excitatory neurotransmitters is only partially mediated by w-CgTx-sensitive calcium channels and is not sensitive to the action of PTX. In contrast, the release of GABA is fully blocked by w-CgTx and an intracellular mechanism linked to GABA release may involve a PTX-sensitive G-protein.

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Acknowledgements. This work was supported by a grant from Bayer Pharma, France.

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