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Effects of postsynaptic GABAB receptor activation on epileptiform activity in hippocampal slices
Neuropharmacology 40 (2001) 131–138 www.elsevier.com/locate/neuropharm
Effects of postsynaptic GABAB receptor activation on epileptiform activity in hippocampal slices Zhi-Qi Xiong, Janet L. Stringer
*
Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA Received 7 March 2000; received in revised form 22 May 2000; accepted 13 June 2000
Abstract GABAB receptor agonists have been reported to have both pro- and antiepileptic properties. Here, the effects of a GABAB receptor agonist, baclofen, were studied on epileptiform activity induced in the absence of synaptic transmission — to focus on the postsynaptic effects. Perfusion of hippocampal slices with 0-added calcium and high potassium induced field bursts in CA1 and the dentate gyrus. Addition of baclofen caused a transient suppression of the field bursts in CA1 and the dentate gyrus. The duration of the suppression was dependent on the concentration of baclofen and when the bursts reappeared they had a larger amplitude than before baclofen. Baclofen also suppressed the multiple population spikes evoked by antidromic stimulation in the dentate gyrus. This effect also decreased with continued baclofen perfusion. The effects of baclofen on the amplitude of the spontaneous field bursts and on the stimulation-induced multiple population spikes were blocked by the GABAB receptor antagonist SCH 50911, suggesting that these effects of baclofen are mediated by GABAB receptor activation. Baclofen significantly increased the peak extracellular K + concentration during each field burst in the dentate gyrus but did not change the baseline level of K + between field bursts. The results suggest that postsynaptic GABAB receptor activation by baclofen has transient antiepileptic effects followed by a rebound increase in excitability. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Baclofen; Field bursts; Postsynaptic GABAB receptor; CA1; Dentate gyrus
1. Introduction Two main receptor subtypes are activated by the inhibitory neurotransmitter, GABA: GABAA receptors and GABAB receptors. GABAA receptors are ionotropic receptors and mediate a fast postsynaptic inhibition via an increase in Cl⫺ conductance (Nicoll et al., 1990; Sivilotti and Nistri, 1991). Postsynaptic GABAB receptors are metabotropic receptors that mediate a slow postsynaptic inhibition via G-protein activation. GABAB receptors are also located presynaptically where they inhibit neurotransmitter release (Nicoll et al., 1990; Thompson, 1994). GABAB receptor agonists, such as baclofen, have been reported to have both antiepileptic and proepileptic effects in a variety of in vitro and in vivo systems. Interictal dis-
* Corresponding author. Tel.: +1-713-798-7937; fax: 1-713-7983145. E-mail address: [email protected] (J.L. Stringer).
charges in the CA3 area of the hippocampus induced by high K + (Ault et al., 1986; Swartzwelder et al., 1986a), bicuculline (Ault et al., 1986; Brady and Swann, 1984; Swartzwelder et al., 1986a), kainic acid (Ault et al., 1986), or stimulus trains (Swartzwelder et al., 1986a,b) are suppressed by baclofen. In intact animals, baclofen can prevent rapid amygdala kindling (Wurpel, 1994) and suppress flurothyl- (Garant et al., 1993), pentylenetetrazol(Veliskova et al., 1996), or stimulus train- (Stringer and Lothman, 1990) induced seizures. The proepileptic effects of baclofen have also been reported in both in vivo and in vitro situations. Baclofen can elicit spontaneous rhythmic sharp waves in hippocampal slices (Lewis et al., 1989). In hippocampal slices perfused with low Mg2+ or 4-aminopyridine, baclofen suppresses interictal activity but causes, or allows, seizure (or ictal) activity (Dreier and Heinemann, 1991; Motalli et al., 1999; Swartzwelder et al., 1987; Watts and Jefferys, 1993). In vivo, GABAB receptor activation has been shown to play a significant role in absence seizures (Hosford et al., 1992; Snead, 1992; Vergnes et al., 1997).
0028-3908/01/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 0 ) 0 0 1 2 0 - 9
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In vitro seizure-like activity, termed field bursts, appears in the absence of synaptic transmission, usually after perfusion with 0-added calcium and elevated levels of potassium (for review see Jefferys, 1995; Dudek et al., 1999). Field bursts were first described in the CA1 region (Jefferys and Haas, 1982; Taylor and Dudek, 1982); however, field bursts have been observed in all hippocampal regions (Snow and Dudek, 1984). Induction of field bursts in the dentate gyrus requires higher extracellular K + levels compared to CA1 (Schweitzer et al., 1992), but once present, the field bursts in the dentate gyrus have a larger amplitude and longer duration than those in CA1. The reasons for these regional differences are not clear. In this study, field bursts in CA1 and the dentate gyrus were used to isolate and study the postsynaptic effects (Haas and Jefferys, 1984) of baclofen on seizure activity.
2. Methods Hippocampal slices from Sprague-Dawley rats were prepared (100–200 g, both sexes). After anesthetizing the rats (ketamine 25 mg/kg, xylazine 5 mg/kg, acepromazine 0.8 mg/kg, ip), the brains were removed. Transverse slices (450–500 µm) through the hippocampus were cut with a Vibratome (Technical Products International, Inc.). Slices were placed in an interface-type chamber and continuously perfused with artificial cerebrospinal fluid (ACSF) at 32°C under a stream of humidified 95% O2/5% CO2. Composition of the ACSF was (in mM): NaCl 127, KCl 2, MgSO4 1.5, KH2PO4 1.1, NaHCO3 26, CaCl2 2 and glucose 10. All solutions were bubbled constantly with 95% O2/5% CO2. Slices were allowed to equilibrate for 1 h before electrophysiological recording. Recording electrodes were made of microfilament capillary thin-walled glass (A-M Systems, 0.9 mm ID, 1.2 mm OD) pulled on a micropipette puller (P-87, Sutter Instruments). Electrodes were filled with 2 M NaCl and had impedances between 4 and 10 M⍀. Recording electrodes were placed in the cell body layer of CA1, CA3, or the dorsal dentate gyrus. For antidromic stimulation of the dentate gyrus, a bipolar tungsten electrode was positioned in the hilar area (600–800 µA, 0.3 ms biphasic). Nonsynaptic epileptiform activity was induced in the hippocampus by changing to ACSF containing 0-added calcium and high potassium. The potassium was raised to 6 mM to induce epileptiform activity in the CA1 region and was raised to 8 mM to induce epileptiform activity in the dentate gyrus. These alterations in calcium and potassium do not significantly change the osmolality of the perfusing solution. In both regions, this non-synaptic epileptiform activity can take more than an hour to appear, but once it appears, the interval between field
bursts and the burst duration remain stable for many hours (Bikson et al., 1999; Pan and Stringer, 1996). Measurements of extracellular potassium concentrations ([K +]o) were carried out with double-barreled ion sensitive electrodes (Stringer and Lothman, 1989). One barrel was silanized with 15% tri-N-butylchlorosilane (Alfrebro, Monroe, OH) in chloroform, and the tip was filled with a potassium selective resin (Fluka Cocktail “B”). The electrode was then backfilled with 1 M potassium acetate. The reference barrel was filled with 2 M NaCl. The reference and potassium signals were amplified and displayed on a chart recorder. Every electrode was calibrated before the experiment in a series of standard solutions in ACSF (2, 3, 5, 10, and 20 mM potassium) at 32°C and the individual calibrations were used to determine the [K +]o for each experiment. The electrode tip diameter was 2–3 µm and electrodes had an impedance of around 108 ⍀. Electrodes were not used for this study unless they had at least a 50 mV change between the 2 and 20 mM potassium standards. During each experiment, the electrode was also calibrated by moving from the slice to the perfusion solution and comparing the bath and tissue potentials. Recordings in the slices were made 100–200 µm below the slice surface. The maximal [K +]o reached during field bursts was taken as the peak level. The [K +]o level exactly halfway between two field bursts (at least 20 s apart) was taken as the baseline level. All compounds were purchased from Sigma Chemical Co. (St Louis, MO), except that SCH 50911 was from Schering-Plough Research Institute (Kenilworth, NJ). All chemicals were dissolved directly into the perfusing solution. For statistical analysis, a paired Student’s t-test was used to compare measurements before and after treatment. The significance level was set at P⬍0.05. Data are expressed as means±SE.
3. Results Hippocampal slices were perfused with 8 mM K + and 0-added Ca2+ ACSF until spontaneous regular field bursts appeared in the dentate gyrus. These field bursts are characterized by bursts of large-amplitude (10–40 mV) population spikes associated with a negative shift of the extracellular DC potential. Twenty minutes after the appearance of the recurrent field bursts, baclofen was added to the perfusing solution. Baclofen caused a transient block of the field bursts, but with continued perfusion with baclofen, spontaneous field bursts reappeared. The duration of the blocking effect of baclofen was dose-dependent. At 5 µM, the period during which the field bursts were blocked, measured from the end of the last field burst before baclofen application to the beginning of the first field burst in the presence of continuous baclofen perfusion, was 5.5±0.5 min (n=12). At
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20 µM, the field bursts were initially blocked for 8.5±1.5 min (n=20); while at 100 µM, they were blocked for 15.3±2.0 min (n=8). When the field bursts reappeared, they had a larger amplitude than before addition of baclofen and this effect of baclofen was also dosedependent. The amplitude was measured at two points within each field burst (5 s after the initiation and 5 s before the termination) and these two measurements were averaged. At 5 µM, the reappearance of field bursts in the presence of baclofen was not associated with significant changes in the amplitude of the population spikes within the field burst (before: 15.8±1.3 mV; after baclofen: 16.2±1.2 mV; P⬎0.05). At 20 µM (before: 15.2±1.4 mV; after baclofen: 21.0±1.6 mV; P⬍0.01, Fig. 1) and 100 µM (before: 16.1±1.2 mV; after
Fig. 1. Effects of baclofen on spontaneous field bursts in the dentate gyrus induced by 0 Ca2+/8 mM K + medium. Field potential recordings from the dorsal granule cell body layer of a hippocampal slice before and after baclofen (20 µM) are shown. Baclofen caused a transient suppression of spontaneous field burst. When the field bursts reappeared they had a larger amplitude and small spikes were present between field bursts.
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baclofen: 38.6±2.2 mV; P⬍0.01, Fig. 2), the amplitudes were significantly increased. In about half (range 40– 63%) of the slices treated with 20 and 100 µM baclofen small amplitude spikes appeared between the field bursts (Fig. 1). These small spikes between the field bursts were never observed after lower doses of baclofen. The effects of baclofen were reversible after prolonged (up to 1 h) washout or were blocked by 20 µM of the GABAB antagonist SCH 50911 (Fig. 2, n=5). SCH 50911 alone had no effects on the frequency (interval between field bursts, before 55.3±5.6 s, after 52.4±5.4 s, P⬎0.05) or amplitude (before 15.9±2.3 mV, after 15.8±2.5 mV, P⬎0.05) of the spontaneous field bursts (n=5). Similar results were obtained from recordings in the CA1 area (Fig. 3). Hippocampal slices were perfused with 0 Ca2+/6 mM K + medium until spontaneous field bursts appeared in CA1. Compared with the dentate gyrus, the field bursts in CA1 were of shorter duration and smaller amplitude. In 60% of the slices, the field bursts consisted of a negative DC shift without population spikes. After the spontaneous field bursts stabilized, baclofen was added to the perfusing solution. Similar to the results in the dentate gyrus, baclofen, at 20 µM caused a transient blockade of the spontaneous field bursts (5.5±1.5 min, n=6). When the field bursts reappeared, they had a significantly larger amplitude compared with before the baclofen (before: 5.1±1.1 mV; baclofen: 24.8±3.0 mV, P⬍0.01). The effects of baclofen in CA1 were also suppressed by SCH 50911 (Fig. 3). In additional slices, simultaneous recordings were performed in CA1, CA3 and the dentate gyrus (Fig. 4). In slices perfused with 0 Ca2+/6 mM K +, spontaneous field bursts developed in CA1 in 100% of slices tested, in CA3 in 15% of slices and in the dentate gyrus in none of the slices tested. When the [K +]o was increased from 6 to 8 mM, spontaneous field bursts appeared in the dentate gyrus, but stopped in the CA1 region and CA3 region (if present before addition of baclofen). Addition of baclofen, 20 µM, to the 0 Ca2+/8 mM K + perfusing solution caused the appearance of field bursts in both CA1 and the dentate gyrus that were of larger amplitude than in the absence of baclofen. The CA3 area displayed either organized field bursts (33% of the slices) or continuous firing (66%) after addition of baclofen (Fig. 4, n=6). The effects of baclofen on antidromically induced multiple population spikes in the dentate gyrus were determined (Fig. 5). Hippocampal slices were perfused with 0 Ca2+/5 mM K + medium and responses to single antidromic stimulation to the hilus were recorded. Multiple population spikes developed in the dentate granule cell body layer within 40 min of perfusion in the 0 Ca2+/5 mM medium. Baclofen, at 5 µM, transiently reduced the number of these multiple spikes, but this
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Fig. 2. Baclofen increased the amplitude of spontaneous field bursts. Examples of field bursts from the dentate gyrus of a single slice are shown before baclofen, after perfusion for 15 min with 100 µM baclofen and 15 min after addition of SCH 50911 (20 µM). Population spikes during the later part of the field burst are shown below on an expanded time base.
Fig. 3. Effects of baclofen on spontaneous field bursts in the CA1 area induced by 0 Ca2+/6 mM K + medium. Field bursts in the CA1 pyramidal cell body layer were induced by perfusion with 0 Ca2+/6 mM K + medium (before). Immediately after the addition of baclofen (20 µM) the field bursts were suppressed. The bursts reappeared between 10 and 15 min later, in spite of continued perfusion with baclofen. The field bursts recorded 20 min after continuous baclofen perfusion (20 µM) are shown. The effects of baclofen are partially reversed by SCH 50911 (10 µM). The field bursts recorded 20 min after addition of SCH50911 are shown.
suppression decreased after 30 min of continuous baclofen perfusion (Fig. 5a, n=5). After perfusion with 20 µM baclofen, antidromic stimulation only evoked a single population spike. Addition of the GABAB antagonist, SCH 50911 (20 µM), reversed the effect of baclofen (Fig. 5b, n=5). Because both GABAB receptors and adenosine A1 receptors are G-protein-coupled receptors that activate
an inwardly rectifying potassium current (Luscher et al., 1997), the effects of adenosine were compared with those for baclofen (Fig. 6). Like baclofen, adenosine at 50 µM (n=5) or 100 µM (n=9) caused an initial depression of the spontaneous field bursts induced by 0 Ca2+/8 mM K + medium in the dentate gyrus (Fig. 6a). Spontaneous field bursts reappeared within 10 min in the continued presence of adenosine. Unlike baclofen, adenosine did not significantly (before: 16.6±1.2 mV; after: 17.2±1.2 mV; n=14, P⬎0.05) increase the amplitude of field bursts and did not cause small spikes between the field bursts. The effects of adenosine on antidromically induced multiple spikes were also similar to those of baclofen: an initial depression with recovery within 20 min in the continued presence of adenosine (Fig. 6b, n=8). Because postsynaptic GABAB receptor activation is coupled to an increase in K + conductance (Luscher et al., 1997) and baclofen has been reported to evoke changes in [K +]o (Obrocea and Morris, 1998), the possibility that the effects of baclofen on field bursts are mediated by changes in [K +]o was tested using K +-sensitive microelectrodes (Fig. 7). Hippocampal slices were perfused with 0 Ca2+/8 mM K + medium until recurrent field bursts were recorded. Field bursts in the dentate gyrus were associated with a change in [K +]o from a baseline 8.1 mM to a peak (the highest) level of 14.5±0.2 mM. After perfusing for 30 min with baclofen at 20 µM, the baseline [K +]o (before: 8.1±0.1 mM; after: 8.2±0.2 mM) did not change, but the peak level during field bursts significantly increased to 16.9±0.2 mM (P⬍0.05, n=12). Before baclofen treatment, the ceiling level of [K +]o was relatively constant during a field burst, but after baclofen, the peak [K +]o actually declined during the field burst, while the amplitude of the field burst was increasing (Fig. 7d). The effect of baclofen on the responses of [K +]o was reversed by SCH 50911 (20 µM, n=8).
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Fig. 5. Effects of baclofen on antidromic population responses in the dentate gyrus in the 0 Ca2+/5 mM K + medium. The dentate gyrus was stimulated from the hilus and the evoked responses were recorded in the granule cell body layer. (a) The effects of a low dose of baclofen (5 µM) are shown. (b) A higher dose of baclofen (20 µM) caused complete suppression of the multiple evoked population spikes. Addition of the GABAB receptor antagonist (SCH 50911, 20 µM) reversed the effect of baclofen. Fig. 4. Effects of baclofen on spontaneous field bursts induced by 0 Ca2+/high K + in the hippocampus. Simultaneous chart recordings from the CA1 (top), CA3 (middle) and dentate gyrus (bottom) regions of a single hippocampal slice. (a) The slice was initially perfused with 0 Ca2+/6 mM K+ medium and spontaneous field bursts appeared only in CA1. (b) The [K +]o in the perfusion was increased to 8 mM and spontaneous field bursts appeared only in the dentate gyrus. (c) The addition of baclofen (20 µM) to the perfusing solution caused a transient disappearance (for 10–15 min) of the field bursts in the dentate gyrus and caused field bursts to appear in CA1. The tracings shown in (c) were obtained 20 min after addition of baclofen.
4. Discussion The results of this study suggest that postsynaptic GABAB receptor activation by baclofen has both antiepileptic and proepileptic properties. Baclofen caused a transient dose-dependent suppression of low Ca2+induced spontaneous field bursts and antidromically evoked multiple population spikes, an effect that disappeared with continued perfusion with baclofen. When the spontaneous field bursts reappeared in the presence of baclofen, the amplitudes of the bursts were increased and there was a higher peak [K +]o level during the field burst compared with before the addition of baclofen. The effects of baclofen on the field bursts and multiple
population spikes appear to be mediated by GABAB receptor activation because the effects were antagonized by a potent and selective GABAB antagonist, SCH 50911 (Bolser et al., 1995). In addition, in the 0 Ca2+ conditions synaptic transmission is blocked (Dudek et al., 1999; Jefferys, 1995), suggesting that the effects of baclofen are, most likely, mediated by postsynaptic receptor activation. It is not possible to entirely rule out activation of presynaptic receptors, but the finding that the GABAB antagonist had no effect on the baseline activity suggests that little, or no, presynaptic activation accounts for the results in this study. Postsynaptic GABAB receptor activation usually leads to hyperpolarization that decreases excitability of principal cells (Newberry and Nicoll, 1984). The suppressive effects of baclofen on spontaneous field bursts and antidromic stimulus-induced multiple population spikes can thus be explained by a postsynaptic GABAB receptor mediated hyperpolarization. Adenosine, which also activates a Gprotein coupled receptor linked to an inwardly rectifying potassium channel (Luscher et al., 1997), also transiently suppressed the field bursts and multiple antidromic population spikes, adding support to the idea that this effect is mediated by postsynaptic hyperpolarization.
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Fig. 6. Effects of adenosine on spontaneous field bursts and antidromic population responses in the dentate gyrus. (a) Field potential recordings from the dorsal granule cell body layer of a hippocampal slice in 0 Ca2+/8 mM K + medium before and after adenosine (0.1 mM) are shown. Adenosine caused a transient suppression of the spontaneous field bursts. (b) Adenosine (0.1 mM) also caused a transient suppression of the multiple population spikes evoked by antidromic stimulation.
The loss of the suppressive effects of baclofen and adenosine in spite of continued perfusion with the drug is difficult to explain, although there are several possibilities. One is that there are two separate processes going on — a transient suppression and an increase in excitability (at least for baclofen). The second is that there is only one process that evolves over time. Finally, there could be multiple processes activated concurrently and mediated through multiple GABAB receptors coupled to different membrane channels or to a variety of second messengers and biochemical processes (for review see Kerr and Ong, 1995). The data suggest that the first hypothesis is more likely. The transient suppression occurred after either baclofen or adenosine administration, but the increase in amplitude of the field bursts occurred only after baclofen. This fits better with the two-process hypothesis. The transient suppression may be a time-dependent change in the receptor state, such as desensitization. Indeed, desensitization to baclofen (Malcangio et al., 1992; Yoshimura et al., 1995) and adenosine (Hosseinzadeh and Stone, 1994; Porter et al., 1988) has been previously reported. For this hypothesis to work, the desensitization would have to be concentration-dependent and have a time course that matches the results of the present experiments and this has not yet been determined. The loss of the blocking effect of baclofen on the
spontaneous field bursts is coincident with the appearance of larger amplitude field bursts. Because extracellular K + has been shown to be involved in epileptogenesis and postsynaptic GABAB receptors are coupled to potassium channels, the loss of the blocking effects might be due to an increase in the baseline [K +]o, producing an increase in excitability (Obrocea and Morris, 1998). However, the [K +]o measurements indicate that baclofen did not significantly change the baseline [K +]o level. The increase in amplitude of the field bursts also does not appear to be due to an increase in [K +]o. During the later part of the field burst, the [K +]o was actually falling while the amplitude of the bursts was increasing. The increase in amplitude of field bursts and peak [K +]o level in the presence of baclofen may reflect an increase in neuronal synchronization, but confirmation of this will require further experimentation. Early in the field burst or before addition of baclofen some principal neurons may be not firing synchronously with the field population spikes within the bursts. This is probably especially relevant in CA1 where the field bursts may consist only of DC shifts and no population spikes, even though neurons are firing action potentials (Bikson et al., 1999). In the present experiments, CA1 and the dentate gyrus had large amplitude population spikes in the presence of baclofen, suggesting that baclofen may recruit more neurons and/or increase neuronal synchronization
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Acknowledgements This work was supported by a grant from NINDS to JLS, NS39941. We thank Dr John G.R. Jefferys for helpful comments and discussion.
References
Fig. 7. Effects of baclofen on [K +]o regulation during and after field bursts in the dentate gyrus. Chart recordings of the reference electrode recording of the extracellular field potentials in the granule cell layer of the dentate gyrus (f.p.) and differential recording of the extracellular potassium concentration ([K +]o) before (a), after 20 min of baclofen (20 µM) perfusion (b), and after 20 min of both baclofen and SCH 50911 (20 µM) (c) are shown. In (d), the tracings of the changes in [K +]o during the field bursts before and after baclofen are superimposed for comparison.
during the field burst, especially during the later part of the field burst. Interestingly the amplitudes of the field bursts did not completely return to baseline after washout of baclofen. This suggests that baclofen may be producing a long-lasting increase in excitability and synchronization. However, we have previously observed that when field bursts have been suppressed by drug application or ionic changes, when the field bursts return they have an increased amplitude. The mechanism behind this long-lasting increase in amplitude is not known, but is unlikely to be specific for the mechanism of baclofen.
Ault, B., Gruenthal, M., Armstrong, D.R., Nadler, J.V., Wang, C.M., 1986. Baclofen suppresses bursting activity induced in hippocampal slices by differing convulsant treatments. European Journal of Pharmacology 126, 289–292. Bikson, M., Ghai, R.S., Baraban, S.C., Durand, D.M., 1999. Modulation of burst frequency, duration, and amplitude in the zero-Ca2+ model of epileptiform activity. Journal of Neurophysiology 82, 2262–2270. Bolser, D.C., Blythin, D.J., Chapman, R.W., Egan, R.W., Hey, J.A., Rizzo, C., Kuo, S.C., Kreutner, W., 1995. The pharmacology of SCH 50911: a novel, orally-active GABAB receptor antagonist. Journal of Pharmacology and Experimental Therapeutics 274, 1393–1398. Brady, R.J., Swann, J.W., 1984. Postsynaptic actions of baclofen associated with its antagonism of bicuculline-induced epileptogenesis in hippocampus. Cellular and Molecular Neurobiology 4, 403–408. Dreier, J.P., Heinemann, U., 1991. Regional and time dependent variations of low Mg2+ induced epileptiform activity in rat temporal cortex slices. Experimental Brain Research 87, 581–596. Dudek, F.E., Patrylo, P.R., Wuarin, J.P., 1999. Mechanisms of neuronal synchronization during epileptiform activity. Advances in Neurology 79, 699–708. Garant, D.S., Xu, S.G., Sperber, E.F., Moshe´, S.L., 1993. The influence of thalamic GABA transmission on the susceptibility of adult rats to flurothyl induced seizures. Epilepsy Research 15, 185–192. Haas, H.L., Jefferys, J.G., 1984. Low-calcium field burst discharges of CA1 pyramidal neurones in rat hippocampal slices. Journal of Physiology (London) 354, 185–201. Hosford, D.A., Clark, S., Cao, Z., Wilson, W.A. Jr., Lin, F.H., Morrisett, R.A., Huin, A., 1992. The role of GABAB receptor activation in absence seizures of lethargic (lh/lh) mice. Science 257, 398–401. Hosseinzadeh, H., Stone, T.W., 1994. The effect of calcium removal on the suppression by adenosine of epileptiform activity in the hippocampus: demonstration of desensitization. British Journal of Pharmacology 112, 316–322. Jefferys, J.G., Haas, H.L., 1982. Synchronized bursting of CA1 hippocampal pyramidal cells in the absence of synaptic transmission. Nature 300, 448–450. Jefferys, J.G., 1995. Nonsynaptic modulation of neuronal activity in the brain: electric currents and extracellular ions. Physiological Review 75, 689–723. Kerr, D.I., Ong, J., 1995. GABAB receptors. Pharmacological Therapy 67, 187–246. Lewis, D.V., Jones, L.S., Mott, D.D., 1989. Baclofen induces spontaneous, rhythmic sharp waves in the rat hippocampal slice. Experimental Neurology 106, 181–186. Luscher, C., Jan, L.Y., Stoffel, M., Malenka, R.C., Nicoll, R.A., 1997. G protein-coupled inwardly rectifying K + channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron 19, 687–695. Malcangio, M., Malmberg-Aiello, P., Giotti, A., Ghelardini, C., Bartolini, A., 1992. Desensitization of GABAB receptors and antagonism by CGP 35348, prevent bicuculline- and picrotoxin-induced antinociception. Neuropharmacology 31, 783–791. Motalli, R., Louvel, J., Tancredi, V., Kurcewicz, I., Wan-Chow-Wah, D., Pumain, R., Avoli, M., 1999. GABAB receptor activation pro-
138
Z.-Q. Xiong, J.L. Stringer / Neuropharmacology 40 (2001) 131–138
motes seizure activity in the juvenile rat hippocampus. Journal of Neurophysiology 82, 638–647. Newberry, N.R., Nicoll, R.A., 1984. Direct hyperpolarizing action of baclofen on hippocampal pyramidal cells. Nature 308, 450–452. Nicoll, R.A., Malenka, R.M., Kauer, J.A., 1990. Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiological Review 70, 513–565. Obrocea, G.V., Morris, M.E., 1998. Changes in [K +]o evoked by baclofen in guinea pig hippocampus. Canadian Journal of Physiology and Pharmacology 76, 148–154. Pan, E., Stringer, J.L., 1996. Burst characteristics of dentate gyrus granule cells: evidence for endogenous and nonsynaptic properties. Journal of Neurophysiology 75, 124–132. Porter, N.M., Radulovacki, M., Green, R.D., 1988. Desensitization of adenosine and dopamine receptors in rat brain after treatment with adenosine analogs. Journal of Pharmacology and Experimental Therapeutics 244, 218–225. Schweitzer, J.S., Patrylo, P.R., Dudek, F.E., 1992. Prolonged field bursts in the dentate gyrus: dependence on low calcium, high potassium, and nonsynaptic mechanisms. Journal of Neurophysiology 68, 2016–2025. Sivilotti, L., Nistri, A., 1991. GABA receptor mechanisms in the central nervous system. Progress in Neurobiology 36, 35–92. Snead, O.C., 1992. Evidence for GABAB-mediated mechanisms in experimental generalized absence seizures. European Journal of Pharmacology 213, 343–349. Snow, R.W., Dudek, F.E., 1984. Synchronous epileptiform bursts without chemical transmission in CA2, CA3 and dentate areas of the hippocampus. Brain Research 298, 382–385. Stringer, J.L., Lothman, E.W., 1989. Model of spontaneous hippocampal epilepsy in the anesthetized rat: electrographic, [K +]o, and [Ca2+]o response patterns. Epilepsy Research 4, 177–186. Stringer, J.L., Lothman, E.W., 1990. Pharmacological evidence indi-
cating a role of GABAergic systems in termination of limbic seizures. Epilepsy Research 7, 197–204. Swartzwelder, H.S., Bragdon, A.C., Sutch, C.P., Ault, B., Wilson, W.A., 1986a. Baclofen suppresses hippocampal epileptiform activity at low concentrations without suppressing synaptic transmission. Journal of Pharmacology and Experimental Therapeutics 237, 881–887. Swartzwelder, H.S., Sutch, C.P., Wilson, W.A., 1986b. Attenuation of epileptiform bursting by baclofen: reduced potency in elevated potassium. Experimental Neurology 94, 726–734. Swartzwelder, H.S., Lewis, D.V., Anderson, W.W., Wilson, W.A., 1987. Seizure-like events in brain slices: suppression by interictal activity. Brain Research 410, 362–366. Taylor, C.P., Dudek, F.E., 1982. Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses. Science 218, 810–812. Thompson, S.M., 1994. Modulation of inhibitory synaptic transmission in the hippocampus. Progress in Neurobiology 42, 575–609. Veliskova, J., Velisek, L., Moshe´, S.L., 1996. Age-specific effects of baclofen on pentylenetetrazol-induced seizures in developing rats. Epilepsia 37, 718–722. Vergnes, M., Boehrer, A., Simler, S., Bernasconi, R., Marescaux, C., 1997. Opposite effects of GABAB receptor antagonists on absences and convulsive seizures. European Journal of Pharmacology 332, 245–255. Watts, A.E., Jefferys, J.G., 1993. Effects of carbamazepine and baclofen on 4-aminopyridine-induced epileptic activity in rat hippocampal slices. British Journal of Pharmacology 108, 819–823. Wurpel, J.N., 1994. Baclofen prevents rapid amygdala kindling in adult rats. Experientia 50, 475–478. Yoshimura, M., Yoshida, S., Taniyama, K., 1995. Desensitization by cyclic AMP-dependent protein kinase of GABAB receptor expressed in Xenopus oocytes. Life Sciences 57, 2397–2401.
Effects of postsynaptic GABAB receptor activation on epileptiform activity in hippocampal slices Zhi-Qi Xiong, Janet L. Stringer
*
Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA Received 7 March 2000; received in revised form 22 May 2000; accepted 13 June 2000
Abstract GABAB receptor agonists have been reported to have both pro- and antiepileptic properties. Here, the effects of a GABAB receptor agonist, baclofen, were studied on epileptiform activity induced in the absence of synaptic transmission — to focus on the postsynaptic effects. Perfusion of hippocampal slices with 0-added calcium and high potassium induced field bursts in CA1 and the dentate gyrus. Addition of baclofen caused a transient suppression of the field bursts in CA1 and the dentate gyrus. The duration of the suppression was dependent on the concentration of baclofen and when the bursts reappeared they had a larger amplitude than before baclofen. Baclofen also suppressed the multiple population spikes evoked by antidromic stimulation in the dentate gyrus. This effect also decreased with continued baclofen perfusion. The effects of baclofen on the amplitude of the spontaneous field bursts and on the stimulation-induced multiple population spikes were blocked by the GABAB receptor antagonist SCH 50911, suggesting that these effects of baclofen are mediated by GABAB receptor activation. Baclofen significantly increased the peak extracellular K + concentration during each field burst in the dentate gyrus but did not change the baseline level of K + between field bursts. The results suggest that postsynaptic GABAB receptor activation by baclofen has transient antiepileptic effects followed by a rebound increase in excitability. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Baclofen; Field bursts; Postsynaptic GABAB receptor; CA1; Dentate gyrus
1. Introduction Two main receptor subtypes are activated by the inhibitory neurotransmitter, GABA: GABAA receptors and GABAB receptors. GABAA receptors are ionotropic receptors and mediate a fast postsynaptic inhibition via an increase in Cl⫺ conductance (Nicoll et al., 1990; Sivilotti and Nistri, 1991). Postsynaptic GABAB receptors are metabotropic receptors that mediate a slow postsynaptic inhibition via G-protein activation. GABAB receptors are also located presynaptically where they inhibit neurotransmitter release (Nicoll et al., 1990; Thompson, 1994). GABAB receptor agonists, such as baclofen, have been reported to have both antiepileptic and proepileptic effects in a variety of in vitro and in vivo systems. Interictal dis-
* Corresponding author. Tel.: +1-713-798-7937; fax: 1-713-7983145. E-mail address: [email protected] (J.L. Stringer).
charges in the CA3 area of the hippocampus induced by high K + (Ault et al., 1986; Swartzwelder et al., 1986a), bicuculline (Ault et al., 1986; Brady and Swann, 1984; Swartzwelder et al., 1986a), kainic acid (Ault et al., 1986), or stimulus trains (Swartzwelder et al., 1986a,b) are suppressed by baclofen. In intact animals, baclofen can prevent rapid amygdala kindling (Wurpel, 1994) and suppress flurothyl- (Garant et al., 1993), pentylenetetrazol(Veliskova et al., 1996), or stimulus train- (Stringer and Lothman, 1990) induced seizures. The proepileptic effects of baclofen have also been reported in both in vivo and in vitro situations. Baclofen can elicit spontaneous rhythmic sharp waves in hippocampal slices (Lewis et al., 1989). In hippocampal slices perfused with low Mg2+ or 4-aminopyridine, baclofen suppresses interictal activity but causes, or allows, seizure (or ictal) activity (Dreier and Heinemann, 1991; Motalli et al., 1999; Swartzwelder et al., 1987; Watts and Jefferys, 1993). In vivo, GABAB receptor activation has been shown to play a significant role in absence seizures (Hosford et al., 1992; Snead, 1992; Vergnes et al., 1997).
0028-3908/01/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 0 ) 0 0 1 2 0 - 9
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In vitro seizure-like activity, termed field bursts, appears in the absence of synaptic transmission, usually after perfusion with 0-added calcium and elevated levels of potassium (for review see Jefferys, 1995; Dudek et al., 1999). Field bursts were first described in the CA1 region (Jefferys and Haas, 1982; Taylor and Dudek, 1982); however, field bursts have been observed in all hippocampal regions (Snow and Dudek, 1984). Induction of field bursts in the dentate gyrus requires higher extracellular K + levels compared to CA1 (Schweitzer et al., 1992), but once present, the field bursts in the dentate gyrus have a larger amplitude and longer duration than those in CA1. The reasons for these regional differences are not clear. In this study, field bursts in CA1 and the dentate gyrus were used to isolate and study the postsynaptic effects (Haas and Jefferys, 1984) of baclofen on seizure activity.
2. Methods Hippocampal slices from Sprague-Dawley rats were prepared (100–200 g, both sexes). After anesthetizing the rats (ketamine 25 mg/kg, xylazine 5 mg/kg, acepromazine 0.8 mg/kg, ip), the brains were removed. Transverse slices (450–500 µm) through the hippocampus were cut with a Vibratome (Technical Products International, Inc.). Slices were placed in an interface-type chamber and continuously perfused with artificial cerebrospinal fluid (ACSF) at 32°C under a stream of humidified 95% O2/5% CO2. Composition of the ACSF was (in mM): NaCl 127, KCl 2, MgSO4 1.5, KH2PO4 1.1, NaHCO3 26, CaCl2 2 and glucose 10. All solutions were bubbled constantly with 95% O2/5% CO2. Slices were allowed to equilibrate for 1 h before electrophysiological recording. Recording electrodes were made of microfilament capillary thin-walled glass (A-M Systems, 0.9 mm ID, 1.2 mm OD) pulled on a micropipette puller (P-87, Sutter Instruments). Electrodes were filled with 2 M NaCl and had impedances between 4 and 10 M⍀. Recording electrodes were placed in the cell body layer of CA1, CA3, or the dorsal dentate gyrus. For antidromic stimulation of the dentate gyrus, a bipolar tungsten electrode was positioned in the hilar area (600–800 µA, 0.3 ms biphasic). Nonsynaptic epileptiform activity was induced in the hippocampus by changing to ACSF containing 0-added calcium and high potassium. The potassium was raised to 6 mM to induce epileptiform activity in the CA1 region and was raised to 8 mM to induce epileptiform activity in the dentate gyrus. These alterations in calcium and potassium do not significantly change the osmolality of the perfusing solution. In both regions, this non-synaptic epileptiform activity can take more than an hour to appear, but once it appears, the interval between field
bursts and the burst duration remain stable for many hours (Bikson et al., 1999; Pan and Stringer, 1996). Measurements of extracellular potassium concentrations ([K +]o) were carried out with double-barreled ion sensitive electrodes (Stringer and Lothman, 1989). One barrel was silanized with 15% tri-N-butylchlorosilane (Alfrebro, Monroe, OH) in chloroform, and the tip was filled with a potassium selective resin (Fluka Cocktail “B”). The electrode was then backfilled with 1 M potassium acetate. The reference barrel was filled with 2 M NaCl. The reference and potassium signals were amplified and displayed on a chart recorder. Every electrode was calibrated before the experiment in a series of standard solutions in ACSF (2, 3, 5, 10, and 20 mM potassium) at 32°C and the individual calibrations were used to determine the [K +]o for each experiment. The electrode tip diameter was 2–3 µm and electrodes had an impedance of around 108 ⍀. Electrodes were not used for this study unless they had at least a 50 mV change between the 2 and 20 mM potassium standards. During each experiment, the electrode was also calibrated by moving from the slice to the perfusion solution and comparing the bath and tissue potentials. Recordings in the slices were made 100–200 µm below the slice surface. The maximal [K +]o reached during field bursts was taken as the peak level. The [K +]o level exactly halfway between two field bursts (at least 20 s apart) was taken as the baseline level. All compounds were purchased from Sigma Chemical Co. (St Louis, MO), except that SCH 50911 was from Schering-Plough Research Institute (Kenilworth, NJ). All chemicals were dissolved directly into the perfusing solution. For statistical analysis, a paired Student’s t-test was used to compare measurements before and after treatment. The significance level was set at P⬍0.05. Data are expressed as means±SE.
3. Results Hippocampal slices were perfused with 8 mM K + and 0-added Ca2+ ACSF until spontaneous regular field bursts appeared in the dentate gyrus. These field bursts are characterized by bursts of large-amplitude (10–40 mV) population spikes associated with a negative shift of the extracellular DC potential. Twenty minutes after the appearance of the recurrent field bursts, baclofen was added to the perfusing solution. Baclofen caused a transient block of the field bursts, but with continued perfusion with baclofen, spontaneous field bursts reappeared. The duration of the blocking effect of baclofen was dose-dependent. At 5 µM, the period during which the field bursts were blocked, measured from the end of the last field burst before baclofen application to the beginning of the first field burst in the presence of continuous baclofen perfusion, was 5.5±0.5 min (n=12). At
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20 µM, the field bursts were initially blocked for 8.5±1.5 min (n=20); while at 100 µM, they were blocked for 15.3±2.0 min (n=8). When the field bursts reappeared, they had a larger amplitude than before addition of baclofen and this effect of baclofen was also dosedependent. The amplitude was measured at two points within each field burst (5 s after the initiation and 5 s before the termination) and these two measurements were averaged. At 5 µM, the reappearance of field bursts in the presence of baclofen was not associated with significant changes in the amplitude of the population spikes within the field burst (before: 15.8±1.3 mV; after baclofen: 16.2±1.2 mV; P⬎0.05). At 20 µM (before: 15.2±1.4 mV; after baclofen: 21.0±1.6 mV; P⬍0.01, Fig. 1) and 100 µM (before: 16.1±1.2 mV; after
Fig. 1. Effects of baclofen on spontaneous field bursts in the dentate gyrus induced by 0 Ca2+/8 mM K + medium. Field potential recordings from the dorsal granule cell body layer of a hippocampal slice before and after baclofen (20 µM) are shown. Baclofen caused a transient suppression of spontaneous field burst. When the field bursts reappeared they had a larger amplitude and small spikes were present between field bursts.
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baclofen: 38.6±2.2 mV; P⬍0.01, Fig. 2), the amplitudes were significantly increased. In about half (range 40– 63%) of the slices treated with 20 and 100 µM baclofen small amplitude spikes appeared between the field bursts (Fig. 1). These small spikes between the field bursts were never observed after lower doses of baclofen. The effects of baclofen were reversible after prolonged (up to 1 h) washout or were blocked by 20 µM of the GABAB antagonist SCH 50911 (Fig. 2, n=5). SCH 50911 alone had no effects on the frequency (interval between field bursts, before 55.3±5.6 s, after 52.4±5.4 s, P⬎0.05) or amplitude (before 15.9±2.3 mV, after 15.8±2.5 mV, P⬎0.05) of the spontaneous field bursts (n=5). Similar results were obtained from recordings in the CA1 area (Fig. 3). Hippocampal slices were perfused with 0 Ca2+/6 mM K + medium until spontaneous field bursts appeared in CA1. Compared with the dentate gyrus, the field bursts in CA1 were of shorter duration and smaller amplitude. In 60% of the slices, the field bursts consisted of a negative DC shift without population spikes. After the spontaneous field bursts stabilized, baclofen was added to the perfusing solution. Similar to the results in the dentate gyrus, baclofen, at 20 µM caused a transient blockade of the spontaneous field bursts (5.5±1.5 min, n=6). When the field bursts reappeared, they had a significantly larger amplitude compared with before the baclofen (before: 5.1±1.1 mV; baclofen: 24.8±3.0 mV, P⬍0.01). The effects of baclofen in CA1 were also suppressed by SCH 50911 (Fig. 3). In additional slices, simultaneous recordings were performed in CA1, CA3 and the dentate gyrus (Fig. 4). In slices perfused with 0 Ca2+/6 mM K +, spontaneous field bursts developed in CA1 in 100% of slices tested, in CA3 in 15% of slices and in the dentate gyrus in none of the slices tested. When the [K +]o was increased from 6 to 8 mM, spontaneous field bursts appeared in the dentate gyrus, but stopped in the CA1 region and CA3 region (if present before addition of baclofen). Addition of baclofen, 20 µM, to the 0 Ca2+/8 mM K + perfusing solution caused the appearance of field bursts in both CA1 and the dentate gyrus that were of larger amplitude than in the absence of baclofen. The CA3 area displayed either organized field bursts (33% of the slices) or continuous firing (66%) after addition of baclofen (Fig. 4, n=6). The effects of baclofen on antidromically induced multiple population spikes in the dentate gyrus were determined (Fig. 5). Hippocampal slices were perfused with 0 Ca2+/5 mM K + medium and responses to single antidromic stimulation to the hilus were recorded. Multiple population spikes developed in the dentate granule cell body layer within 40 min of perfusion in the 0 Ca2+/5 mM medium. Baclofen, at 5 µM, transiently reduced the number of these multiple spikes, but this
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Fig. 2. Baclofen increased the amplitude of spontaneous field bursts. Examples of field bursts from the dentate gyrus of a single slice are shown before baclofen, after perfusion for 15 min with 100 µM baclofen and 15 min after addition of SCH 50911 (20 µM). Population spikes during the later part of the field burst are shown below on an expanded time base.
Fig. 3. Effects of baclofen on spontaneous field bursts in the CA1 area induced by 0 Ca2+/6 mM K + medium. Field bursts in the CA1 pyramidal cell body layer were induced by perfusion with 0 Ca2+/6 mM K + medium (before). Immediately after the addition of baclofen (20 µM) the field bursts were suppressed. The bursts reappeared between 10 and 15 min later, in spite of continued perfusion with baclofen. The field bursts recorded 20 min after continuous baclofen perfusion (20 µM) are shown. The effects of baclofen are partially reversed by SCH 50911 (10 µM). The field bursts recorded 20 min after addition of SCH50911 are shown.
suppression decreased after 30 min of continuous baclofen perfusion (Fig. 5a, n=5). After perfusion with 20 µM baclofen, antidromic stimulation only evoked a single population spike. Addition of the GABAB antagonist, SCH 50911 (20 µM), reversed the effect of baclofen (Fig. 5b, n=5). Because both GABAB receptors and adenosine A1 receptors are G-protein-coupled receptors that activate
an inwardly rectifying potassium current (Luscher et al., 1997), the effects of adenosine were compared with those for baclofen (Fig. 6). Like baclofen, adenosine at 50 µM (n=5) or 100 µM (n=9) caused an initial depression of the spontaneous field bursts induced by 0 Ca2+/8 mM K + medium in the dentate gyrus (Fig. 6a). Spontaneous field bursts reappeared within 10 min in the continued presence of adenosine. Unlike baclofen, adenosine did not significantly (before: 16.6±1.2 mV; after: 17.2±1.2 mV; n=14, P⬎0.05) increase the amplitude of field bursts and did not cause small spikes between the field bursts. The effects of adenosine on antidromically induced multiple spikes were also similar to those of baclofen: an initial depression with recovery within 20 min in the continued presence of adenosine (Fig. 6b, n=8). Because postsynaptic GABAB receptor activation is coupled to an increase in K + conductance (Luscher et al., 1997) and baclofen has been reported to evoke changes in [K +]o (Obrocea and Morris, 1998), the possibility that the effects of baclofen on field bursts are mediated by changes in [K +]o was tested using K +-sensitive microelectrodes (Fig. 7). Hippocampal slices were perfused with 0 Ca2+/8 mM K + medium until recurrent field bursts were recorded. Field bursts in the dentate gyrus were associated with a change in [K +]o from a baseline 8.1 mM to a peak (the highest) level of 14.5±0.2 mM. After perfusing for 30 min with baclofen at 20 µM, the baseline [K +]o (before: 8.1±0.1 mM; after: 8.2±0.2 mM) did not change, but the peak level during field bursts significantly increased to 16.9±0.2 mM (P⬍0.05, n=12). Before baclofen treatment, the ceiling level of [K +]o was relatively constant during a field burst, but after baclofen, the peak [K +]o actually declined during the field burst, while the amplitude of the field burst was increasing (Fig. 7d). The effect of baclofen on the responses of [K +]o was reversed by SCH 50911 (20 µM, n=8).
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Fig. 5. Effects of baclofen on antidromic population responses in the dentate gyrus in the 0 Ca2+/5 mM K + medium. The dentate gyrus was stimulated from the hilus and the evoked responses were recorded in the granule cell body layer. (a) The effects of a low dose of baclofen (5 µM) are shown. (b) A higher dose of baclofen (20 µM) caused complete suppression of the multiple evoked population spikes. Addition of the GABAB receptor antagonist (SCH 50911, 20 µM) reversed the effect of baclofen. Fig. 4. Effects of baclofen on spontaneous field bursts induced by 0 Ca2+/high K + in the hippocampus. Simultaneous chart recordings from the CA1 (top), CA3 (middle) and dentate gyrus (bottom) regions of a single hippocampal slice. (a) The slice was initially perfused with 0 Ca2+/6 mM K+ medium and spontaneous field bursts appeared only in CA1. (b) The [K +]o in the perfusion was increased to 8 mM and spontaneous field bursts appeared only in the dentate gyrus. (c) The addition of baclofen (20 µM) to the perfusing solution caused a transient disappearance (for 10–15 min) of the field bursts in the dentate gyrus and caused field bursts to appear in CA1. The tracings shown in (c) were obtained 20 min after addition of baclofen.
4. Discussion The results of this study suggest that postsynaptic GABAB receptor activation by baclofen has both antiepileptic and proepileptic properties. Baclofen caused a transient dose-dependent suppression of low Ca2+induced spontaneous field bursts and antidromically evoked multiple population spikes, an effect that disappeared with continued perfusion with baclofen. When the spontaneous field bursts reappeared in the presence of baclofen, the amplitudes of the bursts were increased and there was a higher peak [K +]o level during the field burst compared with before the addition of baclofen. The effects of baclofen on the field bursts and multiple
population spikes appear to be mediated by GABAB receptor activation because the effects were antagonized by a potent and selective GABAB antagonist, SCH 50911 (Bolser et al., 1995). In addition, in the 0 Ca2+ conditions synaptic transmission is blocked (Dudek et al., 1999; Jefferys, 1995), suggesting that the effects of baclofen are, most likely, mediated by postsynaptic receptor activation. It is not possible to entirely rule out activation of presynaptic receptors, but the finding that the GABAB antagonist had no effect on the baseline activity suggests that little, or no, presynaptic activation accounts for the results in this study. Postsynaptic GABAB receptor activation usually leads to hyperpolarization that decreases excitability of principal cells (Newberry and Nicoll, 1984). The suppressive effects of baclofen on spontaneous field bursts and antidromic stimulus-induced multiple population spikes can thus be explained by a postsynaptic GABAB receptor mediated hyperpolarization. Adenosine, which also activates a Gprotein coupled receptor linked to an inwardly rectifying potassium channel (Luscher et al., 1997), also transiently suppressed the field bursts and multiple antidromic population spikes, adding support to the idea that this effect is mediated by postsynaptic hyperpolarization.
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Fig. 6. Effects of adenosine on spontaneous field bursts and antidromic population responses in the dentate gyrus. (a) Field potential recordings from the dorsal granule cell body layer of a hippocampal slice in 0 Ca2+/8 mM K + medium before and after adenosine (0.1 mM) are shown. Adenosine caused a transient suppression of the spontaneous field bursts. (b) Adenosine (0.1 mM) also caused a transient suppression of the multiple population spikes evoked by antidromic stimulation.
The loss of the suppressive effects of baclofen and adenosine in spite of continued perfusion with the drug is difficult to explain, although there are several possibilities. One is that there are two separate processes going on — a transient suppression and an increase in excitability (at least for baclofen). The second is that there is only one process that evolves over time. Finally, there could be multiple processes activated concurrently and mediated through multiple GABAB receptors coupled to different membrane channels or to a variety of second messengers and biochemical processes (for review see Kerr and Ong, 1995). The data suggest that the first hypothesis is more likely. The transient suppression occurred after either baclofen or adenosine administration, but the increase in amplitude of the field bursts occurred only after baclofen. This fits better with the two-process hypothesis. The transient suppression may be a time-dependent change in the receptor state, such as desensitization. Indeed, desensitization to baclofen (Malcangio et al., 1992; Yoshimura et al., 1995) and adenosine (Hosseinzadeh and Stone, 1994; Porter et al., 1988) has been previously reported. For this hypothesis to work, the desensitization would have to be concentration-dependent and have a time course that matches the results of the present experiments and this has not yet been determined. The loss of the blocking effect of baclofen on the
spontaneous field bursts is coincident with the appearance of larger amplitude field bursts. Because extracellular K + has been shown to be involved in epileptogenesis and postsynaptic GABAB receptors are coupled to potassium channels, the loss of the blocking effects might be due to an increase in the baseline [K +]o, producing an increase in excitability (Obrocea and Morris, 1998). However, the [K +]o measurements indicate that baclofen did not significantly change the baseline [K +]o level. The increase in amplitude of the field bursts also does not appear to be due to an increase in [K +]o. During the later part of the field burst, the [K +]o was actually falling while the amplitude of the bursts was increasing. The increase in amplitude of field bursts and peak [K +]o level in the presence of baclofen may reflect an increase in neuronal synchronization, but confirmation of this will require further experimentation. Early in the field burst or before addition of baclofen some principal neurons may be not firing synchronously with the field population spikes within the bursts. This is probably especially relevant in CA1 where the field bursts may consist only of DC shifts and no population spikes, even though neurons are firing action potentials (Bikson et al., 1999). In the present experiments, CA1 and the dentate gyrus had large amplitude population spikes in the presence of baclofen, suggesting that baclofen may recruit more neurons and/or increase neuronal synchronization
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Acknowledgements This work was supported by a grant from NINDS to JLS, NS39941. We thank Dr John G.R. Jefferys for helpful comments and discussion.
References
Fig. 7. Effects of baclofen on [K +]o regulation during and after field bursts in the dentate gyrus. Chart recordings of the reference electrode recording of the extracellular field potentials in the granule cell layer of the dentate gyrus (f.p.) and differential recording of the extracellular potassium concentration ([K +]o) before (a), after 20 min of baclofen (20 µM) perfusion (b), and after 20 min of both baclofen and SCH 50911 (20 µM) (c) are shown. In (d), the tracings of the changes in [K +]o during the field bursts before and after baclofen are superimposed for comparison.
during the field burst, especially during the later part of the field burst. Interestingly the amplitudes of the field bursts did not completely return to baseline after washout of baclofen. This suggests that baclofen may be producing a long-lasting increase in excitability and synchronization. However, we have previously observed that when field bursts have been suppressed by drug application or ionic changes, when the field bursts return they have an increased amplitude. The mechanism behind this long-lasting increase in amplitude is not known, but is unlikely to be specific for the mechanism of baclofen.
Ault, B., Gruenthal, M., Armstrong, D.R., Nadler, J.V., Wang, C.M., 1986. Baclofen suppresses bursting activity induced in hippocampal slices by differing convulsant treatments. European Journal of Pharmacology 126, 289–292. Bikson, M., Ghai, R.S., Baraban, S.C., Durand, D.M., 1999. Modulation of burst frequency, duration, and amplitude in the zero-Ca2+ model of epileptiform activity. Journal of Neurophysiology 82, 2262–2270. Bolser, D.C., Blythin, D.J., Chapman, R.W., Egan, R.W., Hey, J.A., Rizzo, C., Kuo, S.C., Kreutner, W., 1995. The pharmacology of SCH 50911: a novel, orally-active GABAB receptor antagonist. Journal of Pharmacology and Experimental Therapeutics 274, 1393–1398. Brady, R.J., Swann, J.W., 1984. Postsynaptic actions of baclofen associated with its antagonism of bicuculline-induced epileptogenesis in hippocampus. Cellular and Molecular Neurobiology 4, 403–408. Dreier, J.P., Heinemann, U., 1991. Regional and time dependent variations of low Mg2+ induced epileptiform activity in rat temporal cortex slices. Experimental Brain Research 87, 581–596. Dudek, F.E., Patrylo, P.R., Wuarin, J.P., 1999. Mechanisms of neuronal synchronization during epileptiform activity. Advances in Neurology 79, 699–708. Garant, D.S., Xu, S.G., Sperber, E.F., Moshe´, S.L., 1993. The influence of thalamic GABA transmission on the susceptibility of adult rats to flurothyl induced seizures. Epilepsy Research 15, 185–192. Haas, H.L., Jefferys, J.G., 1984. Low-calcium field burst discharges of CA1 pyramidal neurones in rat hippocampal slices. Journal of Physiology (London) 354, 185–201. Hosford, D.A., Clark, S., Cao, Z., Wilson, W.A. Jr., Lin, F.H., Morrisett, R.A., Huin, A., 1992. The role of GABAB receptor activation in absence seizures of lethargic (lh/lh) mice. Science 257, 398–401. Hosseinzadeh, H., Stone, T.W., 1994. The effect of calcium removal on the suppression by adenosine of epileptiform activity in the hippocampus: demonstration of desensitization. British Journal of Pharmacology 112, 316–322. Jefferys, J.G., Haas, H.L., 1982. Synchronized bursting of CA1 hippocampal pyramidal cells in the absence of synaptic transmission. Nature 300, 448–450. Jefferys, J.G., 1995. Nonsynaptic modulation of neuronal activity in the brain: electric currents and extracellular ions. Physiological Review 75, 689–723. Kerr, D.I., Ong, J., 1995. GABAB receptors. Pharmacological Therapy 67, 187–246. Lewis, D.V., Jones, L.S., Mott, D.D., 1989. Baclofen induces spontaneous, rhythmic sharp waves in the rat hippocampal slice. Experimental Neurology 106, 181–186. Luscher, C., Jan, L.Y., Stoffel, M., Malenka, R.C., Nicoll, R.A., 1997. G protein-coupled inwardly rectifying K + channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron 19, 687–695. Malcangio, M., Malmberg-Aiello, P., Giotti, A., Ghelardini, C., Bartolini, A., 1992. Desensitization of GABAB receptors and antagonism by CGP 35348, prevent bicuculline- and picrotoxin-induced antinociception. Neuropharmacology 31, 783–791. Motalli, R., Louvel, J., Tancredi, V., Kurcewicz, I., Wan-Chow-Wah, D., Pumain, R., Avoli, M., 1999. GABAB receptor activation pro-
138
Z.-Q. Xiong, J.L. Stringer / Neuropharmacology 40 (2001) 131–138
motes seizure activity in the juvenile rat hippocampus. Journal of Neurophysiology 82, 638–647. Newberry, N.R., Nicoll, R.A., 1984. Direct hyperpolarizing action of baclofen on hippocampal pyramidal cells. Nature 308, 450–452. Nicoll, R.A., Malenka, R.M., Kauer, J.A., 1990. Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiological Review 70, 513–565. Obrocea, G.V., Morris, M.E., 1998. Changes in [K +]o evoked by baclofen in guinea pig hippocampus. Canadian Journal of Physiology and Pharmacology 76, 148–154. Pan, E., Stringer, J.L., 1996. Burst characteristics of dentate gyrus granule cells: evidence for endogenous and nonsynaptic properties. Journal of Neurophysiology 75, 124–132. Porter, N.M., Radulovacki, M., Green, R.D., 1988. Desensitization of adenosine and dopamine receptors in rat brain after treatment with adenosine analogs. Journal of Pharmacology and Experimental Therapeutics 244, 218–225. Schweitzer, J.S., Patrylo, P.R., Dudek, F.E., 1992. Prolonged field bursts in the dentate gyrus: dependence on low calcium, high potassium, and nonsynaptic mechanisms. Journal of Neurophysiology 68, 2016–2025. Sivilotti, L., Nistri, A., 1991. GABA receptor mechanisms in the central nervous system. Progress in Neurobiology 36, 35–92. Snead, O.C., 1992. Evidence for GABAB-mediated mechanisms in experimental generalized absence seizures. European Journal of Pharmacology 213, 343–349. Snow, R.W., Dudek, F.E., 1984. Synchronous epileptiform bursts without chemical transmission in CA2, CA3 and dentate areas of the hippocampus. Brain Research 298, 382–385. Stringer, J.L., Lothman, E.W., 1989. Model of spontaneous hippocampal epilepsy in the anesthetized rat: electrographic, [K +]o, and [Ca2+]o response patterns. Epilepsy Research 4, 177–186. Stringer, J.L., Lothman, E.W., 1990. Pharmacological evidence indi-
cating a role of GABAergic systems in termination of limbic seizures. Epilepsy Research 7, 197–204. Swartzwelder, H.S., Bragdon, A.C., Sutch, C.P., Ault, B., Wilson, W.A., 1986a. Baclofen suppresses hippocampal epileptiform activity at low concentrations without suppressing synaptic transmission. Journal of Pharmacology and Experimental Therapeutics 237, 881–887. Swartzwelder, H.S., Sutch, C.P., Wilson, W.A., 1986b. Attenuation of epileptiform bursting by baclofen: reduced potency in elevated potassium. Experimental Neurology 94, 726–734. Swartzwelder, H.S., Lewis, D.V., Anderson, W.W., Wilson, W.A., 1987. Seizure-like events in brain slices: suppression by interictal activity. Brain Research 410, 362–366. Taylor, C.P., Dudek, F.E., 1982. Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses. Science 218, 810–812. Thompson, S.M., 1994. Modulation of inhibitory synaptic transmission in the hippocampus. Progress in Neurobiology 42, 575–609. Veliskova, J., Velisek, L., Moshe´, S.L., 1996. Age-specific effects of baclofen on pentylenetetrazol-induced seizures in developing rats. Epilepsia 37, 718–722. Vergnes, M., Boehrer, A., Simler, S., Bernasconi, R., Marescaux, C., 1997. Opposite effects of GABAB receptor antagonists on absences and convulsive seizures. European Journal of Pharmacology 332, 245–255. Watts, A.E., Jefferys, J.G., 1993. Effects of carbamazepine and baclofen on 4-aminopyridine-induced epileptic activity in rat hippocampal slices. British Journal of Pharmacology 108, 819–823. Wurpel, J.N., 1994. Baclofen prevents rapid amygdala kindling in adult rats. Experientia 50, 475–478. Yoshimura, M., Yoshida, S., Taniyama, K., 1995. Desensitization by cyclic AMP-dependent protein kinase of GABAB receptor expressed in Xenopus oocytes. Life Sciences 57, 2397–2401.