Suppression of pilocarpine-induced ictal oscillations in the
Transkript
Suppression of pilocarpine-induced ictal oscillations in the
Epilepsy Research 49 (2002) 61 – 71 www.elsevier.com/locate/epilepsyres Suppression of pilocarpine-induced ictal oscillations in the hippocampal slice Eldad J. Hadar b, Yili Yang a, Umit Sayin a, Paul A. Rutecki a,b,c,d,* a Department of Neurology, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA Department of Neurosurgery, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA c Department of Neuroscience Training Program, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA d William Middleton VA Hospital, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA b Received 29 October 2001; received in revised form 21 January 2002; accepted 28 January 2002 Abstract Activation of muscarinic cholinergic receptors produces oscillations in the hippocampal slice that resemble the theta rhythm, but also may produce abnormal synchronous activity that is more characteristic of epileptiform activity. We used pilocarpine, a muscarinic agonist and convulsant, and an elevation in extracellular potassium (5 – 7.5 mM) to produce synchronous neuronal activity that was prolonged ( \ 2 s) and mimicked synchronization noted during seizures in vivo (ictal activity). In the CA3 region of adult rat hippocampal slices, prolonged ictal oscillations consisted of rhythmic field potentials occurring at 4–10 Hz for up to 30 s (ictal duration) that occurred in a regular periodic pattern every 12–166 s (ictal interval). The duration and interval between ictal oscillations were measured before and after application of drugs to define determinants of ictal occurrence. High threshold calcium channel antagonists (nifedipine and verapamil) blocked ictal activity. Release of calcium from intracellular stores also appeared to be important for ictal synchronization because ictal activity was blocked by dantrolene, an inhibitor of calcium release from intracellular stores, and by thapsigargin which blocks the ATPase that maintains intracellular calcium stores. These suppressive effects appeared to be postsynaptic because nifedipine, dantrolene, and thapsigargin had no effect on evoked fEPSPs. Enhancement of presynaptic inhibition by activation of GABAB or adenosine A1 receptors suppressed ictal activity and depressed the amplitude of evoked population synaptic potentials. The results point to an important role for high threshold calcium channels and release of calcium from intracellular stores in addition to strength of synaptic connections in generation of prolonged oscillations that underlie seizure activity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: L-type calcium channel; Dantrolene; Thapsigargin; GABAB; Adenosine; CA3 1. Introduction * Corresponding author. Tel.: +1-608-280-7057; fax: + 1608-263-0412. E-mail address: rutecki@neurology.wisc.edu (P.A. Rutecki). Neuronal synchronization manifest as rhythmic activity recorded extracellularly occurs during normal and abnormal conditions. The hippocampus demonstrates a variety of normal oscillatory 0920-1211/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 0 - 1 2 1 1 ( 0 2 ) 0 0 0 1 6 - 5 62 E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 activity (Bland, 1986; Fisahn et al., 1998; Traub et al., 1998), but is susceptible to abnormal synchronization that is epileptiform in character (Traub et al., 1992; Williams and Kauer, 1997; Rutecki and Yang, 1998). Interictal epileptiform activity consists of brief (B500 ms) recurrent discharges that underlie the interictal spike or sharp wave recorded in the EEG. More prolonged synchronous activity characterizes a seizure or ictal discharge. The transition from a pattern of interictal epileptiform activity to an ictal pattern is the critical determinant of seizure occurrence. Studies of epileptiform activity in the hippocampal slice support the hypothesis that the interictal discharge is driven by synchronous synaptic transmission (Johnston and Brown, 1982; Miles and Wong, 1983; Rutecki et al., 1985). Synchronous synaptic input underlies prolonged ictal-like activity produced by pilocarpine and elevated [K+]o (Traynelis and Dingledine, 1988; Rutecki and Yang, 1998). Fewer studies have defined factors that control the transition from an interictal to an ictal pattern of epileptiform activity (Anderson et al., 1986; Swartzwelder et al., 1987; Traynelis and Dingledine, 1988; Swann et al., 1993; Nagao et al., 1996; Barbarosie and Avoli, 1997; Rutecki and Yang, 1998). In the hippocampal slice, ictal patterns depend on the concentration of extracellular potassium ([K+]o) (Traynelis and Dingledine, 1988; Rutecki and Yang, 1998), a reduction in extracellular space (Traynelis and Dingledine, 1989; Rutecki and Yang, 1998) or other nonsynaptic mechanisms of synchronization (Schweitzer et al., 1992; Jefferys, 1995), the occurrence of prolonged synchronized synaptic input (Swann et al., 1993; Rutecki and Yang, 1998), and in some cases GABAergic synaptic transmission (Avoli et al., 1996; Williams and Kauer, 1997; Kohling et al., 2000). Pilocarpine, a muscarinic agonist and convulsant, activates phosphotidyl inositol metabolism and the production of inositol triphosphate (IP3) that can cause release of calcium from intracellular stores (Irving and Collingridge, 1998; Nakamura et al., 1999). Calcium may also be released from intracellular stores by calcium-dependent mechanisms (Simpson et al., 1995; Emptage et al., 1999). Release from intracellular stores can induce cal- cium oscillations (Charles et al., 1993; Simpson et al., 1995; Berridge, 1998; Irving and Collingridge, 1998) and the potential role of calcium release from intracellular stores in synchronizing a network of neurons is not clear (Berridge, 1998). Modeling studies have suggested that regenerative calcium currents and calcium influx in dendrites of CA3 neurons favor more prolonged ictal patterns of activity (Traub et al., 1993, 1996). Furthermore, non-inactivating dendritic calcium current was used in modeling to create ictal discharges (Traub et al., 1993). Voltage-dependent calcium channels exist in the dendrites of CA3 neurons (Wong and Prince, 1979) and may be activated by synaptic potentials or back-propagating action potentials (Jaffe et al., 1992; Avery and Johnston, 1996; Magee et al., 1996). In the present study, we evaluated the effects of calcium channel blockade and manipulations that alter the release of calcium from intracellular stores. These effects are compared to those produced by activation of presynaptic receptors that inhibit synaptic transmission (GABAB and adenosine A1). Our findings point to the important role of synaptic transmission as well activation of high threshold L-type calcium channels and release of calcium from intracellular stores in the generation of recurring ictal oscillations produced by pilocarpine and elevated [K+]o. 2. Methods 2.1. Slice preparation Hippocampal slices were prepared from young adult male Sprague–Dawley rats that weighed 125–300 g. Rats were anesthetized with ether or pentobarbital (60–75 mg/kg given i.p.), decapitated, and the hippocampus was removed and transferred to iced artificial cerebrospinal fluid (ACSF). Transverse slices (400–500 m thick) were prepared with either a McIllwain tissue chopper or vibratome (Technical Products International) and transferred to an interface chamber. Slices were incubated for 1 h while perfused (0.3–0.5 ml/min) with ACSF that contained (in mM): NaCl 124, KCl 5, NaH2PO4 1.25, CaCl2 2, E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 63 MgSO4 2, NaHCO3 26, glucose 10. The bathing solution was then changed to one containing 10 mM pilocarpine and an extracellular potassium concentration ([K+]o) of either 5 or 7.5 mM. Most (85 of 90 slices) of the ictal recordings were made in 7.5 mM [K+]o, a concentration that favors ictal activity (Rutecki and Yang, 1998), and all interictal recordings were made in 7.5 mM [K+]o ACSF. Recordings began after a 1-h exposure to the pilocarpine containing saline. 2.2. Recording techniques, characterization, and quantification of epileptiform acti6ity Extracellular recordings were made from the CA3b or CA3c subfield using either a Getting amplifier (Model 5A) or an Axoclamp 2 amplifier in current clamp mode. Glass microelectrodes of 2– 10 MV resistance filled with 2 mM NaCl were positioned in stratum pyramidale and adjusted to obtain a maximal amplitude signal. Spontaneously occurring activity was monitored and characterized as desynchronized unit activity or epileptiform activity. The epileptiform activity was characterized as either recurrent, brief (B 500 ms duration) interictal or ictal discharges that consisted of recurrent, prolonged oscillations that lasted greater than 2 s occurring between 4 and 10 Hz (Rutecki and Yang, 1998) (Fig. 1). Some slices displayed a combination of interictal and ictal activity but were classified as ictal (Fig. 1). Slices with epileptiform activity were recorded from before and after ascending concentrations of drugs were added to the bathing solution. Each concentration (between 2 and 4) was applied for 30– 60 min before the next higher concentration was applied. Multiple slices were monitored and care was taken to record from the same area of CA3 for any given slice. This required movement of the recording electrode. Although the amplitude often changed, the pattern of activity did not appear to differ between multiple recordings from the same slice bathed in the same solution. Three–five minute epochs of activity were digitized using a Digidata 1200 interface (Axon Instruments). The duration of ictal discharges and the interval between ictal discharges were measured from digital records (see Fig. 1). A conver- Fig. 1. High threshold calcium channel blockade suppressed ictal activity. (A) Top trace shows the ictal pattern of epileptiform activity produced by 10 mM pilocarpine in 7.5 mM [K+]o. The recording was made in CA3 stratum pyramidale and demonstrates population activity occurring at 4 – 6 Hz. The second trace shows that ictal activity and its recurring pattern. The solid line demonstrates the ictal duration measurement, and the dashed line the ictal interval measurement. At 0.1 mM, nifedipine suppressed the occurrence of ictal discharges and at 1 mM stopped their occurrence. (B) Both nifedipine and verapamil decreased the occurrence of ictal discharges in a dose-dependent fashion. At higher concentrations both high threshold calcium channel blockers reduced the number of slices demonstrating ictal patterns and hence the large SEMs. The changes were significant by Kruskal – Wallis (P =0.0011 for nifedipine, P= 0.0038 for verapamil). (C) The ictal duration was also reduced in a dose-dependent manner (PB 0.05 post hoc comparisons for all concentrations of nifedipine and concentrations of 10 mM or greater for verapamil). 64 E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 sion of an ictal pattern to an exclusively interictal pattern was noted and the ‘ictal duration’ was considered to be zero. The rate of interictal discharges in slices only displaying interictal activity were measured and compared to the rate after a drug application. To assess synaptic transmission, association/ commissural fibers were stimulated in stratum radiatum and the resultant field excitatory synaptic response (fEPSP) was measured in stratum radiatum in the CA3b subfield. Stimulation was delivered through a bipolar electrode with a pulse duration of 50 ms. The stimulus intensity was adjusted to just below population spike threshold as judged by contamination of a population spike in the synaptic sink waveform. The position of the field and stimulating electrode were maintained before and after solution changes. Stimulation was given at 0.1 Hz, and five responses were averaged every 5 min. After a 15-min baseline was obtained, the solution was changed to one containing the effective concentration of a agent that altered ictal activity. Comparisons were made at 20–30 min following bath change and in the case of thapsigargin following a 30-min wash to pilocarpine and high [K+]o. 2.3. Chemicals and solutions All drugs were obtained from Sigma except for thapsigargin that was obtained from Calbiochem; nifedipine, N6-cyclopentyladenosine (CPA), dantrolene, and verapamil from RBI; and 2-hydroxysaclofen from Tocris. All drugs were made as a stock solution, frozen, thawed, and then added to a larger volume of ASCF to achieve the desired concentration. Nifedipine, dantrolene, and thapsigargin were prepared in a DMSO vehicle, and in these cases DMSO was added to the control ACSF containing pilocarpine. 2.4. Statistics Parametric testing (paired t-test or ANOVA) was used when the data were normally distributed, and non-parametric testing (Mann– Whitney, Krushkal Wallace) when the distribution was not normal. Significance was set at less than 0.05. 3. Results 3.1. High threshold calcium channel antagonists block ictal discharges Two L-type calcium channel blockers were evaluated for effects on the pattern of interictal or ictal discharges produced by elevated [K+]o and pilocarpine. Nifedipine, a dihyropyridine derivative, or verapamil, a phenylalkylamine compound, both suppressed the occurrence of ictal discharges in a dose-dependent manner. In the presence of 0.3 mM nifedipine, 10 of 16 slices that initially demonstrated ictal activity converted to an interictal pattern of activity. At 1 mM, nifedipine converted four of the six slices with residual ictal discharges to only interictal patterns of activity (Fig. 1A). Verapamil (30 mM) changed an ictal pattern to an interictal pattern in 11 of 14 slices. The interval between ictal discharges was more than doubled at lower concentrations of nifedipine (0.3 mM) and verapamil (10 mM) (Fig. 1B). Both compounds also decreased the duration of the ictal discharge in a dose-dependent manner (Fig. 1C). In slices that only displayed interictal discharges initially, nifedipine did not significantly change the rate of interictal discharges (control rate of 0.309 0.02 and 0.249 0.02 Hz for 1 mM nifedipine, n= 17). The suppressant effect of L-type calcium channel blockade could be explained by a depression in synaptic transmission; however, nifedipine at a concentration 10-fold greater than that which blocked ictal activity did not reduce the amplitude of the fEPSP (see below, Table 1). 3.2. Role of intracellular calcium release and ictal acti6ity To assess the potential role of intracellular calcium release on the production of ictal activity, we evaluated the effects of two compounds that depress intracellular calcium release, dantrolene and thapsigargin. Dantrolene prevents the release of calcium from intracellular stores (Frandsen and Schousboe, 1991; Charles et al., 1993). At 30 mM, dantrolene prolonged the interval between ictal discharges by more than 100% (Fig. 2). At 100 E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 65 Table 1 Effect of drugs that suppress ictal activity on evoked population EPSPs % Control evoked population EPSP9 SEM Baclofen 1 mM CPA 100 nM Nifedipine 10 mM Dantrolene 100 mM Thapsigargin 5 mM Acute Wash 52.49 6.1a 56.29 8.5a 1059 9.2 1069 3.8 10894.1 10694.9 All measurements were compared to control; measurements made 30 min after drug exposure except for thapsigargin, which included acute 30 min exposure, followed by a 30 min wash. CPA refers to N6-cyclopentylodenosine. a Significantly different from other experiments, F = 13.3, PB0.0001, ANOVA and PB0.05 by post hoc analysis. N= 5 slices for all experiments. mM, dantrolene converted the pattern of epileptiform activity from ictal to interictal in seven of 11 slices. The effects on interval between discharges and ictal duration were dose-dependent (Fig. 2B and C). Thapsigargin (1 mM), an irreversible blocker of the ATP-dependent calcium pump that creates the concentration gradient for endoplasmic reticulum calcium storage (Irving and Collingridge, 1998), converted the ictal pattern of activity to an interictal pattern in three of 12 slices and caused one slice to stop having spontaneous epileptiform activity. In the eight slices that continued to demonstrate ictal patterns, the duration of the ictal discharge was decreased significantly (16.99 2.5 – 7.3 90.8 s) and the interval between discharges was shortened (from 42.997.3 to 23.69 1.8 s, P B0.05, Fig. 3). After a 30– 40 min exposure, 50% of all slices stopped having spontaneous activity and five of eight slices with ictal activity converted to an interictal pattern. Changing the solution back to pilocarpine and 7.5 mM [K+]o did not reverse the effect. At a concentration of 5 mM, thapsigargin converted the ictal pattern observed in five slices to an interictal pattern in two slices and spontaneous activity stopped in another slice. With prolonged exposure (\ 30 min) fol- Fig. 2. Dantrolene effects on ictal activity. (A) The top trace represents control ictal activity in the presence of pilocarpine and elevated potassium. Dantrolene (30 mM) suppressed ictal occurrence and interictal discharges were observed. (B) Dantrolene increased the interval between ictal discharges in a dose-dependent fashion (P= 0.003, Kruskal – Wallis, significant for a concentration of 100 mM by Dunn’s method of comparisons). (C) Dantrolene also reduced the ictal duration as a function of concentration (P =0.015, Kruskal – Wallis, significant for 100 mM by Dunn’s method). lowed by return to pilocarpine and 7.5 mM [K+]o, only one slice displayed spontaneously occurring interictal discharges. Fig. 3. Thapsigargin alters ictal discharges in a time-dependent manner. Top trace shows ictal activity produced by pilocarpine and high [K+]o. The ictal discharges became more frequent but shorter in the presence of 1 mM thapsigargin. The ictal activity was replaced by interictal activity that continued despite wash to control solution (10 mM pilocarpine and 7.5 mM [K+]o). 66 E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 nM concentration of CPA resulted in ictal suppression in four of the six slices that continued to display ictal discharges in 30 nM CPA. 3.4. Effects on fEPSP Fig. 4. GABAA or adenosine A1 receptor activation block ictal discharges. (A) Baclofen at a concentration of 1 mM suppressed ictal discharges. (B) The dose-dependent effects of CPA on ictal discharges. At a concentration of 10 nM, CPA had a minimal effect on ictal discharges, but 30 nM totally suppressed ictal discharges. 3.3. Enhanced presynaptic inhibition suppressed ictal discharges Baclofen, a GABAB agonist reduces synaptic transmission at both GABA and glutamate containing synapses and activates a potassium channel conductance that creates a long lasting synaptic inhibition (Bowery et al., 1980; Newberry and Nicoll, 1984; Thompson and Gahwiler, 1992). Baclofen (0.3–1 mM) converted the ictal pattern of activity produced by pilocarpine to an interictal pattern in six of seven slices (Fig. 4A). In five slices that displayed an interictal pattern of activity initially, baclofen slowed the rate of interictal discharges (0.2890.06 – 0.05 90.02 Hz, P B 0.005 paired t-test). The GABAB antagonist 2-hydroxysaclofen did not alter the pattern of pilocarpine-induced ictal activity. In pilocarpine control saline, 10 of 13 slices demonstrated ictal activity, and nine of 13 slices still demonstrated ictal activity in the presence of 300 mM 2-hydroxysaclofen. No significant change in the duration (6.89 0.6 vs. 6.390.5 s) or interval between discharges (55.49 14.7 vs. 43.6 913.7 s) was noted. CPA is an adenosine A1 agonist and results in presynaptic inhibition and postsynaptic potassium conductance activated by a G protein (Dragunow, 1988; Yoon and Rothman, 1991). Like baclofen, CPA suppressed ictal discharges in a dose-dependent manner (Fig. 4B). At a concentration of 30 nM, CPA suppressed the occurrence of ictal discharges in eight of 14 slices. An increase to 100 The effects of baclofen and CPA may be due to presynaptic inhibition and reduction in the strength of recurrent synaptic connections. We assessed the effects of CPA and baclofen on the fEPSP evoked by stimulating stratum radiatum. Both compounds reduced the fEPSP by about 50% (Table 1). The other manipulations that resulted in suppression of ictal activity were evaluated for effects on the evoked population EPSP. We found that nifedipine (10 mM) did not change the amplitude of the fEPSP recorded in stratum radiatum of CA3 that was evoked by stratum radiatum stimulation (Table 1). Dantrolene and thapsigargin, at concentrations that inhibit ictal discharges, also had no effect on the population EPSP (Table 1). 4. Discussion 4.1. High threshold calcium channel blockers inhibit ictal patterns The dihydropyridine high threshold L-type calcium channel blocker nifedipine, and verapamil, another L-type channel blocker with different molecular characteristics from nifedipine, depressed ictal discharges in a dose-dependent manner. Nifedipine, at a concentration of 10 mM, did not change the population EPSP and others have not found a significant effect of L-type calcium channel blockers on synaptic transmission in the hippocampus (Wheeler et al., 1994). Instead the effect is more likely on dendritic or somatic high threshold calcium channels. The role of dendritic calcium spikes in generation of epileptiform activity has been postulated since the 1960s (Purpura et al., 1966; Wong and Prince, 1979; Schwartzkroin and Wyler, 1980). Others have described antiepileptic effects of high threshold calcium channel blockers on interictal epileptiform activity in the slice (Vezzani et al., 1988; Bing- E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 mann and Speckmann, 1989; Aicardi and Schwartzkroin, 1990), and some anticonvulsants block calcium currents at high therapeutic concentrations (Macdonald and Kelly, 1993). High threshold calcium channels blockers may inhibit ictal patterns of activity by reducing calcium influx at dendritic calcium channels activated by either synaptic depolarization or back-propagation of action potentials. A persistent dendritic calcium current has been used in modeling studies to produce ictal-like discharges and a long-lasting calcium current has been described in CA3 neurons (Traub et al., 1993; Avery and Johnston, 1996). Additionally, muscarinic activation produces a sustained inward current blocked by L-type channel blockade (Fraser and MacVicar, 1996) and such a current could add to the sustained activity that occurs during prolonged ictal discharges. High threshold calcium channel blockade inhibits pilocarpine-induced seizures in vivo and reduces associated cellular loss (Marinho et al., 1997). 4.2. Releasable intracellular calcium stores Our results also point to an important role of calcium release from intracellular stores in the occurrence of ictal discharges. A variety of cells, including neurons and glia are capable of generating intracellular calcium oscillations that are dependent on the release of calcium from intracellular stores. These stores include those released by interaction of IP3 and its receptor or calcium with the ryanodine receptor (Simpson et al., 1995; Berridge, 1998). Our results do not differentiate between these receptors. Thapsigargin produced an irreversible depression of ictal discharges and no change in the evoked population EPSP. The effects of thapsigargin were initially to enhance the frequency of occurrence of ictal discharges, albeit with an associated decrease in ictal duration. This initial enhancement may relate to a decrease in the endoplasmic buffering capability produced by blockade of the ATPase. With time the shortening and eventual suppression of ictal discharges probably results from a loss of releasable calcium from intracellular stores. 67 Dantrolene decreases the release of calcium from intracellular stores in a variety of preparations including calcium release produced by carbachol in acinar cells (Zhang and Melvin, 1993). Dantrolene depresses calcium oscillations in glia (Charles et al., 1993) and can protect against neuronal death following ischemia (Zhang et al., 1993) or high intensity electrical stimulation (Pelletier et al., 1999). In addition, dantrolene has been reported to suppress seizures produced by metabotropic glutamate receptor activation (McDonald et al., 1993), and protects against epileptiform activity that follows trauma in neocortical slices (Yang and Benardo, 1997). The fEPSP was not affected by dantrolene or thapsigargin so the effects do not appear to be related to dampening of synaptic transmission but rather to action at calcium release sites in the soma or dendrites. Calcium entry from the extracellular space has also been hypothesized to replenish intracellular calcium stores (Simpson et al., 1995; Berridge, 1998; Irving and Collingridge, 1998). High threshold calcium channel blockade may have an indirect effect by limiting a source of calcium to replenish stores. Arguing against this hypothesis is that blockade of NMDA channel opening and another potential source of extracellular calcium influx had no effect on ictal patterns (Rutecki and Yang, 1998). Alternatively, calcium entering from the extracellular space through high-threshold channels could lead to calcium-dependent calcium release. Muscarinic receptor activation has been shown to result in an increase in dendritic calcium (Muller and Connor, 1991; Irving and Collingridge, 1998). Although an enhancement in release of calcium mediated by IP3 is expected following application of pilocarpine, calcium entering from high threshold plasma membrane channels may also participate in calcium-dependent calcium release or enhance IP3-mediated calcium release (Irving and Collingridge, 1998; Nakamura et al., 1999). The synchronous oscillation that comprises the ictal discharge should have an associated calcium flux from voltage- and synaptic-dependent mechanisms. Depressing the release of calcium from intracellular stores by dantrolene or thapsigargin and the 68 E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 resultant suppression of ictal activity point to a role of oscillations of intracellular calcium as a mechanism that supports neuronal synchronization that underlies ictal activity. Calcium oscillations typically have a time course of seconds and hence are more likely to influence the duration of ictal events and the period between them (HarrisWhite et al., 1998; Irving and Collingridge, 1998). We hypothesize that the period between ictal discharges is controlled by activation of calcium-dependent potassium currents. Our results suggest that calcium release form intracellular stores may entrain a network of neurons connected synaptically. One possible mechanism for this effect is the activation of a calcium-dependent non-specific cationic current that maintains depolarization above action potential threshold (Fraser and MacVicar, 1996). 4.3. Presynaptic inhibition, role of synaptic dri6e Both interictal and ictal patterns of epileptiform activity are driven by synchronous synaptic drive (Johnston and Brown, 1982; Miles and Wong, 1983; Swann et al., 1993; Rutecki and Yang, 1998) and some convulsants appear to act by enhancing both excitatory and inhibitory synaptic transmission (Rutecki et al., 1987). Transmitters that inhibit synaptic transmission presynaptically should depress synaptic synchronization. The GABAB agonist baclofen was effective in stopping ictal discharges at concentrations that reduced evoked EPSPs. The A1 adenosine agonist CPA has similar properties and adenosine has been considered to be an endogenous anticonvulsant (Dragunow, 1988). These receptors also mediate slower postsynaptic inhibition and such action should also be anticonvulsant. The concentrations of transmitter agonists that inhibited ictal discharges also depressed synaptic transmission measured at a population level. This is in contrast to the action of L-type channel blockers or inhibition of release of calcium from intracellular stores. Paradoxically, muscarinic activation inhibits synaptic transmission in the CA3 region (Valentino and Dingledine, 1981; Williams and Johnston, 1990; Sayin and Rutecki, 1997). Recent studies hypothesize that epileptiform activity in the CA3 region may be limited in duration by the availability of releasable transmitter stores (Staley et al., 1998). In the low magnesium model of epileptiform discharges, baclofen enhances the occurrence of ictal discharges (Swartzwelder et al., 1987), and this may be explained by presynaptic inhibition that limits release so that a long duration pattern of epileptiform activity may occur (Staley et al., 1998). The degree of presynaptic inhibition required for a longer duration discharge may be produced by exposure to pilocarpine and further inhibition by GABAB or adenosine A1 activation results in loss of ictal and interictal activity. In the present study, antagonism of the GABAB receptor with 2-hydroxysaclofen did not alter the pattern of epileptiform activity, suggesting that endogenous activation of GABAB receptors does not influence the generation or termination of ictal discharges. In rhythmic slow activity produced by carbachol, neither GABAA nor GABAB synaptic transmission appeared to be a critical determinant of synchronization (Macvicar and Tse, 1989; Traub et al., 1992, but see Williams and Kauer, 1997). Action potentials generated from ectopic sites including axon terminals may contribute to hippocampal synchronization (Stasheff et al., 1992; Traub et al., 1995, 1996). Activation of presynaptic receptors that act through G-proteins to decrease calcium currents, increase potassium currents, or create a shunt would be expected to decrease ectopic action potential generation near the terminal. GABAB and adenosine A1 receptor activation could depress ictal transitions by this mechanism in combination with depression of synaptic transmission. 4.4. Determinants of ictal epileptiform acti6ity Previous studies have demonstrated a relationship between extracellular potassium concentration, extracellular space, and the pattern of epileptiform activity (Traynelis and Dingledine, 1988, 1989; Schweitzer et al., 1992; Rutecki and Yang, 1998). In this study, the role for L-type calcium channel activation and release of calcium E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71 from intracellular stores appear to be critical variables involved in ictal oscillations. The possible role of changes in intracellular calcium concentrations in glia elements could also effect network synchronization and our experiments do not distinguish between effects on glia or neurons. Calcium release from intracellular stores appears to be necessary for the sustained ictal oscillation, but the mechanism by which such stores synchronize a synaptic network is not obvious. Synaptic transmission has been shown to cause calcium release from postsynaptic ryanodine-sensitive intracellular stores (Emptage et al., 1999), and postsynaptic calcium increases enhance synaptic strength (Yeckel et al., 1999). Pilocarpine and activation of group I metabotropic glutamate receptors both result in IP3-mediated calcium release from intracellular stores and result in ictal patterns of epileptiform activity in the hippocampal slice (Bianchi and Wong, 1994; Jaffe and Brown, 1994; Taylor et al., 1995; Irving and Collingridge, 1998; Bianchi et al., 1999). Muscarinic activation plays a role in generation of theta oscillations and activates a set of second messenger systems. Similar second messengers are activated by glutamate acting at the group I class of metabotropic glutamate receptors. Group I metabotropic receptor activation produces faster frequency oscillations (\20 Hz) and also produces ictal discharges in the hippocampal slice (Taylor et al., 1995; Traub et al., 1998). Activation of either muscarinic or group I metabotropic glutamate receptors results in a significant increase in intracellular calcium that may result from release from intracellular stores or by depolarization (Muller and Connor, 1991; Bianchi and Wong, 1994; Jaffe and Brown, 1994; Taylor et al., 1995; Irving and Collingridge, 1998; Bianchi et al., 1999). Depression of L-type high threshold calcium channels and release of calcium from intracellular stores both appear to be targets for antiepileptic drug development. 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