Inhibition of different GABA transporter systems is required to attenuate epileptiform activity in the CA3 region of the immature rat hippocampus

Epilepsy Research (2014) 108, 182—189

journal homepage: www.elsevier.com/locate/epilepsyres

Inhibition of different GABA transporter systems is required to attenuate epileptiform activity in the CA3 region of the immature rat hippocampus Salim Sharopov, Rongqing Chen, Haiyan Sun, Sergei N. Kolbaev, Sergei Kirischuk, Heiko J. Luhmann, Werner Kilb ∗ Institute of Physiology, University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, D-55120 Mainz, Germany Received 8 May 2013; received in revised form 30 September 2013; accepted 21 November 2013 Available online 1 December 2013

KEYWORDS Field potential recording; Development; Epilepsy; Seizure; Tiagabine

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Summary GABA transporters (GATs) are an essential element of the GABAergic system, which regulate excitability in the central nervous system and are thus used as targets for anticonvulsive therapy. However, in the immature nervous system the functions of the GABAergic system and the expression profile of GATs are distinct from the adult situation, obscuring to predict how different GAT isoforms influence epileptiform activity. Therefore we analyzed the effects of subtype specific GAT inhibitors on repetitive epileptiform discharges using field potential and whole-cell patch-clamp recordings in the CA3 region of hippocampal slices of immature (postnatal days 4—7) rats. These experiments revealed that inhibition of GAT-1 with either tiagabine (30 ␮M) or NO-711 (10 ␮M) exhibited only a minor anticonvulsive effect on repetitive epileptiform discharges. Blockade of GAT-2/3 with SNAP-5114 (40 ␮M) had no anticonvulsive effect, but significantly prolonged the decay of spontaneous GABAergic postsynaptic currents. In contrast, the combined application of 10 ␮M NO-711 and 40 ␮M SNAP-5114 blocked epileptiform activity in 33% of all slices and reduced the occurrence of epileptiform discharges by 54% in the remaining slices. In addition, the input resistance decreased by 10.5 ± 1.0% under this condition. These results indicate that both GAT-1 and GAT-2/3 are functional in the immature hippocampus and that only the combined inhibition of GAT 1—3 is sufficient to promote a considerable anticonvulsive effect. We conclude from these results that both GAT-1 and GAT-2/3 act synergistically to regulate the excitability in the immature hippocampus. © 2013 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +49 211 3926101; fax: +49 211 3926102. E-mail address: [email protected] (W. Kilb).

0920-1211/$ — see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2013.11.019

GABA transporters and epileptic activity

Introduction The amino acid ␥-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the adult brain and mediates its action via two distinct classes of receptors: ionotropic GABAA (and GABAC ) and metabotropic GABAB receptors (Farrant and Kaila, 2007). In the immature rodent hippocampus, however, activation of GABAA receptors elicits a depolarizing and in many cases excitatory response (Mueller et al., 1984; Ben-Ari et al., 1989; Lamsa et al., 2000; Achilles et al., 2007). These depolarizing GABAergic responses are caused by Cl− efflux via GABAA receptors due to the high intracellular Cl− concentration in developing neurons (Blaesse et al., 2009; Ben Ari et al., 2012). A variety of studies suggest that such a depolarizing GABAergic action is essential to generate spontaneous activity transients, which are required for the adequate development of the brain (see Ben-Ari et al., 2007; Kilb et al., 2011 for review). The higher incidence for epileptic seizures during the first postnatal weeks in human development has been attributed to these depolarizing GABAergic responses (Sanchez and Jensen, 2001; Ben Ari et al., 2007), although there is no clear evidence for depolarizing GABAergic responses in human fullterm newborns (see Loscher et al., 2013 for critical review) and a variety of studies demonstrated that the GABAergic system can mediate an inhibitory action already in the immature rodent brain with clear depolarizing actions (Baram and Snead, 1990; Khalilov et al., 1997; Wells et al., 2000; Kilb et al., 2007; Richter et al., 2010). In addition, the poor pharmacological responsiveness of childhood seizures (Booth and Evans, 2004; Silverstein and Jensen, 2007) has been attributed to the distinct molecular and functional properties of the GABAergic system during early development. Beside depolarizing GABAergic responses, the late development of GABAB receptors (Fukuda et al., 1993; Gaiarsa et al., 1995), different properties of synaptic and extrasynaptic GABAergic currents (Marchionni et al., 2007; Kolbaev et al., 2012) as well as an altered molecular composition of GABAA receptors (Fritschy et al., 1994; Taketo and Yoshioka, 2000) can also contribute to the higher seizure susceptibility and the poor pharmacological control in the immature brain. Another important element of the GABAergic system is GABA transporters (GATs), which mediate the sequestration of released GABA from the synaptic cleft (Borden, 1996), but also directly regulate interstitial GABA levels (Richerson and Wu, 2003). GATs belong to the superfamily of solute carriers and mediate the symport of 1 molecule GABA with 1 Cl− ion and 2 Na+ ions (Nelson, 1998). Four different GAT isoforms are identified in rodents and humans, with GAT-1, GAT-2 and GAT-3 described in the CNS (Borden, 1996). In the adult brain GAT-1 is preferentially synaptically located, suggesting a role for fast removal of GABA from the synaptic cleft and GABA uptake to the presynaptic terminal, while GAT2 immunoreactivity was mainly detected in leptomeninges, suggesting that GAT-2 may regulate GABA levels in the cerebrospinal fluid, and GAT-3 is predominantly expressed in distal astrocytic processes (Conti et al., 2004). According to their essential function within the GABAergic system, GATs have been linked to the etiology of epilepsy (Allen et al., 2004; Kim et al., 2011) and blockers of these transporters are used for antiepileptic medication (Gram, 1994; Dalby,

183 2003). However, during postnatal development substantial changes in the expression of GATs occur. In the rat neocortex GAT-1 expression is rather low at birth and reaches adult levels approximately by the end of third postnatal week, while GAT-2 and GAT-3 expression is already relatively high at birth and reaches the adult levels during second postnatal week (Minelli et al., 2003; Conti et al., 2004). In addition, during this developmental period an expression of GAT-1 in astrocytes and of GAT-3 in neurons has been reported (Minelli et al., 2003; Conti et al., 2004). Given this distinct GAT expression and the heterogeneous GABA actions in the immature brain, the consequences of GAT blockade on the excitability of the immature brain are difficult to predict. To address the question, how different GATs influence the excitability of the immature hippocampus, we investigated the effects of specific blockers of GAT subtypes on repetitive epileptiform discharges using field potential recordings in the CA3 region of hippocampal slices of immature rats. In addition, we performed whole-cell patch-clamp recordings from CA3 pyramidal cells to analyze the effect of GABA transport blockers on isolated GABAergic postsynaptic currents.

Experimental procedures Slice preparation All experiments were conducted in accordance with EU directive 86/609/EEC for the use of animals in research and were approved by the local ethical committee. All efforts were made to minimize the number of animals and their suffering. Wistar rat pups of postnatal days 4—7 (P4-7) were obtained from the local breeding facility and were deeply anesthetized by enflurane (Ethrane, Abbot Laboratories, Wiesbaden, Germany). The brains were quickly removed and immersed for 2—3 min in ice-cold standard artificial cerebrospinal fluid (ACSF, composition see below). Coronal slices (400—600 ␮m thickness) including the hippocampus were cut on a vibratome (Vibroslicer 752 M, Campden Instruments Ltd., Leicester, UK, or HR2, Sigmann Elektronik, Hüffenhardt, Germany). The slices were transferred to an interface-type recording chamber where they were continuously superfused with ACSF at a rate of 1—2 ml/min at 31 ± 1 ◦ C. The slices were allowed to recover for at least 1 h under these conditions. For the patch-clamp experiments 400 ␮m thick slices were stored in an incubation chamber filled with oxygenated ACSF at room temperature for at least 1 h before they were transferred to a submerged recording chamber.

Data acquisition and analysis Extracellular field potentials were recorded with tungsten microelectrodes (impedance 4—5 M; FHC, Bowdoinham, ME) in the stratum radiatum of the hippocampal CA3 region as described before (Kilb et al., 2006). Signals were amplified by a purpose built amplifier in AC mode (cutoff frequency 3.8 Hz), low-pass filtered at 3 kHz and stored on a PC using an AD/DA board (ITC-16, HEKA, Lamprecht, Germany) and TIDA software (HEKA). Extracellular field potentials were recorded simultaneously from maximally 4 separate

184 hippocampal slices and were analyzed independently. Slices that did not respond (in total 10 of 118 investigated slices) with epileptiform activity upon the application of 10—50 ␮M 4-AP in low-Mg2+ solution were discarded from analysis. All recordings were analyzed using the program MiniAnalysis 4.3.3 (Synaptosoft, Leonia, NJ). Epileptiform events were identified according to their amplitude and shape by setting the parameters of the MiniAnalysis program. The epileptiform events identified by the program were inspected by eye, unless the number of events were >1000 and the error rate in the first 100 inspected events was less than 3%. Epileptiform activity was characterized by the occurrence of repetitive epileptiform discharges as well as by the amplitude, frequency and number of spikes within such a repetitive epileptiform event. Bursts, which consisted of more that ca. 50 spikes were classified by eye as ictal-like discharges and bursts that consisted of 2—30 spikes and repeated regularly with a similar number and frequency of spikes at interburst intervals between ∼3 and 30 s were classified as recurrent epileptiform discharges (RED). Whole-cell patch-clamp recordings were performed as described previously (Achilles et al., 2007) at 31 ± 1 ◦ C in a submerged-type recording chamber attached to the fixed stage of a microscope (BX51 WI, Olympus or Axioscope II, Zeiss). Pyramidal neurons in the stratum radiatum of the CA3 region were identified by their location and morphological appearance in infrared differential interference contrast image. Patch-pipettes (5—12 M) were pulled from borosilicate glass capillaries (2.0 mm outside, 1.16 mm inside diameter, Science Products, Hofheim, Germany) on a vertical puller (PP-830, Narishige) and filled with pipette solution containing (in mM) 86 K-gluconate, 44KCl, 1CaCl2 , 2MgCl2 , 11EGTA, 10HEPES, 2Na2 -ATP, 0.5Na-GTP (pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose), resulting in a Cl− concentration in the pipette ([Cl− ]p ) of 50 mM. For some experiments the [Cl− ]p was reduced to 10 mM by replacing 40 mM KCl with 40 mM K-gluconate. Potentials were corrected for liquid-junction potentials of 9.1 mV. Most experiments were performed at a holding potential of −69 mV. Isolated tonic GABAergic currents were analyzed at a holding potential of −66 mV for a [Cl− ]p of 50 mM and at −106 mV for [Cl− ]p of 10 mM to yield comparable electromotive forces on Cl− ions. These experiments were performed in the continuous presence of 100 ␮M 4Aminopyridine, 2 mM CsCl, and 2 mM BaCl2 to reduce the background conductances. Signals were recorded with a discontinuous voltage-clamp/current-clamp amplifier (SEC05L, NPI, Tamm, Germany), low-pass filtered at 3 kHz and stored and analyzed using an ITC-1600 AD/DA board (HEKA) and TIDA software. For statistical analysis Wilcoxon rank-sum and Sign tests were used (Systat 11, Point Richmond, CA). Significance was assigned at levels of 0.05 (*), 0.01 (**) and 0.001 (***).

Solutions and drugs Solutions were prepared from 10× concentrated stock solutions. Glucose was added to the solutions on the day of the experiment. All solutions were equilibrated with 95% O2 /5% CO2 at least 1 h before use. Standard ACSF consisted of (in mM) 126NaCl, 26NaHCO3 , 1.25NaH2 PO5 ,

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Figure 1 Activation of GABAA receptors mediates an inhibitory action in the immature hippocampus. (A) Typical field potential registration illustrating the development of epileptiform activity induced by low-Mg2+ solution containing 20 ␮M 4-AP. After a few ictal like discharges epileptiform activity changed to a recurrent epileptiform discharges pattern. (B) Representative trace illustrating two typical recurrent epileptiform discharges (RED). Note the similarity between both discharges. (C) Bath application of the GABAA receptor agonist muscimol suppressed RED, suggesting that GABA mediates an inhibitory action under this condition.

1MgCl2 , 2CaCl2 , 2.5KCl, 10glucose (pH 7.4, osmolarity 306 mOsm). For low-Mg2+ solutions MgCl2 was replaced by 1 mM CaCl2 . Tiagabine ((−)-(R)-1-[4,4-bis(3-methyl-2thienyl)-3-butenyl] nipecotic acid hydrochloride), NO-711 (1-[2-[[(diphenylmethylene)imino]oxy-ethyl]-1,2,5,6tetrahydro-3-pyridinecarboxylic acid hydrochloride), SNAP-5114 (1-[2-[tris(4-methoxyphenyl) methoxy]ethyl](S)-3-piperidinecarboxylic acid) and 4-aminopyridine (4-AP) were purchased from Sigma-Aldrich (Taufkirchen, Germany) and DL-2-amino-5-phosphonopentanoic acid (APV), 6-cyano7-nitroquinoxaline-2,3-dione (CNQX), and muscimol from Biotrend. 4-AP, APV, CNQX, picrotoxin, gabazine, tiagabine, and SNAP-5114 were dissolved in dimethylsulfoxide (DMSO, Sigma). Muscimol, NO-711 and GABA were dissolved in distilled water. All substances were added to the solutions shortly before the experiment. The DMSO concentration of the final solution never exceeds 0.2%.

Results Properties of epileptiform activity In the present study we used a combined 4-AP/low-Mg2+ solution to provoke highly repetitive epileptiform discharges, which enabled us to monitor even subtle changes in epileptiform activity (Kilb et al., 2006, 2007). Bath application of 10—50 ␮M 4-AP in low-Mg2+ solution reliably induced epileptiform activity in hippocampal slices (Fig. 1A and B). This epileptiform activity started in 62% of the recordings with one to few ictal-like discharges and developed within 24 ± 5.6 min (n = 67) into recurrent epileptiform discharges (RED) consisting of 11 ± 1.8 spikes (n = 108) appearing at a frequency of 10.8 ± 1.2 Hz with an average amplitude of 356 ± 26 ␮V (Fig. 1B). The average occurrence of RED was 15.5 ± 0.8 min−1 (n = 108). In accordance with previous in vitro and in vivo studies demonstrating that GABAA receptors already mediate a global inhibitory effect in the early postnatal brain (Baram and Snead, 1990; Khalilov et al., 1997; Wells et al., 2000;

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Kilb et al., 2007; Kolbaev et al., 2012), bath application of the GABAA receptor agonist muscimol (2—3 ␮M) completely suppressed epileptiform activity in all 12 slices investigated (Fig. 1C). We conclude from this response that anticonvulsive actions indicate an activation of GABAA receptors.

Effect of GAT-1 and GAT-2/3 inhibitors on epileptiform activity First, we investigated whether inhibition of GAT-1 attenuates epileptiform activity (Fig. 2A—C and E). Bath application of the specific GAT-1 inhibitor tiagabine (30 ␮M) had no significant effect on the occurrence of epileptiform discharges (105 ± 5.4%, n = 50, p = 0.203), slightly augmented the amplitude of spikes by 8.1 ± 3.1% (n = 50, p = 0.039), and considerably reduced the number of spikes/discharge by 17 ± 4.4% (n = 50, p = 0.016). Similar results were obtained with the GAT-1 specific antagonist NO-711 (Fig. 2E). In the presence of 10 ␮M NO-711 the number of spikes per RED was significantly (p = 0.0391) reduced by 29 ± 9.4% (n = 9), while neither the occurrence of RED (108 ± 11.1%) nor the amplitude of the spikes (112 ± 14.9%) was affected. In order to investigate whether GAT-2/3 influences epileptiform activity, we used the GAT-2/3 specific blocker SNAP-5114 (Soudijn and van Wijngaarden, 2000). In the presence of 40 ␮M SNAP-5114 neither the occurrence of RED (101.2 ± 9.6%, n = 16, p = 0.498), nor the amplitude (106.8 ± 4.1%, p = 0.136) or the number of spikes within a discharge (114.1 ± 16.4, p = 0.625) was significantly altered (Fig. 2D and E). In summary, these results suggest that inhibition of GAT-1 has a slight anticonvulsant effect, while GAT-2/3 inhibition does not attenuate epileptiform activity. Next we investigated the effect of an inhibition of both GAT-1 and GAT 2/3. The combined application of 10 ␮M NO-711 and 40 ␮M SNAP-511 massively suppressed epileptiform discharges, in contrast to the single application of these GAT inhibitors. In 17 out of 51 investigated slices, epileptiform activity ceased in 18 ± 2.1 min after the washin of NO-711 and SNAP-5114 (Fig. 3A). In the remaining 34 slices the occurrence of RED was significantly (p = 0.0006) reduced by 54 ± 6.2% (Fig. 3B and C). Neither the amplitude (117.5 ± 7.5%, n = 34) nor the number of spikes per discharge (92.2 ± 10.6%) was significantly altered under this condition

Figure 2 Effect of GAT inhibitors on epileptiform activity. (A) Typical field potential registration illustrating that bath application of the GAT-1 inhibitor tiagabine had no obvious effect on the amplitude and frequency of RED. (B and C) Typical RED, as marked in A, illustrating the decreased number of spikes per RED in the presence of 30 ␮M tiagabine. (D) Typical field potential recording illustrating that bath application of 40 ␮M SNAP 5114 had no effect on RED. (E) Statistical analyses of the effects of GAT-1 and GAT-2/3 inhibitors on the occurrence and number of spikes per RED. Bars represent mean ± SEM. Note that tiagabine and NO-711 decreased the number of spikes per RED, while SNAP-5114 had no significant effect.

(Fig. 3C). In the presence of 3 ␮M gabazine, application of NO-711/SNAP-5114 containing solutions did not block epileptiform activity (n = 14). Similarly, addition of 100 ␮M PTX to NO-711/SNAP-5114 containing solutions restored epileptiform activity in all slices that showed a complete

Figure 3 Effect of combined application of GAT-1 and GAT-2/3 inhibitors on RED. (A) Typical field potential registration of an experiment in which the combined application of 10 ␮M NO-711 and 40 ␮M SNAP 5114 induced a complete block of RED. (B) Typical field potential recording of an experiment in which the combined application of 10 ␮M NO-711 and 40 ␮M SNAP 5114 induced a reduction of RED. (C) Statistical analyses illustrating the effect of the combined NO-711/SNAP 5114 application on the occurrence, amplitude and number of spikes per RED in experiments where no complete block of RED was observed. Bars represent mean ± SEM.

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block in the presence of NO-711/SNAP-5114 (n = 5). In summary, these results demonstrate that a complete blockade of GAT-1 and GAT-2/3 considerably suppresses epileptiform discharges, suggesting that only under this condition the interstitial GABA levels are high enough to suppress excitability in the immature hippocampus.

Effect of GAT-1 and GAT-2 inhibitors on GABAergic activity In addition, we investigated the effect of these GAT inhibitors on GABAergic postsynaptic currents (GABA-PSCs), isolated in the presence of 10 ␮M CNQX and 60 ␮M APV, and tonic GABAergic currents in CA3 pyramidal neurons using whole-cell patch-clamp recordings. These experiments revealed that GABA-PSCs with an average amplitude of 77.9 ± 7.7 pA (n = 24 cells), a mean charge transfer of 1.2 ± 0.15 pC, a rise time of 4.1 ± 0.3 ms, and a decay time of 16.3 ± 1.3 ms occurred at a frequency of 3.2 ± 0.5 Hz. In the presence of 10 ␮M NO711 the frequency of the GABA-PSCs was significantly (p = 0.0215) reduced by 25 ± 8.1% (n = 10 cells), while the amplitude (78.4 ± 15.5 pA), the mean charge transfer (1.3 ± 0.3 pC), the rise time (4.0 ± 0.5 ms) and the decay time (17.4 ± 2.2 ms) were not significantly different from control conditions (Fig. 4A). Bath application of 10 ␮M NO711 did not significantly change the holding current of CA3 pyramidal neurons (−0.9 ± 0.4 pA shift, n = 6 cells), suggesting that tonic GABAergic currents were unaffected. Since this observation was different from a previous publication (Sipila et al., 2007), we performed additional experiments that resembled the conditions of the latter study. These experiments revealed that bath application of 10 ␮M NO771 did not significantly alter the holding current if a [Cl− ]p of 50 mM was used (−3.1 ± 2.5 pA, n = 7 cells), but at a low [Cl− ]p of 10 mM significantly (p = 0.0313) changed the holding current (−9.2 ± 1.4 pA, n = 6 cells). This result suggests that the intracellular Cl− concentration may influence the capacity of GAT-1 transport. Application of the GAT-2/3 inhibitor SNAP-5114 revealed different effects on GABA-PSCs (Fig. 4B). In the presence of 40 ␮M SNAP-5114, the amplitude of GABA-PSCs amounted to 74.1 ± 5.5 pA (n = 13 cells), the mean charge transfer to 1.3 ± 0.15 pC, and the frequency to 2.1 ± 0.5 Hz. While these values were comparable to the control experiments, the decay time was significantly (p = 0.0034) reduced by 25.7 ± 6.8% to a value of 18.6 ± 1.6 ms (n = 13), suggesting that GAT-2/3 contribute to the re-uptake of synapticallyreleased GABA. On the other hand, 40 ␮M SNAP-5114 did not significantly influence the holding current (−0.9 ± 1.4 pA, n = 7 cells). Under conditions that resemble the conditions used by Sipila et al. (2007), SNAP-5114 did not induce significant shifts in the holding current at a [Cl− ]p of 50 mM (−3.1 ± 2.5 pA, n = 7 cells) or at a [Cl− ]p of 10 mM (−2 ± 3.1 pA, n = 6 cells). These results indicate, that tonic GABAergic currents were unaffected by GAT-2/3 inhibition. The combined application of 10 ␮M NO-711 and 40 ␮M SNAP-5114 significantly reduced the frequency of GABAPSCs by 46.3 ± 8.2% (n = 19, p = 0.0044) and their amplitude by 14.6 ± 3.9% (n = 19, p = 0.0192), while the rise time (104 ± 3.9%) and the decay time constant (109 ± 5.6%) were

Figure 4 Effect of the GAT inhibitors on spontaneous GABAergic postsynaptic currents (GABA-PSCs) and membrane properties. (A) Typical current traces from voltage-clamp recordings of a CA3 pyramidal neurons showing pharmacologically isolated GABA-PSCs recorded under control conditions (black trace) and after the application of 10 ␮M NO-711 (gray trace). Note the slightly reduced frequency of GABA-PSCs. The right panel represents averaged, normalized GABA-PSCs recorded under control conditions (black trace) and in the presence of 10 ␮M NO-711 (gray trace). (B) Pharmacologically isolated GABA-PSCs in a CA3 pyramidal neuron recorded under control conditions (black trace) and after the application of 40 ␮M SNAP 5114 (gray trace). Note the prolonged decay in the presence of SNAP 5114 in the right panel displaying averaged, normalized GABA-PSCs. (C) GABA-PSCs recorded under control conditions (black trace) and in the presence of 10 ␮M NO-711 and 40 ␮M SNAP 5114 (gray trace). Note the reduced amplitude and frequency of GABA-PSCs. (D) Typical current responses upon a 20 mV hyperpolarizing voltage step under control conditions (black trace) or in the presence of 10 ␮M NO-711 and 40 ␮M SNAP 5114 (gray trace). Note the increased holding current and the larger current step amplitude, which indicates a reduced input resistance.

not significantly affected (Fig. 4C). In addition, in the presence of 10 ␮M NO-711 and 40 ␮M SNAP-5114 the holding current of CA3 pyramidal neurons was slightly augmented by 1.6 ± 0.8 pA, n = 19, p = 0.0636), in accordance with a significant (p = 0.0156) decrease of the input resistance by 10.5 ± 1.0% (n = 7) under this condition (Fig. 4D). In summary, these results indicate that tonic GABAergic currents were enhanced if both GAT-1 and GAT 2/3 were blocked.

Discussion The main result of our present study is that inhibition of both GAT-1 and GAT-2/3 provided a substantial anticonvulsant effect in vitro, while neither inhibition of GAT-1 nor of GAT-2/3 alone considerably attenuated epileptiform

GABA transporters and epileptic activity discharges. On the other hand, inhibition of GAT-1 changed the number of spikes per discharge and inhibition of GAT-2/3 decelerated the decay of GABAergic PSCs. In summary, these results indicate that both GAT-1 and GAT-2/3 are functional in the early postnatal hippocampus and suggest that these transporters act synergistically to regulate the interstitial GABA concentration in the immature hippocampus. In the present study we observed that the activation of GABAA receptors by muscimol suppresses epileptiform activity. This observation corroborates numerous previous reports that demonstrated a general inhibitory action of GABA already in the immature rodent CNS (Baram and Snead, 1990; Khalilov et al., 1997; Wells et al., 2000; Kilb et al., 2007; Richter et al., 2010), although we like to emphasize here that the cellular action of GABA during the early postnatal interval is complex and thus depolarizing GABAergic responses can also contribute to the generation of epileptiform activity (Dzhala and Staley, 2003; Dzhala et al., 2005; Kolbaev et al., 2012). However, we propose from our observation that anticonvulsive effects in general indicate an enhanced GABAergic drive. Here we demonstrate, to our knowledge for the first time, that only the combined application of GAT-1 and GAT2/3 inhibitors is sufficient to attenuate or even suppress epileptiform activity in the immature hippocampus. This observation strongly suggests that GAT-1 and GAT-2/3 act synergistically to maintain a low interstitial GABA concentration. Such an synergistic effect of different GAT subtypes has already been shown in the CNS, where under resting conditions only the combined application of GAT-1 and GAT-2/3 inhibitors substantially enhance tonic conductances (Keros and Hablitz, 2005; Kirmse et al., 2009; Kersante et al., 2013; Song et al., 2013). In accordance with these publications, we assume that an increased tonic conductance is responsible for the anticonvulsant effect observed after inhibition of GAT-1 and GAT-2/3. This assumption was substantiated by our observation, that a considerable tonic current and a decrease in the input resistance could be observed after inhibition of GAT-1 and GAT-2/3. In our experiments we found that a specific blockade of GAT-1 by tiagabine or NO-711 mediated only a slight anticonvulsive effect in the CA3 region of the immature hippocampus, in accordance with previous findings, which also demonstrated only a minor anticonvulsive action of tiagabine on epileptiform activity in the CA1 region of the immature hippocampus (Sabau et al., 1999). In contrast, in the adult hippocampus GAT-1 inhibitors mediate a considerably anticonvulsive effect in vitro (Pfeiffer et al., 1996, but see Sabau et al., 1999) and in vivo (Dalby, 2000; Fueta et al., 2002; Kubova et al., 1998). In addition to this lack of a clear anticonvulsant effect, we also found that GAT-1 inhibitors did not affect the properties of spontaneous GABA-PSCs in early postnatal hippocampal CA3 pyramidal neurons, in agreement with previous reports in immature CA3 pyramidal neurons (Sipila et al., 2004) and granule cells (Draguhn and Heinemann, 1996). On the other hand, application of NO-711 decreased the frequency of GABA-PSCs, an effect that was enhanced by the coapplication of NO-711 and SNAP5114. This observation may be caused by a specific inhibition of GABAergic interneurons (Semyanov et al., 2003), by an excitatory effect of moderate tonic currents (Song et al., 2011) or by an interaction of the increased interstitial GABA

187 concentration with presynaptically located GABAB receptors (Kirmse and Kirischuk, 2006; Lei and McBain, 2003). Our further experiments performed under conditions that provide a comparable Cl− driving force between both pipette solutions, revealed that GAT-1 inhibition induced significant tonic current only at a low, but not at a high intracellular Cl− concentration. A higher intracellular Cl− concentration directly influences GAT mediated GABA transport by determining the reversal potential of this Cl− dependent transmembrane transporter (Richerson and Wu, 2003; Kirischuk and Kilb, 2012). Therefore we assume that GATs in the immature nervous system mediate a less efficient uptake than in mature nervous system with a lower intracellular Cl− concentration. It has been assumed that during early postnatal development of rodents GAT-3 may account for the largest fraction of GABA transport in the neocortex (Conti et al., 2004). Accordingly, the GAT-2/3 blocker SNAP-5114 mediates a slight anticonvulsant effect in the early postnatal neocortex (Richter et al., 2010) and impeded the migration of neuronal progenitors (Bolteus and Bordey, 2004). Here, we show that SNAP-5114 decelerated the decay of GABA PSCs, thus demonstrating for the first time, that GAT-2 or GAT3 are functional also in the early postnatal hippocampus. Since it has been reported that GAT-2 expression is mainly localized in leptomenigneal cells already during early postnatal development, with only a faint expression in the brain parenchyma (Minelli et al., 2003), we assume that SNAP5114 mediates its effect predominantly via inhibition of GAT-3 in our preparations. In parallel to the lack of a major anticonvulsive effect of GAT-1 inhibition, SNAP-5114 also had no anticonvulsant effect on the immature hippocampus. In summary, our results indicate that in addition to GAT-1 also GAT-3 must be blocked in order to mediate a substantial anticonvulsive effect in the developing brain. Strategies involving two or more GATs as pharmacological targets have been already successfully used in animal models (Madsen et al., 2009). Therefore such strategies may also be useful to solve the problem of the poor pharmacological responsiveness of early childhood epilepsies (Booth and Evans, 2004; Silverstein and Jensen, 2007), although possible adverse effects associated with any interference with the GABAergic system during development must be considered (Bolteus and Bordey, 2004; Manent et al., 2007).

Acknowledgements The authors thank Sigrid Stroh-Kaffei for excellent technical assistance. The work was supported by the DFG grants KI 835/2 to WK and by the MAIFOR-program of the University Mainz.

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