The Journal of Physiology Neuroscience

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1 J Physiol (2013) pp The Journal of Physiology Neuroscience Genetic and pharmacological modulation of giant depolarizing potentials in the neonatal hippocampus associates with increased seizure susceptibility Ernesto Vargas 1,StevenPetrou 1,2,3 and Christopher A. Reid 1 1 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Melbourne, Australia 2 Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Melbourne, Australia 3 Centre for Neural Engineering, University of Melbourne, Parkville, Melbourne, Australia Key points Our earlier work established that seizure susceptibility in the GABA A γ2 R43Q epilepsy mouse may be in part due to the developmental consequences of impaired GABA- A receptor function. Giant depolarizing potentials (GDP) are brain network activity that involve GABA transmission, and they are thought to be important for the wiring of the developing brain. The GABA A γ2 R43Q epilepsy mutation may have an impact on this function. To test this, we measured GPD events in a mouse model with the human mutation and showed a significantly lower frequency of these events. We also reduced GDPs using a drug called bumetanide and showed an increased seizure susceptibility. Our data suggest that both genetic and pharmacological reductions in GDP expression can increase the likelihood of having a seizure when exposed to heat. Abstract The expression of Na + K + 2Cl cotransporter (NKCC1) is responsible for high intracellular Cl resulting in the excitatory action of GABA A receptor activation in the developing brain. Giant depolarizing potentials (GDPs) are spontaneous network oscillations that involve GABA A receptors and are thought to be important in establishing neuronal circuit wiring. Earlier work established that seizure susceptibility in the GABA A γ2 R43Q epilepsy mouse is impacted by developmental consequences of impaired GABA A receptor function. We investigated the potential mechanism of the developmental influence by recording GDPs in the CA3 pyramidal neurons from brain slices of the neonatal GABA A γ2 R43Q mouse. Interestingly, the number of GPDs was significantly lower in slices from mutant mouse compared with wild-type control, suggesting an involvement in setting seizure susceptibility. To test this idea we blocked NKCC1 with bumetanide in neonatal mice and reduced the number of GDPs to a level similar to that seen in the mutant mice. We found that neonatal treatment with bumetanide resulted in a similar level of susceptibility to thermally induced seizures as described for the GABA A γ2 R43Q mouse. These results provide evidence that a human GABA A receptor epilepsy mutation exerts a developmental influence by modulating the number of GDPs. It also draws attention to the potential risk of early treatment with bumetanide. (Received 30 April 2012; accepted after revision 24 September 2012; first published online 24 September 2012) Corresponding author C. Reid: Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010, Australia. careid@unimelb.edu.au Abbreviations ACSF, artificial cerebrospinal fluid; GDP, giant depolarizing potential; NKCC1, Na + K + 2Cl cotransporter; WT, wild-type. DOI: /jphysiol

2 58 E. Vargas and others J Physiol Introduction GABA A receptors have a well-recognized acute role setting the real time excitability of neurons and networks. However, they also play an important developmental role where the activation of these receptors is thought to be critical for defining how neuronal networks form. In the developing brain GABA promotes excitation/depolarization of neurons (Ben-Ari et al. 1989) due to the high intracellular concentration of Cl that depends on the function of the Na + K + 2Cl cotransporter (NKCC1; Russell, 2000; Blaesse et al. 2009). The NKCC1 blocker bumetanide has been proposed as a potentially effective anti-epileptic agent in the neonatal brain by shifting the reversal potential of Cl in immature neurons (Dzhala et al. 2005; Ben-Ari et al. 2011, but see also Vanhatalo et al. 2009; Brandt et al. 2010). Further, recent evidence suggests that bumetanide treatment at this early developmental stage leads to synaptic and morphological changesinthecortexthatcouldresultinsocio-behavioural consequences (Wang & Kriegstein, 2011). Dysfunction in the GABAergic system in early development may also have long-lasting effects on seizure susceptibility. For example, prenatal exposure to diazepam, a positive modulator of the GABA A receptor, alters susceptibility to a proconvulsant challenge in adult rats (Nicosia et al. 2003). We have also demonstrated in a conditional mouse model of generalized epilepsy that the GABA A γ2 R43Q mutated protein acts in early development to increase long-term seizure susceptibility (Chiu et al. 2008). The underlying cellular basis of these early developmental alterations is yet to be fully established. Giant depolarizing potentials (GDP) are normal spontaneous network oscillations that occur in the neonatal brain (Ben-Ari et al. 1989), although recently others have challenged this view (Bregestovski & Bernard, 2012, but see also Ben-Ari et al. 2012). GABA has been suggested to promote GDPs by directly contributing an excitatory current to target cells (Ben-Ari et al. 1989) and/or by depolarizing CA3 pyramidal neurons (Sipila et al. 2006). Early developmental network synchronization is thought to be important for the establishment and refinement of network circuitry (Zhang & Poo, 2001). The GABA A γ2 subunit is expressed in the developmental time window during which GDPs occur (Laurie et al. 1992). Therefore, manipulations that alter GABA A γ2 function, such as the GABA A γ2 R43Q mutation, may be expected to influence GDP expression. It is well documented that bumetamide, through its effects on Cl reversal in immature neurons, disrupts GDPs (Sipila et al. 2006; Nardou et al. 2009; Tyzio et al. 2011). In this study we explore the impact of the GABA A γ2 R43Q mutation on GDP expression. We also investigate if the GDP modulator, bumetamide, given during the neonatal period influences longer-term seizure susceptibility. Methods Brain slice electrophysiology All experiments were approved by the Animal Ethics Committee at the Florey Neuroscience Institute of Neuroscience and Mental Health, and conform to the guidelines of The Journal of Physiology. Mice were killed by rapid cervical dislocation before decapitation. A total of 60 animals was assigned to this project. Horizontal 400 μm-thick brain slices were cut from P3 P5 C57/Bl6 (n > 10) heterozygous GABA A γ2 R43Q and wild-type (WT) littermates, as described previously (Tan et al. 2007). For bumetanide experiments, slices were obtained from a separate cohort of C57/Bl6 mice. Slices were perfused either with normal artificial cerebrospinal fluid (ACSF; in mm: NaCl, 125; NaHCO 3, 26; KCl, 2.5; CaCl 2, 2.4; NaH 2 PO 4, 1.2; MgCl 2, 1.3; glucose, 10) or ACSF containing 10 μm bumetanide for 5 min before beginning recording. All recordings were made at room temperature. Whole-cell voltage-clamp recording were made using an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA) and Axograph acquisition software (Axograph, Australia) from CA3 pyramidal neurons held at 70 mv. Signals were sampled at 5 khz and low-pass filtered at 3 khz. Pipettes were filled with (in mm): CsCl, 120; KCl, 10; MgCl, 1; Hepes, 10; glucose, 4; ATP, 2; GTP, 0.3; ph 7.3. Experiments only proceeded when the online estimate of series resistance was <20 M. The average estimate of Rs was not statistically different in recordings from GABA A γ2 R43Q and WT neurons (P = 0.6). Statistical comparisons were made using an unpaired t test unless specified (StatPro, UK). Values are reported as mean ± SEM. Genotyping was done post hoc from tail DNA as previously described (Tan et al. 2007). Drugs and salts were obtained from Sigma-Aldrich (Castle Hill, Australia). Event detection GDPs were automatically detected in Axograph with a user-defined template of variable amplitude. The template was derived from the average of 10 visually identified GDPs (n = 3 cells) with a morphology similar to that presented in Fig. 1. Once defined the same template was used to detect GDPs under all conditions. Captured events with amplitudes of less than 200 pa were excluded. Detection of spontaneous spscs was also done in Axograph using the variable-amplitude sliding template algorithm. The function template had a 2 ms rise time and 15 ms time constant decay. Events detection signal-to-noise ratio threshold was set to 3 SDs. Detected events from each cell were averaged, and rise and decay times calculated from this average. Rise time was measured from 10 90% of peak current, and decay time with a single

3 J Physiol GDP modulation influences seizure susceptibility 59 exponential from peak both measured using Axograph analysis tools. Febrile seizure test Bumetanide stock was prepared in DMSO at 50 mg ml 1 and diluted in sterile saline solution (0.9% NaCl) to 0.1 mg ml 1 for injection. The equivalent volume of DMSO was added to saline acting as the vehicle control. Daily I.P. injections of bumetanide (2 mg kg 1 )orvehicle control were given from P1 to P5. At P17 the pups were tested for susceptibility to heat-induced seizures (Schuchmann et al. 2006). Only mice in the 7 9 g range were used to reduce variability associated with different heating rates due to size. Pups were placed in a heated chamber at 42 C, and the time to first seizure was recorded. Survival curves were plotted for the control and the bumetanide-injected groups, and the Mantel Cox test (Graph Pad Prism 5, San Diego, USA) was used to test for significant differences. Results GDP frequency is lower in the R43Q mutant GDPs could be reliably recorded from CA3 pyramidal neurons, and were classified by their large amplitude and distinctive morphology (Fig. 1). Whole-cell voltage-clamp recordings were used to measure GDPs and record spontaneous synaptic activity (see below). Although recordings are in voltage-clamp, we have used the term GDP as a more accurate description of the neuronal network event. A significantly smaller average number of GDPs per minute was observed in the GABA A γ2 R43Q mice when compared with WT littermates (Fig. 1C; 0.33 ± 0.07 GDPs min 1, n = 28 (GABA A γ2 R43Q ) vs ± 0.09 GDPs min 1, n = 26 (WT), P < 0.05). Bicuculline (10 μm) completely abolishes GDPs in both GABA A γ2 R43Q and WT slices (Fig. 1D; n = 4 for both genotypes), confirming their dependence on GABA A receptor-mediated transmission. Smaller amplitude, reduced frequency of spscs in the GABA A γ2 R43Q mouse spscs were also recorded from CA3 neurons (Fig. 2). Bicuculline (10 μm) essentially abolished spontaneous events in both WT (not shown) and mutant neurons (Fig. 1D), suggesting that the depolarizing action of GABA A receptor-mediated transmission is an important arbiter of excitation in the immature hippocampus. A reduction in the mean peak amplitudes was observed for spsc recorded from GABA A γ2 R43Q neurons (Fig. 2C; 63 ± 3 pa, n = 33 (GABA A γ2 R43Q ) Figure 1. A decrease in giant depolarizing potentials (GDPs) frequency is evident in the GABA A γ2 R43Q epilepsy mouse Representative whole-cell recordings from CA3 pyramidal neurons in acute neonatal hippocampal slices from A wild-type (WT; RR) and B GABA A γ 2 R43Q (RQ) mice. Stars represent GDP events detected using the automatic algorithm. The expanded time scale of CA3 pyramidal recordings is shown below. C, mean pooled data of GDP frequency. P < D, example recording indicating the effect of bicuculline (10 μm; Bic) in a GABA A γ 2 R43Q hippocampal slice.

4 60 E. Vargas and others J Physiol vs. 78 ± 4 pa, n = 37 (WT), P < 0.05). A reduction in the frequency of spsc events was also evident (Fig. 2C; 0.38 ± 0.05 event s 1, n = 33 (GABA A γ2 R43Q ) vs ± 0.06 event s 1, n = 37 (WT), P < 0.05). The morphologies of the spsc events were different, with a slightly slower rise time (2.09 ± 0.09 ms, n = 33 (GABA A γ2 R43Q ) vs ± 0.07 ms, n = 37 (WT), P < 0.05) but comparable decay time (21.7 ± 0.9 ms, n = 33 vs ± 0.7 ms, n = 37) of the spsc recorded from the mutant animal. Bumetanide reduces GDP events frequency Bumetanide modifies the Cl gradient of immature neurons by blocking NKCC1 and, as a consequence, reduces GDP frequency (Dzhala et al. 2005; Figure 2. Spontaneous spscs are reduced in the GABA A γ2 R43Q epilepsy mouse A, representative whole-cell recording from CA3 pyramidal neurons in neonatal hippocampal slices from wild-type (WT) and mutant GABA A γ 2 R43Q mice. B, average postsynaptic events recorded from a single CA3 pyramidal neuron from a WT and mutant mouse. C, mean pooled data of frequency, amplitude, and rise and decay times of spsc from WT and GABA A γ 2 R43Q mice. P < 0.05.

5 J Physiol GDP modulation influences seizure susceptibility 61 Sipila et al. 2006; Tyzio et al. 2011). We tested the effect of bumetanide on hippocampal slices cut from P3 5 WT mice to allow direct comparison with data obtained for the GABA A γ2 R43Q mouse. In a similar manner to previous reports (Sipila et al. 2006; Nardou et al. 2009), the frequency of GDP events was significantly less in the presence of bumetanide (10 μm) when compared with slices in ACSF alone (Fig. 3; 0.60 ± 0.13 GDPs min 1, n = 33 (ACSF) vs ± 0.06 GDPs min 1, n = 26 (bumetanide), P < 0.05). There was no statistical difference between GDP occurrence in slices from GABA A γ2 R43Q mice and bumetanide (10 μm)-treated WT slices (0.33 ± 0.07 GDPs min 1, n = 26 (GABA A γ2 R43Q ) vs ± 0.06 GDPs min 1, n = 26 (bumetanide), P > 0.05). Bumetanide reduces the frequency of spscs The frequency of spscs was reduced in bumetanide (Fig. 4; 0.74 ± 0.05 event s 1, n = 29 (ACSF) vs ± 0.09 event s 1, n = 19 (bumetanide), P < 0.05), consistent with a reduction in hippocampal network excitability. However, neither mean peak amplitudes (Fig. 4; 83 ± 4 pa, n = 29 (ACSF) vs. 87 ± 3 pa, n = 19 (bumetanide), P > 0.05), rise time (2.3 ± 0.1 ms vs. 2.3 ± 0.1 ms, P > 0.05) nor decay time (19 ± 1msvs. 21 ± 1ms, P > 0.05) were different, as expected, given that the Cl concentration in the recorded cell was essentially clamped to the concentration of the internal solution and that ion flux is through WT channels. Early postnatal bumetanide increases susceptibility to thermogenic seizures Bumetanide is proposed to be an effective anti-seizure agent in neonates (Tyzio et al. 2011). However, reducing GDPs can have an enduring impact on network formation (Wang & Kriegstein, 2011). A key phenotype of the GABA A γ2 R43Q mouse is that it develops seizures at a lower body temperature (Hill et al. 2011), suggestive of heightened febrile seizure susceptibility as seen in the patients (Wallace et al. 2001). Given the similar impact of bumetanide and the GABA A γ2 R43Q on GDP expression, we tested if the injection of postnatal bumetanide (2 mg kg 1, I.P., P2 P5) also altered the susceptibility to thermogenic seizures. At P18 the latency to the first clonic-tonic seizure for the antenatal bumetanide-treated mice was significantly faster than for injected-control mice (Fig. 5; P = 0.02, Mantel Cox test). Discussion Perturbation of the GABAergic system in early development may have a long-lasting impact on neuron Figure 3. Bumetanide reduces giant depolarizing potentials (GDPs) frequency in WT mice Representative whole-cell recordings from CA3 pyramidal neurons in acute neonatal hippocampal slices perfused with: A, normal artificial cerebrospinal fluid (ACSF); and B, 10 μm bumetanide. Stars represent GDP events detected using the automatic algorithm. Expanded time scale of CA3 pyramidal recordings is shown below. C, mean pooled data of GDP frequency. P < 0.05.

6 62 E. Vargas and others J Physiol circuitry, potentially influencing a range of behaviours (Nicosia et al. 2003; Wang & Kriegstein, 2011). We have focused on GDPs that rely on the depolarizing action of the GABA A receptor. Hippocampal slices cut from neonatal GABA A γ2 R43Q epilepsy mice have a lower frequency of GDPs when compared with WT littermates. As expected, bumetanide reduced GDP frequency in neonatal WT slice. Increased susceptibility to thermogenic seizures is also common to both antenatal bumetanide-treated mice and the GABA A γ2 R43Q mouse model (Hill et al. 2011). We have used a standard concentration of bumetanide (10 μm), consistent with most in vitro studies (e.g. Dzhala et al. 2005; Sipila et al. 2006). A wide range of bumetanide doses is used in rodent in vivo models, ranging from Figure 4. Bumetanide reduces spontaneous spscs A, representative whole-cell recording from CA3 pyramidal neurons in neonatal hippocampal slices in normal artificial cerebrospinal fluid (ACSF) or in bumetanide (10 μm). B, average postsynaptic events recorded from CA3 pyramidal neurons in ASCF and bumetanide. C, mean pooled data of frequency, amplitude, and rise and decay times. P < 0.05.

7 J Physiol GDP modulation influences seizure susceptibility mg kg 1 (Wang & Kriegstein, 2011) to >10 mg kg 1 (Brandt et al. 2010). Dosing is complicated with the brain pharmacokinetic profile age, and is potentially species specific (discussed in Brandt et al. 2010). We have used amediumdoseinthisstudyof2mgkg 1. Assuming the very simplified situation of equal distribution throughout the animal body, the concentration would be predicted to be 6 μm. The precise brain penetration and elimination of bumetanide under our conditions is not known. However, we feel that the concentration of 10 μm used in vitro is a reasonable upper estimate of brain concentrations expected in vivo. It is important to note that both the impact of the GABA A γ2 R43Q mutation and the influence of bumetanide are not limited to simply influencing GDP expression, and that the impact of both these manipulations on other cellular processes may be responsible for a change in brain state. However, our data support the idea that reduced GDP expression in the early neonatal period may cause enduring changes to neuronal networks that increase the susceptibility of thermogenic seizures. GDPs are a neuronal network phenomenon that rely on GABA A receptor-mediated transmission (Ben-Ari et al. 1989, 1997). The GABA A receptor antagonist, bicuculline, abolishes GDPs, confirming this in our preparation. Bicuculline also essentially blocked spontaneous activity onto CA3 pyramidal neurons. This is consistent with the idea that GABA A receptor activation depolarizes neurons at this developmental age (Sipila et al. 2005). Analysis of the spsc revealed smaller peak amplitude and a lower event frequency in the GABA A γ2 R43Q mouse. Although we have not pharmacologically isolated spontaneous GABA A receptor-mediated PSCs in these experiments, the results are consistent with smaller inhibitory PSCs seen in cortical neurons of older animals (Tan et al. 2007). By blocking NKCC1, bumetanide shifts the reversal Figure 5. Neonatal exposure to bumetanide increases susceptibility to thermogenic seizures Survival curve of the latency to first tonic-clonic seizure of mice injected with saline or bumetanide (2 mg kg 1 ). P < 0.05, Mantel Cox test. potential of Cl such that GABA A receptor activation is no longer excitatory (Wang & Kriegstein, 2011). The reduction in GDP frequency with bumetanide is consistent with other research groups (Sipila et al. 2006; Nardou et al. 2009; Tyzio et al. 2011). As well as reducing GDP frequency, bumetanide also reduced the frequency of spscs to a similar level as that observed in the mutant slices. GDPs are probabilistic events, and we propose that areductioningaba A receptor-mediated depolarization in the epilepsy model and in bumetanide-treated slices results in a reduction in the number of times that the GDP activation threshold is crossed. This is in accordance with that proposed by Sipila et al. (2005), where GABA increases the general level of network excitation facilitating GDP occurrence. We have previously shown that the suppression of disease allele reduces long-term seizure susceptibility in a conditional mouse model based on the GABA A γ2 R43Q mutation (Chiu et al. 2008). Suppression of the disease allele occurred over a wide developmental range that includes the time at which GDPs are expressed. Reduced GDP expression in the GABA A γ2 R43Q mouse is therefore well placed to alter micro-circuitry, potentially leading to increased long-term seizure susceptibility. Here we report that bumetanide given in the early neonatal period has an enduring impact, increasing thermogenic seizure susceptibility weeks after exposure. Several studies have implicated neonatal stress as a contributing risk factor for acquired epilepsy frequently through changes in GABA A receptor expression and function (Koe et al. 2009). Further, drugs used during a neonatal period that act through GABA A receptors (e.g. benzodiazepines) influence seizure susceptibility into adulthood (Nicosia et al. 2003). Early developmental modulation of GABAergic transmission is likely to have broader implications. For instance, early developmental exposure to bumetanide can lead to permanent alterations of cortical circuits, potentially leading to socio-behavioural deficits (Wang & Kriegstein, 2011). Together, this and our data strongly support the idea that altered network activity in early development, driven through GABA A receptor activation, plays a critical role in defining neuronal circuits, and that disruption in this function may result in disease. How do GDPs modulate circuit formation? GDP-mediated Ca 2+ flux in neurons due to the activation of NMDA receptors and voltage-gated calcium channels may activate signalling pathways that help in the establishment and refinement of network circuitry (Zhang & Poo, 2001). For example, GDPs are proposed to contribute to the synaptic refinement at the CA3 mossy fibre synapses (Kasyanov et al. 2004). Additionally, reduced frequency of GDPs leads to delay in the synaptic switch from GABAergic to glutamatergic during the first postnatal week, and affects the expression of AMPA and NMDA receptors (Pfeffer et al. 2009). GABA-signalling

8 64 E. Vargas and others J Physiol stimulates the migration of embryonic cortical cells in vitro with concentration-dependent effects (Behar et al. 1996; Cuzon et al. 2006). Which of these mechanisms, if any, is central to defining seizure susceptibility is yet to be determined. We can conclude that impairment of the GABA A receptor-dependent maturation of neuronal circuits, caused either by genetic dysfunction (i.e. γ2 R43Q )orby pharmacological modulation (bumetanide) can lead to enduring changes in neuronal networks that may result in enhanced seizure susceptibility. References Behar TN, Li YX, Tran HT, Ma W, Dunlap V, Scott C & Barker JL (1996). GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms. JNeurosci16, Ben-Ari Y, Cherubini E, Corradetti R & Gaiarsa JL (1989). Giant synaptic potentials in immature rat CA3 hippocampal neurones. JPhysiol416, Ben-Ari Y, Khazipov R, Leinekugel X, Caillard O & Gaiarsa JL (1997). GABA A, NMDA and AMPA receptors: a developmentally regulated menage a trois. Trends Neurosci 20, Ben-Ari Y, Tyzio R & Nehlig A (2011). Excitatory action of GABA on immature neurons is not due to absence of ketone bodies metabolites or other energy substrates. Epilepsia 52, Ben-Ari Y, Woodin MA, Sernagor E, Cancedda L, Vinay L, Rivera C, Legendre P, Luhmann HJ, Bordey A, Wenner P, Fukuda A, van den Pol AN, Gaiarsa JL & Cherubini E (2012). Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever. Front Cell Neurosci 6, 35. Blaesse P, Airaksinen MS, Rivera C & Kaila K (2009). Cation-chloride cotransporters and neuronal function. Neuron 61, Brandt C, Nozadze M, Heuchert N, Rattka M & Loscher W (2010). Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy. JNeurosci30, Bregestovski P & Bernard C (2012). Excitatory GABA: how a correct observation may turn out to be an experimental artifact. Front Pharmacol 3, 65. Chiu C, Reid CA, Tan HO, Davies PJ, Single FN, Koukoulas I, Berkovic SF, Tan SS, Sprengel R, Jones MV & Petrou S (2008). Developmental impact of a familial GABAA receptor epilepsy mutation. Ann Neurol 64, Cuzon VC, Yeh PW, Cheng Q & Yeh HH (2006). Ambient GABA promotes cortical entry of tangentially migrating cells derived from the medial ganglionic eminence. Cereb Cortex 16, Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE & Staley KJ (2005). NKCC1 transporter facilitates seizures in the developing brain. Nat Med 11, Hill EL, Hosie S, Mulligan RS, Richards KL, Davies PJ, Dube CM, Baram TZ, Reid CA, Jones MV & Petrou S (2011). Temperature elevation increases GABA (A) -mediated cortical inhibition in a mouse model of genetic epilepsy. Epilepsia 52, Kasyanov AM, Safiulina VF, Voronin LL & Cherubini E (2004). GABA-mediated giant depolarizing potentials as coincidence detectors for enhancing synaptic efficacy in the developing hippocampus. Proc Natl Acad Sci U S A 101, Koe AS, Jones NC & Salzberg MR (2009). Early life stress as an influence on limbic epilepsy: an hypothesis whose time has come? Front Behav Neurosci 3, 24. Laurie DJ, Seeburg PH & Wisden W (1992). The distribution of 13 GABA A receptor subunit mrnas in the rat brain. II. Olfactory bulb and cerebellum. JNeurosci12, Nardou R, Ben-Ari Y & Khalilov I (2009). Bumetanide, an NKCC1 antagonist, does not prevent formation of epileptogenic focus but blocks epileptic focus seizures in immature rat hippocampus. J Neurophysiol 101, Nicosia A, Giardina L, Di Leo F, Medico M, Mazzola C, Genazzani AA & Drago F (2003). Long-lasting behavioral changes induced by pre- or neonatal exposure to diazepam in rats. Eur J Pharmacol 469, Pfeffer CK, Stein V, Keating DJ, Maier H, Rinke I, Rudhard Y, Hentschke M, Rune GM, Jentsch TJ & Hubner CA (2009). NKCC1-dependent GABAergic excitation drives synaptic network maturation during early hippocampal development. JNeurosci29, Russell JM (2000). Sodium-potassium-chloride cotransport. Physiol Rev 80, Schuchmann S, Schmitz D, Rivera C, Vanhatalo S, Salmen B, Mackie K, Sipila ST, Voipio J & Kaila K (2006). Experimental febrile seizures are precipitated by a hyperthermia-induced respiratory alkalosis. Nat Med 12, Sipila ST, Huttu K, Soltesz I, Voipio J & Kaila K (2005). Depolarizing GABA acts on intrinsically bursting pyramidal neurons to drive giant depolarizing potentials in the immature hippocampus. JNeurosci25, Sipila ST, Schuchmann S, Voipio J, Yamada J & Kaila K (2006). The cation-chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus. JPhysiol573, Tan HO, Reid CA, Single FN, Davies PJ, Chiu C, Murphy S, Clarke AL, Dibbens L, Krestel H, Mulley JC, Jones MV, Seeburg PH, Sakmann B, Berkovic SF, Sprengel R & Petrou S (2007). Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy. Proc Natl Acad Sci U S A 104, Tyzio R, Allene C, Nardou R, Picardo MA, Yamamoto S, Sivakumaran S, Caiati MD, Rheims S, Minlebaev M, Milh M, Ferre P, Khazipov R, Romette JL, Lorquin J, Cossart R, Khalilov I, Nehlig A, Cherubini E & Ben-Ari Y (2011). Depolarizing actions of GABA in immature neurons depend neither on ketone bodies nor on pyruvate. JNeurosci31, Vanhatalo S, Hellstrom-Westas L & De Vries LS (2009). Bumetanide for neonatal seizures: based on evidence or enthusiasm? Epilepsia 50,

9 J Physiol GDP modulation influences seizure susceptibility 65 Wallace RH, Marini C, Petrou S, Harkin LA, Bowser DN, Panchal RG, Williams DA, Sutherland GR, Mulley JC, Scheffer IE & Berkovic SF (2001). Mutant GABA (A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures. Nat Genet 28, Wang DD & Kriegstein AR (2011). Blocking early GABA depolarization with bumetanide results in permanent alterations in cortical circuits and sensorimotor gating deficits. Cereb Cortex 21, Zhang LI & Poo MM (2001). Electrical activity and development of neural circuits. Nat Neurosci 4Suppl, Author contributions E.V., S.P. and C.A.R. designed experiments, developed analysis and were involved in drafting of the manuscript. E.V. completed all experimental work at the Florey Institute of Neuroscience and Mental Health. Acknowledgements This study was supported by NHMRC project grant to C.A.R., and a NHMRC program grant to S.P. C.A.R. also acknowledges the support of an ARC Future Fellowship (FT ). SP is a NHMRC Senior Research Fellow. The authors have no financial conflict to declare with regards to this research.