Progressive NKCC1-Dependent Neuronal Chloride Accumulation During Neonatal Seizures
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The Journal of Neuroscience, September 1, 2010 • 30(35):11745–11761 • 11745 Neurobiology of Disease Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures Volodymyr I. Dzhala,1* Kishore V. Kuchibhotla,1,3* Joseph C. Glykys,1 Kristopher T. Kahle,2 Waldemar B. Swiercz,1 Guoping Feng,4 Thomas Kuner,4 George J. Augustine,4 Brian J. Bacskai,1 and Kevin J. Staley1 Departments of 1Neurology and 2Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, 3Program in Biophysics, Harvard University, Cambridge, Massachusetts 02138, and 4Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710 Seizures induce excitatory shifts in the reversal potential for GABAA-receptor-mediated responses, which may contribute to the intrac- tability of electro-encephalographic seizures and preclude the efficacy of widely used GABAergic anticonvulsants such as phenobarbital. We now report that, in intact hippocampi prepared from neonatal rats and transgenic mice expressing Clomeleon, recurrent seizures Ϫ progressively increase the intracellular chloride concentration ([Cl ]i ) assayed by Clomeleon imaging and invert the net effect of GABAA receptor activation from inhibition to excitation assayed by the frequency of action potentials and intracellular Ca 2ϩ transients. These changes correlate with increasing frequency of seizure-like events and reduction in phenobarbital efficacy. The Na ϩ–K ϩ–2Cl Ϫ (NKCC1) cotransporter blocker bumetanide inhibited seizure-induced neuronal Cl Ϫ accumulation and the consequent facilitation of recurrent Ϫ seizures. Our results demonstrate a novel mechanism by which seizure activity leads to [Cl ]i accumulation, thereby increasing the probability of subsequent seizures. This provides a potential mechanism for the early crescendo phase of neonatal seizures. Introduction neonatal seizures (Staley and Smith, 2001), and depolarized val- Neurons respond to a variety of stimuli with long-term shifts in ues of EGABA contribute to the reduced efficacy of GABA- enhancing anticonvulsants (Dzhala et al., 2005, 2008) that are the reversal potential for GABAA receptor (GABAA-R)-mediated postsynaptic currents (EGABA). For example, epileptogenic inju- first-line agents in the treatment of neonatal seizures (Rennie and ries alter neuronal chloride transport, which is the principal de- Boylan, 2003; Carmo and Barr, 2005). NKCC1 is selectively terminant of EGABA (Jin et al., 2005; Pathak et al., 2007). EGABA is blocked by low micromolar concentrations of the loop diuretic persistently shifted to more positive (depolarizing) potentials by bumetanide (Isenring et al., 1998; Hannaert et al., 2002). By re- prolonged seizure activity (Khalilov et al., 2003) or trains of ac- ducing intracellular chloride accumulation, this diuretic shifts tion potentials without (Fiumelli and Woodin, 2007; Brumback EGABA to negative potentials, resulting in more effective inhibi- and Staley, 2008) or with concomitant GABAA receptor activa- tion (Yamada et al., 2004; Dzhala et al., 2005, 2008; Sipila et al., tion (Woodin et al., 2003). In addition, other events, including 2006; Rheims et al., 2008). The increase in inhibition has a signif- oxygen-glucose deprivation (Pond et al., 2006) and trauma (van icant anticonvulsant effect that is synergistic with anticonvul- den Pol et al., 1996), shift EGABA to more depolarized potentials. sants such as barbiturates that prolong the open probability of the Depolarized values of EGABA reduce the efficacy of inhibition and GABAA-receptor-mediated chloride channels (Twyman et al., contribute to seizure activity (Dzhala and Staley, 2003; Khazipov 1989; Dzhala et al., 2005, 2008). et al., 2004; Dzhala et al., 2005). Several recent reports regarding the experimental treatment Immature neurons express high levels of NKCC1 (Delpire, of neonatal seizures have reported conflicting results (Table 1). 2000; Wang et al., 2002; Dzhala et al., 2005), an electroneutral For example, if administered soon after the onset of seizures, ϩ ϩ Ϫ cotransporter that imports Na ,K , and Cl . This raises the phenobarbital is relatively more effective (Quilichini et al., 2003; intracellular chloride concentration, resulting in a depolarized Raol et al., 2009) and bumetanide is less effective (Kilb et al., EGABA. This shift in EGABA can initiate a positive feedback cycle in 2007) as an anticonvulsant. In contrast, experiments in which drugs are administered after many seizures demonstrate reduced phenobarbital efficacy and increased efficacy of drugs such as Received April 6, 2010; revised July 7, 2010; accepted July 12, 2010. bumetanide that block NKCC1-mediated shifts in EGABA (Dzhala This work was supported by the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke Grant NS 40109-06 (K.J.S.), NIH Grants EB000768 (B.J.B.) and NS580752 (K.V.K.), an American Epilepsy et al., 2005, 2008; Mazarati et al., 2009). Here we consider the Society grant (J.C.G.), and a Hearst Foundation grant (V.I.D.). possibility that activity-dependent, NKCC1-dependent changes *V.I.D. and K.V.K. contributed equally to this work. in EGABA help determine the time course of neonatal seizures. Correspondence should be addressed to Dr. Kevin J. Staley, Department of Neurology, MassGeneral Institute for This hypothesis could explain the substantial variations in the NeurodegenerativeDisease,MassachusettsGeneralHospital,16thStreet,CNYB-114,Room2600,Charlestown,MA efficacy of GABAergic anticonvulsants in the experimental set- 02129. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.1769-10.2010 tings described in Table 1. This hypothesis is also of considerable Copyright © 2010 the authors 0270-6474/10/3011745-17$15.00/0 clinical importance because it provides an additional impetus for 11746 • J. Neurosci., September 1, 2010 • 30(35):11745–11761 Dzhala et al. • Inverted Action of GABA in Neonatal Seizures Table 1. Efficacy of GABAergic anticonvulsants in animal models of neonatal seizures # Preparation/model Age Anticonvulsant (AED) Concentration Application Efficacy Authors 1 In vivo/flurothyl P9, P15 Phenobarbital 20 mg/kg Pretreatment (i.p) High (Velísek et al., 1995) 2 In vivo/pentylenetetrazol P7, P12 Clobazam 0.5–7.5 mg/kg Pretreatment (i.p) High (Haugvicová et al., 1999) 2ϩ 3 In vitro/0 mM Mg P7–P8 Phenobarbital 30 M After 3 ILDs Low (Quilichini et al., 2003) Phenobarbital 100 M High ϩ 4 In vitro; in vivo/K ϩ gabazine P8–P12, P17–P21 Diazepam 5 M Coinjection (before ILD) High (Isaev et al., 2007) 2ϩ 5 In vitro/0 mM Mg P4–P6 Phenobarbital 100 M After 7 ILDs Low (Dzhala et al., 2008) ϩ 6 In vitro/8.5 mM K ; in vivo/kainic acid P5–P7 Phenobarbital 100 M After 7 ILDs pretreatment (i.p) Low (Dzhala et al., 2005) P9–P12 Phenobarbital 25 mg/kg Modest 7 In vivo/kainic acid; flurothyl P10 Phenobarbital 50 mg/kg Stage 5 seizures (i.p) Significant (Raol et al., 2009) P10 Diazepam 4–16 mg/kg Transient ILD, Ictal-like discharges; AED, antiepileptic drug. immediate and aggressive treatment of neonatal seizures: the Group measures are expressed as mean Ϯ SEM; error bars also indi- more seizures that occur, the more EGABA will shift, and the lower cate SEM. Statistical tests of significance were assessed with Student’s t, 2 the probability that the seizures will respond to available anti- ANOVA, , and Mann–Whitney tests as indicated in Results. The level of significance was set at p Ͻ 0.05. convulsants such as phenobarbital, particularly during the Ϫ Clomeleon imaging and [Cl ]i determination. Clomeleon is a fusion crescendo phase of neonatal seizures (Painter et al., 1999). A Ϫ protein comprising the Cl -sensitive yellow fluorescent protein (YFP) corollary of this hypothesis is that, by blocking NKCC1, bu- Ϫ and the Cl -insensitive cyan fluorescent protein (CFP). Transgenic metanide should prevent or reduce the seizure-induced shift CLM-1 mice expressing Clomeleon were received from Duke University in EGABA, potentially ameliorating the crescendo pattern of Medical Center (Durham, NC) and housed at Massachusetts General neonatal seizures. Finally, enhancement of the efficacy of Hospital Center for Comparative Medicine (Charlestown, MA). Intact GABAergic anticonvulsants by bumetanide should increase hippocampi were prepared from neonatal (P5–P7) mice as described with the duration of the previous seizure activity. These hy- previously (Dzhala et al., 2008). The hippocampi were incubated in ox- potheses are tested in whole hippocampal preparations from ygenated ACSF at room temperature (20–22°C) for at least 1 h before neonatal rats and CLM-1 mice. use. For optical imaging and simultaneous extracellular field potential recordings, the hippocampus was placed into a conventional sub- Materials and Methods merged chamber on a precision x–y stage mounted on the microscope Animals. All animal-use protocols conformed to the guidelines of the and continuously superfused with oxygenated ACSF at 32°C and at a National Institutes of Health and the Massachusetts General Hospital flow rate 4 ml/min. Two-photon Clomeleon imaging was performed on Center for Comparative Medicine on the use of laboratory animals. an Olympus Fluoview 1000MPE with pre-chirp excitation optics and a In vitro electrophysiology. Intact hippocampal formations were pre- fast acousto-optical modulator mounted on an Olympus BX61WI up- ϫ pared from neonatal postnatal day 5 (P5) to P7 male and female CLM-1 right microscope using an Olympus 20 water-immersion objective mice pups and P3–P6 Sprague Dawley rat male pups as described previ- (XLUMPLFL 20xW; numerical aperture, 0.95). A mode-locked titani- ously (Dzhala et al., 2008). Animals were anesthetized and decapitated. um/sapphire laser (MaiTai; Spectra Physics) generated two-photon flu- The brain was rapidly removed to oxygenated (95% O –5% CO ) ice- orescence with 860 nm excitation. Emitted light passed through a 2 2 dichroic mirror (460 nm cutoff) and was bandpass filtered through one cold (2–5°C) artificial CSF (ACSF) containing the following (in mM): 126 of two emission filters 480 Ϯ 15 nm (D480/30) for CFP and 535 Ϯ 20 nM NaCl, 3.5 KCl, 2 CaCl2, 1.3 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11 glucose, pH 7.4.