Roles of the cation-chloride cotransporters in neurological disease

In the nervous system, the intracellular chloride concentration ([Cl(-)](i)) determines the strength and polarity of gamma-aminobutyric acid (GABA)-mediated neurotransmission. [Cl(-)](i) is determined, in part, by the activities of the SLC12 cation-chloride cotransporters (CCCs). These transporters include the Na-K-2Cl cotransporter NKCC1, which mediates chloride influx, and various K-Cl cotransporters--such as KCC2 and KCC3-that extrude chloride. A precise balance between NKCC1 and KCC2 activity is necessary for inhibitory GABAergic signaling in the adult CNS, and for excitatory GABAergic signaling in the developing CNS and the adult PNS. Altered chloride homeostasis, resulting from mutation or dysfunction of NKCC1 and/or KCC2, causes neuronal hypoexcitability or hyperexcitability; such derangements have been implicated in the pathogenesis of seizures and neuropathic pain. [Cl(-)](i) is also regulated to maintain normal cell volume. Dysfunction of NKCC1 or of swelling-activated K-Cl cotransporters has been implicated in the damaging secondary effects of cerebral edema after ischemic and traumatic brain injury, as well as in swelling-related neurodegeneration. CCCs represent attractive therapeutic targets in neurological disorders the pathogenesis of which involves deranged cellular chloride homoestasis.     

Developmental Patterns in the Regulation of Chloride Homeostasis and GABAA Receptor Signaling by Seizures

Summary:  GABA_A receptors have dual functions during development. They depolarize immature neurons but hyperpolarize more mature neurons. This functional switch has been attributed to age-related differences in the relative abundance of cation chloride cotransporters, such as KCC2 and NKCC1, which regulate chloride homeostasis. Certain insults, such as trauma, ischemia, and seizures, if they occur when GABA_Aergic signaling is hyperpolarizing, such as in the adult brain, can lead to reappearance of the immature, depolarizing synaptic responses to GABA_A receptor activation. In certain cases, this has been associated with either reduced expression of KCC2 or increase in NKCC1. In epilepsy, the depolarizing effects of GABA_A receptors have been proposed to be important for the acquisition and/or maintenance of the epileptic state. Using the kainic acid model of status epilepticus, we have studied the effects of repetitive neonatal episodes of status epilepticus on the expression of cation chloride cotransporter KCC2 in the neonatal hippocampus. In contrast to adults, seizures increased KCC2 mRNA expression in the CA3 region of the neonatal hippocampus. The contrasting patterns of regulation of KCC2 by seizures in mature and immature neurons may be one of the age-related factors that protect the neonatal brain against the development of epilepsy.     

Acute TrkB inhibition rescues phenobarbital‐resistant seizures in a mouse model of neonatal ischemia

Neonatal seizures are commonly associated with hypoxic–ischemic encephalopathy. Phenobarbital (PB) resistance is common and poses a serious challenge in clinical management. Using a newly characterized neonatal mouse model of ischemic seizures, this study investigated a novel strategy for rescuing PB resistance. A small‐molecule TrkB antagonist, ANA12, used to selectively and transiently block post‐ischemic BDNF‐TrkB signaling in vivo, determined whether rescuing TrkB‐mediated post‐ischemic degradation of the K⁺–Cl^? co‐transporter (KCC2) rescued PB‐resistant seizures. The anti‐seizure efficacy of ANA12 + PB was quantified by (i) electrographic seizure burden using acute continuous video‐electroencephalograms and (ii) post‐treatment expression levels of KCC2 and NKCC1 using Western blot analysis in postnatal day (P)7 and P10 CD1 pups with unilateral carotid ligation. ANA12 significantly rescued PB‐resistant seizures at P7 and improved PB efficacy at P10. A single dose of ANA12 + PB prevented the post‐ischemic degradation of KCC2 for up to 24 h. As anticipated, ANA12 by itself had no anti‐seizure properties and was unable to prevent KCC2 degradation at 24 h without follow‐on PB. This indicates that unsubdued seizures can independently lead to KCC2 degradation via non‐TrkB‐dependent pathways. This study, for the first time as a proof‐of‐concept, reports the potential therapeutic value of KCC2 modulation for the management of PB‐resistant seizures in neonates. Future investigations are required to establish the mechanistic link between ANA12 and the prevention of KCC2 degradation.     

Patterns of cation-chloride cotransporter expression during embryonic rodent CNS development

Intracellular Cl- plays a key role in cellular volume regulation, cell cycle control and shaping the polarity of inhibitory postsynaptic responses mediated by anion-permeable GABA and glycine receptors. In this study, we have investigated the expression patterns of members of the cation-chloride cotransporters (CCCs), including the K-Cl cotransporters KCC1-4 and the Na-K-2 Cl cotranporter NKCC1 during rodent embryonic brain development. At the time of neurogenesis (embryonic days; E12.5-14.5), KCC1 was only detectable in the developing choroid plexus. KCC2 mRNA was detectable as early as E12.5 in the ventral part of the (cervical) spinal cord, and by E14.5, the expression had spread to TUJ1-positive differentiating regions of the rhombencephalon, diencephalon and olfactory bulb, in parallel with neuronal maturation. KCC3 mRNA was scarce in the cortical plate at E14.5, and slightly up-regulated at birth. In contrast, KCC4 mRNA was abundantly expressed in the ventricular zone and was down-regulated perinatally. At E14.5, NKCC1 was highly expressed in the vimentin-positive radial glia of the proliferative zone of the subcortical region. At later embryonic stages, during gliogenesis (E17-P0), there was a shift in NKCC1 expression to the neuron specific Class III beta-tubulin (betaIII) positive region of the cortical plate. These unique spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl- regulatory mechanisms are critically involved in the control of neuronal development.     

Inflammation alters cation chloride cotransporter expression in sensory neurons

Cation chloride cotransporters have been proposed to play a role in the modulation of neuronal responses to gamma-aminobutyric acid (GABA). In conditions of neuronal damage, where neuronal excitability is increased, the expression of the KCC2 transporter is decreased. This is also seen in spinal cord in models of neuropathic pain. We have investigated the expression of the Na-K-Cl, and K-Cl cotransporters NKCC1 and KCC2, in dorsal root ganglion (DRG) and spinal sensory neurons during arthritis, a condition in which neuronal excitability is also increased. NKCC1 was expressed in control DRG neurons, and its expression was decreased in arthritis. Both NKCC1 and KCC2 were expressed in sensory neurons in the spinal cord. In acute arthritis, both NKCC1 and KCC2 mRNA increased in superficial but not deep dorsal horn, and this was accompanied by an increase in protein expression. In chronic arthritis, NKCC1 expression remained raised, but KCC2 mRNA and protein expression returned to control levels. Altered KCC2 and NKCC1 expression in arthritis may contribute to the control of spinal cord excitability and may represent novel therapeutic targets in the treatment of inflammatory pain.     

Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum

Cation chloride cotransporters are considered to play pivotal roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function. To establish how these cotransporters are involved in cerebellum development, we investigated the expression of KCC1, KCC2 and NKCC1 mRNAs in the developing rat cerebellum using in situ hybridization histochemistry. In the external germinal layer, where premature cells exist, we found substantial KCC1 and NKCC1 mRNA expression on P7 and P14, while KCC2 mRNA was not detected. In contrast, KCC2 mRNA was already expressed in Purkinje cells on P1. We also observed KCC2 mRNA expression in postmigratory granule cells after P7. The expression of KCC1, KCC2, and NKCC1 mRNAs reached adult patterns by P21. In the adult cerebellum, KCC2 mRNA was expressed in most neurons, including Purkinje cells, granule cells, and stella/basket cells, while KCC1 and NKCC1 mRNAs were only detected in granule cells and glial cells. These findings suggest that in the rat cerebellum KCC2 mRNA expression is induced when neurons arrive their final destinations.     

GABAA receptor-mediated activation of L-type calcium channels induces neuronal excitation in surgically resected human hypothalamic hamartomas.

PURPOSE: The human hypothalamic hamartoma (HH) is a rare, intrinsically epileptogenic lesion associated with gelastic seizures, but the underlying mechanisms remain unclear. Here, we examined the role of GABAA receptors in surgically resected HH tissue. METHODS: HH tissue slices (350 microm) were studied using cellular electrophysiological, calcium imaging, and immunocytochemical techniques. RESULTS: Two neuronal cell types were seen: small (10-16 microm) spontaneously firing GABAergic neurons and large (20-28 microm) quiescent neurons. In gramicidin-perforated patch recordings, muscimol (30 microM) induced membrane depolarization in 70% of large (but not small) neurons and a concomitant rise in intracellular calcium. These responses were blocked by bicuculline methiodide (50 microM). Depolarizing neurons also exhibited more positive reversal potentials (Emuscimol) and significantly higher intracellular chloride concentrations compared to those that hyperpolarized. The cation chloride co-transporters NKCC1 and KCC2 were coexpressed in the majority of large neurons, but fluorometric measurements revealed that 84% of large HH neurons expressed solely or relatively more NKCC1. Bumetanide (20 microM), a NKCC1 antagonist, partially suppressed muscimol-induced excitation in large neurons. Concordant with robust expression of CaV1.2 and CaV1.3 subunits in HH neurons, the L-type calcium channel blocker nifedipine (100 microM) prevented muscimol-induced neuronal excitation. CONCLUSIONS: GABAA receptor-mediated excitation, due in part to differential expression of NKCC1 and KCC2 and subsequent activation of L-type calcium channels, may contribute to seizure genesis in HH tissue. Given the ready availability of L-type calcium channel blockers, our results have clinical ramifications for the treatment of seizures associated with HH lesions.     

Ketogenic diet does not change NKCC1 and KCC2 expression in rat hippocampus

In control rats, we examined the effects of ketogenic diet on NKCC1 and KCC2 expression levels in hippocampus. Neither the number of NKCC1 immunoreactive cells nor the intensity of labeling of KCC2 was found to modify in hippocampus of the rats after ketogenic diet treatment. These results indicate that ketogenic diet by itself does not modify the expression of these cation chloride cotransporters.     

Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus

Intracellular chloride concentration, [Cl(-)](i), determines the polarity of GABA(A)-induced neuronal Cl(-) currents. In neurons, [Cl(-)](i) is set by the activity of Na(+), K(+), 2Cl(-) cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl(-) in the cell, and Cl(-) extruding K(+), Cl(-) cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex. NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCC-immunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.     

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

Salt‐loading (SL) impairs GABA_A inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down‐regulating the activity of K⁺/Cl^? co‐transporter2 (KCC2) and up‐regulating Na⁺/K⁺/Cl^? co‐transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl^?]_i in SON AVP neurones. The study combined virally‐mediated chloride imaging with ClopHensorN with a single‐cell western blot analysis. An adeno‐associated virus with ClopHensorN and a vasopressin promoter (AAV2‐0VP1‐ClopHensorN) was bilaterally injected in the SON of adult male Sprague‐Dawley rats that were either euhydrated (Eu) or salt‐loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl^?]_i in response to focal application of the GABA_A agonist muscimol (100 μmol L^‐1). SON AVP neurones from Eu rats showed muscimol‐induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β‐actin. Neurones that did not respond during chloride imaging did not show KCC2 and β‐actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L^‐1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L^‐1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol‐induced Cl^? influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA‐12) (50 μmol L^‐1) and protein kinase inhibitor (K252a) (100 nmol L^‐1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt‐loading increases [Cl^?]_i in SON AVP neurones via a TrKB‐KCC2‐NKCC1‐dependent mechanism in rats.     

NKCC1、KCC2及mTOR相关蛋白4E-BP1在外伤性癫痫中的表达及意义

目的探讨主要表达于神经元细胞膜上的NA⁺-K⁺-2CL^-转运体（NKCC1）、K⁺-CL^-转运体（KCC2）及表达于神经元细胞质中mTOR通路信号蛋白4E结合蛋白1（4E-BP1）在外伤性癫痫（PTE）癫痫灶中的表达及临床意义。 方法收集2010年1月至2015年12月福建医科大学附属第一医院神经外科外伤性癫痫患者术后脑组织标本14例作为实验组;选取脑外伤患者行减压或清创手术获取的和各种病变患者手术入路不可避免要切除的正常脑组织8例作为对照组。用Western blot和实时定量荧光PCR（RT-PCR）检测14例PTE癫痫脑组织、8例对照组"正常脑组织"NKCC1、KCC2、4E-BP1的表达。 结果Western blot显示PTE病灶脑组织中4E-BP1、NKCC1相对灰度值（0.61±0.12、0.92±0.19）高于正常脑组织（0.27±0.05、0.67±0.66）,差异有统计学意义（P<0.05）。PTE病灶脑组织中KCC2相对灰度值（0.58±0.99）低于正常脑组织（0.72±0.06）,差异有统计学意义（P<0.05）。RT-PCR结果显示,PTE病灶脑组织中4E-BP1、NKCC1相对灰度值（30.84±1.32、27.81±1.92）高于正常脑组织（26.94±1.24、23.52±0.74）,差异有统计学意义（P<0.05）。PTE病灶脑组织中KCC2相对灰度值（21.55±1.01）低于正常脑组织（24.59±1.02）,差异有统计学意义（P<0.05）。PTE组中NKCC1/KCC2比值（1.29±0.11）高于对照组（0.96±0.26）,差异有统计学意义（P<0.05）。 结论NKCC1、KCC2和mTOR通路信号蛋白4E-BP1的异常改变,可能是外伤后脑组织组织学改变及反复癫痫发作的重要分子机制。     

Neonatal seizures: dilemmas in workup and management

There is a pressing need for consistent, evidence-based guidelines in the management of neonatal seizures by pediatric neurologists and neonatologists. Israeli pediatric neurologists and neonatologists completed a 20-item, self-administered questionnaire on choices of antiepileptic drugs, treatment of intractable neonatal seizures (unremitting seizures after 3 medications), treatment duration, and recommended workup. The responding 36/55 (65%) neurologists and 66/112 (59%) neonatologists made similar antiepileptic drug choices (phenobarbital as first line, phenytoin as second line, and benzodiazepines as third line). Antiepileptic treatment duration was similar for both groups, but varied considerably within them (range, 1-52 weeks). Neurologists tended to recommend longer treatment for seizures secondary to asphyxia or hemorrhage. Neurologists and neonatologists recommended different antiepileptic drugs for intractable neonatal seizures: valproic acid and topiramate by neurologists, vs lidocaine and benzodiazepines by neonatologists (P = 0.0023). Fewer neurologists recommended continuous electroencephalography monitoring after asphyxia than neonatologists (40% vs 70.5%, P = 0.013). These responses reflect both similarities and inconsistencies of the two groups in diagnosing and treating neonatal seizures. Our findings call for controlled clinical trials to establish protocols for (1) diagnosing neonatal seizures, (2) studying the efficacy and safety of new-generation antiepileptic drugs, and (3) determining optimal duration of drug administration.     

Regulation of KCC2 and NKCC during development: membrane insertion and differences between cell types

The developmental switch of GABA's action from excitation to inhibition is likely due to a change in intracellular chloride concentration from high to low. Here we determined if the GABA switch correlates with the developmental expression patterns of KCC2, the chloride extruder K+-Cl- cotransporter, and NKCC, the chloride accumulator Na+-K+-Cl- cotransporter. Immunoblots of ferret retina showed that KCC2 upregulated in an exponential manner similar to synaptophysin (a synaptic marker). In contrast, NKCC, which was initially expressed at a constant level, upregulated quickly between P14 and P28, and finally downregulated to an adult level that was greater than the initial phase. At the cellular level, immunocytochemistry showed that in the inner plexiform layer KCC2's density increased gradually and its localization within ganglion cells shifted from being primarily in the cytosol (between P1-13) to being in the plasma membrane (after P21). In the outer plexiform layer, KCC2 was detected as soon as this layer started to form and increased gradually. Interestingly, however, KCC2 was initially restricted to photoreceptor terminals, while in the adult it was restricted to bipolar dendrites. Thus, the overall KCC2 expression level in ferret retina increases with age, but the time course differs between cell types. In ganglion cells the upregulation of KCC2 by itself cannot explain the relatively fast switch in GABA's action; additional events, possibly KCC2's integration into the plasma membrane and downregulation of NKCC, might also contribute. In photoreceptors the transient expression of KCC2 suggests a role for this transporter in development.     

NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension

Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.     

Increased excitability and molecular changes in adult rats after a febrile seizure

Both early life inflammation and prolonged febrile seizures have been associated with increased excitation in the adult brain. We hypothesized this may be due in part to changes in the cation‐chloride cotransporter system. Rat pups received saline or lipopolysaccharide/kainic acid (LPS/KA) resulting in inflammation, followed by a behavioral febrile seizure (FS) in approximately 50% of rats. Adult animals from the saline, inflammation, or inflammation + FS groups underwent the following: (1) in vitro electrophysiologic studies; (2) Western blotting or polymerase chain reaction; or (3) application of the Na‐K‐Cl cotransporter 1 (NKCC1) blocker bumetanide to determine its effect on reversing increased excitability in vitro. The inflammation and inflammation + FS groups demonstrated increased excitability in vitro and increased hippocampal protein expression of NR2B and GABA_A α5 receptor subunits and mRNA expression of NKCC1. The inflammation + FS group also had decreased protein expression of GluR2 and GABA_A α1 receptor subunits and mRNA and protein expression of KCC2. Bumetanide decreased in vitro 4‐aminopyridine‐induced inter‐ictal activity in the inflammation and inflammation + FS groups. The results demonstrate early‐life inflammation with or without a behavioral FS can lead to long‐lasting molecular changes and increased excitability in the adult rat hippocampus, although some changes are more extensive when inflammation is accompanied by behavioral seizure activity. Bumetanide is effective in reversing increased excitability in vitro, providing evidence for a causal role for cation‐chloride cotransporters and suggesting this drug may prove useful for treating epilepsy that develops after a FS.     

Alterations in the expression of neuronal chloride transporters may contribute to schizophrenia

During brain development, neuronal stem cells and immature neurons express high and low levels of, respectively, the Cl⁻ transporters NKCC1 and KCC2, which results in high intracellular Cl⁻ concentrations. Under these circumstances chloride-flux through the GABA-A channel is from intracellular to extracellular and consequently GABA depolarizes rather than hyperpolarizes immature cells. This excitatory response is essential for neurodevelopment since it affects proliferation of the neuronal progenitor pool, neuronal differentiation, dendrite and synapse formation and integration into the existing neuronal network. In animal experiments, seizures were found to increase NKCC1 expression, lower the KCC2 expression and accelerate neuronal differentiation. An increased expression of NKCC1 and mutations of the gene have been associated with schizophrenia. Stimulation of nicotinic α-7 receptors on mouse hippocampal neurons increases the expression of KCC2. A microdeletion in the genomic area 15q13-14 containing the nicotine α7 receptor has been described in patients with mental retardation, schizophrenia and juvenile epilepsy. It is conceivable that haplotype-insufficiency of the nicotinic α7 receptor might lead to a reduction in KCC2 protein levels. The data indicate that all three schizophrenia risk factors, i.e. seizures, mutations in NKCC1 and nicotinic α-7 receptors haplotype-insufficiency contribute to higher intracellular Cl⁻ concentrations, increased neuronal excitability and accelerated neuronal differentiation. Since also several other genetic risk factors for schizophrenia seem to accelerate neuronal maturation, it is hypothesized that the structural, cognitive and behavioral deficits of schizophrenia are caused be a too fast brain maturation process.     

Age- and region-specific effects of anticonvulsants and bumetanide on 4-aminopyridine-induced seizure-like events in immature rat hippocampal-entorhinal cortex slices

Purpose: Seizure‐like events (SLEs) induced by 4‐aminopyridine in rat organotypic slices cultures, which are prepared early after birth, are resistant to standard antiepileptic drugs. In this study we tested the hypothesis that pharmacoresistance may be an intrinsic property of the immature brain. Methods: Frequently recurring SLEs presumably representing status epilepticus were induced by 4‐aminopyridine in acute rat hippocampal–entorhinal cortex slices obtained from postnatal day 3–19 (P3–P19), and the effects of carbamazepine, phenytoin, valproic acid, and phenobarbital were examined. In addition, bumetanide was tested, which blocks the Na⁺‐K⁺‐2Cl⁻ (NKCC1) cotransporter, and also acetazolamide, which blocks the carbonic anhydrase and thereby the accumulation of bicarbonate inside neurons. Results: The efficacy of all antiepileptic drugs in blocking SLEs was dependent on postnatal age, with low efficacy in P3–P5 slices. Antiepileptic drugs suppressed SLEs more readily in the medial entorhinal cortex (ECm) than in the CA3. In P3–P5 slices, valproic acid and phenobarbital increased both tonic and clonic seizure‐like activities in the CA3, whereas phenytoin and carbamazepine blocked tonic‐like but prolonged clonic‐like activity. In P3–P5 slices, bumetanide often blocked SLEs in the CA3, but was not as effective in the ECm. Like with other antiepileptic drugs, the seizure‐suppressing effects of acetazolamide increased with postnatal age. Conclusion: We conclude that pharmacoresistance may be inherent to very immature tissue and suggest that expression of the NKCC1 cotransporter might contribute to pharmacoresistance.     

Hemispheric polymicrogyria and neonatal seizures: a potentially life‐threatening combination

Polymicrogyria (PMG) is a heterogeneous malformation of cortical development characterized by excessive gyration and abnormal cortical lamination. Typically, bilateral forms have more severe developmental delay and early‐onset epilepsy, but the full spectrum of severity remains ill‐defined. We report two cases of right hemispheric PMG and neonatal‐onset, drug‐resistant seizures culminating in early death. Case 1 began having seizures on Day 1 of life that intensified in severity and proved resistant to numerous antiepileptic drugs. He underwent right functional hemispherectomy but died three weeks post‐operatively due to ongoing seizures. Case 2 presented with seizures on Day 3 of life and required respiratory support for prolonged ictal apnoeas. Seizures were resistant to antiepileptic drugs and eventually led to respiratory arrest, once aggressive resuscitative measures were withdrawn. In both cases, seizures seemingly originated independently in both hemispheres. These cases represent a severe phenotype of unilateral hemispheric PMG with bilateral seizures.