NKCC1 activity modulates formation of functional inhibitory synapses in cultured neocortical neurons

Intracellular Cl(-) concentration ([Cl(-)](i)) in immature neurons is higher than that expected for a passive distribution, therefore the equilibrium potential for chloride is more positive than the resting membrane potential, and the resulting GABA renders immature neurons depolarization. The higher [Cl(-)](i) in immature neurons is thought to be attributed to the uptake of Cl(-) mediated by NKCC1 (Na(+), K(+)-2Cl(-) cotransporter). Thus, a dysfunction of this transporter could affect synaptic development through a GABA(A) receptor-mediated pathway. To test this possibility, we examined the effects of a Cl(-)-uptake inhibitor on the development of synaptic activities of rat neocortical neurons in culture. Chronic treatment with bumetanide at 10 microM during the culture diminished the amplitude of synaptically-driven rhythmic depolarizing potentials (RDPs) in neurons and also decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but not of spontaneous excitatory postsynaptic currents (sEPSCs). Chronic treatment with bumetanide decreased vesicular GABA transporter (VGAT)-immunopositive particles without affecting paired-pulse ratio of evoked IPSCs (eIPSCs), indicating decrease in the number of functional GABAergic synapses. Acute treatment with bumetanide (10 microM) decreased neuronal [Cl(-)](i), the amplitude of RDPs, and neuronal excitability, while bumetanide had no effect on RDPs and neuronal excitability in the presence of bicuculline. These results suggest that the uptake of Cl(-) by NKCC1 affects the development of inhibitory synapses by promoting a depolarizing GABA-mediated response.     

Chronic hyperosmotic stress converts GABAergic inhibition into excitation in vasopressin and oxytocin neurons in the rat

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.     

Consequences of inhibition of bumetanide metabolism in rodents on brain penetration and effects of bumetanide in chronic models of epilepsy

The diuretic bumetanide, which acts by blocking the Na–K–Cl cotransporter (NKCC), is widely used to inhibit neuronal NKCC1, particularly when NKCC1 expression is abnormally increased in brain diseases such as epilepsy. However, bumetanide poorly penetrates into the brain and, in rodents, is rapidly eliminated because of extensive oxidation of its N‐butyl sidechain, reducing the translational value of rodent experiments. Inhibition of oxidation by piperonyl butoxide (PBO) has previously been reported to increase the half‐life and diuretic activity of bumetanide in rats. Here we studied whether inhibition of bumetanide metabolism by PBO also increases brain levels of bumetanide in rats, and whether this alters pharmacodynamic effects in the kindling model of epilepsy. Furthermore, we studied the effects of PBO in mice. Mice eliminated bumetanide less rapidly than rats (elimination half‐life 47 min vs. 13 min). Pretreatment with PBO increased the half‐life in mice to average values (70 min) previously determined in humans, and markedly elevated brain levels of bumetanide. In rats, the increase in plasma and brain levels of bumetanide by PBO was less marked than in mice. PBO significantly increased the diuretic activity of bumetanide in rats and, less effectively, in mice. In epileptic mice, bumetanide (with PBO) did not suppress spontaneous seizures. In the rat kindling model, bumetanide (with or without PBO) did not exert anticonvulsant effects on fully kindled seizures, but dose‐dependently altered kindling development. These data indicate that PBO offers a simple means to enhance the translational properties of rodent experiments with bumetanide, particularly when using mice.     

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.     

NKCC1 and KCC2 prevent hyperexcitability in the mouse hippocampus

During postnatal development of the central nervous system (CNS), the response of GABA_A receptors to its agonist undergoes maturation from depolarizing to hyperpolarizing. This switch in polarity is due to the developmental decrease of the intracellular Cl concentration in neurons. Here we show that absence of NKCC1 in P9–P13 CA3 pyramidal neurons, through genetic manipulation or through bumetanide inhibition, results in a significant increase in cell excitability. Furthermore, the pro-convulsant agent 4-aminopyridine induces seizure-like events in NKCC1-null mice but not in wild-type mice. Measurements of muscimol responses in the presence and absence of NKCC1 shows that the Na–K–2Cl cotransporter only marginally affects intracellular Cl⁻ in P9–P13 CA3 principal neurons. However, large increases in intracellular Cl⁻ are observed in CA3 pyramidal neurons following increased hyperexcitability, indicating that P9–P13 CA3 pyramidal neurons lack robust mechanisms to regulate intracellular Cl⁻ during high synaptic activity. This increase in the Cl⁻ concentration is network-driven and activity-dependent, as it is blocked by the non-NMDA glutamate receptor antagonist DNQX. We also show that expression of the outward K–Cl cotransporter, KCC2, prevents the development of hyperexcitability, as a reduction of KCC2 expression by half results in increased susceptibility to seizure under control and 4-AP conditions.     

The extracellular space and epileptic activity in the adult brain: explaining the antiepileptic effects of furosemide and bumetanide

Treatments that modulate the size of the extracellular space (ECS) also block epileptiform activity in adult brain tissue. This includes the loop diuretics furosemide and bumetanide, and alterations of the osmolarity of the ECS. These treatments block epileptiform activity in a variety of laboratory adult seizure models regardless of the underlying synaptic and physiologic mechanisms generating the seizure activity. Optical imaging studies on adult hippocampal slices show that the blockade of epileptiform activity by these treatments is concomitant with their blockade of activity-driven changes of the ECS. Here we develop and analyze the hypothesis that activity-driven changes in the size of the ECS are necessary for the maintenance of hypersynchronous epileptiform activity. In support of this hypothesis is an accumulation of data from a number of studies suggesting that furosemide and bumetanide mediate antiepileptic effects through their blockade of cell swelling, dependent on their antagonism of the glial Na+-K-2Cl cotransporter (NKCC1).     

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.     

KCC2 was downregulated in small neurons localized in epileptogenic human focal cortical dysplasia

Focal cortical dysplasia (FCD), which is characterized histologically by disorganized cortical lamination and large abnormal cells, is one of the major causes of intractable epilepsies. γ-aminobutyric acid (GABA)(A) receptor-mediated synchronous depolarizing potentials have been observed in FCD tissue. Since alterations in Cl(-) homeostasis might underlie these depolarizing actions of GABA, cation-Cl(-) cotransporters could play critical roles in the generation of these abnormal actions. We examined the expression patterns of NKCC1 and KCC2 by in situ hybridization histochemistry and immunohistochemistry in FCD tissue obtained by surgery from patients with intractable epilepsy. KCC2 mRNA and protein were expressed not only in non-dysplastic neurons in histologically normal portions located in the periphery of the excised cortex, but also in dysplastic cells in FCD tissue. The levels of KCC2 mRNA and protein were significantly decreased in the neurons around large abnormal neurons (giant neurons), but not in giant neurons, compared with non-dysplastic neurons. The neurons localized only around giant neurons significantly smaller than non-dysplastic neurons. However NKCC1 expression did not differ among these cell types. These results suggest that the intracellular Cl(-) concentration ([Cl(-)](i)) of small neurons might increase, so that depolarizing GABA actions could occur in the FCD tissue of epileptic foci.     

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.     

Postnatal maturation of Na+, K+, 2Cl- cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl- through GABAA-gated anion channels. The outward Cl- gradient that provides the driving force for Cl- efflux might be generated and maintained by the Na+, K+, 2Cl- cotransporter (NKCC) that keeps intracellular Cl- concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.     

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.     

Long‐term health‐related quality of life in drug‐resistant temporal lobe epilepsy after anterior temporal lobectomy

Epilepsy surgery is beneficial to patients suffering from drug‐resistant temporal lobe epilepsy in the short term, but fewer reports of long‐term outcomes have been published. To clarify the long‐term outcomes of seizure control and health‐related quality of life after epilepsy surgery, we enrolled 48 patients suffering from drug‐resistant temporal lobe epilepsy. All of the patients received comprehensive presurgical evaluations, including the Quality of Life in Epilepsy Inventory‐89 (QOLIE‐89) questionnaire to measure their health‐related quality of life. Among the patients, 28 patients received surgery (surgical group) and 20 patients remained under medication (medical group). Eight years later, the seizure frequency and QOLIE‐89 were evaluated. The seizure‐free rate was much higher in the surgical group (53.6%) than in the medical group (5%), eight years after the initial evaluation. The follow‐up QOLIE‐89 score was significantly higher in the surgical group than in the medical group. Moreover, the seizure frequency inversely correlated to the QOLIE‐89 score, regardless of the treatment group. Our results provide evidence that epilepsy surgery confers benefits with respect to seizure control and health‐related quality of life for drug‐resistant temporal lobe epilepsy patients based on long‐term follow‐up.     

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).     

The role of Na-K-Cl co-transporter in cerebral ischemia

The electroneutral Na-K-Cl co-transporter (NKCC) protein transports Na(+), K(+) and Cl(-) into cells under physiological conditions with a stoichiometry of 1Na(+) :1K(+) :2Cl(-). NKCC is characteristically inhibited by the sulfamoylbenzoic acid "loop' diuretics, such as bumetanide and furosemide. To date, only two distinct isoforms, NKCC1 and NKCC2, have been identified. NKCC1 has a broad tissue distribution, while the NKCC2 isoform is only found in vertebrate kidney. NKCC serves multiple functions, including ion and fluid movements in secreting or reabsorbing epithelia and cell volume regulation. However, understanding the role of NKCC1 in the central nervous system has just begun. NKCC1 protein is expressed in neurons throughout the brain. Dendritic localization of NKCC1 is found in both pyramidal and non-pyramidal neurons. NKCC1 is important in the maintenance of intracellular Cl(-) in neurons and contributes to GABA-mediated depolarization in immature neurons. Thus, NKCC1 may affect neuronal excitability through regulation of intracellular Cl(-) concentration. Expression of NKCC1 protein has also been found in astrocytes and oligodendrocytes. In addition to its role in the accumulation of Cl(-), NKCC1 may also contribute to K(+) clearance and maintenance of intracellular Na(+) in glia. Our recent studies suggest that NKCC1 activation leads to high [K(+)](o(-)) induced astrocyte swelling and glutamate release, as well as neuronal Na(+) , and Cl(-) influx during acute excitotoxicity. Inhibition of NKCC1 activity significantly reduces infarct volume and cerebral edema following cerebral focal ischemia.     

The search for NKCC1-selective drugs for the treatment of epilepsy: Structure–function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A

The Na⁺–K⁺–Cl⁻ cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl⁻ concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~ 5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure–function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC₅₀s) of bumetanide were 0.68 (hNKCC1A) and 4.0 μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC₅₀s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.     

Low frequency stimulation of ventral hippocampal commissures reduces seizures in a rat model of chronic temporal lobe epilepsy

Purpose: To investigate the effects of low frequency stimulation (LFS) of a fiber tract for the suppression of spontaneous seizures in a rat model of human temporal lobe epilepsy. Methods: Stimulation electrodes were implanted into the ventral hippocampal commissure (VHC) in a rat post‐status epilepticus (SE) model of human temporal lobe epilepsy (n = 7). Two recording electrodes were placed in the CA3 regions bilaterally and neural data were recorded for a minimum of 6 weeks. LFS (60 min train of 1 Hz biphasic square wave pulses, each 0.1 ms in duration and 200 μA in amplitude, followed by 15 min of rest) was applied to the VHC for 2 weeks, 24 h a day. Key Findings: The baseline mean seizure frequency of the study animals was 3.7 seizures per day. The seizures were significantly reduced by the application of LFS in every animal (n = 7). By the end of the 2‐week period of stimulation, there was a significant, 90% (<1 seizure/day) reduction of seizure frequencies (p < 0.05) and a 57% reduction during the period following LFS (p < 0.05) when compared to baseline. LFS also resulted in a significant reduction of hippocampal interictal spike frequency (71%, p < 0.05), during 2 weeks of LFS session. The hippocampal histologic analysis showed no significant difference between rats that received LFS and SE induction and those that had received only SE‐induction. None of the animals showed any symptomatic hemorrhage, infection, or complication. Significance: Low frequency stimulation applied at a frequency of 1 Hz significantly reduced both the excitability of the neural tissue as well as the seizure frequency in a rat model of human temporal lobe epilepsy. The results support the hypothesis that LFS of fiber tracts can be an effective method for the suppression of spontaneous seizures in a temporal lobe model of epilepsy in rats and could lead to the development of a new therapeutic modality for human patients with temporal lobe epilepsy.     

Coincident pre- and postsynaptic activity downregulates NKCC1 to hyperpolarize E(Cl) during development

In the mature CNS, coincident pre- and postsynaptic activity decreases the strength of gamma-aminobutyric acid (GABA)(A)-mediated inhibition through a Ca2+-dependent decrease in the activity of the neuron-specific K+-Cl- cotransporter KCC2. In the present study we examined whether coincident pre- and postsynaptic activity can also modulate immature GABAergic synapses, where the Na+-K+-2Cl- (NKCC1) cotransporter maintains a relatively high level of intracellular chloride ([Cl-](i)). Dual perforated patch-clamp recordings were made from cultured hippocampal neurons prepared from embryonic Sprague-Dawley rats. These recordings were used to identify GABAergic synapses where the reversal potential for Cl- (ECl) was hyperpolarized with respect to the action potential threshold but depolarized with respect to the resting membrane potential. At these synapses, repetitive postsynaptic spiking within +/- 5 ms of GABAergic synaptic transmission resulted in a hyperpolarizing shift of ECl by 10.03 +/- 1.64 mV, increasing the strength of synaptic inhibition. Blocking the inward transport of Cl- by NKCC1 with bumetanide (10 microm) hyperpolarized ECl by 16.14 +/- 4.8 mV, and prevented this coincident activity-induced shift of ECl. The bumetanide-induced hyperpolarization of ECl occluded furosemide, a K+-Cl- cotransporter antagonist, from producing further shifts in ECl. Together, this indicates that brief coincident pre- and postsynaptic activity strengthens inhibition through a regulation of NKCC1. This study further demonstrates ionic plasticity as a mechanism underlying inhibitory synaptic plasticity.     

Pharmacotherapeutic targeting of cation‐chloride cotransporters in neonatal seizures

Seizures are a common manifestation of acute neurologic insults in neonates and are often resistant to the standard antiepileptic drugs that are efficacious in children and adults. The paucity of evidence‐based treatment guidelines, coupled with a rudimentary understanding of disease pathogenesis, has made the current treatment of neonatal seizures empiric and often ineffective, highlighting the need for novel therapies. Key developmental differences in γ‐aminobutyric acid (GABA)ergic neurotransmission between the immature and mature brain, and trauma‐induced alterations in the function of the cation‐chloride cotransporters (CCCs) NKCC1 and KCC2, probably contribute to the poor efficacy of standard antiepileptic drugs used in the treatment of neonatal seizures. Although CCCs are attractive drug targets, bumetanide and other existing CCC inhibitors are suboptimal because of pharmacokinetic constraints and lack of target specificity. Newer approaches including isoform‐specific NKCC1 inhibitors with increased central nervous system penetration, and direct and indirect strategies to enhance KCC2‐mediated neuronal chloride extrusion, might allow therapeutic modulation of the GABAergic system for neonatal seizure treatment. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here .     

Interictal Behavioral Alterations and Cerebrospinal Fluid Amino Acid Changes in a Chronic Seizure Model of Temporal Lobe Epilepsy

This study extends our previous work in which we described the presence of an interictal behavioral disturbance in a chronic animal model of temporal lobe epilepsy (TLE). In this study, we investigated the cerebrospinal fluid (CSF) neurotransmitter changes underlying the development of chronic recurrent seizures of temporal lobe origin and interictal behavioral disturbance in cats made epileptic after intrahippocampal injection of kainic acid (KA). Using high-performance liquid chromatography, we measured 22 putative neurotransmitter amino acids. After intrahippocampal KA injection, cats developed an initial acute period of intense seizure activity. Cisternal CSF amino acids, which were repeatedly sampled during the acute period through a permanent indwelling cannula, were unchanged apart from a mild elevation in CSF alanine. The high-level seizure activity gradually decreased, and cats entered a chronic epileptic period characterized by recurrent yet intermittent temporal lobe seizures. CSF GABA levels during the chronic epileptic period were significantly decreased. In contrast, CSF levels of other amino acids--alanine, tyrosine, taurine, aspartic acid, and glutamic acid--did not change significantly. Behavioral testing also showed a heightened interictal defensive reactivity during the chronic epileptic period. To the extent that CSF GABA concentration reflects brain GABA concentration, this study suggests that a decrease in brain GABA may contribute both to the epilepsy and interictal emotional lability of animals with a chronic seizure disorder of temporal lobe origin.