The mRNA levels of NKCC1, an inwardly directed Na ⁺ , K ⁺ -2Cl ⁻ cotransporter that facilitates the accumulation of intracellular Cl ⁻ , and of KCC2, an outwardly directed K ⁺ -Cl ⁻ cotransporter that extrudes Cl ⁻ , were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative RT-PCR analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an up-regulation of NKCC1 mRNA and a down-regulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl ⁻ transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABA _A receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potential ( E _GABA ) compared with the hippocampus proper or the neocortex. The NKCC1 blocker bumetanide or a temperature decrease of 10°C shifted the GABA-current E _GABA more negative in oocytes injected with membranes from TLE hippocampal subiculum, matching the E _GABA of TL neocortex-injected oocytes. We conclude that the anomalous expression of both Cl ⁻ transporters, KCC1 and NKCC2, in TLE hippocampal subiculum probably causes altered Cl ⁻ transport in the “epileptic” neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly “exciting,” a feature that may contribute to the precipitation of epileptic seizures.

Summary: A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. Most importantly, the same inflammatory pathways have also been found in surgically resected brain tissue from patients with treatment‐resistant epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti‐ictogenesis, antiepileptogenesis, and/or disease‐modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof‐of‐concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries.

Purpose: Several factors contribute to epileptogenesis in patients with brain tumors, including reduced γ-aminobutyric acid (GABA)ergic inhibition. In particular, changes in Cl⁻ homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl⁻ homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl⁻ concentration ([Cl⁻]_i) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl⁻]_i. Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors. Methods: Membranes from peritumoral cortical tissues of epileptic patients afflicted by gliomas (from II to IV WHO grade) and from cortical tissues of nonepileptic patients were injected into Xenopus oocytes leading to the incorporation of functional GABA_A receptors. The GABA-evoked currents were recorded using standard two-microelectrode voltage-clamp technique. In addition, immunoblot analysis and immunohistochemical staining were carried out on membranes and tissues from the same patients. Key Findings: We found that in oocytes injected with epileptic peritumoral cerebral cortex, the GABA-evoked currents had a more depolarized reversal potential (E_GABA) compared to those from nonepileptic healthy cortex. This difference of E_GABA was abolished by the NKCC1 blocker bumetanide or unblocking of KCC2 with the Zn²⁺ chelator TPEN. Moreover, Western blot analysis revealed an increased expression of NKCC1, and more modestly, of KCC2 transporters in epileptic peritumoral tissues compared to nonepileptic control tissues. In addition, NKCC1 immunoreactivity was strongly increased in peritumoral cortex with respect to nonepileptic cortex, with a prominent expression in neuronal cells. Significance: We report that the positive shift of E_GABA in epileptic peritumoral human cortex is due to an altered expression of NKCC1 and KCC2, perturbing Cl⁻ homeostasis, which might lead to a consequent reduction in GABAergic inhibition. These findings point to a key role of Cl⁻ transporters KCC2 and NKCC1 in tumor-related epilepsy, suggesting a more specific drug therapy and surgical approaches for the epileptic patients afflicted by brain tumors.

Summary: Purpose: The chemokine fractalkine/CX3CL1 and its receptor CX3CR1 are widely expressed in the central nervous system (CNS). Recent evidence showed that CX3CL1 participates in inflammatory responses that are common features of CNS disorders, such as epilepsy. Mesial temporal lobe epilepsy (MTLE) is the prevalent form of focal epilepsy in adults, and hippocampal sclerosis (HS) represents the most common underlying pathologic abnormality, as demonstrated at autopsy and postresection studies. Relevant features of MTLE are a characteristic pattern of neuronal loss, as are astrogliosis and microglia activation. Several factors affect epileptogenesis in patients with MTLE, including a lack of γ‐aminobutyric acid (GABA)ergic inhibitory efficacy. Therefore, experiments were designed to investigate whether, in MTLE brain tissues, CX3CL1 may influence GABA_A receptor (GABA_AR) mediatedtransmission, with a particular focus on the action of CX3CL1 on the use‐dependent decrease (rundown) of the GABA‐evoked currents (I_GABA), a feature underlying the reduction of GABAergic function in epileptic tissue.Methods: Patch‐clamp recordings were obtained from cortical pyramidal neurons in slices from six MTLE patients after surgery. Alternatively, the cell membranes from epileptic brain tissues of 17 MTLE patients or from surgical samples and autopsies of nonepileptic patients were microtransplanted into Xenopus oocytes, and I_GABA were recorded using the standard two‐microelectrode voltage‐clamp technique. Immunohistochemical staining and double‐labeling studies were carried out on the same brain tissues to analyze CX3CR1 expression.Key Findings: In native pyramidal neurons from cortical slices of patients with MTLE, CX3CL1 reduced I_GABA rundown and affected the recovery of I_GABA amplitude from rundown. These same effects were confirmed in oocytes injected with cortical and hippocampal MTLE membranes, whereas CX3CL1 did not influence I_GABA in oocytes injected with nonepileptic tissues. Consistent with a specific effect of CX3CL1 on tissues from patients with MTLE, CX3CR1 immunoreactivity was higher in MTLE sclerotic hippocampi than in control tissues, with a prominent expression in activated microglial cells.Significance: These findings indicate a role for CX3CL1 in MTLE, supporting recent evidence on the relevance of brain inflammation in human epilepsies. Our data demonstrate that in MTLE tissues the reduced GABAergic function can be modulated by CX3CL1. The increased CX3CR1 expression in microglia and the modulation by CX3CL1 of GABAergic currents in human epileptic brain suggests new therapeutic approaches for drug‐resistant epilepsies based on the evidence that the propagation of seizures can be influenced by inflammatory processes.

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl ⁻ channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to γ-aminobutyric acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.