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  1. Johan Pallud, Michel Le Van Quyen, Franck Bielle, Christophe Pellegrino, Pascale Varlet, Marianne Labussiere, Noémie Cresto, Marie-Joseph Dieme, Michel Baulac, Charles Duyckaerts, Nazim Kourdougli, Geneviève Chazal, Bertrand Devaux, Claudio Rivera, Richard Miles, Laurent Capelle and Gilles Huberfeld.
    Cortical GABAergic excitation contributes to epileptic activities around human glioma.. Science translational medicine 6(244):244ra89, July 2014.
    Abstract Brain gliomas are highly epileptogenic. Excitatory glutamatergic mechanisms are involved in the generation of epileptic activities in the neocortex surrounding gliomas. However, chloride homeostasis is known to be perturbed in glioma cells. Thus, the contribution of $\gamma$-aminobutyric acidergic (GABAergic) mechanisms that depend on intracellular chloride merits closer study. We studied the occurrence, networks, cells, and signaling basis of epileptic activities in neocortical slices from the peritumoral surgical margin resected around human brain gliomas. Postoperative glioma tissue from 69% of patients spontaneously generated interictal-like discharges, synchronized, with a high-frequency oscillation signature, in superficial layers of neocortex around areas of glioma infiltration. Interictal-like events depended both on glutamatergic AMPA receptor-mediated transmission and on depolarizing GABAergic signaling. GABA released by interneurons depolarized 65% of pyramidal cells, in which chloride homeostasis was perturbed because of changes in expression of neuronal chloride cotransporters: KCC2 (K-Cl cotransporter 2) was reduced by 42% and expression of NKCC1 (Na-K-2Cl cotransporter 1) increased by 144%. Ictal-like activities were initiated by convulsant stimuli exclusively in these epileptogenic areas. This study shows that epileptic activities are sustained by excitatory effects of GABA in human peritumoral neocortex, as reported in temporal lobe epilepsies, suggesting that both glutamate and GABA signaling and cellular chloride regulation processes, all also involved in oncogenesis as already shown, induce an imbalance between synaptic excitation and inhibition underlying epileptic discharges in glioma patients. Thus, the control of chloride in neurons and glioma cells may provide a therapeutic target for patients with epileptogenic gliomas.
    URL, DOI BibTeX

    @article{Pallud2014,
    	abstract = "Brain gliomas are highly epileptogenic. Excitatory glutamatergic mechanisms are involved in the generation of epileptic activities in the neocortex surrounding gliomas. However, chloride homeostasis is known to be perturbed in glioma cells. Thus, the contribution of $\gamma$-aminobutyric acidergic (GABAergic) mechanisms that depend on intracellular chloride merits closer study. We studied the occurrence, networks, cells, and signaling basis of epileptic activities in neocortical slices from the peritumoral surgical margin resected around human brain gliomas. Postoperative glioma tissue from 69\% of patients spontaneously generated interictal-like discharges, synchronized, with a high-frequency oscillation signature, in superficial layers of neocortex around areas of glioma infiltration. Interictal-like events depended both on glutamatergic AMPA receptor-mediated transmission and on depolarizing GABAergic signaling. GABA released by interneurons depolarized 65\% of pyramidal cells, in which chloride homeostasis was perturbed because of changes in expression of neuronal chloride cotransporters: KCC2 (K-Cl cotransporter 2) was reduced by 42\% and expression of NKCC1 (Na-K-2Cl cotransporter 1) increased by 144\%. Ictal-like activities were initiated by convulsant stimuli exclusively in these epileptogenic areas. This study shows that epileptic activities are sustained by excitatory effects of GABA in human peritumoral neocortex, as reported in temporal lobe epilepsies, suggesting that both glutamate and GABA signaling and cellular chloride regulation processes, all also involved in oncogenesis as already shown, induce an imbalance between synaptic excitation and inhibition underlying epileptic discharges in glioma patients. Thus, the control of chloride in neurons and glioma cells may provide a therapeutic target for patients with epileptogenic gliomas.",
    	author = "Pallud, Johan and {Le Van Quyen}, Michel and Bielle, Franck and Pellegrino, Christophe and Varlet, Pascale and Labussiere, Marianne and Cresto, No\'{e}mie and Dieme, Marie-Joseph and Baulac, Michel and Duyckaerts, Charles and Kourdougli, Nazim and Chazal, Genevi\`{e}ve and Devaux, Bertrand and Rivera, Claudio and Miles, Richard and Capelle, Laurent and Huberfeld, Gilles",
    	doi = "10.1126/scitranslmed.3008065",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Pallud et al. - 2014 - Cortical GABAergic excitation contributes to epileptic activities around human glioma.pdf:pdf",
    	issn = "1946-6242",
    	journal = "Science translational medicine",
    	keywords = "Action Potentials,Brain Neoplasms,Brain Neoplasms: pathology,Brain Neoplasms: physiopathology,Chlorides,Chlorides: metabolism,Epilepsy,Epilepsy: pathology,Epilepsy: physiopathology,Glioma,Glioma: pathology,Glioma: physiopathology,Glutamates,Glutamates: metabolism,Humans,Interneurons,Interneurons: pathology,Neocortex,Neocortex: pathology,Neocortex: physiopathology,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: metabolism",
    	month = "jul",
    	number = 244,
    	pages = "244ra89",
    	pmid = 25009229,
    	title = "{Cortical GABAergic excitation contributes to epileptic activities around human glioma.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4409113\&tool=pmcentrez\&rendertype=abstract",
    	volume = 6,
    	year = 2014
    }
    
  2. L L Jantzie, P M Getsy, D J Firl, C G Wilson, R H Miller and S Robinson.
    Erythropoietin attenuates loss of potassium chloride co-transporters following prenatal brain injury.. Molecular and cellular neurosciences 61:152–62, 2014.
    Abstract Therapeutic agents that restore the inhibitory actions of $\gamma$-amino butyric acid (GABA) by modulating intracellular chloride concentrations will provide novel avenues to treat stroke, chronic pain, epilepsy, autism, and neurodegenerative and cognitive disorders. During development, upregulation of the potassium-chloride co-transporter KCC2, and the resultant switch from excitatory to inhibitory responses to GABA guide the formation of essential inhibitory circuits. Importantly, maturation of inhibitory mechanisms is also central to the development of excitatory circuits and proper balance between excitatory and inhibitory networks in the developing brain. Loss of KCC2 expression occurs in postmortem samples from human preterm infant brains with white matter lesions. Here we show that late gestation brain injury in a rat model of extreme prematurity impairs the developmental upregulation of potassium chloride co-transporters during a critical postnatal period of circuit maturation in CA3 hippocampus by inducing a sustained loss of oligomeric KCC2 via a calpain-dependent mechanism. Further, administration of erythropoietin (EPO) in a clinically relevant postnatal dosing regimen following the prenatal injury protects the developing brain by reducing calpain activity, restoring oligomeric KCC2 expression and attenuating KCC2 fragmentation, thus providing the first report of a safe therapy to address deficits in KCC2 expression. Together, these data indicate it is possible to reverse abnormalities in KCC2 expression during the postnatal period, and potentially reverse deficits in inhibitory circuit formation central to cognitive impairment and epileptogenesis.
    URL, DOI BibTeX

    @article{Jantzie2014,
    	abstract = "Therapeutic agents that restore the inhibitory actions of $\gamma$-amino butyric acid (GABA) by modulating intracellular chloride concentrations will provide novel avenues to treat stroke, chronic pain, epilepsy, autism, and neurodegenerative and cognitive disorders. During development, upregulation of the potassium-chloride co-transporter KCC2, and the resultant switch from excitatory to inhibitory responses to GABA guide the formation of essential inhibitory circuits. Importantly, maturation of inhibitory mechanisms is also central to the development of excitatory circuits and proper balance between excitatory and inhibitory networks in the developing brain. Loss of KCC2 expression occurs in postmortem samples from human preterm infant brains with white matter lesions. Here we show that late gestation brain injury in a rat model of extreme prematurity impairs the developmental upregulation of potassium chloride co-transporters during a critical postnatal period of circuit maturation in CA3 hippocampus by inducing a sustained loss of oligomeric KCC2 via a calpain-dependent mechanism. Further, administration of erythropoietin (EPO) in a clinically relevant postnatal dosing regimen following the prenatal injury protects the developing brain by reducing calpain activity, restoring oligomeric KCC2 expression and attenuating KCC2 fragmentation, thus providing the first report of a safe therapy to address deficits in KCC2 expression. Together, these data indicate it is possible to reverse abnormalities in KCC2 expression during the postnatal period, and potentially reverse deficits in inhibitory circuit formation central to cognitive impairment and epileptogenesis.",
    	author = "Jantzie, L L and Getsy, P M and Firl, D J and Wilson, C G and Miller, R H and Robinson, S",
    	doi = "10.1016/j.mcn.2014.06.009",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Jantzie et al. - 2014 - Erythropoietin attenuates loss of potassium chloride co-transporters following prenatal brain injury.pdf:pdf",
    	issn = "1095-9327",
    	journal = "Molecular and cellular neurosciences",
    	keywords = "Age Factors,Animals,Animals, Newborn,Brain-Derived Neurotrophic Factor,Brain-Derived Neurotrophic Factor: genetics,Brain-Derived Neurotrophic Factor: metabolism,Brain-Derived Neurotrophic Factor: pharmacology,Carbazoles,Carbazoles: pharmacology,Enzyme Inhibitors,Enzyme Inhibitors: pharmacology,Erythropoietin,Erythropoietin: immunology,Erythropoietin: therapeutic use,Excitatory Amino Acid Agonists,Excitatory Amino Acid Agonists: pharmacology,Female,Gene Expression Regulation, Developmental,Gene Expression Regulation, Developmental: drug ef,Gene Expression Regulation, Developmental: physiol,Hippocampus,Hippocampus: drug effects,Hippocampus: metabolism,In Vitro Techniques,Indole Alkaloids,Indole Alkaloids: pharmacology,Ischemic Attack, Transient,Ischemic Attack, Transient: drug therapy,Ischemic Attack, Transient: etiology,Ischemic Attack, Transient: metabolism,Male,N-Methylaspartate,N-Methylaspartate: pharmacology,Neuroprotective Agents,Neuroprotective Agents: therapeutic use,Pregnancy,Rats,Rats, Sprague-Dawley,Symporters,Symporters: metabolism",
    	month = "",
    	pages = "152--62",
    	pmid = 24983520,
    	title = "{Erythropoietin attenuates loss of potassium chloride co-transporters following prenatal brain injury.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4134983\&tool=pmcentrez\&rendertype=abstract",
    	volume = 61,
    	year = 2014
    }
    
  3. James W Grau, Russell J Huie, Kuan H Lee, Kevin C Hoy, Yung-Jen Huang, Joel D Turtle, Misty M Strain, Kyle M Baumbauer, Rajesh M Miranda, Michelle A Hook, Adam R Ferguson and Sandra M Garraway.
    Metaplasticity and behavior: how training and inflammation affect plastic potential within the spinal cord and recovery after injury.. Frontiers in neural circuits 8:100, January 2014.
    Abstract Research has shown that spinal circuits have the capacity to adapt in response to training, nociceptive stimulation and peripheral inflammation. These changes in neural function are mediated by physiological and neurochemical systems analogous to those that support plasticity within the hippocampus (e.g., long-term potentiation and the NMDA receptor). As observed in the hippocampus, engaging spinal circuits can have a lasting impact on plastic potential, enabling or inhibiting the capacity to learn. These effects are related to the concept of metaplasticity. Behavioral paradigms are described that induce metaplastic effects within the spinal cord. Uncontrollable/unpredictable stimulation, and peripheral inflammation, induce a form of maladaptive plasticity that inhibits spinal learning. Conversely, exposure to controllable or predictable stimulation engages a form of adaptive plasticity that counters these maladaptive effects and enables learning. Adaptive plasticity is tied to an up-regulation of brain derived neurotrophic factor (BDNF). Maladaptive plasticity is linked to processes that involve kappa opioids, the metabotropic glutamate (mGlu) receptor, glia, and the cytokine tumor necrosis factor (TNF). Uncontrollable nociceptive stimulation also impairs recovery after a spinal contusion injury and fosters the development of pain (allodynia). These adverse effects are related to an up-regulation of TNF and a down-regulation of BDNF and its receptor (TrkB). In the absence of injury, brain systems quell the sensitization of spinal circuits through descending serotonergic fibers and the serotonin 1A (5HT 1A) receptor. This protective effect is blocked by surgical anesthesia. Disconnected from the brain, intracellular Cl(-) concentrations increase (due to a down-regulation of the cotransporter KCC2), which causes GABA to have an excitatory effect. It is suggested that BDNF has a restorative effect because it up-regulates KCC2 and re-establishes GABA-mediated inhibition.
    URL, DOI BibTeX

    @article{Grau2014,
    	abstract = "Research has shown that spinal circuits have the capacity to adapt in response to training, nociceptive stimulation and peripheral inflammation. These changes in neural function are mediated by physiological and neurochemical systems analogous to those that support plasticity within the hippocampus (e.g., long-term potentiation and the NMDA receptor). As observed in the hippocampus, engaging spinal circuits can have a lasting impact on plastic potential, enabling or inhibiting the capacity to learn. These effects are related to the concept of metaplasticity. Behavioral paradigms are described that induce metaplastic effects within the spinal cord. Uncontrollable/unpredictable stimulation, and peripheral inflammation, induce a form of maladaptive plasticity that inhibits spinal learning. Conversely, exposure to controllable or predictable stimulation engages a form of adaptive plasticity that counters these maladaptive effects and enables learning. Adaptive plasticity is tied to an up-regulation of brain derived neurotrophic factor (BDNF). Maladaptive plasticity is linked to processes that involve kappa opioids, the metabotropic glutamate (mGlu) receptor, glia, and the cytokine tumor necrosis factor (TNF). Uncontrollable nociceptive stimulation also impairs recovery after a spinal contusion injury and fosters the development of pain (allodynia). These adverse effects are related to an up-regulation of TNF and a down-regulation of BDNF and its receptor (TrkB). In the absence of injury, brain systems quell the sensitization of spinal circuits through descending serotonergic fibers and the serotonin 1A (5HT 1A) receptor. This protective effect is blocked by surgical anesthesia. Disconnected from the brain, intracellular Cl(-) concentrations increase (due to a down-regulation of the cotransporter KCC2), which causes GABA to have an excitatory effect. It is suggested that BDNF has a restorative effect because it up-regulates KCC2 and re-establishes GABA-mediated inhibition.",
    	author = "Grau, James W and Huie, J Russell and Lee, Kuan H and Hoy, Kevin C and Huang, Yung-Jen and Turtle, Joel D and Strain, Misty M and Baumbauer, Kyle M and Miranda, Rajesh M and Hook, Michelle A and Ferguson, Adam R and Garraway, Sandra M",
    	doi = "10.3389/fncir.2014.00100",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Grau et al. - 2014 - Metaplasticity and behavior how training and inflammation affect plastic potential within the spinal cord and recov.pdf:pdf",
    	issn = "1662-5110",
    	journal = "Frontiers in neural circuits",
    	keywords = "Animals,Brain-Derived Neurotrophic Factor,Brain-Derived Neurotrophic Factor: genetics,Brain-Derived Neurotrophic Factor: metabolism,Humans,Inflammation,Inflammation: physiopathology,Learning,Learning Disorders,Learning Disorders: physiopathology,Learning: physiology,Neuronal Plasticity,Neuronal Plasticity: physiology,Receptors, Glutamate,Receptors, Glutamate: metabolism,Recovery of Function,Recovery of Function: physiology,Spinal Cord Injuries,Spinal Cord Injuries: pathology,Spinal Cord Injuries: physiopathology",
    	month = "jan",
    	pages = 100,
    	pmid = 25249941,
    	title = "{Metaplasticity and behavior: how training and inflammation affect plastic potential within the spinal cord and recovery after injury.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4157609\&tool=pmcentrez\&rendertype=abstract",
    	volume = 8,
    	year = 2014
    }
    
  4. Florian Gackière and Laurent Vinay.
    Serotonergic modulation of post-synaptic inhibition and locomotor alternating pattern in the spinal cord.. Frontiers in neural circuits 8:102, January 2014.
    Abstract The central pattern generators (CPGs) for locomotion, located in the lumbar spinal cord, are functional at birth in the rat. Their maturation occurs during the last few days preceding birth, a period during which the first projections from the brainstem start to reach the lumbar enlargement of the spinal cord. Locomotor burst activity in the mature intact spinal cord alternates between flexor and extensor motoneurons through reciprocal inhibition and between left and right sides through commisural inhibitory interneurons. By contrast, all motor bursts are in phase in the fetus. The alternating pattern disappears after neonatal spinal cord transection which suppresses supraspinal influences upon the locomotor networks. This article will review the role of serotonin (5-HT), in particular 5-HT2 receptors, in shaping the alternating pattern. For instance, pharmacological activation of these receptors restores the left-right alternation after injury. Experiments aimed at either reducing the endogenous level of serotonin in the spinal cord or blocking the activation of 5-HT2 receptors. We then describe recent evidence that the action of 5-HT2 receptors is mediated, at least in part, through a modulation of chloride homeostasis. The postsynaptic action of GABA and glycine depends on the intracellular concentration of chloride ions which is regulated by a protein in the plasma membrane, the K(+)-Cl(-) cotransporter (KCC2) extruding both K(+) and Cl(-) ions. Absence or reduction of KCC2 expression leads to a depolarizing action of GABA and glycine and a marked reduction in the strength of postsynaptic inhibition. This latter situation is observed early during development and in several pathological conditions, such as after spinal cord injury, thereby causing spasticity and chronic pain. It was recently shown that specific activation of 5-HT2A receptors is able to up-regulate KCC2, restore endogenous inhibition and reduce spasticity.
    URL, DOI BibTeX

    @article{Gackiere2014,
    	abstract = "The central pattern generators (CPGs) for locomotion, located in the lumbar spinal cord, are functional at birth in the rat. Their maturation occurs during the last few days preceding birth, a period during which the first projections from the brainstem start to reach the lumbar enlargement of the spinal cord. Locomotor burst activity in the mature intact spinal cord alternates between flexor and extensor motoneurons through reciprocal inhibition and between left and right sides through commisural inhibitory interneurons. By contrast, all motor bursts are in phase in the fetus. The alternating pattern disappears after neonatal spinal cord transection which suppresses supraspinal influences upon the locomotor networks. This article will review the role of serotonin (5-HT), in particular 5-HT2 receptors, in shaping the alternating pattern. For instance, pharmacological activation of these receptors restores the left-right alternation after injury. Experiments aimed at either reducing the endogenous level of serotonin in the spinal cord or blocking the activation of 5-HT2 receptors. We then describe recent evidence that the action of 5-HT2 receptors is mediated, at least in part, through a modulation of chloride homeostasis. The postsynaptic action of GABA and glycine depends on the intracellular concentration of chloride ions which is regulated by a protein in the plasma membrane, the K(+)-Cl(-) cotransporter (KCC2) extruding both K(+) and Cl(-) ions. Absence or reduction of KCC2 expression leads to a depolarizing action of GABA and glycine and a marked reduction in the strength of postsynaptic inhibition. This latter situation is observed early during development and in several pathological conditions, such as after spinal cord injury, thereby causing spasticity and chronic pain. It was recently shown that specific activation of 5-HT2A receptors is able to up-regulate KCC2, restore endogenous inhibition and reduce spasticity.",
    	author = "Gacki\`{e}re, Florian and Vinay, Laurent",
    	doi = "10.3389/fncir.2014.00102",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Gacki\`{e}re, Vinay - 2014 - Serotonergic modulation of post-synaptic inhibition and locomotor alternating pattern in the spinal cord.pdf:pdf",
    	issn = "1662-5110",
    	journal = "Frontiers in neural circuits",
    	keywords = "Animals,Humans,Locomotion,Locomotion: physiology,Neural Inhibition,Neural Inhibition: drug effects,Neural Inhibition: physiology,Rats,Receptors, Serotonin,Receptors, Serotonin: metabolism,Serotonin,Serotonin: metabolism,Serotonin: pharmacology,Spinal Cord,Spinal Cord Injuries,Spinal Cord Injuries: chemically induced,Spinal Cord Injuries: pathology,Spinal Cord Injuries: physiopathology,Spinal Cord: cytology,Spinal Cord: physiology,Synapses,Synapses: drug effects,Synapses: physiology",
    	month = "jan",
    	pages = 102,
    	pmid = 25221477,
    	title = "{Serotonergic modulation of post-synaptic inhibition and locomotor alternating pattern in the spinal cord.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4148025\&tool=pmcentrez\&rendertype=abstract",
    	volume = 8,
    	year = 2014
    }
    
  5. Sanaz Eftekhari, Soraya Mehrabi, Mansooreh Soleimani, Gholamreza Hassanzadeh, Amene Shahrokhi, Hossein Mostafavi, Parisa Hayat, Mahmood Barati, Hajar Mehdizadeh, Reza Rahmanzadeh, Mahmoud Reza Hadjighassem and Mohammad Taghi Joghataei.
    BDNF modifies hippocampal KCC2 and NKCC1 expression in a temporal lobe epilepsy model.. Acta neurobiologiae experimentalis 74(3):276–87, January 2014.
    Abstract Excitatory GABA actions, induced by altered expression of chloride transporters (KCC2/NKCC1), can contribute to seizure generation in temporal lobe epilepsy. In the present study, we evaluated whether BDNF administration can affect KCC2/NKCC1 expression, ictogenesis and behavioral alterations in this paradigm. Status epilepticus was induced in male rats with pilocarpine, followed by a treatment of either a single high dose or multiple injections of BDNF during the latent phase of temporal lobe epilepsy. Chloride transporters expression, spontaneous recurrent seizures, and hyperexcitability post-seizural behaviors were evaluated after treatment. NKCC1 protein expression was markedly upregulated, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Application of BDNF (both single high dose and multiple injections) increased KCC2 expression in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyperexcitability behaviors were ameliorated for a short period of time. To prevent GABA-A mediated depolarization and design appropriate treatment strategies for temporal lobe epilepsy, chloride transporters can be considered as a target. Future studies are warranted to investigate any possible therapeutic effects of BDNF via altering chloride transporters expression.
    URL BibTeX

    @article{Eftekhari2014,
    	abstract = "Excitatory GABA actions, induced by altered expression of chloride transporters (KCC2/NKCC1), can contribute to seizure generation in temporal lobe epilepsy. In the present study, we evaluated whether BDNF administration can affect KCC2/NKCC1 expression, ictogenesis and behavioral alterations in this paradigm. Status epilepticus was induced in male rats with pilocarpine, followed by a treatment of either a single high dose or multiple injections of BDNF during the latent phase of temporal lobe epilepsy. Chloride transporters expression, spontaneous recurrent seizures, and hyperexcitability post-seizural behaviors were evaluated after treatment. NKCC1 protein expression was markedly upregulated, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Application of BDNF (both single high dose and multiple injections) increased KCC2 expression in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyperexcitability behaviors were ameliorated for a short period of time. To prevent GABA-A mediated depolarization and design appropriate treatment strategies for temporal lobe epilepsy, chloride transporters can be considered as a target. Future studies are warranted to investigate any possible therapeutic effects of BDNF via altering chloride transporters expression.",
    	author = "Eftekhari, Sanaz and Mehrabi, Soraya and Soleimani, Mansooreh and Hassanzadeh, Gholamreza and Shahrokhi, Amene and Mostafavi, Hossein and Hayat, Parisa and Barati, Mahmood and Mehdizadeh, Hajar and Rahmanzadeh, Reza and Hadjighassem, Mahmoud Reza and Joghataei, Mohammad Taghi",
    	issn = "1689-0035",
    	journal = "Acta neurobiologiae experimentalis",
    	keywords = "Animals,Brain-Derived Neurotrophic Factor,Brain-Derived Neurotrophic Factor: administration ,Brain-Derived Neurotrophic Factor: pharmacology,Disease Models, Animal,Epilepsy, Temporal Lobe,Epilepsy, Temporal Lobe: drug therapy,Hippocampus,Hippocampus: drug effects,Hippocampus: metabolism,Male,Neurons,Neurons: drug effects,Neurons: metabolism,Pilocarpine,Pilocarpine: pharmacology,Rats, Wistar,Solute Carrier Family 12, Member 2,Solute Carrier Family 12, Member 2: metabolism,Symporters,Symporters: metabolism,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: drug effects,gamma-Aminobutyric Acid: metabolism",
    	month = "jan",
    	number = 3,
    	pages = "276--87",
    	pmid = 25231847,
    	title = "{BDNF modifies hippocampal KCC2 and NKCC1 expression in a temporal lobe epilepsy model.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/25231847",
    	volume = 74,
    	year = 2014
    }
    
  6. Chicheng Sun, Lei Zhang and Gong Chen.
    An unexpected role of neuroligin-2 in regulating KCC2 and GABA functional switch.. Molecular brain 6:23, January 2013.
    Abstract BACKGROUND: GABAA receptors are ligand-gated Cl- channels, and the intracellular Cl- concentration governs whether GABA function is excitatory or inhibitory. During early brain development, GABA undergoes functional switch from excitation to inhibition: GABA depolarizes immature neurons but hyperpolarizes mature neurons due to a developmental decrease of intracellular Cl- concentration. This GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl- outside neurons. However, the upstream factor that regulates KCC2 expression is unclear. RESULTS: We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. Gramicidin-perforated patch clamp recordings confirm that NL2 directly regulates the GABA equilibrium potential. We further demonstrate that knockdown of NL2 decreases dendritic spines through down-regulating KCC2. CONCLUSIONS: Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.
    URL, DOI BibTeX

    @article{Sun2013,
    	abstract = "BACKGROUND: GABAA receptors are ligand-gated Cl- channels, and the intracellular Cl- concentration governs whether GABA function is excitatory or inhibitory. During early brain development, GABA undergoes functional switch from excitation to inhibition: GABA depolarizes immature neurons but hyperpolarizes mature neurons due to a developmental decrease of intracellular Cl- concentration. This GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl- outside neurons. However, the upstream factor that regulates KCC2 expression is unclear. RESULTS: We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. Gramicidin-perforated patch clamp recordings confirm that NL2 directly regulates the GABA equilibrium potential. We further demonstrate that knockdown of NL2 decreases dendritic spines through down-regulating KCC2. CONCLUSIONS: Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.",
    	author = "Sun, Chicheng and Zhang, Lei and Chen, Gong",
    	doi = "10.1186/1756-6606-6-23",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Sun, Zhang, Chen - 2013 - An unexpected role of neuroligin-2 in regulating KCC2 and GABA functional switch.pdf:pdf",
    	issn = "1756-6606",
    	journal = "Molecular brain",
    	keywords = "Animals,Cell Adhesion Molecules, Neuronal,Cell Adhesion Molecules, Neuronal: metabolism,Cerebral Cortex,Cerebral Cortex: cytology,Dendritic Spines,Dendritic Spines: metabolism,Female,Gene Knockdown Techniques,Glutamates,Glutamates: metabolism,HEK293 Cells,Humans,Ion Channel Gating,Male,Mice,Mice, Inbred C57BL,Models, Biological,Nerve Tissue Proteins,Nerve Tissue Proteins: metabolism,Neurons,Neurons: cytology,Neurons: metabolism,Rats,Receptors, GABA-A,Receptors, GABA-A: metabolism,Symporters,Symporters: metabolism,Synapses,Synapses: metabolism,Synaptic Transmission,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: metabolism",
    	month = "jan",
    	pages = 23,
    	pmid = 23663753,
    	title = "{An unexpected role of neuroligin-2 in regulating KCC2 and GABA functional switch.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3661362\&tool=pmcentrez\&rendertype=abstract",
    	volume = 6,
    	year = 2013
    }
    
  7. Masahiro Shibasaki, Daiki Masukawa, Kazunori Ishii, Yui Yamagishi, Tomohisa Mori and Tsutomu Suzuki.
    Involvement of the K+-Cl- co-transporter KCC2 in the sensitization to morphine-induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system.. Journal of neurochemistry 125(5):747–55, 2013.
    Abstract Benzodiazepines are commonly used as sedatives, sleeping aids, and anti-anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K(+) -Cl(-) co-transporter 2 (KCC2) in the sensitization to morphine-induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of $\gamma$-aminobutyric acid A-type receptor (GABAA R) $\alpha$1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine-induced hyperlocomotion, which is accompanied by the up-regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down-regulation of protein phosphatase-1 (PP-1) as well as the up-regulation of phosphorylated protein kinase C $\gamma$ (pPKC$\gamma$). Furthermore, PP-1 directly associated with KCC2 and pPKC$\gamma$, whereas pPKC$\gamma$ did not associate with KCC2. On the other hand, pre-treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine-induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKC$\gamma$-PP-1-KCC2 pathway by chronic treatment with zolpidem.
    URL, DOI BibTeX

    @article{Shibasaki2013,
    	abstract = "Benzodiazepines are commonly used as sedatives, sleeping aids, and anti-anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K(+) -Cl(-) co-transporter 2 (KCC2) in the sensitization to morphine-induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of $\gamma$-aminobutyric acid A-type receptor (GABAA R) $\alpha$1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine-induced hyperlocomotion, which is accompanied by the up-regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down-regulation of protein phosphatase-1 (PP-1) as well as the up-regulation of phosphorylated protein kinase C $\gamma$ (pPKC$\gamma$). Furthermore, PP-1 directly associated with KCC2 and pPKC$\gamma$, whereas pPKC$\gamma$ did not associate with KCC2. On the other hand, pre-treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine-induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKC$\gamma$-PP-1-KCC2 pathway by chronic treatment with zolpidem.",
    	author = "Shibasaki, Masahiro and Masukawa, Daiki and Ishii, Kazunori and Yamagishi, Yui and Mori, Tomohisa and Suzuki, Tsutomu",
    	doi = "10.1111/jnc.12258",
    	issn = "1471-4159",
    	journal = "Journal of neurochemistry",
    	keywords = "Animals,Drug Synergism,Limbic System,Limbic System: drug effects,Limbic System: metabolism,Male,Mice,Mice, Inbred ICR,Morphine,Morphine: administration \& dosage,Psychomotor Agitation,Psychomotor Agitation: metabolism,Pyridines,Pyridines: administration \& dosage,Symporters,Symporters: biosynthesis,Symporters: physiology,Treatment Outcome,Up-Regulation,Up-Regulation: drug effects,Up-Regulation: physiology",
    	month = "",
    	number = 5,
    	pages = "747--55",
    	pmid = 23565710,
    	title = "{Involvement of the K+-Cl- co-transporter KCC2 in the sensitization to morphine-induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/23565710",
    	volume = 125,
    	year = 2013
    }
    
  8. Wolfgang Liedtke, Michele Yeo, Hongbo Zhang, Yiding Wang, Michelle Gignac, Sara Miller, Ken Berglund and Jie Liu.
    Highly conductive carbon nanotube matrix accelerates developmental chloride extrusion in central nervous system neurons by increased expression of chloride transporter KCC2.. Small (Weinheim an der Bergstrasse, Germany) 9(7):1066–75, 2013.
    Abstract Exceptional mechanical and electrical properties of carbon nanotubes (CNT) have attracted neuroscientists and neural tissue engineers aiming to develop novel devices that interface with nervous tissues. In the central nervous system (CNS), the perinatal chloride shift represents a dynamic change that forms the basis for physiological actions of $\gamma$-aminobutyric acid (GABA) as an inhibitory neurotransmitter, a process of fundamental relevance for normal functioning of the CNS. Low intra-neuronal chloride concentrations are maintained by a chloride-extruding transporter, potassium chloride cotransporter 2 (KCC2). KCC2's increasing developmental expression underlies the chloride shift. In neural injury, repressed KCC2 expression plays a co-contributory role by corrupting inhibitory neurotransmission. Mechanisms of Kcc2 up-regulation are thus pertinent because of their medical relevance, yet they remain elusive. Here, it is shown that primary CNS neurons originating from the cerebral cortex, cultured on highly-conductive few-walled-CNT (fwCNT) have a strikingly accelerated chloride shift caused by increased KCC2 expression. KCC2 upregulation is dependent on neuronal voltage-gated calcium channels (VGCC) and, furthermore, on calcium/calmodulin-dependent kinase II, which is linked to VGCC-mediated calcium-influx. It is also demonstrated that accelerated Kcc2 transcription in brain-slices prepared from genetically-engineered reporter mice, in which Kcc2 promoter drives luciferase, when the cerebral cortex of these mice is exposed to fwCNT-coated devices. Based on these findings, whether fwCNT can enhance neural engineering devices for the benefit of neural injury conditions associated with elevated neuronal intracellular chloride concentration-such as pain, epilepsy, traumatic neural injury and ischemia-can now be addressed. Taken together, our novel insights illustrate how fwCNTs can promote low neuronal chloride in individual neurons and thus inhibitory transmission in neural circuits.
    URL, DOI BibTeX

    @article{Liedtke2013,
    	abstract = "Exceptional mechanical and electrical properties of carbon nanotubes (CNT) have attracted neuroscientists and neural tissue engineers aiming to develop novel devices that interface with nervous tissues. In the central nervous system (CNS), the perinatal chloride shift represents a dynamic change that forms the basis for physiological actions of $\gamma$-aminobutyric acid (GABA) as an inhibitory neurotransmitter, a process of fundamental relevance for normal functioning of the CNS. Low intra-neuronal chloride concentrations are maintained by a chloride-extruding transporter, potassium chloride cotransporter 2 (KCC2). KCC2's increasing developmental expression underlies the chloride shift. In neural injury, repressed KCC2 expression plays a co-contributory role by corrupting inhibitory neurotransmission. Mechanisms of Kcc2 up-regulation are thus pertinent because of their medical relevance, yet they remain elusive. Here, it is shown that primary CNS neurons originating from the cerebral cortex, cultured on highly-conductive few-walled-CNT (fwCNT) have a strikingly accelerated chloride shift caused by increased KCC2 expression. KCC2 upregulation is dependent on neuronal voltage-gated calcium channels (VGCC) and, furthermore, on calcium/calmodulin-dependent kinase II, which is linked to VGCC-mediated calcium-influx. It is also demonstrated that accelerated Kcc2 transcription in brain-slices prepared from genetically-engineered reporter mice, in which Kcc2 promoter drives luciferase, when the cerebral cortex of these mice is exposed to fwCNT-coated devices. Based on these findings, whether fwCNT can enhance neural engineering devices for the benefit of neural injury conditions associated with elevated neuronal intracellular chloride concentration-such as pain, epilepsy, traumatic neural injury and ischemia-can now be addressed. Taken together, our novel insights illustrate how fwCNTs can promote low neuronal chloride in individual neurons and thus inhibitory transmission in neural circuits.",
    	author = "Liedtke, Wolfgang and Yeo, Michele and Zhang, Hongbo and Wang, Yiding and Gignac, Michelle and Miller, Sara and Berglund, Ken and Liu, Jie",
    	doi = "10.1002/smll.201201994",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Liedtke et al. - 2013 - Highly conductive carbon nanotube matrix accelerates developmental chloride extrusion in central nervous system.pdf:pdf",
    	issn = "1613-6829",
    	journal = "Small (Weinheim an der Bergstrasse, Germany)",
    	keywords = "Animals,Cells, Cultured,Central Nervous System,Central Nervous System: drug effects,Central Nervous System: metabolism,Immunohistochemistry,Mice,Nanotubes, Carbon,Nanowires,Nanowires: chemistry,Oxides,Oxides: chemistry,Rats,Silicon Compounds,Silicon Compounds: chemistry,Symporters,Symporters: genetics,Symporters: metabolism",
    	month = "",
    	number = 7,
    	pages = "1066--75",
    	pmid = 23229576,
    	title = "{Highly conductive carbon nanotube matrix accelerates developmental chloride extrusion in central nervous system neurons by increased expression of chloride transporter KCC2.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3822771\&tool=pmcentrez\&rendertype=abstract",
    	volume = 9,
    	year = 2013
    }
    
  9. Rémi Bos, Karina Sadlaoud, Pascale Boulenguez, Dorothée Buttigieg, Sylvie Liabeuf, Cécile Brocard, Georg Haase, Hélène Bras and Laurent Vinay.
    Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.. Proceedings of the National Academy of Sciences of the United States of America 110(1):348–53, January 2013.
    Abstract In healthy adults, activation of $\gamma$-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).
    URL, DOI BibTeX

    @article{Bos2013,
    	abstract = "In healthy adults, activation of $\gamma$-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).",
    	author = "Bos, R\'{e}mi and Sadlaoud, Karina and Boulenguez, Pascale and Buttigieg, Doroth\'{e}e and Liabeuf, Sylvie and Brocard, C\'{e}cile and Haase, Georg and Bras, H\'{e}l\`{e}ne and Vinay, Laurent",
    	doi = "10.1073/pnas.1213680110",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Bos et al. - 2013 - Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.pdf:pdf",
    	issn = "1091-6490",
    	journal = "Proceedings of the National Academy of Sciences of the United States of America",
    	keywords = "Animals,Bicyclo Compounds,Bicyclo Compounds: pharmacology,Blotting, Western,Chlorides,Chlorides: metabolism,Gene Expression Regulation,Gene Expression Regulation: drug effects,H-Reflex,Immunohistochemistry,Inhibitory Postsynaptic Potentials,Inhibitory Postsynaptic Potentials: physiology,Methylamines,Methylamines: pharmacology,Motor Neurons,Motor Neurons: metabolism,Muscle Spasticity,Muscle Spasticity: drug therapy,Muscle Spasticity: etiology,Rats,Receptor, Serotonin, 5-HT2A,Receptor, Serotonin, 5-HT2A: metabolism,Serotonin,Serotonin 5-HT2 Receptor Agonists,Serotonin 5-HT2 Receptor Agonists: pharmacology,Serotonin: metabolism,Serotonin: pharmacology,Spinal Cord Injuries,Spinal Cord Injuries: complications,Symporters,Symporters: metabolism",
    	month = "jan",
    	number = 1,
    	pages = "348--53",
    	pmid = 23248270,
    	title = "{Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3538195\&tool=pmcentrez\&rendertype=abstract",
    	volume = 110,
    	year = 2013
    }
    
  10. Zeng-You Ye, De-Pei Li, Hee Sun Byun, Li Li and Hui-Lin Pan.
    NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension.. The Journal of neuroscience : the official journal of the Society for Neuroscience 32(25):8560–8, June 2012.
    Abstract 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.
    URL, DOI BibTeX

    @article{Ye2012,
    	abstract = "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.",
    	author = "Ye, Zeng-You and Li, De-Pei and Byun, Hee Sun and Li, Li and Pan, Hui-Lin",
    	doi = "10.1523/JNEUROSCI.1346-12.2012",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Ye et al. - 2012 - NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic driv.pdf:pdf",
    	issn = "1529-2401",
    	journal = "The Journal of neuroscience : the official journal of the Society for Neuroscience",
    	keywords = "Animals,Blood Pressure,Blood Pressure: physiology,Blotting, Western,Cell Membrane,Cell Membrane: metabolism,Chlorides,Chlorides: metabolism,Electrophysiological Phenomena,Ganglia, Sympathetic,Ganglia, Sympathetic: physiology,Ganglionectomy,Glycosylation,Homeostasis,Homeostasis: genetics,Hypertension,Hypertension: metabolism,Hypertension: physiopathology,Hypothalamus,Hypothalamus: metabolism,Male,Neurons,Neurons: physiology,Paraventricular Hypothalamic Nucleus,Paraventricular Hypothalamic Nucleus: metabolism,Paraventricular Hypothalamic Nucleus: physiopathol,Rats,Rats, Inbred SHR,Rats, Inbred WKY,Sodium-Potassium-Chloride Symporters,Sodium-Potassium-Chloride Symporters: biosynthesis,Solute Carrier Family 12, Member 2,Sympathetic Nervous System,Sympathetic Nervous System: physiopathology,Synapses,Synapses: physiology,Up-Regulation,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: physiology",
    	month = "jun",
    	number = 25,
    	pages = "8560--8",
    	pmid = 22723696,
    	title = "{NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3390258\&tool=pmcentrez\&rendertype=abstract",
    	volume = 32,
    	year = 2012
    }
    
  11. R A Saadi, K He, K A Hartnett, K Kandler, M Hershfinkel and E Aizenman.
    SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro.. Neuroscience 210:38–46, May 2012.
    Abstract The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABA(A) receptor channels. In a recent study, we showed that Zn(2+) regulates GABA(A) reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn(2+) initiates this process by activating the Gq-coupled metabotropic Zn(2+) receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca(2+) indicator and exposing cells to Zn(2+), which triggered consistent Ca(2+) responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-$\alpha$-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn(2+) treatment under these conditions did not increase the intracellular concentrations of Zn(2+) itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH(4)(+) influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn(2+) pretreatment induced a Ca(2+)-dependent increase in KCC2 activity. The effects of Zn(2+) on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39(-/-) mice, suggesting that Zn(2+) acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn(2+)-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn(2+). These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn(2+) stimulation of mZnR/GPR39.
    URL, DOI BibTeX

    @article{Saadi2012,
    	abstract = "The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABA(A) receptor channels. In a recent study, we showed that Zn(2+) regulates GABA(A) reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn(2+) initiates this process by activating the Gq-coupled metabotropic Zn(2+) receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca(2+) indicator and exposing cells to Zn(2+), which triggered consistent Ca(2+) responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-$\alpha$-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn(2+) treatment under these conditions did not increase the intracellular concentrations of Zn(2+) itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH(4)(+) influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn(2+) pretreatment induced a Ca(2+)-dependent increase in KCC2 activity. The effects of Zn(2+) on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39(-/-) mice, suggesting that Zn(2+) acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn(2+)-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn(2+). These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn(2+) stimulation of mZnR/GPR39.",
    	author = "Saadi, R A and He, K and Hartnett, K A and Kandler, K and Hershfinkel, M and Aizenman, E",
    	doi = "10.1016/j.neuroscience.2012.03.001",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Saadi et al. - 2012 - SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor acti.pdf:pdf",
    	issn = "1873-7544",
    	journal = "Neuroscience",
    	keywords = "Animals,Blotting, Western,Cerebral Cortex,Cerebral Cortex: metabolism,Immunoblotting,In Vitro Techniques,Mice,Mice, Knockout,Neurons,Neurons: metabolism,Rats,Rats, Sprague-Dawley,Receptors, G-Protein-Coupled,Receptors, G-Protein-Coupled: metabolism,SNARE Proteins,SNARE Proteins: metabolism,Signal Transduction,Signal Transduction: physiology,Symporters,Symporters: metabolism,Transfection,Up-Regulation,Zinc,Zinc: metabolism",
    	month = "may",
    	pages = "38--46",
    	pmid = 22441041,
    	title = "{SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3358579\&tool=pmcentrez\&rendertype=abstract",
    	volume = 210,
    	year = 2012
    }
    
  12. C Morgado, P Pereira-Terra, C D Cruz and I Tavares.
    Minocycline completely reverses mechanical hyperalgesia in diabetic rats through microglia-induced changes in the expression of the potassium chloride co-transporter 2 (KCC2) at the spinal cord.. Diabetes, obesity & metabolism 13(2):150–9, 2011.
    Abstract AIM: neuronal hyperactivity at the spinal cord during mechanical hyperalgesia induced by diabetes may result from a decrease in the local expression of the potassium chloride co-transporter 2 (KCC2), which shifts the action of the neurotransmitter $\gamma$-amminobutiric acid (GABA) from inhibitory to excitatory. In this study, we evaluated the effects of spinal microglia inhibition or brain-derived neurotrophic factor (BDNF) blockade on KCC2 expression, spinal neuronal activity and mechanically induced pain responses of streptozotocin (STZ)-diabetic rats. METHODS: four weeks after induction of diabetes, the STZ-diabetic rats received daily intrathecal injections, for 3 days, of minocycline (microglia inhibitor), TrkB/Fc (BDNF sequester) or saline. Behavioural responses to mechanical nociceptive stimulation of STZ-diabetic rats were evaluated by the Randall-Selitto test. The lumbar spinal cord was immunoreacted against the Fos protein (marker of neuronal activation) or KCC2, which was also quantified by western blotting. BDNF levels at the spinal cord were quantified by an enzyme-linked immunosorbent assay (ELISA). RESULTS: minocycline treatment reversed the mechanical hyperalgesia, increased Fos expression and decreased the KCC2 expression detected in STZ-diabetic rats to control levels. Treatment with TrkB/Fc was less effective, inducing moderate effects in mechanical hyperalgesia and Fos expression and only a partial correction of KCC2 expression. BDNF levels were not increased in STZ-diabetic rats. CONCLUSIONS: this study demonstrates that the microglial activation at the spinal cord contributes to mechanical hyperalgesia and spinal neuronal hyperactivity induced by diabetes, apparently by regulating the KCC2 expression. These effects do not seem to be mediated by BDNF, which is an important difference from other chronic pain conditions. New targets directed to prevent spinal microglia activation should be considered for the treatment of mechanical hyperalgesia induced by diabetes.
    URL, DOI BibTeX

    @article{Morgado2011,
    	abstract = "AIM: neuronal hyperactivity at the spinal cord during mechanical hyperalgesia induced by diabetes may result from a decrease in the local expression of the potassium chloride co-transporter 2 (KCC2), which shifts the action of the neurotransmitter $\gamma$-amminobutiric acid (GABA) from inhibitory to excitatory. In this study, we evaluated the effects of spinal microglia inhibition or brain-derived neurotrophic factor (BDNF) blockade on KCC2 expression, spinal neuronal activity and mechanically induced pain responses of streptozotocin (STZ)-diabetic rats. METHODS: four weeks after induction of diabetes, the STZ-diabetic rats received daily intrathecal injections, for 3 days, of minocycline (microglia inhibitor), TrkB/Fc (BDNF sequester) or saline. Behavioural responses to mechanical nociceptive stimulation of STZ-diabetic rats were evaluated by the Randall-Selitto test. The lumbar spinal cord was immunoreacted against the Fos protein (marker of neuronal activation) or KCC2, which was also quantified by western blotting. BDNF levels at the spinal cord were quantified by an enzyme-linked immunosorbent assay (ELISA). RESULTS: minocycline treatment reversed the mechanical hyperalgesia, increased Fos expression and decreased the KCC2 expression detected in STZ-diabetic rats to control levels. Treatment with TrkB/Fc was less effective, inducing moderate effects in mechanical hyperalgesia and Fos expression and only a partial correction of KCC2 expression. BDNF levels were not increased in STZ-diabetic rats. CONCLUSIONS: this study demonstrates that the microglial activation at the spinal cord contributes to mechanical hyperalgesia and spinal neuronal hyperactivity induced by diabetes, apparently by regulating the KCC2 expression. These effects do not seem to be mediated by BDNF, which is an important difference from other chronic pain conditions. New targets directed to prevent spinal microglia activation should be considered for the treatment of mechanical hyperalgesia induced by diabetes.",
    	author = "Morgado, C and Pereira-Terra, P and Cruz, C D and Tavares, I",
    	doi = "10.1111/j.1463-1326.2010.01333.x",
    	issn = "1463-1326",
    	journal = "Diabetes, obesity \& metabolism",
    	keywords = "Animals,Diabetes Mellitus, Experimental,Diabetic Neuropathies,Diabetic Neuropathies: diet therapy,Diabetic Neuropathies: metabolism,Diabetic Neuropathies: physiopathology,Hyperalgesia,Hyperalgesia: drug therapy,Hyperalgesia: metabolism,Hyperalgesia: physiopathology,Male,Microglia,Microglia: drug effects,Minocycline,Minocycline: administration \& dosage,Minocycline: pharmacology,Potassium Chloride,Potassium Chloride: metabolism,Rats,Rats, Wistar,Spinal Cord,Spinal Cord: drug effects,Spinal Cord: metabolism,Spinal Cord: physiopathology,Symporters,Synaptic Transmission,Synaptic Transmission: physiology,Up-Regulation,Up-Regulation: physiology",
    	month = "",
    	number = 2,
    	pages = "150--9",
    	pmid = 21199267,
    	title = "{Minocycline completely reverses mechanical hyperalgesia in diabetic rats through microglia-induced changes in the expression of the potassium chloride co-transporter 2 (KCC2) at the spinal cord.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/21199267",
    	volume = 13,
    	year = 2011
    }
    
  13. Anastasia Ludwig, Pavel Uvarov, Christophe Pellegrino, Judith Thomas-Crusells, Sebastian Schuchmann, Mart Saarma, Matti S Airaksinen and Claudio Rivera.
    Neurturin evokes MAPK-dependent upregulation of Egr4 and KCC2 in developing neurons.. Neural plasticity 2011:1–8, January 2011.
    Abstract The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.
    URL, DOI BibTeX

    @article{Ludwig2011,
    	abstract = "The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.",
    	author = "Ludwig, Anastasia and Uvarov, Pavel and Pellegrino, Christophe and Thomas-Crusells, Judith and Schuchmann, Sebastian and Saarma, Mart and Airaksinen, Matti S and Rivera, Claudio",
    	doi = "10.1155/2011/641248",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Ludwig et al. - 2011 - Neurturin evokes MAPK-dependent upregulation of Egr4 and KCC2 in developing neurons.pdf:pdf",
    	issn = "1687-5443",
    	journal = "Neural plasticity",
    	keywords = "Animals,Animals, Newborn,Cells, Cultured,Early Growth Response Transcription Factors,Early Growth Response Transcription Factors: biosy,Early Growth Response Transcription Factors: physi,Hippocampus,Hippocampus: metabolism,MAP Kinase Signaling System,MAP Kinase Signaling System: physiology,Mice,Mitogen-Activated Protein Kinases,Mitogen-Activated Protein Kinases: physiology,Neurons,Neurons: metabolism,Neurturin,Neurturin: physiology,Organ Culture Techniques,Symporters,Symporters: biosynthesis,Symporters: physiology,Up-Regulation,Up-Regulation: physiology",
    	month = "jan",
    	pages = "1--8",
    	pmid = 21837281,
    	title = "{Neurturin evokes MAPK-dependent upregulation of Egr4 and KCC2 in developing neurons.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3151499\&tool=pmcentrez\&rendertype=abstract",
    	volume = 2011,
    	year = 2011
    }
    
  14. Ehud Chorin, Ofir Vinograd, Ilya Fleidervish, David Gilad, Sharon Herrmann, Israel Sekler, Elias Aizenman and Michal Hershfinkel.
    Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnR/GPR39 receptor.. The Journal of neuroscience : the official journal of the Society for Neuroscience 31(36):12916–26, 2011.
    Abstract Vesicular Zn(2+) regulates postsynaptic neuronal excitability upon its corelease with glutamate. We previously demonstrated that synaptic Zn(2+) acts via a distinct metabotropic zinc-sensing receptor (mZnR) in neurons to trigger Ca(2+) responses in the hippocampus. Here, we show that physiological activation of mZnR signaling induces enhanced K(+)/Cl(-) cotransporter 2 (KCC2) activity and surface expression. As KCC2 is the major Cl(-) outward transporter in neurons, Zn(2+) also triggers a pronounced hyperpolarizing shift in the GABA(A) reversal potential. Mossy fiber stimulation-dependent upregulation of KCC2 activity is eliminated in slices from Zn(2+) transporter 3-deficient animals, which lack synaptic Zn(2+). Importantly, activity-dependent ZnR signaling and subsequent enhancement of KCC2 activity are also absent in slices from mice lacking the G-protein-coupled receptor GPR39, identifying this protein as the functional neuronal mZnR. Our work elucidates a fundamentally important role for synaptically released Zn(2+) acting as a neurotransmitter signal via activation of a mZnR to increase Cl(-) transport, thereby enhancing inhibitory tone in postsynaptic cells.
    URL, DOI BibTeX

    @article{Chorin2011,
    	abstract = "Vesicular Zn(2+) regulates postsynaptic neuronal excitability upon its corelease with glutamate. We previously demonstrated that synaptic Zn(2+) acts via a distinct metabotropic zinc-sensing receptor (mZnR) in neurons to trigger Ca(2+) responses in the hippocampus. Here, we show that physiological activation of mZnR signaling induces enhanced K(+)/Cl(-) cotransporter 2 (KCC2) activity and surface expression. As KCC2 is the major Cl(-) outward transporter in neurons, Zn(2+) also triggers a pronounced hyperpolarizing shift in the GABA(A) reversal potential. Mossy fiber stimulation-dependent upregulation of KCC2 activity is eliminated in slices from Zn(2+) transporter 3-deficient animals, which lack synaptic Zn(2+). Importantly, activity-dependent ZnR signaling and subsequent enhancement of KCC2 activity are also absent in slices from mice lacking the G-protein-coupled receptor GPR39, identifying this protein as the functional neuronal mZnR. Our work elucidates a fundamentally important role for synaptically released Zn(2+) acting as a neurotransmitter signal via activation of a mZnR to increase Cl(-) transport, thereby enhancing inhibitory tone in postsynaptic cells.",
    	author = "Chorin, Ehud and Vinograd, Ofir and Fleidervish, Ilya and Gilad, David and Herrmann, Sharon and Sekler, Israel and Aizenman, Elias and Hershfinkel, Michal",
    	doi = "10.1523/JNEUROSCI.2205-11.2011",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Chorin et al. - 2011 - Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnRGPR39 receptor.pdf:pdf",
    	issn = "1529-2401",
    	journal = "The Journal of neuroscience : the official journal of the Society for Neuroscience",
    	keywords = "Animals,Blotting, Western,CA3 Region, Hippocampal,CA3 Region, Hippocampal: cytology,CA3 Region, Hippocampal: physiology,Electrophysiological Phenomena,Excitatory Postsynaptic Potentials,Excitatory Postsynaptic Potentials: physiology,Female,Genotype,In Vitro Techniques,Male,Mice,Mice, Knockout,Microscopy, Fluorescence,Mossy Fibers, Hippocampal,Mossy Fibers, Hippocampal: physiology,Patch-Clamp Techniques,Receptors, Cell Surface,Receptors, Cell Surface: metabolism,Receptors, G-Protein-Coupled,Receptors, G-Protein-Coupled: drug effects,Receptors, G-Protein-Coupled: genetics,Receptors, GABA-A,Receptors, GABA-A: drug effects,Reverse Transcriptase Polymerase Chain Reaction,Symporters,Symporters: biosynthesis,Symporters: physiology,Synapses,Synapses: metabolism,Synaptic Transmission,Synaptic Transmission: drug effects,Up-Regulation,Up-Regulation: drug effects,Zinc,Zinc: metabolism,Zinc: pharmacology",
    	month = "",
    	number = 36,
    	pages = "12916--26",
    	pmid = 21900570,
    	title = "{Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnR/GPR39 receptor.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3227684\&tool=pmcentrez\&rendertype=abstract",
    	volume = 31,
    	year = 2011
    }
    
  15. Dominique Arion and David A Lewis.
    Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia.. Archives of general psychiatry 68(1):21–31, 2011.
    Abstract CONTEXT: Disturbances in markers of cortical $\gamma$-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of $\gamma$-aminobutyric acid neurotransmission (hyperpolarizing or depolarizing) depends on the local intracellular chloride concentration. In the central nervous system, the intracellular chloride level is determined by the activity of 2 cation-chloride transporters, NKCC1 and KCC2. The activities of these transporters are in turn regulated by a network of serine-threonine kinases that includes OXSR1, STK39, and the WNK kinases WNK1, WNK3, and WNK4. OBJECTIVE: To compare the levels of NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts in prefrontal cortex area 9 between subjects with schizophrenia and healthy comparison subjects. DESIGN: Real-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex. SETTING: Human brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania. PARTICIPANTS: Postmortem brain specimens from 42 subjects with schizophrenia and 42 matched healthy comparison subjects. Brain specimens from 18 macaque monkeys exposed to haloperidol, olanzapine, or sham long-term. MAIN OUTCOME MEASURES: Relative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts. RESULTS: OXSR1 and WNK3 transcripts were substantially overexpressed in subjects with schizophrenia relative to comparison subjects. In contrast, NKCC1, KCC2, STK39, WNK1, and WNK4 transcript levels did not differ between subject groups. OXSR1 and WNK3 transcript expression levels were not changed in antipsychotic-exposed monkeys and were not affected by potential confounding factors in the subjects with schizophrenia. CONCLUSION: In schizophrenia, increased expression levels, and possibly increased kinase activities, of OXSR1 and WNK3 may shift the balance of chloride transport by NKCC1 and KCC2 and alter the nature of $\gamma$-aminobutyric acid neurotransmission in the prefrontal cortex.
    URL, DOI BibTeX

    @article{Arion2011,
    	abstract = "CONTEXT: Disturbances in markers of cortical $\gamma$-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of $\gamma$-aminobutyric acid neurotransmission (hyperpolarizing or depolarizing) depends on the local intracellular chloride concentration. In the central nervous system, the intracellular chloride level is determined by the activity of 2 cation-chloride transporters, NKCC1 and KCC2. The activities of these transporters are in turn regulated by a network of serine-threonine kinases that includes OXSR1, STK39, and the WNK kinases WNK1, WNK3, and WNK4. OBJECTIVE: To compare the levels of NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts in prefrontal cortex area 9 between subjects with schizophrenia and healthy comparison subjects. DESIGN: Real-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex. SETTING: Human brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania. PARTICIPANTS: Postmortem brain specimens from 42 subjects with schizophrenia and 42 matched healthy comparison subjects. Brain specimens from 18 macaque monkeys exposed to haloperidol, olanzapine, or sham long-term. MAIN OUTCOME MEASURES: Relative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts. RESULTS: OXSR1 and WNK3 transcripts were substantially overexpressed in subjects with schizophrenia relative to comparison subjects. In contrast, NKCC1, KCC2, STK39, WNK1, and WNK4 transcript levels did not differ between subject groups. OXSR1 and WNK3 transcript expression levels were not changed in antipsychotic-exposed monkeys and were not affected by potential confounding factors in the subjects with schizophrenia. CONCLUSION: In schizophrenia, increased expression levels, and possibly increased kinase activities, of OXSR1 and WNK3 may shift the balance of chloride transport by NKCC1 and KCC2 and alter the nature of $\gamma$-aminobutyric acid neurotransmission in the prefrontal cortex.",
    	author = "Arion, Dominique and Lewis, David A",
    	doi = "10.1001/archgenpsychiatry.2010.114",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Arion, Lewis - 2011 - Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia.pdf:pdf",
    	issn = "1538-3636",
    	journal = "Archives of general psychiatry",
    	keywords = "Adult,Autopsy,Case-Control Studies,Chlorides,Chlorides: metabolism,Diagnostic and Statistical Manual of Mental Disord,Female,Humans,Intracellular Signaling Peptides and Proteins,Ion Transport,Ion Transport: genetics,Male,Middle Aged,Prefrontal Cortex,Prefrontal Cortex: metabolism,Protein-Serine-Threonine Kinases,Protein-Serine-Threonine Kinases: genetics,Protein-Serine-Threonine Kinases: metabolism,RNA, Messenger,Reverse Transcriptase Polymerase Chain Reaction,Schizophrenia,Schizophrenia: genetics,Schizophrenia: metabolism,Sodium-Potassium-Chloride Symporters,Sodium-Potassium-Chloride Symporters: genetics,Sodium-Potassium-Chloride Symporters: metabolism,Solute Carrier Family 12, Member 1,Solute Carrier Family 12, Member 2,Up-Regulation,Up-Regulation: genetics",
    	month = "",
    	number = 1,
    	pages = "21--31",
    	pmid = 20819979,
    	title = "{Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3015012\&tool=pmcentrez\&rendertype=abstract",
    	volume = 68,
    	year = 2011
    }
    
  16. Anastasia Shulga, Anne Blaesse, Kai Kysenius, Henri J Huttunen, Kimmo Tanhuanpää, Mart Saarma and Claudio Rivera.
    Thyroxin regulates BDNF expression to promote survival of injured neurons.. Molecular and cellular neurosciences 42(4):408–18, December 2009.
    Abstract A growing amount of evidence indicates that neuronal trauma can induce a recapitulation of developmental-like mechanisms for neuronal survival and regeneration. Concurrently, ontogenic dependency of central neurons for brain-derived neurotrophic factor (BDNF) is lost during maturation but is re-acquired after injury. Here we show in organotypic hippocampal slices that thyroxin, the thyroid hormone essential for normal CNS development, induces up-regulation of BDNF upon injury. This change in the effect of thyroxin is crucial to promote survival and regeneration of damaged central neurons. In addition, the effect of thyroxin on the expression of the K-Cl cotransporter (KCC2), a marker of neuronal maturation, is changed from down to up-regulation. Notably, previous results in humans have shown that during the first few days after traumatic brain injury or spinal cord injury, thyroid hormone levels are often diminished. Our data suggest that maintaining normal levels of thyroxin during the early post-traumatic phase of CNS injury could have a therapeutically positive effect.
    URL, DOI BibTeX

    @article{Shulga2009,
    	abstract = "A growing amount of evidence indicates that neuronal trauma can induce a recapitulation of developmental-like mechanisms for neuronal survival and regeneration. Concurrently, ontogenic dependency of central neurons for brain-derived neurotrophic factor (BDNF) is lost during maturation but is re-acquired after injury. Here we show in organotypic hippocampal slices that thyroxin, the thyroid hormone essential for normal CNS development, induces up-regulation of BDNF upon injury. This change in the effect of thyroxin is crucial to promote survival and regeneration of damaged central neurons. In addition, the effect of thyroxin on the expression of the K-Cl cotransporter (KCC2), a marker of neuronal maturation, is changed from down to up-regulation. Notably, previous results in humans have shown that during the first few days after traumatic brain injury or spinal cord injury, thyroid hormone levels are often diminished. Our data suggest that maintaining normal levels of thyroxin during the early post-traumatic phase of CNS injury could have a therapeutically positive effect.",
    	author = {Shulga, Anastasia and Blaesse, Anne and Kysenius, Kai and Huttunen, Henri J and Tanhuanp\"{a}\"{a}, Kimmo and Saarma, Mart and Rivera, Claudio},
    	doi = "10.1016/j.mcn.2009.09.002",
    	issn = "1095-9327",
    	journal = "Molecular and cellular neurosciences",
    	keywords = "Animals,Axotomy,Biological Markers,Biological Markers: metabolism,Brain Injuries,Brain Injuries: metabolism,Brain Injuries: pathology,Brain-Derived Neurotrophic Factor,Brain-Derived Neurotrophic Factor: genetics,Brain-Derived Neurotrophic Factor: metabolism,Caspase 3,Caspase 3: metabolism,Cell Survival,Cell Survival: physiology,Female,Hippocampus,Hippocampus: cytology,Hippocampus: metabolism,Hippocampus: pathology,Humans,Male,Mice,Nerve Regeneration,Nerve Regeneration: physiology,Neurons,Neurons: cytology,Neurons: pathology,Neurons: physiology,RNA, Messenger,RNA, Messenger: genetics,RNA, Messenger: metabolism,Receptor, trkB,Receptor, trkB: metabolism,Symporters,Symporters: genetics,Symporters: metabolism,Thyroxine,Thyroxine: genetics,Thyroxine: metabolism,Up-Regulation",
    	month = "dec",
    	number = 4,
    	pages = "408--18",
    	pmid = 19765661,
    	title = "{Thyroxin regulates BDNF expression to promote survival of injured neurons.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/19765661",
    	volume = 42,
    	year = 2009
    }
    
  17. Mamoru Fukuchi, Takuya Nii, Naoki Ishimaru, Aya Minamino, Daichi Hara, Ichiro Takasaki, Akiko Tabuchi and Masaaki Tsuda.
    Valproic acid induces up- or down-regulation of gene expression responsible for the neuronal excitation and inhibition in rat cortical neurons through its epigenetic actions.. Neuroscience research 65(1):35–43, September 2009.
    Abstract Valproic acid (VPA), a drug used to treat epilepsy and bipolar mood disorder, inhibits histone deacetylase (HDAC), which is associated with the epigenetic regulation of gene expression. Using a microarray, we comprehensively examined which genes are affected by stimulating cultured rat cortical neurons with VPA, and found that the VPA-treatment markedly altered gene expression (up-regulated; 726 genes, down-regulated; 577 genes). The mRNA expression for brain-derived neurotrophic factor (BDNF) and the alpha4 subunit of the GABA(A) receptor (GABA(A)Ralpha4), known to be involved in epileptogenesis, was up-regulated, with the increase in BDNF exon I-IX mRNA expression being remarkable, whereas that for GABA(A)Rgamma2, GAD65 and 67, and the K(+)/Cl(-) co-transporter KCC2, which are responsible for the development of GABAergic inhibitory neurons, was down-regulated. The number of GAD67-positive neurons decreased upon VPA-treatment. Similar changes of up- and down-regulation were obtained by trichostatin A. VPA did not affect the intracellular Ca(2+) concentration and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting its direct action on HDAC. The acetylation of histones H3 and H4 was increased in the promoters of up-regulated but not down-regulated genes. Thus, VPA may disrupt a balance between excitatory and inhibitory neuronal activities through its epigenetic effect.
    URL, DOI BibTeX

    @article{Fukuchi2009,
    	abstract = "Valproic acid (VPA), a drug used to treat epilepsy and bipolar mood disorder, inhibits histone deacetylase (HDAC), which is associated with the epigenetic regulation of gene expression. Using a microarray, we comprehensively examined which genes are affected by stimulating cultured rat cortical neurons with VPA, and found that the VPA-treatment markedly altered gene expression (up-regulated; 726 genes, down-regulated; 577 genes). The mRNA expression for brain-derived neurotrophic factor (BDNF) and the alpha4 subunit of the GABA(A) receptor (GABA(A)Ralpha4), known to be involved in epileptogenesis, was up-regulated, with the increase in BDNF exon I-IX mRNA expression being remarkable, whereas that for GABA(A)Rgamma2, GAD65 and 67, and the K(+)/Cl(-) co-transporter KCC2, which are responsible for the development of GABAergic inhibitory neurons, was down-regulated. The number of GAD67-positive neurons decreased upon VPA-treatment. Similar changes of up- and down-regulation were obtained by trichostatin A. VPA did not affect the intracellular Ca(2+) concentration and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting its direct action on HDAC. The acetylation of histones H3 and H4 was increased in the promoters of up-regulated but not down-regulated genes. Thus, VPA may disrupt a balance between excitatory and inhibitory neuronal activities through its epigenetic effect.",
    	author = "Fukuchi, Mamoru and Nii, Takuya and Ishimaru, Naoki and Minamino, Aya and Hara, Daichi and Takasaki, Ichiro and Tabuchi, Akiko and Tsuda, Masaaki",
    	doi = "10.1016/j.neures.2009.05.002",
    	issn = "1872-8111",
    	journal = "Neuroscience research",
    	keywords = "Animals,Blotting, Western,Brain-Derived Neurotrophic Factor,Brain-Derived Neurotrophic Factor: metabolism,Calcium Signaling,Cells, Cultured,Cerebral Cortex,Cerebral Cortex: cytology,Cerebral Cortex: drug effects,Cerebral Cortex: metabolism,Chromatin Immunoprecipitation,Down-Regulation,Epigenesis, Genetic,Gene Expression Profiling,Glutamate Decarboxylase,Glutamate Decarboxylase: metabolism,Histones,Histones: metabolism,Neurons,Neurons: drug effects,Neurons: metabolism,Oligonucleotide Array Sequence Analysis,Rats,Rats, Sprague-Dawley,Receptors, GABA-A,Receptors, GABA-A: metabolism,Symporters,Symporters: metabolism,Up-Regulation,Valproic Acid,Valproic Acid: pharmacology",
    	month = "sep",
    	number = 1,
    	pages = "35--43",
    	pmid = 19463867,
    	title = "{Valproic acid induces up- or down-regulation of gene expression responsible for the neuronal excitation and inhibition in rat cortical neurons through its epigenetic actions.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/19463867",
    	volume = 65,
    	year = 2009
    }
    
  18. Stefan Titz, Sheriar Hormuzdi, Andrea Lewen, Hannah Monyer and Ulrich Misgeld.
    Intracellular acidification in neurons induced by ammonium depends on KCC2 function.. The European journal of neuroscience 23(2):454–64, January 2006.
    Abstract The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.
    URL, DOI BibTeX

    @article{Titz2006,
    	abstract = "The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.",
    	author = "Titz, Stefan and Hormuzdi, Sheriar and Lewen, Andrea and Monyer, Hannah and Misgeld, Ulrich",
    	doi = "10.1111/j.1460-9568.2005.04583.x",
    	issn = "0953-816X",
    	journal = "The European journal of neuroscience",
    	keywords = "Animals,Bumetanide,Bumetanide: pharmacology,Calcium,Calcium Channel Agonists,Calcium Channel Agonists: pharmacology,Calcium: metabolism,Cells, Cultured,Diagnostic Imaging,Diagnostic Imaging: methods,Dose-Response Relationship, Drug,Drug Interactions,Embryo, Mammalian,Female,Furosemide,Furosemide: pharmacology,Green Fluorescent Proteins,Green Fluorescent Proteins: metabolism,Hippocampus,Hippocampus: cytology,Hydrogen-Ion Concentration,Hydrogen-Ion Concentration: drug effects,Immunohistochemistry,Immunohistochemistry: methods,Intracellular Space,Intracellular Space: physiology,Mesencephalon,Mesencephalon: cytology,Neuroglia,Neuroglia: drug effects,Neuroglia: metabolism,Neurons,Neurons: cytology,Neurons: drug effects,Potassium Chloride,Potassium Chloride: pharmacology,Pregnancy,Pyrroles,Pyrroles: pharmacology,Quaternary Ammonium Compounds,Quaternary Ammonium Compounds: pharmacology,Rats,Rats, Wistar,Sodium Potassium Chloride Symporter Inhibitors,Sodium Potassium Chloride Symporter Inhibitors: ph,Symporters,Symporters: physiology,Time Factors,Transfection,Transfection: methods,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: pharmacology",
    	month = "jan",
    	number = 2,
    	pages = "454--64",
    	pmid = 16420452,
    	title = "{Intracellular acidification in neurons induced by ammonium depends on KCC2 function.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/16420452",
    	volume = 23,
    	year = 2006
    }
    
  19. Claudio Rivera, Juha Voipio and Kai Kaila.
    Two developmental switches in GABAergic signalling: the K+-Cl- cotransporter KCC2 and carbonic anhydrase CAVII.. The Journal of physiology 562(Pt 1):27–36, January 2005.
    Abstract GABAergic signalling has the unique property of 'ionic plasticity', which is based on short-term and long-term changes in the Cl(-) and HCO(3)(-) ion concentrations in the postsynaptic neurones. While short-term ionic plasticity is caused by activity-dependent, channel-mediated anion shifts, long-term ionic plasticity depends on changes in the expression patterns and kinetic regulation of molecules involved in anion homeostasis. During development the efficacy and also the qualitative nature (depolarization/excitation versus hyperpolarization/inhibition) of GABAergic transmission is influenced by the neuronal expression of two key molecules: the chloride-extruding K(+)-Cl(-) cotransporter KCC2, and the cytosolic carbonic anhydrase (CA) isoform CAVII. In rat hippocampal pyramidal neurones, a steep up-regulation of KCC2 accounts for the 'developmental switch', which converts depolarizing and excitatory GABA responses of immature neurones to classical hyperpolarizing inhibition by the end of the second postnatal week. The immature hippocampus generates large-scale network activity, which is abolished in parallel by the up-regulation of KCC2 and the consequent increase in the efficacy of neuronal Cl(-) extrusion. At around postnatal day 12 (P12), an abrupt, steep increase in intrapyramidal CAVII expression takes place, promoting excitatory responses evoked by intense GABAergic activity. This is largely caused by a GABAergic potassium transient resulting in spatially widespread neuronal depolarization and synchronous spike discharges. These facts point to CAVII as a putative target of CA inhibitors that are used as antiepileptic drugs. KCC2 expression in adult rat neurones is down-regulated following epileptiform activity and/or neuronal damage by BDNF/TrkB signalling. The lifetime of membrane-associated KCC2 is very short, in the range of tens of minutes, which makes KCC2 ideally suited for mediating GABAergic ionic plasticity. In addition, factors influencing the trafficking and kinetic modulation of KCC2 as well as activation/deactivation of CAVII are obvious candidates in the ionic modulation of GABAergic responses. The down-regulation of KCC2 under pathophysiological conditions (epilepsy, damage) in mature neurones seems to reflect a 'recapitulation' of early developmental mechanisms, which may be a prerequisite for the re-establishment of connectivity in damaged brain tissue.
    URL, DOI BibTeX

    @article{Rivera2005,
    	abstract = "GABAergic signalling has the unique property of 'ionic plasticity', which is based on short-term and long-term changes in the Cl(-) and HCO(3)(-) ion concentrations in the postsynaptic neurones. While short-term ionic plasticity is caused by activity-dependent, channel-mediated anion shifts, long-term ionic plasticity depends on changes in the expression patterns and kinetic regulation of molecules involved in anion homeostasis. During development the efficacy and also the qualitative nature (depolarization/excitation versus hyperpolarization/inhibition) of GABAergic transmission is influenced by the neuronal expression of two key molecules: the chloride-extruding K(+)-Cl(-) cotransporter KCC2, and the cytosolic carbonic anhydrase (CA) isoform CAVII. In rat hippocampal pyramidal neurones, a steep up-regulation of KCC2 accounts for the 'developmental switch', which converts depolarizing and excitatory GABA responses of immature neurones to classical hyperpolarizing inhibition by the end of the second postnatal week. The immature hippocampus generates large-scale network activity, which is abolished in parallel by the up-regulation of KCC2 and the consequent increase in the efficacy of neuronal Cl(-) extrusion. At around postnatal day 12 (P12), an abrupt, steep increase in intrapyramidal CAVII expression takes place, promoting excitatory responses evoked by intense GABAergic activity. This is largely caused by a GABAergic potassium transient resulting in spatially widespread neuronal depolarization and synchronous spike discharges. These facts point to CAVII as a putative target of CA inhibitors that are used as antiepileptic drugs. KCC2 expression in adult rat neurones is down-regulated following epileptiform activity and/or neuronal damage by BDNF/TrkB signalling. The lifetime of membrane-associated KCC2 is very short, in the range of tens of minutes, which makes KCC2 ideally suited for mediating GABAergic ionic plasticity. In addition, factors influencing the trafficking and kinetic modulation of KCC2 as well as activation/deactivation of CAVII are obvious candidates in the ionic modulation of GABAergic responses. The down-regulation of KCC2 under pathophysiological conditions (epilepsy, damage) in mature neurones seems to reflect a 'recapitulation' of early developmental mechanisms, which may be a prerequisite for the re-establishment of connectivity in damaged brain tissue.",
    	author = "Rivera, Claudio and Voipio, Juha and Kaila, Kai",
    	doi = "10.1113/jphysiol.2004.077495",
    	file = ":C$\backslash$:/Users/riku/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Rivera, Voipio, Kaila - 2005 - Two developmental switches in GABAergic signalling the K-Cl- cotransporter KCC2 and carbonic anhydrase CA.pdf:pdf",
    	issn = "0022-3751",
    	journal = "The Journal of physiology",
    	keywords = "Animals,Carbonic Anhydrases,Carbonic Anhydrases: physiology,Homeostasis,Homeostasis: physiology,Humans,Isoenzymes,Isoenzymes: physiology,Neuronal Plasticity,Neuronal Plasticity: physiology,Signal Transduction,Signal Transduction: physiology,Symporters,Symporters: physiology,Up-Regulation,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: physiology",
    	month = "jan",
    	number = "Pt 1",
    	pages = "27--36",
    	pmid = 15528236,
    	title = "{Two developmental switches in GABAergic signalling: the K+-Cl- cotransporter KCC2 and carbonic anhydrase CAVII.}",
    	url = "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1665491\&tool=pmcentrez\&rendertype=abstract",
    	volume = 562,
    	year = 2005
    }
    
  20. Elizabeth Leitch, Julie Coaker, Carol Young, Vandana Mehta and Evelyne Sernagor.
    GABA type-A activity controls its own developmental polarity switch in the maturing retina.. The Journal of neuroscience : the official journal of the Society for Neuroscience 25(19):4801–5, 2005.
    Abstract In the developing CNS, GABA(A) responses switch from early excitation to late mature inhibition. The developmental factors that induce the polarity switch remain to be unraveled. Here, we bring the first experimental evidence in vivo in the retina that chronic activation of GABA(A) receptors is necessary for the switch to occur and for the chloride extrusion mechanism (through the K+/Cl- cotransporter KCC2) to develop. Using a turtle model and calcium imaging, we investigated how chronic blockade of GABA(A) receptors with bicuculline during the period of the GABAergic polarity switch (from 1 week before hatching until 4 weeks after hatching) influences developmental changes in the patterns of spontaneously generated electrical activity in the retinal ganglion cell (RGC) layer. During that period, spontaneous activity normally switches from propagating waves to stationary patches of coactive cells, until correlated activity completely disappears. These changes in activity patterns coincide with the switch of GABA(A) responses from excitation to inhibition. When GABA(A) receptors are chronically blocked, GABA(A) responses remain excitatory and spontaneous waves keep propagating across the RGC layer. Concomitantly, the developmental upregulation of KCC2 is inhibited on dendritic processes in the inner plexiform layer, suggesting that the intracellular concentration of chloride remains higher, as in younger cells. This study presents the first demonstration in vivo that GABA autoregulates its developmental polarity switch, emphasizing the importance of GABAergic activity in controlling activity patterns in the maturing retina.
    URL, DOI BibTeX

    @article{Leitch2005,
    	abstract = "In the developing CNS, GABA(A) responses switch from early excitation to late mature inhibition. The developmental factors that induce the polarity switch remain to be unraveled. Here, we bring the first experimental evidence in vivo in the retina that chronic activation of GABA(A) receptors is necessary for the switch to occur and for the chloride extrusion mechanism (through the K+/Cl- cotransporter KCC2) to develop. Using a turtle model and calcium imaging, we investigated how chronic blockade of GABA(A) receptors with bicuculline during the period of the GABAergic polarity switch (from 1 week before hatching until 4 weeks after hatching) influences developmental changes in the patterns of spontaneously generated electrical activity in the retinal ganglion cell (RGC) layer. During that period, spontaneous activity normally switches from propagating waves to stationary patches of coactive cells, until correlated activity completely disappears. These changes in activity patterns coincide with the switch of GABA(A) responses from excitation to inhibition. When GABA(A) receptors are chronically blocked, GABA(A) responses remain excitatory and spontaneous waves keep propagating across the RGC layer. Concomitantly, the developmental upregulation of KCC2 is inhibited on dendritic processes in the inner plexiform layer, suggesting that the intracellular concentration of chloride remains higher, as in younger cells. This study presents the first demonstration in vivo that GABA autoregulates its developmental polarity switch, emphasizing the importance of GABAergic activity in controlling activity patterns in the maturing retina.",
    	author = "Leitch, Elizabeth and Coaker, Julie and Young, Carol and Mehta, Vandana and Sernagor, Evelyne",
    	doi = "10.1523/JNEUROSCI.0172-05.2005",
    	issn = "1529-2401",
    	journal = "The Journal of neuroscience : the official journal of the Society for Neuroscience",
    	keywords = "Action Potentials,Action Potentials: drug effects,Action Potentials: physiology,Animals,Bicuculline,Bicuculline: pharmacology,Calcium,Calcium: metabolism,Dextrans,Dextrans: metabolism,Diagnostic Imaging,Diagnostic Imaging: methods,Embryo, Nonmammalian,GABA Antagonists,GABA Antagonists: pharmacology,Gene Expression Regulation, Developmental,Gene Expression Regulation, Developmental: physiol,Immunohistochemistry,Immunohistochemistry: methods,Indoles,Indoles: diagnostic use,Neural Inhibition,Neural Inhibition: physiology,Polyvinyls,Polyvinyls: metabolism,Receptors, GABA-A,Receptors, GABA-A: drug effects,Receptors, GABA-A: physiology,Retina,Retina: cytology,Retina: growth \& development,Retinal Ganglion Cells,Retinal Ganglion Cells: physiology,Symporters,Symporters: metabolism,Turtles,gamma-Aminobutyric Acid,gamma-Aminobutyric Acid: pharmacology",
    	month = "",
    	number = 19,
    	pages = "4801--5",
    	pmid = 15888655,
    	title = "{GABA type-A activity controls its own developmental polarity switch in the maturing retina.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/15888655",
    	volume = 25,
    	year = 2005
    }
    
  21. Aristea S Galanopoulou and Solomon L Moshé.
    Role of sex hormones in the sexually dimorphic expression of KCC2 in rat substantia nigra.. Experimental neurology 184(2):1003–9, 2003.
    Abstract KCC2 is a neuronal-specific potassium chloride cotransporter. The level of KCC2 expression is a factor determining whether GABA(A) receptor agonists depolarize or hyperpolarize neurons. Substantia nigra reticulata (SNR) neurons of male postnatal day 15 (PN15) rats have low KCC2 mRNA expression and respond to GABA(A) receptor activation with depolarization and activation of calcium-regulated gene expression. Female PN15 SNR neurons have high KCC2 mRNA expression and GABA(A) receptor agonists cannot activate calcium-dependent signaling processes. We investigate whether sex hormones regulate KCC2 mRNA expression in PN15 rat SNR. Using in situ hybridization, we studied the effects of acute (4 h) or prolonged (52 h) subcutaneous (s.c.) administration of testosterone (100 microg), dihydrotestosterone (180 microg) or 17beta-estradiol benzoate (5 microg) on KCC2 mRNA expression in male and female PN15 rat SNR. Different doses of estradiol (1 and 10 microg s.c., 4 h) were also acutely administered in female PN15 rats. Controls received oil injections. Separate groups of PN15 male rats were pretreated with antagonists of L-type voltage-sensitive calcium channels (L-VSCCs) [nifedipine, 100 mg/kg s.c.] or GABA(A) receptors [bicuculline, 2 mg/kg intraperitoneally (i.p.)] or their vehicles, 30 min before estradiol (5 microg s.c., 4 h). Testosterone and dihydrotestosterone upregulated KCC2 mRNA in both sexes. Estradiol downregulated KCC2 mRNA in males but not in females. Both acute and prolonged hormonal administration had similar effects. In male PN15 SNR, nifedipine and bicuculline decreased KCC2 mRNA acutely and prevented further downregulation of KCC2 mRNA by estradiol. Estradiol therefore downregulates KCC2 mRNA in male PN15 SNR, by interacting with the GABA(A) receptor and L-VSCC signaling pathway.
    URL, DOI BibTeX

    @article{Galanopoulou2003a,
    	abstract = "KCC2 is a neuronal-specific potassium chloride cotransporter. The level of KCC2 expression is a factor determining whether GABA(A) receptor agonists depolarize or hyperpolarize neurons. Substantia nigra reticulata (SNR) neurons of male postnatal day 15 (PN15) rats have low KCC2 mRNA expression and respond to GABA(A) receptor activation with depolarization and activation of calcium-regulated gene expression. Female PN15 SNR neurons have high KCC2 mRNA expression and GABA(A) receptor agonists cannot activate calcium-dependent signaling processes. We investigate whether sex hormones regulate KCC2 mRNA expression in PN15 rat SNR. Using in situ hybridization, we studied the effects of acute (4 h) or prolonged (52 h) subcutaneous (s.c.) administration of testosterone (100 microg), dihydrotestosterone (180 microg) or 17beta-estradiol benzoate (5 microg) on KCC2 mRNA expression in male and female PN15 rat SNR. Different doses of estradiol (1 and 10 microg s.c., 4 h) were also acutely administered in female PN15 rats. Controls received oil injections. Separate groups of PN15 male rats were pretreated with antagonists of L-type voltage-sensitive calcium channels (L-VSCCs) [nifedipine, 100 mg/kg s.c.] or GABA(A) receptors [bicuculline, 2 mg/kg intraperitoneally (i.p.)] or their vehicles, 30 min before estradiol (5 microg s.c., 4 h). Testosterone and dihydrotestosterone upregulated KCC2 mRNA in both sexes. Estradiol downregulated KCC2 mRNA in males but not in females. Both acute and prolonged hormonal administration had similar effects. In male PN15 SNR, nifedipine and bicuculline decreased KCC2 mRNA acutely and prevented further downregulation of KCC2 mRNA by estradiol. Estradiol therefore downregulates KCC2 mRNA in male PN15 SNR, by interacting with the GABA(A) receptor and L-VSCC signaling pathway.",
    	author = "Galanopoulou, Aristea S and Mosh\'{e}, Solomon L",
    	doi = "10.1016/S0014-4886(03)00387-X",
    	issn = "0014-4886",
    	journal = "Experimental neurology",
    	keywords = "Animals,Calcium Channel Blockers,Calcium Channel Blockers: pharmacology,Calcium Channels, L-Type,Calcium Channels, L-Type: drug effects,Calcium Channels, L-Type: metabolism,Dihydrotestosterone,Dihydrotestosterone: pharmacology,Estradiol,Estradiol: analogs \& derivatives,Estradiol: pharmacology,Female,GABA Antagonists,GABA Antagonists: pharmacology,Gonadal Steroid Hormones,Gonadal Steroid Hormones: pharmacology,In Situ Hybridization,Male,RNA, Messenger,RNA, Messenger: analysis,Rats,Rats, Sprague-Dawley,Receptors, GABA-A,Receptors, GABA-A: drug effects,Receptors, GABA-A: metabolism,Sex Characteristics,Substantia Nigra,Substantia Nigra: physiology,Symporters,Symporters: biosynthesis,Symporters: drug effects,Testosterone,Testosterone: pharmacology,Time Factors",
    	month = "",
    	number = 2,
    	pages = "1003--9",
    	pmid = 14769394,
    	title = "{Role of sex hormones in the sexually dimorphic expression of KCC2 in rat substantia nigra.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/14769394",
    	volume = 184,
    	year = 2003
    }
    
  22. Aristea S Galanopoulou, Andreas Kyrozis, Olga I Claudio, Patric K Stanton and Solomon L Moshé.
    Sex-specific KCC2 expression and GABA(A) receptor function in rat substantia nigra.. Experimental neurology 183(2):628–37, 2003.
    Abstract GABA(A) receptor activation by muscimol has sex and age specific effects on substantia nigra reticulata (SNR)-mediated control of generalized seizures. GABA(A) receptor agonists depolarize or hyperpolarize neurons depending upon the level of expression of the neuronal specific potassium chloride contransporter KCC2. We studied KCC2 mRNA expression in the SNR as a function of sex and age and correlated KCC2 expression with the in vivo and in vitro effects of muscimol. Methods included in situ hybridization, gramicidin-perforated patch clamp and fura-2 AM imaging of acute SNR slices. KCC2 mRNA expression increased between postnatal days (PN) 15 and 30 in both sexes, and reached adult levels in males by PN30. Female PN15 and PN30 SNR neurons contained more KCC2 mRNA compared with age-matched males. In male PN14-17 rats, bath application of the GABA(A) receptor agonist muscimol in acute SNR slices depolarized neurons and increased intracellular calcium concentration ([Ca(2+)](i)). Furthermore, acute in vivo administration of muscimol upregulated, whereas blockade of L-type voltage sensitive calcium channels with nifedipine downregulated KCC2 mRNA. In contrast, in female PN14-17 rats, bath application of muscimol hyperpolarized SNR neurons and did not alter [Ca(2+)](i). In vivo muscimol administration acutely downregulated KCC2 mRNA expression whereas nifedipine had no effect. The lower expression of KCC2 mRNA in infantile male SNR neurons may explain why muscimol-induced depolarization and [Ca(2+)](i) increases occur only in males. Consequently, GABA(A) receptor activation selectively upregulates the expression of calcium-regulated genes, such as KCC2, in male SNR, promoting the sexual differentiation of the SNR.
    URL BibTeX

    @article{Galanopoulou2003,
    	abstract = "GABA(A) receptor activation by muscimol has sex and age specific effects on substantia nigra reticulata (SNR)-mediated control of generalized seizures. GABA(A) receptor agonists depolarize or hyperpolarize neurons depending upon the level of expression of the neuronal specific potassium chloride contransporter KCC2. We studied KCC2 mRNA expression in the SNR as a function of sex and age and correlated KCC2 expression with the in vivo and in vitro effects of muscimol. Methods included in situ hybridization, gramicidin-perforated patch clamp and fura-2 AM imaging of acute SNR slices. KCC2 mRNA expression increased between postnatal days (PN) 15 and 30 in both sexes, and reached adult levels in males by PN30. Female PN15 and PN30 SNR neurons contained more KCC2 mRNA compared with age-matched males. In male PN14-17 rats, bath application of the GABA(A) receptor agonist muscimol in acute SNR slices depolarized neurons and increased intracellular calcium concentration ([Ca(2+)](i)). Furthermore, acute in vivo administration of muscimol upregulated, whereas blockade of L-type voltage sensitive calcium channels with nifedipine downregulated KCC2 mRNA. In contrast, in female PN14-17 rats, bath application of muscimol hyperpolarized SNR neurons and did not alter [Ca(2+)](i). In vivo muscimol administration acutely downregulated KCC2 mRNA expression whereas nifedipine had no effect. The lower expression of KCC2 mRNA in infantile male SNR neurons may explain why muscimol-induced depolarization and [Ca(2+)](i) increases occur only in males. Consequently, GABA(A) receptor activation selectively upregulates the expression of calcium-regulated genes, such as KCC2, in male SNR, promoting the sexual differentiation of the SNR.",
    	author = "Galanopoulou, Aristea S and Kyrozis, Andreas and Claudio, Olga I and Stanton, Patric K and Mosh\'{e}, Solomon L",
    	issn = "0014-4886",
    	journal = "Experimental neurology",
    	keywords = "Age Factors,Animals,Calcium,Calcium Channel Blockers,Calcium Channel Blockers: pharmacology,Calcium Channels, L-Type,Calcium Channels, L-Type: drug effects,Calcium: metabolism,Female,GABA Agonists,GABA Agonists: pharmacology,GABA-A Receptor Agonists,Gene Expression Regulation, Developmental,Gene Expression Regulation, Developmental: drug ef,Gene Expression Regulation, Developmental: physiol,In Vitro Techniques,Male,Muscimol,Muscimol: pharmacology,Neurons,Neurons: drug effects,Neurons: metabolism,Nifedipine,Nifedipine: pharmacology,Patch-Clamp Techniques,RNA, Messenger,RNA, Messenger: metabolism,Rats,Rats, Sprague-Dawley,Receptors, GABA-A,Receptors, GABA-A: metabolism,Sex Factors,Substantia Nigra,Substantia Nigra: cytology,Substantia Nigra: drug effects,Substantia Nigra: metabolism,Symporters,Symporters: genetics",
    	month = "",
    	number = 2,
    	pages = "628--37",
    	pmid = 14552904,
    	title = "{Sex-specific KCC2 expression and GABA(A) receptor function in rat substantia nigra.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/14552904",
    	volume = 183,
    	year = 2003
    }
    
  23. Nam-Sik Woo, Jianming Lu, Roger England, Robert McClellan, Samuel Dufour, David B Mount, Ariel Y Deutch, David M Lovinger and Eric Delpire.
    Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K-Cl cotransporter gene.. Hippocampus 12(2):258–68, January 2002.
    Abstract Four genes encode electroneutral, Na+-independent, K-Cl cotransporters. KCC2, is exclusively expressed in neurons where it is thought to drive intracellular Cl- to low concentrations and shift the reversal potential for Cl- conductances such as GABA(A) or glycine receptor channels, thus participating in the postnatal development of inhibitory mechanisms in the brain. Indeed, expression of the cotransporter is low at birth and increases postnatally, at a time when the intracellular Cl- concentration in neurons decreases and gamma-aminobutyric acid switches its effect from excitatory to inhibitory. To assert the significance of KCC2 in neuronal function, we disrupted the mouse gene encoding this neuronal-specific K-Cl cotransporter. We demonstrate that animals deficient in KCC2 exhibit frequent generalized seizures and die shortly after birth. We also show upregulation of Fos, the product of the immediate early gene c-fos, and the significant loss of parvalbumin-positive interneurons, both indicative of brain injury. The regions most affected are the hippocampus and temporal and entorhinal cortices. Extracellular field potential measurements in the CA1 hippocampus exhibited hyperexcitability. Application of picrotoxin, a blocker of the GABA(A) receptor, further increased hyperexcitability in homozygous hippocampal sections. Pharmacological treatment of pups showed that diazepam relieved the seizures while phenytoin prevented them between postnatal ages P4-P12. Finally, we demonstrate that adult heterozygote animals show increased susceptibility for epileptic seizure and increased resistance to the anticonvulsant effect of propofol. Taken together, these results indicate that KCC2 plays an important role in controlling CNS excitability during both postnatal development and adult life.
    URL, DOI BibTeX

    @article{Woo2002,
    	abstract = "Four genes encode electroneutral, Na+-independent, K-Cl cotransporters. KCC2, is exclusively expressed in neurons where it is thought to drive intracellular Cl- to low concentrations and shift the reversal potential for Cl- conductances such as GABA(A) or glycine receptor channels, thus participating in the postnatal development of inhibitory mechanisms in the brain. Indeed, expression of the cotransporter is low at birth and increases postnatally, at a time when the intracellular Cl- concentration in neurons decreases and gamma-aminobutyric acid switches its effect from excitatory to inhibitory. To assert the significance of KCC2 in neuronal function, we disrupted the mouse gene encoding this neuronal-specific K-Cl cotransporter. We demonstrate that animals deficient in KCC2 exhibit frequent generalized seizures and die shortly after birth. We also show upregulation of Fos, the product of the immediate early gene c-fos, and the significant loss of parvalbumin-positive interneurons, both indicative of brain injury. The regions most affected are the hippocampus and temporal and entorhinal cortices. Extracellular field potential measurements in the CA1 hippocampus exhibited hyperexcitability. Application of picrotoxin, a blocker of the GABA(A) receptor, further increased hyperexcitability in homozygous hippocampal sections. Pharmacological treatment of pups showed that diazepam relieved the seizures while phenytoin prevented them between postnatal ages P4-P12. Finally, we demonstrate that adult heterozygote animals show increased susceptibility for epileptic seizure and increased resistance to the anticonvulsant effect of propofol. Taken together, these results indicate that KCC2 plays an important role in controlling CNS excitability during both postnatal development and adult life.",
    	author = "Woo, Nam-Sik and Lu, Jianming and England, Roger and McClellan, Robert and Dufour, Samuel and Mount, David B and Deutch, Ariel Y and Lovinger, David M and Delpire, Eric",
    	doi = "10.1002/hipo.10014",
    	issn = "1050-9631",
    	journal = "Hippocampus",
    	keywords = "Animals,Anticonvulsants,Anticonvulsants: pharmacology,Chimera,Convulsants,Entorhinal Cortex,Entorhinal Cortex: cytology,Entorhinal Cortex: growth \& development,Entorhinal Cortex: physiology,Epilepsy,Epilepsy: chemically induced,Epilepsy: drug therapy,Epilepsy: physiopathology,Female,Heterozygote,Hippocampus,Hippocampus: cytology,Hippocampus: growth \& development,Hippocampus: physiology,Homozygote,Male,Mice,Mice, Inbred C57BL,Mutagenesis,Mutagenesis: physiology,Neural Inhibition,Neural Inhibition: physiology,Neurons,Neurons: chemistry,Neurons: physiology,Pentylenetetrazole,Propofol,Propofol: pharmacology,Proto-Oncogene Proteins c-fos,Proto-Oncogene Proteins c-fos: analysis,Symporters,Symporters: genetics,Symporters: metabolism,Synaptic Transmission,Synaptic Transmission: physiology",
    	month = "jan",
    	number = 2,
    	pages = "258--68",
    	pmid = 12000122,
    	title = "{Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K-Cl cotransporter gene.}",
    	url = "http://www.ncbi.nlm.nih.gov/pubmed/12000122",
    	volume = 12,
    	year = 2002
    }