Potassium-chloride Cotransporter Research Articles

Maternal restraint stress delays maturation of cation-chloride cotransporters and GABAA receptor subunits in the hippocampus of rat pups at puberty

The GABAergic synapse undergoes structural and functional maturation during early brain development. Maternal stress alters GABAergic synapses in the pup's brain that are associated with the pathophysiology of neuropsychiatric disorders in adults; however, the mechanism for this is still unclear. In this study, we examined the effects of maternal restraint stress on the development of Cation-Chloride Cotransporters (CCCs) and the GABAA receptor α1 and α5 subunits in the hippocampus of rat pups at different postnatal ages. Our results demonstrate that maternal restraint stress induces a transient but significant increase in the level of NKCC1 (Sodium–Potassium Chloride Cotransporter 1) only at P14, followed by a brief, yet significant increase in the level of KCC2 (Potassium-Chloride Cotransporter 2) at P21, which then decreases from P28 until P40. Thus, maternal stress alters NKCC1 and KCC2 ratio in the hippocampus of rat pups, especially during P14 to P28. Maternal restraint stress also caused biphasic changes in the level of GABAA receptor subunits in the pup's hippocampus. GABAA receptor α1 subunit gradually increased at P14 then decreased thereafter. On the contrary, GABAA receptor α5 subunit showed a transient decrease followed by a long-term increase from P21 until P40. Altogether, our study suggested that the maternal restraint stress might delay maturation of the GABAergic system by altering the expression of NKCC1, KCC2 and GABAA receptor α1 and α5 subunits in the hippocampus of rat pups. These changes demonstrate the dysregulation of inhibitory neurotransmission during early life, which may underlie the pathogenesis of psychiatric diseases at adolescence.

Hyperpolarization-independent maturation and refinement of GABA/glycinergic connections in the auditory brain stem.

During development GABA and glycine synapses are initially excitatory before they gradually become inhibitory. This transition is due to a developmental increase in the activity of neuronal potassium-chloride cotransporter 2 (KCC2), which shifts the chloride equilibrium potential (ECl) to values more negative than the resting membrane potential. While the role of early GABA and glycine depolarizations in neuronal development has become increasingly clear, the role of the transition to hyperpolarization in synapse maturation and circuit refinement has remained an open question. Here we investigated this question by examining the maturation and developmental refinement of GABA/glycinergic and glutamatergic synapses in the lateral superior olive (LSO), a binaural auditory brain stem nucleus, in KCC2-knockdown mice, in which GABA and glycine remain depolarizing. We found that many key events in the development of synaptic inputs to the LSO, such as changes in neurotransmitter phenotype, strengthening and elimination of GABA/glycinergic connection, and maturation of glutamatergic synapses, occur undisturbed in KCC2-knockdown mice compared with wild-type mice. These results indicate that maturation of inhibitory and excitatory synapses in the LSO is independent of the GABA and glycine depolarization-to-hyperpolarization transition.

Tear Production After Bilateral Main Lacrimal Gland Resection in Rabbits.

This study reports tear compensation observed in rabbits with bilateral resection of main lacrimal gland (LG) and explored the potential mechanisms. Dry eye conditions were created by resection of nictitating membrane (NM), Harderian gland (HG), and main LG in eight (16 eyes) male New Zealand White rabbits. In addition to Schirmer test, Periodic acid-Schiff (PAS) staining, and ocular surface staining with fluorescein and rose Bengal, conjunctival impression cytology was employed before and up to 4 months after excision (AE). Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), expression of inflammatory biomarkers (IL-1β, TNF-α, and matrix metalloproteinase-9) were monitored. Further, involvement of ionic and water transporters were investigated in conjunctival epithelium. Significant dry eye phenotypes in rabbits were observed 1 month AE, which corroborated with elevated biomarker mRNA expression. However, Schirmer test score and goblet cell numbers never decreased AE in conjunctival epithelium. Moreover, ocular surface staining, and biomarker expression declined to baseline in over 4 months AE. No upregulation was observed of the following conjunctival ionic transporters: cystic fibrosis transmembrane conductance regulator, sodium potassium chloride cotransporters, sodium potassium ATPase, and epithelial sodium channels. Instead, aquaporin (AQP) 4 and AQP5 were upregulated. Immunolocalization and immune blotting of AQP4 was demonstrated in rabbit conjunctival epithelium. In the absence of NM, HG, and main LG in rabbits, tear secretion was not decreased and significant improvement of dry eye phenotypes observed with time AE. Conjunctival AQPs are possibly involved in a compensatory tear fluid production.

Chloride dysregulation and inhibitory receptor blockade yield equivalent disinhibition of spinal neurons yet are differentially reversed by carbonic anhydrase blockade.

Synaptic inhibition plays a key role in processing somatosensory information. Blocking inhibition at the spinal level is sufficient to produce mechanical allodynia, and many neuropathic pain conditions are associated with reduced inhibition. Disinhibition of spinal neurons can arise through decreased GABAA/glycine receptor activation or through dysregulation of intracellular chloride. We hypothesized that these distinct disinhibitory mechanisms, despite all causing allodynia, are differentially susceptible to therapeutic intervention. Specifically, we predicted that reducing bicarbonate efflux by blocking carbonic anhydrase with acetazolamide (ACTZ) would counteract disinhibition caused by chloride dysregulation without affecting normal inhibition or disinhibition caused by GABAA/glycine receptor blockade. To test this, responses to innocuous tactile stimulation were recorded in vivo from rat superficial dorsal horn neurons before and after different forms of pharmacological disinhibition and again after application of ACTZ. Blocking GABAA or glycine receptors caused hyperresponsiveness equivalent to that caused by blocking the potassium chloride cotransporter KCC2, but, consistent with our predictions, only disinhibition caused by KCC2 blockade was counteracted by ACTZ. ACTZ did not alter responses of neurons with intact inhibition. As pathological downregulation of KCC2 is triggered by brain-derived neurotrophic factor, we also confirmed that ACTZ was effective against brain-derived neurotrophic factor-induced hyperresponsiveness. Our results argue that intrathecal ACTZ has antiallodynic effects only if allodynia arises through chloride dysregulation; therefore, behavioral evidence that ACTZ is antiallodynic in nerve-injured animals affirms the contribution of chloride dysregulation as a key pathological mechanism. Although different disinhibitory mechanisms are not mutually exclusive, these results demonstrate that their relative contribution dictates which specific therapies will be effective.

Erythropoietin Modulates Cerebral and Serum Degradation Products from Excess Calpain Activation following Prenatal Hypoxia-Ischemia

Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies.

Enhanced expression of potassium-chloride cotransporter KCC2 in human temporal lobe epilepsy.

Synaptic reorganization in the epileptic hippocampus involves altered excitatory and inhibitory transmission besides the rearrangement of dendritic spines, resulting in altered excitability, ion homeostasis, and cell swelling. The potassium-chloride cotransporter-2 (KCC2) is the main chloride extruder in neurons and hence will play a prominent role in determining the polarity of GABAA receptor-mediated chloride currents. In addition, KCC2 also interacts with the actin cytoskeleton which is critical for dendritic spine morphogenesis, and for the maintenance of glutamatergic synapses and cell volume. Using immunocytochemistry, we examined the cellular and subcellular levels of KCC2 in surgically removed hippocampi of temporal lobe epilepsy (TLE) patients and compared them to control human tissue. We also studied the distribution of KCC2 in a pilocarpine mouse model of epilepsy. An overall increase in KCC2-expression was found in epilepsy and confirmed by Western blots. The cellular and subcellular distributions in control mouse and human samples were largely similar; moreover, changes affecting KCC2-expression were also alike in chronic epileptic human and mouse hippocampi. At the subcellular level, we determined the neuronal elements exhibiting enhanced KCC2 expression. In epileptic tissue, staining became more intense in the immunopositive elements detected in control tissue, and profiles with subthreshold expression of KCC2 in control samples became labelled. Positive interneuron somata and dendrites were more numerous in epileptic hippocampi, despite severe interneuron loss. Whether the elevation of KCC2-expression is ultimately a pro- or anticonvulsive change, or both-behaving differently during ictal and interictal states in a context-dependent manner-remains to be established.

PC-FACS

PC-FACS

Developmental and degenerative modulation of GABAergic transmission in the mouse hippocampus

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter involved in synaptic plasticity. GABAergic transmission is also implicated in developmental and degenerative processes in the brain. The goal of the present study was to understand the developmental and degenerative regulation of GABAergic transmission in the mouse hippocampus by examining changes in GABA receptor subunit mRNA levels and GABA-related protein expression during postnatal development of the hippocampus and trimethyltin (TMT)-induced neurodegeneration in the juvenile (postnatal day [PD] 24) and adult hippocampus (PD 56). During postnatal development, the mRNA levels of GABA A receptor (GABAAR) subunits, including α1, α4, β1, β2, and δ; GABA B receptor (GABABR) subunit 2; and the expression of GABA-related proteins, including glutamic acid decarboxylase, vesicular GABA transporter (VGAT), and potassium chloride cotransporter 2 increased gradually in the mouse hippocampus. The results of seizure scoring and histopathological findings in the hippocampus revealed a more pronounced response to the same administered TMT dose in juvenile mice, compared with that in adult mice. The mRNA levels of most GABA receptor subunits in the juvenile hippocampus, excluding GABAAR subunit β3, were dynamically altered after TMT treatment. The mRNA levels of GABAAR subunits γ2 and δ decreased significantly in the adult hippocampus following TMT treatment, whereas the level of GABABR subunit 1 mRNA increased significantly. Among the GABA-related proteins, only VGAT decreased significantly in the juvenile and adult mouse hippocampus after TMT treatment. In conclusion, regulation of GABAergic signaling in the mouse hippocampus may be related to maturation of the central nervous system and the degree of neurodegeneration during postnatal development and TMT-induced neurodegeneration in the experimental animals.

Prenatal Hypoxia-Ischemia Induces Abnormalities in CA3 Microstructure, Potassium Chloride Co-Transporter 2 Expression and Inhibitory Tone.

Infants who suffer perinatal brain injury, including those with encephalopathy of prematurity, are prone to chronic neurological deficits, including epilepsy, cognitive impairment, and behavioral problems, such as anxiety, inattention, and poor social interaction. These deficits, especially in combination, pose the greatest hindrance to these children becoming independent adults. Cerebral function depends on adequate development of essential inhibitory neural circuits and the appropriate amount of excitation and inhibition at specific stages of maturation. Early neuronal synaptic responses to γ-amino butyric acid (GABA) are initially excitatory. During the early postnatal period, GABAAR responses switch to inhibitory with the upregulation of potassium-chloride co-transporter KCC2. With extrusion of chloride by KCC2, the Cl− reversal potential shifts and GABA and glycine responses become inhibitory. We hypothesized that prenatal hypoxic–ischemic brain injury chronically impairs the developmental upregulation of KCC2 that is essential for cerebral circuit formation. Following late gestation hypoxia–ischemia (HI), diffusion tensor imaging in juvenile rats shows poor microstructural integrity in the hippocampal CA3 subfield, with reduced fractional anisotropy and elevated radial diffusivity. The loss of microstructure correlates with early reduced KCC2 expression on NeuN-positive pyramidal neurons, and decreased monomeric and oligomeric KCC2 protein expression in the CA3 subfield. Together with decreased inhibitory post-synaptic currents during a critical window of development, we document for the first time that prenatal transient systemic HI in rats impairs hippocampal CA3 inhibitory tone. Failure of timely development of inhibitory tone likely contributes to a lower seizure threshold and impaired cognitive function in children who suffer perinatal brain injury.

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Alterations in Brain Inflammation, Synaptic Proteins, and Adult Hippocampal Neurogenesis during Epileptogenesis in Mice Lacking Synapsin2.

Synapsins are pre-synaptic vesicle-associated proteins linked to the pathogenesis of epilepsy through genetic association studies in humans. Deletion of synapsins causes an excitatory/inhibitory imbalance, exemplified by the epileptic phenotype of synapsin knockout mice. These mice develop handling-induced tonic-clonic seizures starting at the age of about 3 months. Hence, they provide an opportunity to study epileptogenic alterations in a temporally controlled manner. Here, we evaluated brain inflammation, synaptic protein expression, and adult hippocampal neurogenesis in the epileptogenic (1 and 2 months of age) and tonic-clonic (3.5-4 months) phase of synapsin 2 knockout mice using immunohistochemical and biochemical assays. In the epileptogenic phase, region-specific microglial activation was evident, accompanied by an increase in the chemokine receptor CX3CR1, interleukin-6, and tumor necrosis factor-α, and a decrease in chemokine keratinocyte chemoattractant/ growth-related oncogene. Both post-synaptic density-95 and gephyrin, scaffolding proteins at excitatory and inhibitory synapses, respectively, showed a significant up-regulation primarily in the cortex. Furthermore, we observed an increase in the inhibitory adhesion molecules neuroligin-2 and neurofascin and potassium chloride co-transporter KCC2. Decreased expression of γ-aminobutyric acid receptor-δ subunit and cholecystokinin was also evident. Surprisingly, hippocampal neurogenesis was reduced in the epileptogenic phase. Taken together, we report molecular alterations in brain inflammation and excitatory/inhibitory balance that could serve as potential targets for therapeutics and diagnostic biomarkers. In addition, the regional differences in brain inflammation and synaptic protein expression indicate an epileptogenic zone from where the generalized seizures in synapsin 2 knockout mice may be initiated or spread.

KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX.

Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.

Expanding Spectrum of Sodium Potassium Chloride Co-transporters in the Pathophysiology of Diseases.

Sodium potassium chloride co-transporter (NKCC) belongs to cation-dependent chloride co-transporter family, whose activation allows the entry of Na+, K+ and 2Cl- inside the cell. It acts in concert with K+ Cl- co-transporter (KCC), which extrudes K+ and Cl- ions from cell. NKCC1 is widely distributed throughout the body, while NKCC2 is exclusively present in kidney. Protein kinase A, protein kinase C, Ste20-related proline-alanine-rich kinase, oxidative stress responsive kinases, With No K=lysine kinase and protein phosphatase type 1 control the phosphorylation/dephosphorylation of key threonine residues of in regulatory domain of NKCC1. The selective inhibitors of NKCC1 including bumetanide and furosemide are conventionally employed as diuretics. However, recent studies have indicated that NKCC1 may be involved in the pathophysiology of anxiety, cerebral ischemia, epilepsy, neuropathic pain, fragile X syndrome, autism and schizophrenia. The inhibitors of NKCC1 are shown to produce anxiolytic effects; attenuate cerebral ischemia-induced neuronal injury; produce antiepileptic effects and attenuate neuropathic pain. In the early developing brain, GABAA activation primarily produces excitatory actions due to high NKCC1/KCC2 ratio. However, as the development progresses, the ratio of NKCC1/KCC2 ratio reverses and there is switch in the polarity of GABAA actions and latter acquires the inhibitory actions. The recapitulation of developmental-like state during pathological state may be associated with increase in the expression and functioning of NKCC1, which decreases the strength of inhibitory GABAergic neurotransmission. The present review describes the expanding role and mechanism of NKCC1 in the pathophysiology of different diseases.

Differential expression patterns of K(+) /Cl(-) cotransporter 2 in neurons within the superficial spinal dorsal horn of rats.

γ-Aminobutyric acid (GABA)- and glycine-mediated hyperpolarizing inhibition is associated with a chloride influx that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium-chloride cotransporters (KCC2s). KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high-resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the β3-subunit of GABAA receptors and KCC2, GABAA receptors were revealed on dendritic segments with high and also with low KCC2 densities. By measuring the distances between spots immunoreactive for gephyrin (a scaffolding protein of GABAA and glycine receptors) and KCC2 on the surface of neurokinin 1 (NK1) receptor-immunoreactive dendrites, we found that gephyrin-immunoreactive spots were located at various distances from KCC2 cotransporters; 5.7 % of them were recovered in the middle of 4-10-µm-long dendritic segments that were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABAA and glycine receptors along the dendrites of spinal neurons.

Contribution of the potassium-chloride cotransporter KCC2 to the strength of inhibition in the neonatal rodent spinal cord in vitro.

In healthy mature motoneurons (MNs), KCC2 cotransporters maintain the intracellular chloride concentration at low levels, a prerequisite for postsynaptic inhibition mediated by GABA and glycine. KCC2 expression in lumbar MNs is reduced after spinal cord injury (SCI) resulting in a depolarizing shift of the chloride equilibrium potential. Despite modeling studies indicating that such a downregulation of KCC2 function would reduce the strength of postsynaptic inhibition, physiological evidence is still lacking. The present study aimed at investigating the functional impact of a modification of KCC2 function. We focused on a well characterized disynaptic inhibitory pathway responsible for reciprocal inhibition between antagonistic muscles. We performed in vitro extracellular recordings on spinal cords isolated from rodents at the end of the first postnatal week. Genetic reduction of KCC2 expression, pharmacological blockade of KCC2, as well as SCI-induced downregulation of KCC2 all resulted in a reduction of the strength of reciprocal inhibition. We then tried to restore endogenous inhibition after SCI by means of zinc ions that have been shown to boost KCC2 function in other models. Zinc chloride indeed hyperpolarized the chloride equilibrium potential in MNs and increased reciprocal inhibition after neonatal SCI. This study demonstrates that the level of KCC2 function sets the strength of postsynaptic inhibition and suggests that the downregulation of KCC2 after SCI likely contributes to the high occurrence of flexor-extensor cocontractions in SCI patients.

Role of the potassium chloride cotransporter isoform 2-mediated spinal chloride homeostasis in a rat model of visceral hypersensitivity.

Visceral hypersensitivity represents an important hallmark in the pathophysiology of irritable bowel syndrome (IBS), of which the mechanisms remain elusive. The present study was designed to examine whether cation-chloride cotransporter (CCC)-mediated chloride (Cl(-)) homeostasis of the spinal cord is involved in chronic stress-induced visceral hypersensitivity. Chronic visceral hypersensitivity was induced by exposing male Wistar rats to water avoidance stress (WAS). RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of CCCs in the spinal cord. Patch-clamp recordings were performed on adult spinal cord slices to evaluate Cl(-) homeostasis and Cl(-) extrusion capacity of lamina I neurons. Visceral sensitivity was estimated by measuring the abdominal withdrawal reflex in response to colorectal distension (CRD). After 10 days of WAS exposure, levels of both total protein and the oligomeric form of the K(+)-Cl(-) cotransporter isoform 2 (KCC2), but not Na(+)-K(+)-2Cl(-) transporter isoform 1 (NKCC1), were significantly decreased in the dorsal horn of the lumbosacral spinal cord. The downregulation of KCC2 resulted in a depolarizing shifted equilibrium potential of GABAergic inhibitory postsynaptic current and impaired Cl(-) extrusion capacity in lamina I neurons of the lumbosacral spinal cord from WAS rats. Acute noxious CRD disrupted spinal KCC2 expression and function 2 h after the final distention in sham rats, but not in WAS rats. Pharmacological blockade of KCC2 activity by intrathecal injection of a KCC2 inhibitor [(dihydroindenyl)oxy] alkanoic acid enhanced visceral nociceptive sensitivity in sham rats, but not in WAS rats. These results suggest that KCC2 downregulation-mediated impairment of spinal cord Cl(-) homeostasis may play an important role in chronic stress-induced visceral hypersensitivity.

Involvement of medullary GABAergic system in extraterritorial neuropathic pain mechanisms associated with inferior alveolar nerve transection

In order to determine if the functional changes in the GABAergic system in the trigeminal spinal subnucleus caudalis (Vc) are involved in the mechanisms underlying extraterritorial neuropathic pain in the orofacial region following inferior alveolar nerve transection (IANX), mechanical noxious behavior, phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry and single neuronal activity were analyzed in vesicular GABA transporter (VGAT)-VenusA rats expressing fluorescent protein and the VGAT in Vc neurons.The number of VGAT-VenusA positive neurons was significantly reduced in IANX rats than naive and sham rats at 7days after nerve transection. The number of VGAT-VenusA positive pERK-immunoreactive (IR) cells was significantly increased in IANX rats at 21days after IAN transection compared with naive and sham rats. The background activity and mechanical-evoked responses of Vc nociceptive neurons were significantly depressed after intrathecal application of the GABA receptor agonist muscimol in sham rats but not in IANX rats. Furthermore, the expression of potassium-chloride co-transporter 2 (KCC2) in the Vc was significantly reduced in IANX rats compared with sham rats. The head-withdrawal threshold (HWT) to mechanical stimulation of the whisker pad skin was significantly decreased in IANX rats compared with sham rats on days 7 and 21 after IANX. The significant reduction of the HWT and significant increase in the number of VGAT-VenusA negative pERK-IR cells were observed in KCC2 blocker R-DIOA-injected rats compared with vehicle-injected rats on day 21 after sham treatment.These findings revealed that GABAergic Vc neurons might be reduced in their number at the early period after IANX and the functional changes might occur in GABAergic neurons from inhibitory to excitatory at the late period after IANX, suggesting that the neuroplastic changes occur in the GABAergic neuronal network in the Vc due to morphological and functional changes at different time periods following IANX and resulting in the extraterritorial neuropathic pain in the orofacial region following trigeminal nerve injury.

Investigating the stress attenuating potential of furosemide in immobilization and electric foot-shock stress models in mice

The present study was designed to investigate the antistress effect of furosemide (sodium potassium chloride co-transporter inhibitor) in immobilization and foot-shock stress-induced behavioral alterations in the mice. Acute stress was induced in Swiss albino mice either by applying electric foot shocks of 0.6-mA intensity of 1-s duration with 30-s inter-shock interval for 1h or immobilizing for 150min. The acute stress-induced behavioral changes were assessed by using actophotometer, hole board, open-field, and social interaction tests. Biochemically, the corticosterone levels were estimated in the serum as a biomarker of hypothalamus-pituitary-adrenal (HPA) axis. Acute stress resulted in the development of behavioral alterations and elevation of the corticosterone levels. Intraperitoneal administration of furosemide (25 and 50mg/kg) significantly attenuated immobilization and foot-shock stress-induced behavioral changes along with normalization of the corticosterone levels. It may be concluded that furosemide produces beneficial effects in reestablishing the behavioral and biochemical alterations in immobilization and foot-shock-induced acute stress in mice.

BDNF/TRK/KCC2 pathway in nicotine withdrawal-induced hyperalgesia.

PurposeTo investigate the effect of brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase (Trk) on potassium chloride cotransporter 2 (KCC2) in rats following nicotine withdrawal and the roles played by BDNF/Trk/KCC2 pathway in nicotine withdrawal-induced hyperalgesia.MethodsSeventy-eight rats were randomly assigned to five groups: control group (n = 12) without any treatment, normal saline group (NS group, n = 12) and nicotine withdrawal group (NW group, n = 30) receiving a subcutaneous injection of saline or nicotine for 7 days, respectively. The NW + dimethyl sulfoxide (DMSO) (n = 12) and NW+ Trk antagonist K252a groups (n = 12) received an intrathecal injection of DMSO (10 μl) and K252a (10 μg/10 μl) for 3 days after nicotine withdrawal, respectively. Nicotine withdrawal was precipitated by subcutaneous injection of nonselective and noncompetitive antagonist of nicotinic acetylcholine receptors mecamylamine. Pain was tested using thermal withdrawal latency (TWL). A Western blot was used to examine the expression of BDNF and KCC2.ResultsThe TWL was significantly decreased in NW group relative to control and NS groups (P < 0.01). Compared with the NW group, the NW+K252a group manifested a significantly higher latency (P < 0.01). The BDNF expression was increased and KCC2 was decreased in NW group compared with the control group (P < 0.01). K252a reduced KCC2 downregulation.ConclusionBDNF/Trk signaling may contribute to nicotine withdrawal-induced hyperalgesia via downregulation of KCC2.

Ca(2+) is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line.

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.