The effect of repeated stress on KCC2 and NKCC1 immunoreactivity in the hippocampus of female mice.
ABSTRACT: K(+)-Cl(-) co-transporter (KCC2) and Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) are the main regulators of neuronal intracellular chloride concentration; altered expression patterns of KCC2 and NKCC1 have been reported in several neurodegenerative diseases. In this paper, we show the effect of repeated stress on KCC2, NKCC1, and serine 940 phosphorylated KCC2 (pKCC2(ser940)) immunoreactivity. The data were obtained from the hippocampus of female mice using single-plane confocal microscopy images. The mean fluorescence intensity of the perisomatic area of neurons, defined as raw fluorescence intensity (RFI) was calculated. Repeated stress (RS) resulted in a decrease in perisomatic area of immunoreactive (IR)-KCC2 and an increase of the IR-NKCC1. In addition, RS decreased perisomatic IR-pKCC2(ser940), corresponding to that of KCC2. The data in this article support the results of a previous study  and provide the details of immunohistological methods. Interpretation of the data in this article can be found in "Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice" by Tsukahara et al. .
Project description:During the early postnatal period, GABA(B) receptor activation facilitates L-type calcium current in rat hippocampus. One developmental process that L-type current may regulate is the change in expression of the K(+)Cl(-) co-transporter (KCC2) and N(+)K(+)2Cl(-) co-transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L-type current, GABA(B) receptors, and expression of chloride transporters during development. The facilitation of L-type current by GABA(B) receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L-type channel antagonist and a GABA(B) receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L-type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L-type channels in the early postnatal period in hippocampal neurons.
Project description:The thalamus is important for sensory integration with the ventrobasal thalamus (VB) as relay controlled by GABAergic projections from the nucleus reticularis thalami (NRT). Depending on the [Cl-]i primarily set by cation-chloride-cotransporters, GABA is inhibitory or excitatory. There is evidence that VB and NRT differ in terms of GABA action, with classical hyperpolarization in VB due to the expression of the Cl- extruder KCC2 and depolarizing/excitatory GABA action in the NRT, where KCC2 expression is low and Cl- accumulation by the Cl- inward transporter NKCC1 has been postulated. However, data on NKCC1 expression and functional analysis of both transporters are missing. We show that KCC2-mediated Cl- extrusion set the [Cl-]i in VB, while NKCC1 did not contribute substantially to Cl- accumulation and depolarizing GABA action in the NRT. The finding that NKCC1 did not play a major role in NRT neurons is of high relevance for ongoing studies on the therapeutic use of NKCC1 inhibitors trying to compensate for a disease-induced up-regulation of NKCC1 that has been described for various brain regions and disease states like epilepsy and chronic pain. These data suggest that NKCC1 inhibitors might have no major effect on healthy NRT neurons due to limited NKCC1 function.
Project description:<h4>Background</h4>During development the switch from a depolarizing to a hyperpolarizing action of GABA is a consequence of a decrease of the Na+-K+-2Cl- co-transporter (NKCC1, Cl--uptake) and increase of the K+-Cl- co-transporter (KCC2, Cl--extrusion) expression. However albino visual cortex neurons don't show a corresponding decrease in intracellular chloride concentration during development of the visual system as compared to pigmented animals.<h4>Results</h4>Our study revealed that more cells express NKCC1 in albinos compared to pigmented rat visual cortex neurons whereas KCC2 is expressed in all cells in both strains. We determined a positive relationship between the presence of NKCC1 and an inhibitory deficit in single neurons of the albino visual cortex. After pharmacological blockade of NKCC1 function with its specific inhibitor, bumetanide, the reversal potential of electrically evoked GABAA receptor-mediated postsynaptic currents and, as a consequence, [Cl-]i in albino visual cortex neurons shifted to the pigmented rat brain value. In conclusion, our pharmacological experiments and subsequent single cell real time PCR analysis of the co-transporter mRNA demonstrated that the inhibitory deficit present in the albino visual cortical network is almost exclusively mediated by NKCC1.<h4>Conclusion</h4>Our findings suggest that blocking of NKCC1 in albino visual cortex neurons could improve processing in visual cortex and therefore might be beneficial for vision in albinos.
Project description:BACKGROUND:Cognitive deficits after perinatal anesthetic exposure are well established outcomes in animal models. This vulnerability is sex-dependent and associated with expression levels of the chloride transporters NKCC1 and KCC2. The hypothesis was that androgen signaling, NKCC1 function, and the age of isoflurane exposure are critical for the manifestation of anesthetic neurotoxicity in male rats. METHODS:Flutamide, an androgen receptor antagonist, was administered to male rats on postnatal days 2, 4, and 6 before 6?h of isoflurane on postnatal day 7 (ntotal = 26). Spatial and recognition memory were subsequently tested in adulthood. NKCC1 and KCC2 protein levels were measured from cortical lysates by Western blot on postnatal day 7 (ntotal = 20). Bumetanide, an NKCC1 antagonist, was injected immediately before isoflurane exposure (postnatal day 7) to study the effect of NKCC1 inhibition (ntotal = 48). To determine whether male rats remain vulnerable to anesthetic neurotoxicity as juveniles, postnatal day 14 animals were exposed to isoflurane and assessed as adults (ntotal = 30). RESULTS:Flutamide-treated male rats exposed to isoflurane successfully navigated the spatial (Barnes maze probe trial F[1, 151] = 78; P < 0.001; mean goal exploration ± SD, 6.4 ± 3.9?s) and recognition memory tasks (mean discrimination index ± SD, 0.09 ± 0.14; P = 0.003), unlike isoflurane-exposed controls. Flutamide changed expression patterns of NKCC1 (mean density ± SD: control, 1.49 ± 0.69; flutamide, 0.47 ± 0.11; P < 0.001) and KCC2 (median density [25th percentile, 75th percentile]: control, 0.23 [0.13, 0.49]; flutamide, 1.47 [1.18,1.62]; P < 0.001). Inhibiting NKCC1 with bumetanide was protective for spatial memory (probe trial F[1, 162] = 6.6; P = 0.011; mean goal time, 4.6 [7.4] s). Delaying isoflurane exposure until postnatal day 14 in males preserved spatial memory (probe trial F[1, 140] = 28; P < 0.001; mean goal time, 6.1 [7.0] s). CONCLUSIONS:Vulnerability to isoflurane neurotoxicity is abolished by blocking the androgen receptor, disrupting the function of NKCC1, or delaying the time of exposure to at least 2 weeks of age in male rats. These results support a dynamic role for androgens and chloride transporter proteins in perinatal anesthetic neurotoxicity. EDITOR’S PERSPECTIVE:
Project description:Spinal inhibition is required to generate coordinated outputs between antagonistic muscles during locomotion. It relies on low neuronal chloride concentration set by two cation-chloride cotransporters, NKCC1 and KCC2 which, respectively, pumps Cl(-) in or out of neurons. It is generally accepted that NKCC1 is gradually inactivated during development, while KCC2 is upregulated and activated, resulting in low intracellular [Cl(-)]. Newborn opossums are very immature but perform rhythmic and alternate movements of the forelimbs to crawl on the mother's belly and attach to a teat. Their hindlimbs are immobile. The alternation of the forelimbs suggests that mechanisms allowing spinal inhibition are present at birth. We studied the anatomical basis of inhibition in the spinal enlargements of postnatal opossums by immunolocalizing NKCC1 and KCC2. In some specimens, motoneurons and sensory afferents were labeled with TRDA prior to immunolabeling. At birth, both NKCC1 and KCC2 are detected in the presumptive gray and white matter of the ventral and the intermediolateral cord of both enlargements, but are sparse in the dorsal horn, where KCC2 is mostly seen on a small bundle of dendrites along primary afferents. KCC2 labeling is bright and has a mesh-like appearance in the gray matter and a radial appearance in the white matter, whereas NKCC1 is pale and diffuse. The subsequent expression of the cotransporters follows general ventrodorsal and mediolateral gradients, with the lumbar segments slightly lagging the cervical segments, until the mature pattern is observed around the 5th week. At all ages studied, KCC2 labeling is strong in the periphery of neurons. NKCC1 labeling decreases and becomes more uniformly distributed in the cells with age. Despite the significant anatomical and motor differences between the forelimbs and the hindlimbs of neonatal opossums, the maturation of KCC2 and NKCC1 is quite similar in both enlargements.
Project description:Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100 ?mol L-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and ?-actin. Neurones that did not respond during chloride imaging did not show KCC2 and ?-actin protein expression. The KCC2 antagonist (VU0240551,10 ?mol L-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 ?mol L-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 ?mol L-1 ) and protein kinase inhibitor (K252a) (100 nmol L-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.
Project description:The amyloid precursor protein (APP) modulates synaptic activity, resulting from the fine tuning of excitatory and inhibitory neurotransmission. GABAergic inhibitory neurotransmission is affected by modifications in intracellular chloride concentrations regulated by Na<sup>+</sup>-K<sup>+</sup>-2Cl<sup>-</sup> cotransporter 1 (NKCC1) and neuronal K<sup>+</sup>-Cl<sup>-</sup> cotransporter 2 (KCC2), allowing entrance and efflux of chloride, respectively. Modifications in NKCC1 and KCC2 expression during maturation of cortical cells induce a shift in GABAergic signaling. Here, we demonstrated that APP affects this GABA shift. Expression of APP in cortical cells decreased the expression of KCC2, without modifying NKCC1, eliciting a less inhibitory GABA response. Downregulation of KCC2 expression by APP was independent of the APP intracellular domain, but correlated with decreased expression of upstream stimulating factor 1 (USF1), a potent regulator of Slc12a5 gene expression (encoding KCC2). KCC2 was also downregulated in vivo following APP expression in neonatal mouse brain. These results argue for a key role of APP in the regulation of GABAergic neurotransmission.
Project description:<h4>Background</h4>Altered chloride homeostasis has been thought to be a risk factor for several brain disorders, while less attention has been paid to its role in liver disease. We aimed to analyze the involvement and possible mechanisms of altered chloride homeostasis of GABAergic neurons within the substantia nigra pars reticulata (SNr) in the motor deficit observed in a model of encephalopathy caused by acute liver failure, by using glutamic acid decarboxylase 67 - green fluorescent protein knock-in transgenic mice.<h4>Methods</h4>Alterations in intracellular chloride concentration in GABAergic neurons within the SNr and changes in the expression of two dominant chloride homeostasis-regulating genes, KCC2 and NKCC1, were evaluated in mice with hypolocomotion due to hepatic encephalopathy (HE). The effects of pharmacological blockade and/or activation of KCC2 and NKCC1 functions with their specific inhibitors and/or activators on the motor activity were assessed.<h4>Results</h4>In our mouse model of acute liver injury, chloride imaging indicated an increase in local intracellular chloride concentration in SNr GABAergic neurons. In addition, the mRNA and protein levels of KCC2 were reduced, particularly on neuronal cell membranes; in contrast, NKCC1 expression remained unaffected. Furthermore, blockage of KCC2 reduced motor activity in the normal mice and led to a further deteriorated hypolocomotion in HE mice. Blockade of NKCC1 was not able to normalize motor activity in mice with liver failure.<h4>Conclusion</h4>Our data suggest that altered chloride homeostasis is likely involved in the pathophysiology of hypolocomotion following HE. Drugs aimed at restoring normal chloride homeostasis would be a potential treatment for hepatic failure.
Project description:Disturbances in markers of cortical ?-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of ?-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.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.Real-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex.Human brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania.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.Relative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts.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.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 ?-aminobutyric acid neurotransmission in the prefrontal cortex.
Project description:<h4>Background</h4>Cannabis usage is increasing with its widespread legalization. Cannabis use by mothers during lactation transfers active cannabinoids to the developing offspring during this critical period and alters postnatal neurodevelopment. A key neurodevelopmental landmark is the excitatory to inhibitory gamma-aminobutyric acid (GABA) switch caused by reciprocal changes in expression ratios of the K<sup>+</sup>/Cl<sup>-</sup> transporters potassium-chloride cotransporter 2 (KCC2) and sodium-potassium-chloride transporter (NKCC1).<h4>Methods</h4>Rat dams were treated with Δ<sup>9</sup>-tetrahydrocannabinol or a synthetic cannabinoid during the first 10 days of postnatal development, and experiments were then conducted in the offspring exposed to these drugs via lactation. The network influence of GABA transmission was analyzed using cell-attached recordings. KCC2 and NKCC1 levels were determined using Western blot and quantitative polymerase chain reaction analyses. Ultrasonic vocalization and homing behavioral experiments were carried out at relevant time points.<h4>Results</h4>Treating rat dams with cannabinoids during early lactation retards transcriptional upregulation and expression of KCC2, thereby delaying the GABA switch in pups of both sexes. This perturbed trajectory was corrected by the NKCC1 antagonist bumetanide and accompanied by alterations in ultrasonic vocalization without changes in homing behavior. Neurobehavioral deficits were prevented by CB<sub>1</sub> receptor antagonism during maternal exposure, showing that the CB<sub>1</sub> receptor underlies the cannabinoid-induced alterations.<h4>Conclusions</h4>These results reveal how perinatal cannabinoid exposure retards an early milestone of development, delaying the trajectory of GABA's polarity transition and altering early-life communication.