Cl Gradient Research Articles

The impact of hypertonic saline on damage control laparotomy after penetrating abdominal trauma.

The inability to achieve primary fascial closure (PFC) after emergency laparotomy increases the rates of adverse outcomes including fistula formation, incisional hernia, and intraabdominal infection. Hypertonic saline (HTS) infusion improves early PFC rates and decreases time to PFC in patients undergoing damage control laparotomy (DCL) after injury. We hypothesized that in patients undergoing DCL after penetrating abdominal injury, HTS infusion would decrease the time to fascial closure as well as the volume of crystalloid required for resuscitation without inducing clinically relevant acute kidney injury (AKI) or electrolyte derangements. We retrospectively analyzed all penetrating abdominal injury patients undergoing DCL within the University of Pennsylvania Health System (January 2015-December 2018). We compared patients who received 3% HTS at 30mL/h (HTS) to those receiving isotonic fluid (ISO) for resuscitation while the abdominal fascia remained open. Primary outcomes were the rate of early PFC (PFC within 72h) and time to PFC; secondary outcomes included acute kidney injury, sodium derangement, ventilator-free days, hospital length of stay (LOS), and ICU LOS. Intergroup comparisons occurred by ANOVA and Tukey's comparison, and student's t, and Fischer's exact tests, as appropriate. A Shapiro-Wilk test was performed to determine normality of distribution. Fifty-seven patients underwent DCL after penetrating abdominal injury (ISO n = 41, HTS n = 16). There were no significant intergroup differences in baseline characteristics or injury severity score. Mean time to fascial closure was significantly shorter in HTS (36.37h ± 14.21 vs 59.05h ± 50.75, p = 0.02), and the PFC rate was significantly higher in HTS (100% vs 73%, p = 0.01). Mean 24-h fluid and 48-h fluid totals were significantly less in HTS versus ISO (24h: 5.2L ± 1.7 vs 8.6L ± 2.2, p = 0.01; 48h: 1.3L ± 1.1 vs 2.6L ± 2.2, p = 0.008). During the first 72h, peak sodium (Na) concentration (146.2mEq/L ± 2.94 vs 142.8mEq/L ± 3.67, p = 0.0017) as well as change in Na from ICU admission (5.1mEq/L vs 2.3, p = 0.016) were significantly higher in HTS compared to ISO. Patients in the HTS group received significantly more blood in the trauma bay compared to ISO. There were no intergroup differences in intraoperative blood transfusion volume, AKI incidence, change in chloride concentration (△Cl) from ICU admit, Na to Cl gradient (Na:Cl), initial serum creatinine (Cr), peak post-operative Cr, change in creatinine concentration (△Cr) from ICU admission, creatinine clearance (CrCl), initial serum potassium (K), peak ICU K, change in K from ICU admission, initial pH, highest or lowest post-operative pH, mean hospital LOS, ICU LOS, and ventilator-free days. HTS infusion in patients undergoing DCL after penetrating abdominal injury decreases the time to fascial closure and led to 100% early PFC. HTS infusion also decreased resuscitative fluid volume without causing significant AKI or electrolyte derangement. HTS appears to offer a safe and effective fluid management approach in patients who sustain penetrating abdominal injury and DCL to support early PFC without inducing measurable harm. Level III.

Gradients of chemical abundances in the Milky Way from H ii regions: distances derived from Gaia EDR3 parallaxes and temperature inhomogeneities

ABSTRACT We present a reassessment of the radial abundance gradients of He, C, N, O, Ne, S, Cl, and Ar in the Milky Way using the deep optical spectra of 42 H ii regions presented in Arellano-Córdova et al. (2020, 2021) and Méndez-Delgado et al. (2020) exploring the impact of: (1) new distance determinations based on Gaia EDR3 parallaxes and (2) the use of Peimbert’s temperature fluctuations paradigm (t2 > 0) for deriving ionic abundances. We find that distances based on Gaia EDR3 data are more consistent with kinematic ones based on Galactic rotation curves calibrated with radio parallaxes, which give less dispersion and uncertainties than those calibrated with spectrophotometric stellar distances. The distances based on the Gaia parallaxes – DR2 or EDR3 – eliminate the internal flattening observed in previous determinations of the Galactic gradients at smaller distances than ∼7 kpc. Abundances and gradients determined assuming t2 > 0 – not only for O but also for the rest of elements – are not affected by the abundance discrepancy problem and give elemental abundances much consistent with the solar ones for most elements. We find that our radial abundance gradient of He is consistent with the most accurate estimates of the primordial He abundance. We do not find evidence of azimuthal variations in the chemical abundances of our sample. Moreover, the small dispersion in the O gradient – indicator of metallicity in photoionized regions – indicate that the gas of the H ii regions is well mixed in the sampled areas of the Galaxy.

Inflection Points on Groundwater Age and Geochemical Profiles Along Wellbores Light up Hierarchically Nested Flow Systems

AbstractHierarchically nested groundwater flow systems have been widely investigated by numerical modeling and laboratory experiments but seldom recognized from in situ observations and testing. Groundwater age and geochemical profiles were obtained by the authors using a packer system along two wellbores drilled in the Ordos Plateau, China. Groundwater age profiles were constructed from 3H, 85Kr, 14C, and 81Kr data, with the apparent age generally increasing with depth, while exhibiting several inflection points. These inflection points were compared with geochemical profiles and numerical modeling results to delineate groundwater flow systems. Inflection points with significant increases in the gradients of age and Cl− concentration versus depth may indicate interfaces among flow systems. A concurrent decrease‐increase turning of groundwater age and Cl− concentration is influenced by a stagnation point. The groundwater age profiles are quite different from previous studies, indicating that the method can greatly reduce the uncertainty in model construction.

Muscle Ionic Shifts During Exercise: Implications for Fatigue and Exercise Performance.

Exercise causes major shifts in multiple ions (e.g., K+ , Na+ , H+ , lactate- , Ca2+ , and Cl- ) during muscle activity that contributes to development of muscle fatigue. Sarcolemmal processes can be impaired by the trans-sarcolemmal rundown of ion gradients for K+ , Na+ , and Ca2+ during fatiguing exercise, while changes in gradients for Cl- and Cl- conductance may exert either protective or detrimental effects on fatigue. Myocellular H+ accumulation may also contribute to fatigue development by lowering glycolytic rate and has been shown to act synergistically with inorganic phosphate (Pi) to compromise cross-bridge function. In addition, sarcoplasmic reticulum Ca2+ release function is severely affected by fatiguing exercise. Skeletal muscle has a multitude of ion transport systems that counter exercise-related ionic shifts of which the Na+ /K+ -ATPase is of major importance. Metabolic perturbations occurring during exercise can exacerbate trans-sarcolemmal ionic shifts, in particular for K+ and Cl- , respectively via metabolic regulation of the ATP-sensitive K+ channel (KATP ) and the chloride channel isoform 1 (ClC-1). Ion transport systems are highly adaptable to exercise training resulting in an enhanced ability to counter ionic disturbances to delay fatigue and improve exercise performance. In this article, we discuss (i) the ionic shifts occurring during exercise, (ii) the role of ion transport systems in skeletal muscle for ionic regulation, (iii) how ionic disturbances affect sarcolemmal processes and muscle fatigue, (iv) how metabolic perturbations exacerbate ionic shifts during exercise, and (v) how pharmacological manipulation and exercise training regulate ion transport systems to influence exercise performance in humans. © 2021 American Physiological Society. Compr Physiol 11:1895-1959, 2021.

Intricacies of GABAA Receptor Function: The Critical Role of the β3 Subunit in Norm and Pathology

Neuronal intracellular chloride ([Cl−]i) is a key determinant in γ-aminobutyric acid type A (GABA)ergic signaling. γ-Aminobutyric acid type A receptors (GABAARs) mediate both inhibitory and excitatory neurotransmission, as the passive fluxes of Cl− and HCO3− via pores can be reversed by changes in the transmembrane concentration gradient of Cl−. The cation–chloride co-transporters (CCCs) are the primary systems for maintaining [Cl−]i homeostasis. However, despite extensive electrophysiological data obtained in vitro that are supported by a wide range of molecular biological studies on the expression patterns and properties of CCCs, the presence of ontogenetic changes in [Cl−]i—along with the consequent shift in GABA reversal potential—remain a subject of debate. Recent studies showed that the β3 subunit possesses properties of the P-type ATPase that participates in the ATP-consuming movement of Cl− via the receptor. Moreover, row studies have demonstrated that the β3 subunit is a key player in GABAAR performance and in the appearance of serious neurological disorders. In this review, we discuss the properties and driving forces of CCCs and Cl−, HCO3−ATPase in the maintenance of [Cl−]i homeostasis after changes in upcoming GABAAR function. Moreover, we discuss the contribution of the β3 subunit in the manifestation of epilepsy, autism, and other syndromes.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. III. A functional framework for α345 hexamer assembly

We identified a genetic variant, an 8-residue appendage, of the α345 hexamer of collagen IV present in patients with glomerular basement membrane diseases, Goodpasture’s disease and Alport syndrome, and determined the long-awaited crystal structure of the hexamer. We sought to elucidate how variants cause glomerular basement membrane disease by exploring the mechanism of the hexamer assembly. Chloride ions induced in vitro hexamer assembly in a composition-specific manner in the presence of equimolar concentrations of α3, α4, and α5 NC1 monomers. Chloride ions, together with sulfilimine crosslinks, stabilized the assembled hexamer. Furthermore, the chloride ion–dependent assembly revealed the conformational plasticity of the loop-crevice-loop bioactive sites, a critical property underlying bioactivity and pathogenesis. We explored the native mechanism by expressing recombinant α345 miniprotomers in the cell culture and characterizing the expressed proteins. Our findings revealed NC1-directed trimerization, forming protomers inside the cell; hexamerization, forming scaffolds outside the cell; and a Cl gradient–signaled hexamerization. This assembly detail, along with a crystal structure, provides a framework for understanding hexamer dysfunction. Restoration of the native conformation of bioactive sites and α345 hexamer replacement are prospective approaches to therapeutic intervention.

Drivers in AF colocate to sites of electrogram organization and rapidity: Potential synergy between spectral analysis and STAR mapping approaches in prioritizing drivers for ablation.

Stochastic trajectory analysis of ranked signals (STAR) mapping has recently been used to ablate persistent atrial fibrillation (AF) with high rates of AF termination and long-term freedom from AF in small, single-arm studies. We hypothesized that rapidity and organization markers would correlate with early sites of activation (ESA). Patients undergoing persistent AF ablation as part of the STAR mapping study were included. Five-minute unipolar basket recordings used to create STAR maps were used to determine the minimum-cycle length (Min-CL) and CL variability (CLV) at each electrode to identify the site of the fastest Min-CL and lowest CLV across the left atrium (LA). The location of ESA targeted with ablation was compared with these sites. Dominant frequency was assessed at ESA and compared with that of neighboring electrodes to assess for regional gradients. Thirty-two patients were included with 83 ESA ablated, with an ablation response at 73 sites (24 AF termination and 49 CL slowing ≥30 ms). Out of these, 54 (74.0%) and 56 (76.7%) colocated to sites of fastest Min-CL and lowest CLV, respectively. Regional CL and frequency gradients were demonstrable at majority of ESA. ESA colocating to sites of fastest Min-CL and lowest CLV were more likely to terminate AF with ablation (odds ratio, 34 and 29, respectively, P = .02). These showed a moderate sensitivity (74.0% Min-CL and 75.3% CLV) and specificity (66.7% Min-CL and 76.9% CLV) in predicting ESA with an ablation response. ESA correlate with rapidity and organization markers. Further work is needed to clarify any role for spectral analysis in prioritizing driver ablation.

Evaluation of a genetically encoded Cl− and pH sensor (ClopHensor) for monitoring intragranular Cl− in renal endocrine juxtaglomerular (JG) cells.

In most cells, intracellular cytoplasmic Cl− is maintained between 10 and 30 mM by a combination of entry and exit transport pathways. However, in endocrine cells intra‐granule Clin secretory granules is higher than cytoplasmic Cl−. This, is in part due to an intragranular positive H+ gradient driven by V‐ATPases, which generates low intra‐granular pH (4–5), and provides an electrochemical gradient for Cl− entry via CLC‐6/7. The role of V‐ATPases in exocytosis is well known, but less is known about how changes in Cl− contribute. There are very few methods available to measure intra‐granule Cl− concentration and monitor how it changes. ClopHensor is a genetically encoded protein sensor based on a Cl ‐sensitive GFP mutant (E2‐GFP) and DsRed ( Nature Methods, v7, pages 516–518, 2010). When excited by a 457 nm laser, E2‐GFP emission is sensitive to Cl− whereas at 488 nm it is sensitive to pH, whereas DsRed (insensitive to Cl− or pH) serves to control for volume changes. Renal endocrine juxtaglomerular (JG) cells, store renin, the rate‐limiting step enzyme in the production of angiotensin II, which play an essential role in blood pressure control. Renin is stored in large secretory granules and its exocytosis is stimulated primarily by increases in cAMP. We hypothesized that targeting ClopHensor to secretory granules would allow measurement of intra‐granular Cl− in JG cells and changes in its concentration during stimulation. We first tested the ability of ClopHensor to measure intracellular Cl− in M1 cells, clamped at pH 5.5 (reported for secretory granules). M1 cells transduced with adenovirus coding for ClopHensor were permeabilized to H+ with nygericin and bathed at pH 5.5, and permeabilized to Cl−. Cells were imaged by confocal microscopy under 457, 491 and 568 nm laser excitation while simultaneously acquiring emissions at 525 (E2‐GFP) and 590nm (Dsred). Under these conditions increasing Cl− from 0, to 20, 80 and 120 mM caused a logarithmic reduction in the 457/568 emission ratio (Cl‐sensitive) with a half‐max inhibition at 10.7±1.4 mM (n =6). To monitor intra‐granule Cl− we generated adenoviruses expressing NeuroPeptide Y targeting motif (NPY)‐ClopHensor. When primary mouse JG cells were transduced with NPY‐ClopHensor fluorescence was restricted to large granules. We could not permeabilize granules to Cl‐ in live JG cells Thus we measured baseline intra‐granule Cl− immediately after a daily calibration of ClopHensor in M1 cells. These results yielded an intra‐granule Cl− concentration of 77±15 mM (n=8). After measuring baseline Cl− in granules JG cells were treated with forskolin/IBMX to increase cAMP, and then 0 mM extracellular Cl− and bafilomycin. cAMP decreased intra‐granule Cl−, evidenced by a 22±5% increase (p<0.05, n= 11) in 457/568 Cl‐sensitive ratio, which was further increased by inhibiting V‐ATPse with bafilomycin by 40±6% (p<0.05, n=13). Our data show for the first time, measurements of intra‐granule Cl− in renal JG cells and suggest that NPY‐ClopHensor can be used to monitor the effect of agonists, or V‐ATPase regulators on intra‐granule Cl−. The role of intra‐granule Cl− in renin exocytosis and renin‐angiotensin system physiology remains to be studied.Support or Funding InformationHFH Research fund: Pablo A Ortiz

Effect of Salinity on Nitrogen Removal Performance of a Pilot-scale Anaerobic Ammonia Oxidation Process and Its Recovery Kinetics

The denitrification characteristics of anaerobic ammonia oxidizing bacteria (AnAOB) treating high salinity wastewater were investigated in an pilot-scale anaerobic sequencing batch reactor (ASBR, 530 L) by gradually increasing the Cl- concentration. The results showed that AnAOB can adapt to the high salinity (Cl- concentration of 10000 mg·L-1) environment for high-efficiency denitrification by means of gradual salinity acclimation and total nitrogen (TN) removal rate of up to 92.3%. In particular, the denitrification performance was influenced by two gradients of Cl- concentrations, namely 6000 mg·L-1 and 10000 mg·L-1, but it could be gradually recovered as the acclimatization process continued. The modified Boltzmann model accurately fit the activity recovery process of AnAOB after being inhibited by the different salinities, and the correlation coefficient R2 was above 0.96. The fitted recovered median values tc for Cl- concentrations of 6000 mg·L-1 and 10000 mg·L-1 were 28.765 d and 44.495 d. NRRmax for these concentrations was 0.145 kg·(m3·d)-1 and 0.212 kg·(m3·d)-1, and NRRmin was 0.021 kg·(m3·d)-1 and 0.085 kg·(m3·d)-1, respectively. After salinity acclimation, the dominant bacteria of AnAOB were Candidatus Brocadia and Candidatus Jettenia (the abundances were 14.76% and 2.7%, respectively), the granulation degree and sludge density increased to varying degrees, and the sludge color was reddish brown.

Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor.

Most kidney stones (KS) are composed of calcium oxalate, and small increases in urine oxalate affect the stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 (PAT1) plays a crucial role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and related KS, reflecting the importance of understanding regulation of intestinal oxalate transport. We previously showed that ATP and UTP inhibit oxalate transport by human intestinal Caco2-BBE cells (C2). Since ATP is rapidly degraded to adenosine (ADO), we examined whether intestinal oxalate transport is regulated by ADO. We measured [14C]oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, ≥49% of which is PAT1-mediated in C2 cells. We found that ADO significantly inhibited oxalate transport by C2 cells, an effect completely blocked by the nonselective ADO receptor antagonist 8- p-sulfophenyltheophylline. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A2B adenosine receptor (A2B AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A2B AR, phospholipase C, and PKC. ADO inhibits oxalate transport by reducing PAT1 surface expression as shown by biotinylation studies. We conclude that ADO inhibits oxalate transport by lowering PAT1 surface expression in C2 cells through signaling pathways including the A2B AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A2B AR in inflammatory bowel disease (IBD), our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl- efflux and the subsequent paracellular Na+ transport. In this model, the Na+-K+ pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl- transport via basolateral Na+-K+-2Cl- cotransport is generated by the Na+-K+ pump. In addition, the continuous electrochemical gradient for Cl- flow during acinar cell stimulation is maintained by the basolateral K+ efflux. However, using a combination of single-cell electrophysiology and Ca2+-imaging, we demonstrate that photolysis of Ca2+ close to the apical membrane of parotid acinar cells triggered significant K+ current, indicating that a substantial amount of K+ is secreted into the lumen during stimulation. Nevertheless, the K+ content of the primary saliva is relatively low, suggesting that K+ might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na+-K+ pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K+ from and secretes Na+ to the lumen, which can partially supplement the paracellular Na+ pathway.

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl− from acidic organelles in amacrine cells

γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligand-gated ion channels support the influx of Cl- However, the electrochemical gradient for Cl- across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl- levels support inhibition, whereas higher levels of cytosolic Cl- can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl- from acidic organelles and transiently elevates cytosolic Cl-, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl- We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage-clamp recordings of currents through GABA-gated Cl- channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl- To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl- from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl- from acidic organelles.NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl- from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl- levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission.

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl− co-transporter KCC2 in hippocampal neurons

Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ∼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.

Negative News: Cl- and HCO3- in the Vascular Wall.

Cl(-) and HCO3 (-) are the most prevalent membrane-permeable anions in the intra- and extracellular spaces of the vascular wall. Outwardly directed electrochemical gradients for Cl(-) and HCO3 (-) permit anion channel opening to depolarize vascular smooth muscle and endothelial cells. Transporters and channels for Cl(-) and HCO3 (-) also modify vascular contractility and structure independently of membrane potential. Transport of HCO3 (-) regulates intracellular pH and thereby modifies the activity of enzymes, ion channels, and receptors. There is also evidence that Cl(-) and HCO3 (-) transport proteins affect gene expression and protein trafficking. Considering the extensive implications of Cl(-) and HCO3 (-) in the vascular wall, it is critical to understand how these ions are transported under physiological conditions and how disturbances in their transport can contribute to disease development. Recently, sensing mechanisms for Cl(-) and HCO3 (-) have been identified in the vascular wall where they modify ion transport and vasomotor function, for instance, during metabolic disturbances. This review discusses current evidence that transport (e.g., via NKCC1, NBCn1, Ca(2+)-activated Cl(-) channels, volume-regulated anion channels, and CFTR) and sensing (e.g., via WNK and RPTPγ) of Cl(-) and HCO3 (-) influence cardiovascular health and disease.

Differences in chloride gradients allow for three distinct types of synaptic modulation by endocannabinoids.

Endocannabinoids can elicit persistent depression of excitatory and inhibitory synapses, reducing or enhancing (disinhibiting) neural circuit output, respectively. In this study, we examined whether differences in Cl(-) gradients can regulate which synapses undergo endocannabinoid-mediated synaptic depression vs. disinhibition using the well-characterized central nervous system (CNS) of the medicinal leech, Hirudo verbana Exogenous application of endocannabinoids or capsaicin elicits potentiation of pressure (P) cell synapses and depression of both polymodal (Npoly) and mechanical (Nmech) nociceptive synapses. In P synapses, blocking Cl(-) export prevented endocannabinoid-mediated potentiation, consistent with a disinhibition process that has been indicated by previous experiments. In Nmech neurons, which are depolarized by GABA due to an elevated Cl(-) equilibrium potentials (ECl), endocannabinoid-mediated depression was prevented by blocking Cl(-) import, indicating that this decrease in synaptic signaling was due to depression of excitatory GABAergic input (disexcitation). Npoly neurons are also depolarized by GABA, but endocannabinoids elicit depression in these synapses directly and were only weakly affected by disruption of Cl(-) import. Consequently, the primary role of elevated ECl may be to protect Npoly synapses from disinhibition. All forms of endocannabinoid-mediated plasticity required activation of transient potential receptor vanilloid (TRPV) channels. Endocannabinoid/TRPV-dependent synaptic plasticity could also be elicited by distinct patterns of afferent stimulation with low-frequency stimulation (LFS) eliciting endocannabinoid-mediated depression of Npoly synapses and high-frequency stimulus (HFS) eliciting endocannabinoid-mediated potentiation of P synapses and depression of Nmech synapses. These findings demonstrate a critical role of differences in Cl(-) gradients between neurons in determining the sign, potentiation vs. depression, of synaptic modulation under normal physiological conditions.

A glial K(+) /Cl(-) cotransporter modifies temperature-evoked dynamics in Caenorhabditis elegans sensory neurons.

K(+) /Cl(-) cotransporters (KCCs) are known to be crucial in the control of neuronal electrochemical Cl(-) gradient. However, the role of these proteins in glial cells remains largely unexplored despite a number of studies showing expression of KCC proteins in glial cells of many species. Here, we show that the Caenorhabditis elegans K(+) /Cl(-) cotransporter KCC-3 is expressed in glial-like cells and regulates the thermosensory behavior through modifying temperature-evoked activity of a thermosensory neuron. Mutations in the kcc-3 gene were isolated from a genetic screen for mutants defective in thermotaxis. KCC-3 is expressed and functions in the amphid sheath glia that ensheathes the AFD neuron, a major thermosensory neuron known to be required for thermotaxis. A genetic analysis indicated that the regulation of the thermosensory behavior by KCC-3 is mediated through AFD, and we further show that KCC-3 in the amphid sheath glia regulates the dynamics of the AFD activity. Our results show a novel mechanism by which the glial KCC-3 protein non-cell autonomously modifies the stimulus-evoked activity of a sensory neuron and highlights the functional importance of glial KCC proteins in modulating the dynamics of a neural circuitry to control an animal behavior.

Development and regulation of chloride homeostasis in the central nervous system.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl− gradients, which are generated by cation-Cl− co-transporters. An accumulation of Cl− by the Na+-K+-2Cl− co-transporter (NKCC1) increases the intracellular Cl− concentration ([Cl−]i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl−-extruder KCC2, which is a neuron-specific K+-Cl− co-transporter, with or without downregulation of NKCC1, results in low [Cl−]i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl− homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl−]i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl− homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1–4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na+ and K+ ions, topographical interaction with the Na+-K+ ATPase and its modulators such as creatine kinase (CK) should modulate functions of Cl− transporters. Therefore, regional developmental regulation of these regulators and modulators of Cl− transporters may also play a pivotal role in the development of Cl− homeostasis.

Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures

Neto2 is a transmembrane protein that interacts with the neuron-specific K+-Cl− cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl− gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2−/− neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2−/− neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

Neuroimmune Regulation of GABAergic Neurons Within the Ventral Tegmental Area During Withdrawal from Chronic Morphine.

Opioid dependence is accompanied by neuroplastic changes in reward circuitry leading to a negative affective state contributing to addictive behaviors and risk of relapse. The current study presents a neuroimmune mechanism through which chronic opioids disrupt the ventral tegmental area (VTA) dopaminergic circuitry that contributes to impaired reward behavior. Opioid dependence was induced in rodents by treatment with escalating doses of morphine. Microglial activation was observed in the VTA following spontaneous withdrawal from chronic morphine treatment. Opioid-induced microglial activation resulted in an increase in brain-derived neurotrophic factor (BDNF) expression and a reduction in the expression and function of the K+Cl− co-transporter KCC2 within VTA GABAergic neurons. Inhibition of microglial activation or interfering with BDNF signaling prevented the loss of Cl− extrusion capacity and restored the rewarding effects of cocaine in opioid-dependent animals. Consistent with a microglial-derived BDNF-induced disruption of reward, intra-VTA injection of BDNF or a KCC2 inhibitor resulted in a loss of cocaine-induced place preference in opioid-naïve animals. The loss of the extracellular Cl− gradient undermines GABAA-mediated inhibition, and represents a mechanism by which chronic opioid treatments can result in blunted reward circuitry. This study directly implicates microglial-derived BDNF as a negative regulator of reward in opioid-dependent states, identifying new therapeutic targets for opiate addictive behaviors.

The radial abundance gradient of chlorine in the Milky Way

We determine the radial abundance gradient of Cl in the Milky Way from HII regions spectra. For the first time, the Cl/H ratios are computed by simply adding ionic abundances and not using an ionization correction factor (ICF). We use a collection of published very deep spectra of Galactic HII regions. We have re-calculated the physical conditions, ionic and total abundances of Cl and O using the same methodology and updated atomic data for all the objects. We find that the slopes of the radial gradients of Cl and O are identical within the uncertainties: -0.043 dex/kpc. This is consistent with a lockstep evolution of both elements. We obtain that the mean value of the Cl/O ratio across the Galactic disc is log(Cl/O) = -3.42 +/- 0.06. We compare our Cl/H ratios with those determined from Cl++ abundances and using some available ICF schemes of the literature. We find that our total Cl abundances are always lower than the values determined using ICFs, indicating that those correction schemes systematically overestimate the contribution of Cl+ and Cl+++ species to the total Cl abundance. Finally, we propose an empirical ICF(Cl++) to estimate the Cl/H ratio in HII regions.