Mechanically and physiologically optimizing prosthetic elevated vacuum systems in people with transtibial amputation: a pilot study.

Introduction: The most suitable elevated vacuum (EV) pressure may differ for each individual prosthesis user depending on suspension needs, socket fit, prosthetic components, and health. Mechanical and physiological effects of EV were evaluated in an effort to determine the optimal vacuum pressure for three individuals. Methods: Instrumented EV sockets were created based on the participants' regular EV sockets. Inductive distance sensors were embedded into the wall of the socket at select locations to measure limb movement relative to the socket. Each participant conducted an activity protocol while limb movement, limb fluid volume, and user-reported comfort were measured at various socket vacuum pressure settings. Results: Increased socket vacuum pressure resulted in reduced limb-socket displacement for each participant; however, 81-93% of limb movement was eliminated by a vacuum pressure setting of 12 (approximately -9 inHg). Relative limb-socket displacement by sensor location varied for each participant, suggesting distinct differences related to socket fit or residual limb tissue content. The rate of limb fluid volume change and the change in socket comfort did not consistently differ with socket vacuum pressure, suggesting a more complex relationship unique to each individual. Conclusions: Practitioners may use individual responses to optimize socket vacuum pressure settings, balancing mechanical and physiological effects of EV for improved clinical outcomes.

Clearance of activity-evoked K+ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform-selective AQP4 inhibitor.

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4-deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4-/- mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4-expressing Xenopus laevis oocytes monitored by a high-resolution volume recording system, we here demonstrate that the compound TGN-020 is such a selective AQP4 inhibitor. TGN-020 targets the tested species of AQP4 with an IC50 of ~3.5 µM, but displays no inhibitory effect on the other AQPs (AQP1-AQP9). With this tool, we employed rat hippocampal slices and ion-sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN-020-mediated inhibition of AQP4 did not prevent stimulus-induced extracellular space shrinkage, nor did it slow clearance of the activity-evoked K+ transient. These data, obtained with a verified isoform-selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K+ clearance or the associated extracellular space shrinkage.

Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells.

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

Incorporating a Ferrous Polymer Target into Elastomeric Liners for Socket Fit Sensing in Prosthesis Users.

Liner-to-socket distance measurement using inductive sensing may be an effective means to continuously monitor socket fit in people using trans-tibial prostheses. A practical limitation, however, is a means to incorporate a thin uniform-thickness layer of conductive or magnetically permeable target material into the wide range of prosthetic liner products that people with limb amputation commonly use. In this paper, a method is presented whereby a 0.50-mm thickness ferrous polymer made from a SEEPS polymer and iron powder that is formed adjacent to a 0.25-mm thick non-ferrous layer of SEEPS polymer is assembled between two sheets of elastic fabric material. Bench testing showed that the fabrication procedure achieved a root-mean-square error in the thickness of this construct of 58 µm, helping to create a consistent calibration result over the entire surface. The original fabric backing of an off-the-shelf prosthetic liner was removed and replaced with the developed construct. When worn in the shoe of an able-bodied participant for 7.5 h per day for 28 days, the sensor well maintained the shape of its calibration curve at the start of wear, but a distance offset (shifting of the y-intercept) was introduced that increased during the initial approximately 12 days of wear. When the distance offset was corrected, for the primary distance range of clinical interest for this application (0.00-5.00 mm), the sensor maintained its calibration within 4.4%. Before being used in clinical application for liner-to-socket distance monitoring, new ferrous liners may need to be pre-worn so as to achieve a consistent distance reference.

Developmental maturation of activity-induced K+ and pH transients and the associated extracellular space dynamics in the rat hippocampus.

KEY POINTS: Neuronal activity induces fluctuation in extracellular space volume, [K+ ]o and pHo , the management of which influences neuronal function The neighbour astrocytes buffer the K+ and pH and swell during the process, causing shrinkage of the extracellular space In the present study, we report the developmental rise of the homeostatic control of the extracellular space dynamics, for which regulation becomes tighter with maturation and thus is proposed to ensure efficient synaptic transmission in the mature animals The extracellular space dynamics of volume, [K+ ]o and pHo evolve independently with developmental maturation and, although all of them are inextricably tied to neuronal activity, they do not couple directly. ABSTRACT: Neuronal activity in the mammalian central nervous system associates with transient extracellular space (ECS) dynamics involving elevated K+ and pH and shrinkage of the ECS. These ECS properties affect membrane potentials, neurotransmitter concentrations and protein function and are thus anticipated to be under tight regulatory control. It remains unresolved to what extent these ECS dynamics are developmentally regulated as synaptic precision arises and whether they are directly or indirectly coupled. To resolve the development of homeostatic control of [K+ ]o , pH, and ECS and their interaction, we utilized ion-sensitive microelectrodes in electrically stimulated rat hippocampal slices from rats of different developmental stages (postnatal days 3-28). With the employed stimulation paradigm, the stimulus-evoked peak [K+ ]o and pHo transients were stable across age groups, until normalized to neuronal activity (field potential amplitude), in which case the K+ and pH shifted significantly more in the younger animals. By contrast, ECS dynamics increased with age until normalized to the field potential, and thus correlated with neuronal activity. With age, the animals not only managed the peak [K+ ]o better, but also displayed swifter post-stimulus removal of [K+ ]o , in correlation with the increased expression of the α1-3 isoforms of the Na+ /K+ -ATPase, and a swifter return of ECS volume. The different ECS dynamics approached a near-identical temporal pattern in the more mature animals. In conclusion, although these phenomena are inextricably tied to neuronal activity, our data suggest that they do not couple directly.

Thin Magnetically Permeable Targets for Inductive Sensing: Application to Limb Prosthetics.

The purpose of this research was to create a thin ferrous polymer composite to be used as a target for inductive sensing in limb prosthetics. Inductive sensors are used to monitor limb-to-socket distance in prosthetic sockets, which reflects socket fit. A styrene-ethylene-ethylene/propylene-styrene (SEEPS) polymer was mixed with iron powder at three concentrations (75, 77, 85 wt%), and thin disk-shaped samples were fabricated (0.50, 0,75, 1.00 mm thickness). For 85 wt% samples of 0.50 mm thickness, which proved the best combination of high signal strength and low target volume, inductive sensor sensitivity ranged from 3.2E5 counts/mm at 0.00-1.00 mm distances to 7.2E4 counts/mm at 4.00-5.00 mm distances. The application of compressive stress (up to 425 kPa) introduced an absolute measurement error of less than 3.3 µm. Tensile elasticity was 282 kPa, which is comparable to that of commercial elastomeric liners. Durability testing in the shoe of an able-bodied participant demonstrated a change in calibration coefficient of less than 3.8% over two weeks of wear. The ferrous polymer composite may facilitate the development of automatically adjusting sockets that use limb-to-socket distance measurement for feedback control.

Prosthetists' perceptions of information obtained from a lower limb prosthesis monitoring system: a pilot study.

INTRODUCTION: Prosthetists have limited knowledge of their patients' use of a prosthesis outside of the clinical environment. Prosthesis-mounted monitors can be used to directly measure patients' prosthesis use and activity. Prosthetists' opinions regarding potential clinical applications for sensor-based information may inform further development of this technology. A pilot study was conducted to assess prosthetists' perceptions of prosthesis use and activity information obtained by a monitoring system. MATERIALS AND METHODS: Three local prosthetists were recruited to participate in the study. One patient with transtibial amputation from each prosthetist volunteered to wear limb presence and activity monitors for two weeks. Collected data were used to determine prosthesis use and activity. Each prosthetist completed a survey, examined clinical reports of their patient's prosthesis use and activity, and participated in a semi-structured interview. Survey results and interview transcripts were analyzed to identify and compare prosthetists' perceptions. RESULTS: Prosthesis use and activity varied among patients. Prosthetists over- and under-estimated patient activity, relative to measurements recorded by the monitors. All three prosthetists selected multiple clinical applications for the prosthesis use and activity information in the survey, and several additional applications were suggested during the interviews. When presented with multiple report formats, prosthetists found features of each to be clinically useful. CONCLUSIONS: Prosthesis-mounted monitors may provide prosthetists with a better understanding of their patients' prosthesis use and activity. Information provided by the monitoring system may inform clinical decisions and promote evidence-based practices.

Mesenchymal Hox6 function is required for mouse pancreatic endocrine cell differentiation.

Despite significant advances in our understanding of pancreatic endocrine cell development, the function of the pancreatic mesodermal niche in this process is poorly understood. Here we report a novel role for mouse Hox6 genes in pancreatic organogenesis. Hox6 genes are expressed exclusively in the mesoderm of the developing pancreas. Genetic loss of all three Hox6 paralogs (Hoxa6, Hoxb6 and Hoxc6) leads to a dramatic loss of endoderm-derived endocrine cells, including insulin-secreting ß-cells, and to mild delays and disruptions in pancreatic branching and exocrine differentiation. Ngn3-expressing pan-endocrine progenitor cells are specified normally in Hox6 mutant pancreata, but fail to mature into hormone-producing cells. Reduced expression of Wnt5a is observed in mutant pancreatic mesenchyme, leading to subsequent loss of expression of the crucial Wnt inhibitors Sfrp3 and Dkk1 in endocrine progenitor cells. These results reveal a key role for Hox6 genes in establishing Wnt mesenchymal-epithelial crosstalk in pancreatic development.

ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function.

DNA damage activates the ATM and ATR kinases that coordinate checkpoint and DNA repair pathways. An essential step in homology-directed repair (HDR) of DNA breaks is the formation of RAD51 nucleofilaments mediated by PALB2-BRCA2; however, roles of ATM and ATR in this critical step of HDR are poorly understood. Here, we show that PALB2 is markedly phosphorylated in response to genotoxic stresses such as ionizing radiation and hydroxyurea. This response is mediated by the ATM and ATR kinases through three N-terminal S/Q-sites in PALB2, the consensus target sites for ATM and ATR Importantly, a phospho-deficient PALB2 mutant is unable to support proper RAD51 foci formation, a key PALB2 regulated repair event, whereas a phospho-mimicking PALB2 version supports RAD51 foci formation. Moreover, phospho-deficient PALB2 is less potent in HDR than wild-type PALB2. Further, this mutation reveals a separation in PALB2 function, as the PALB2-dependent checkpoint response is normal in cells expressing the phospho-deficient PALB2 mutant. Collectively, our findings highlight a critical importance of PALB2 phosphorylation as a novel regulatory step in genome maintenance after genotoxic stress.

A Method for C2 Acylation of 1,3-Indandiones.

The 1,3-indandione scaffold is an important structural motif used in the preparation of a large number of industrial chemical and pharmaceutical compounds. However, few approaches allow for the direct C2 acylation on these building blocks. A method was developed using DMAP and EDCI, which is mild in reactivity, covers a diverse range of carboxylic acid acylating agents, is compatible with electron releasing and withdrawing substituents on the 1,3-indandione partner, and performs well in a polar aprotic solvent (for solubility reasons) This method cleanly afforded twenty five different products in yields of 32-96%.

An Inductive Sensing System to Measure In-Socket Residual Limb Displacements for People Using Lower-Limb Prostheses.

The objective of this research was to assess the performance of an embedded sensing system designed to measure the distance between a prosthetic socket wall and residual limb. Low-profile inductive sensors were laminated into prosthetic sockets and flexible ferromagnetic targets were created from elastomeric liners with embedded iron particles for four participants with transtibial amputation. Using insights from sensor performance testing, a novel calibration procedure was developed to quickly and accurately calibrate the multiple embedded sensors. The sensing system was evaluated through laboratory tests in which participants wore sock combinations with three distinct thicknesses and conducted a series of activities including standing, walking, and sitting. When a thicker sock was worn, the limb typically moved further away from the socket and peak-to-peak displacements decreased. However, sensors did not measure equivalent distances or displacements for a given sock combination, which provided information regarding the fit of the socket and how a sock change intervention influenced socket fit. Monitoring of limb⁻socket displacements may serve as a valuable tool for researchers and clinicians to quantitatively assess socket fit.

Activity-dependent astrocyte swelling is mediated by pH-regulating mechanisms.

During neuronal activity in the mammalian brain, the K+ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na+ /K+ /2Cl- cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity-dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion-sensitive microelectrodes to measure changes in ECS, [K+ ]o and [H+ ]o /pHo during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K+ and glutamate released into the extracellular space (the K+ /Cl- cotransporter, KCC, glutamate transporters and G protein-coupled receptors) did not modulate the extracellular space dynamics. The HCO3--transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na+ / HCO3- cotransporter 1 (NBCe1), is activated by the K+ -mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by ∼25% without affecting the K+ transients, pointing to NBCe1 as a key contributor to the stimulus-induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like-wise, reduced the ECS shrinkage by ∼25% without compromising the K+ transients. Isosmotic reduction of extracellular Cl- revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus-evoked astrocytic cell swelling does not appear to occur as a direct effect of the K+ clearance, as earlier proposed, but partly via the pH-regulating transport mechanisms activated by the K+ -induced astrocytic depolarization and the activity-dependent metabolism.

The α2ß2 isoform combination dominates the astrocytic Na+ /K+ -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.

Synaptic activity results in transient elevations in extracellular K+ , clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+ /K+ -ATPase. The Na+ /K+ -ATPase consists of an α and a ß subunit, each with several isoforms present in the central nervous system, of which the α2ß2 and α2ß1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2ß2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and ß isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+ /K+ -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both ß1 and ß2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+ /K+ -ATPase is dominated by α2, paired with ß1 or ß2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.

Glutamate transporter activity promotes enhanced Na+ /K+ -ATPase-mediated extracellular K+ management during neuronal activity.

KEY POINTS: Management of glutamate and K+ in brain extracellular space is of critical importance to neuronal function. The astrocytic α2ß2 Na+ /K+ -ATPase isoform combination is activated by the K+ transients occurring during neuronal activity. In the present study, we report that glutamate transporter-mediated astrocytic Na+ transients stimulate the Na+ /K+ -ATPase and thus the clearance of extracellular K+ . Specifically, the astrocytic α2ß1 Na+ /K+ -ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+ . Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+ /K+ -ATPase ß-subunits. ABSTRACT: Neuronal activity is associated with transient [K+ ]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+ /K+ -ATPase of which several subunit isoform combinations exist. The astrocytic Na+ /K+ -ATPase α2ß2 isoform constellation responds directly to increased [K+ ]o but, in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic ß2 has remained elusive as a result of inherent expression of ß1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+ /K+ -ATPase was not fully saturated at basal astrocytic [Na+ ]i , irrespective of isoform constellation, although the ß1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the ß2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2ß1 appeared to be specifically geared to respond to the [Na+ ]i transients associated with activity-induced glutamate transporter activity.

Spacecraft surface charging within geosynchronous orbit observed by the Van Allen Probes.

Using the Helium Oxygen Proton Electron (HOPE) and Electric Field and Waves (EFW) instruments from the Van Allen Probes, we explored the relationship between electron energy fluxes in the eV and keV ranges and spacecraft surface charging. We present statistical results on spacecraft charging within geosynchronous orbit by L and MLT. An algorithm to extract the H+ charging line in the HOPE instrument data was developed to better explore intense charging events. Also, this study explored how spacecraft potential relates to electron number density, electron pressure, electron temperature, thermal electron current, and low-energy ion density between 1 and 210 eV. It is demonstrated that it is imperative to use both EFW potential measurements and the HOPE instrument ion charging line for examining times of extreme spacecraft charging of the Van Allen Probes. The results of this study show that elevated electron energy fluxes and high-electron pressures are present during times of spacecraft charging but these same conditions may also occur during noncharging times. We also show noneclipse significant negative charging events on the Van Allen Probes.

Synthesis and Biological Evaluation of Lactimidomycin and Its Analogues.

The studies culminating in the total synthesis of the glutarimide-containing eukaryote translation elongation inhibitor lactimidomycin are described. The optimized synthetic route features a Zn(II)-mediated intramolecular Horner-Wadsworth-Emmons (HWE) reaction resulting in a highly stereoselective formation of the strained 12-membered macrolactone of lactimidomycin on a 423 mg scale. The presence of the E,Z-diene functionality was found to be key for effective macrocyclizations as a complete removal of these unsaturation units resulted in exclusive formation of the dimer rather than monocyclic enoate. The synthetic route features a late-stage installation of the glutarimide functionality via an asymmetric catalytic Mukaiyama aldol reaction, which allows for a quick generation of lactimidomycin homolog 55 containing two additional carbons in the glutarimide side chain. Similar to lactimidomycin, this analog was found to possess cytotoxicity against MDA-MB-231 breast cancer cells (GI50 =1-3 µM) using in vitro 2D and 3D assays. Although lactimidomycin was found to be the most potent compound in terms of anticancer activity, 55 as well as truncated analogues 50-52 lacking the glutarimide side-chain were found to be significantly less toxic against human mammary epithelial cells.

Regulation and Function of AQP4 in the Central Nervous System.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. Based on studies on AQP4(-/-) mice, AQP4 has been assigned physiological roles in stimulus-induced K(+) clearance, paravascular fluid flow, and brain edema formation. Conflicting data have been presented on the role of AQP4 in K(+) clearance and associated extracellular space shrinkage and on the stroke-induced alterations of AQP4 expression levels during edema formation, raising questions about the functional importance of AQP4 in these (patho)physiological aspects. Phosphorylation-dependent gating of AQP4 has been proposed as a regulatory mechanism for AQP4-mediated osmotic water transport. This paradigm was, however, recently challenged by experimental evidence and molecular dynamics simulations. Regulatory patterns and physiological roles for AQP4 thus remain to be fully explored.

AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at COOH-terminal serine residues.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser(321), and Ser(322). To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel.

Contributions of the Na⁺/K⁺-ATPase, NKCC1, and Kir4.1 to hippocampal K⁺ clearance and volume responses.

Network activity in the brain is associated with a transient increase in extracellular K(+) concentration. The excess K(+) is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na(+)/K(+)/2Cl(-) cotransporter 1 (NKCC1), and/or Na(+)/K(+)-ATPase activity. Their individual contribution to [K(+)]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na(+)/K(+)-ATPase and to resolve their involvement in clearance of extracellular K(+) transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K(+)]o increases above basal levels. Increased [K(+)]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K(+) clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K(+) removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K(+)]o increase. In contrast, inhibition of the different isoforms of Na(+)/K(+)-ATPase reduced post-stimulus clearance of K(+) transients. The astrocyte-characteristic α2ß2 subunit composition of Na(+)/K(+)-ATPase, when expressed in Xenopus oocytes, displayed a K(+) affinity and voltage-sensitivity that would render this subunit composition specifically geared for controlling [K(+)]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na(+)/K(+)-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K(+) recovery in native hippocampal tissue while Kir4.1 and Na(+)/K(+)-ATPase serve temporally distinct roles.