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 α2β2 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. GLIA 2014;62:608–622     

Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning

Initial clearance of extracellular K⁺ ([K⁺]_o) following neuronal excitation occurs by astrocytic uptake, because elevated [K⁺]_o activates astrocytic but not neuronal Na⁺,K⁺-ATPases. Subsequently, astrocytic K⁺ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K⁺]_o increase and allowing neuronal Na⁺,K⁺-ATPase-mediated re-uptake. Na⁺,K⁺-ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K⁺]_o increases to open L-channels for Ca²⁺, activating [K⁺]_o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K⁺]_o is decreased to levels mainly affecting astrocytic membrane potential or Na⁺,K⁺-ATPase or by abnormalities in its α2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K⁺]_o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocytic-oligodendrocytic gap junctions also promotes learning.     

Characteristics of activity-dependent potassium accumulation in mammalian peripheral nerve in vitro

Ion-sensitive microelectrodes were used to study the behavior of extracellular ions in rat sciatic nerve during and following activity. Nerve stimulation produced increases in [K⁺]_o that were dependent upon the frequency and duration of stimulation; no change in extracellular pH occurred with stimulation. Increases in [K⁺]_o dependent on axonal discharge since they were blocked by inhibiting sodium channels with tetrodotoxin. At 22°C, stimulation could induce increases in [K⁺]_o of several mM; at 36°C, stimulation rarely produced increases in [K⁺]_o greater than 1mM. Stimulated increases in [K⁺]_o dissipated very slowly (i.e. t_1/2 = 50–100s) and the rate of dissipation was not significantly affected by anoxia, changes in temperature, changes in extracellular pH, or the application of a blocker of Na⁺, K⁺-ATPase (ouabain) or a K⁺ channel blocker (Ba²⁺). In comparison to the central nervous system, neural activity in rat sciatic nerve produced smaller increases in [K⁺]_o and these increases dissipated much more slowly. The primary mechanism of K⁺ dissipation appeared to be diffusion, probably facilitated by the larger extracellular space in peripheral nerve compared to the central nervous system, but impeded by diffusion barriers imposed by the blood-nerve barrier.     

Background sodium current underlying respiratory rhythm regularity

Rhythm-generating neural circuits underlying diverse behaviors such as locomotion, sleep states, digestion and respiration play critical roles in our lives. Irregularities in these rhythmic behaviors characterize disease states – thus, it is essential that we identify the ionic and/or cellular mechanisms that are necessary for triggering these rhythmic behaviors on a regular basis. Here, we examine which ionic conductances underlie regular or ‘stable’ respiratory activities, which are proposed to underlie eupnea, or normal quiet breathing. We used a mouse in vitro medullary slice preparation containing the rhythmogenic respiratory neural circuit, called the preBötzinger complex (preBötC), that underlies inspiratory respiratory activity. We varied either [K⁺]_o or [Na⁺]_o, or blocked voltage-gated calcium channels, while recording from synaptically isolated respiratory pacemakers, and examined which of these manipulations resulted in their endogenous bursting becoming more irregular. Of these, lowering [Na⁺]_o increased the irregularity of endogenous bursting by synaptically isolated pacemakers. Lowering [Na⁺]_o also decreased the regularity of fictive eupneic activity generated by the ventral respiratory group (VRG) population and hypoglossal motor output. Voltage clamp data indicate that lowering [Na⁺]_o, in a range that results in irregular population rhythm generation, decreased persistent sodium currents, but not transient sodium currents underlying action potentials. Our data suggest that background sodium currents play a major role in determining the regularity of the fictive eupneic respiratory rhythm.     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

β-Adrenergic potentiation of E–C coupling increases force in rat skeletal muscle

We examined the mechanism(s) which allow terbutaline, a β₂-adrenergic agonist, to increase isometric force in bundles of normal and denervated rat soleus fibers. Terbutaline (10 μmol/L) potentiated tetanic contractions during exposure to 1 mmol/L ouabain, 10 μmol/L nifedipine, or 0.5 mmol/L iodoacetate. Terbutafine induced equivalent increases in submaximal potassium (K⁺) contracture and tetanic force: these effects were mimicked by 2 mmol/L dibutyrl-cyclic AMP. Therefore, terbutaline increased force by a cyclic AMP-dependent mechanism other than enhancement of sodium-pump activity, dihydropyridine sensitive Ca²⁺ currents, glycolysis, or action potentials. Pretreatment with 1 mmol/L caffeine induced submaximal potentiation of peak tetanic force but prevented further potentiation by terbutaline. This suggested that terbutaline did not influence the myofilaments, but acted on the sarcoplasmic reticulum (SR) to increase the myoplasmic Ca²⁺ concentration and hence force production. We speculate that force is potentiated following β-adrenoceptor activation by a cyclic AMP-dependent phosphorylation of Ca²⁺ release channels to facilitate SR calcium release during tetanic stimulation. © 1993 John Wiley & Sons, Inc.     

Extracellular magnesium and calcium reduce myotonia in ClC-1 inhibited rat muscle

Loss-of-function mutations in the ClC-1 Cl^? channel trigger skeletal muscle hyperexcitability in myotonia congenita. For reasons that remain unclear, the severity of the myotonic symptoms can vary markedly even among patients with identical ClC-1 mutations, and may become exacerbated during pregnancy and with diuretic treatment. Since both these conditions are associated with hypomagnesemia and hypocalcemia, we explored whether extracellular Mg²⁺ and Ca²⁺ ([Mg²⁺]_o and [Ca²⁺]_o) can affect myotonia. Experimental myotonia was induced in isolated rat muscles by ClC-1 inhibition and effects of [Mg²⁺]_o or [Ca²⁺]_o on myotonic contractions were determined. Both cations dampened myotonia within their physiological concentration ranges. Thus, myotonic contractile activity was 6-fold larger at 0.3 than at 1.2 mM [Mg²⁺]_o and 82-fold larger at 0.3 than at 1.27 mM [Ca²⁺]_o. In intracellular recordings of action potentials, the threshold for action potential excitation was raised by 4–6 mV when [Mg²⁺]_o was elevated from 0.6 to 3 mM, compatible with an increase in the depolarization of the membrane potential necessary to activate the Na⁺ channels. Supporting this notion, mathematical simulations showed that myotonia went from appearing with normal Cl^? channel function to disappearing in the absence of Cl^? channel function when Na⁺ channel activation was depolarized by 6 mV. In conclusion, variation in serum Mg²⁺ and Ca²⁺ may contribute to phenotypic variation in myotonia congenita patients.     

Interstitial potassium concentration, slow depolarization and focal potential responses in the dorsal horn of the rat spinal slice

Extracellular potassium concentration ([K⁺]_o) was measured, and intra- and extracellular recordings made, in the dorsal horn of rat spinal cord slices maintained in vitro during repetitive dorsal root stimulation. In about half of the dorsal horn neurons, the stimulation evoked a possibly substance P-mediated slow depolarization. [K⁺]_o increased during stimulation, reaching its highest values approximately 150 μm from the dorsal surface. The time course of Δ[K⁺]_o was different from that of the slow depolarization. Substance P itself evoked a much smaller Δ[K⁺]_o (0.4 mM) in the dorsal horn. It is concluded that the slow depolarization is not mediated by elevated [K⁺]_o.     

Unidentified neuroglia potentials during propagated seizures in neocortex

Cortical surface and intracellular recording of silent cells (neuroglia) was carried out in the pericruciate cortex of cats during propagated seizures produced by repetitive stimulation of the surface of the opposite homotopic neocortex. The membrane characteristics of these cells were similar to neuroglial cells studied in leech, amphibian, and rat optic nerves, tissue culture, and mammalian cerebral cortex. By varying the parameters of transcallosal stimulation, it was possible to obtain either minor or major propagated seizures. All cells with resting membrane potentials (RMP) greater than 30 mv recorded during minor propagated seizures exhibited a depolarizing response (5–14 mv) during the seizure episode followed by a postictal hyperpolarizing response (1–9 mv) and a slow return to the original resting level. The peak amplitude of the depolarizing response was proportional to the cell's RMP and the amplitude of the seizure waves in the EEG. During major propagated seizures, an augmentation of the depolarizing response to 16–30 mv and the hyperpolarizing response to 10–15 mv was noted. A membrane conductance change during these events was not observed. During major propagated seizures, an increase in [K⁺]_o over the resting [K⁺]_o was calculated to be 10 meq/liter. However, the level of [K⁺]_o reached in the extracellular clefts was probably much higher than this calculated value for reasons which are discussed. A model for seizure propagation is presented. The postictal hyperpolarization most likely represents the effect of a K⁺-sensitive electrogenic pump in the glial membrane.     

The effects of moderate changes of extracellular K and Ca on synaptic and neural function in the CA1 region of the hippocampal slice

The effects of moderate changes of the concentration of ions on the function of mammalian central nervous tissue have not exactly been determined. We placed tissue slices from rat hippocampal formation in an interface chamber for study in vitro. Extracellular potentials were recorded in stratum radiatum and stratum pyramidale in response to stimuli of varying intensity applied to the Schaffer collateral bundle. The overall input-output relationship of excitatory synaptic transmission was gauged by expressing postsynaptic population spike amplitude as a function of presynaptic volley amplitude. The components of the transmission process were also examined by plotting (1) the maximal rate of rise (slope) of the focally recorded synaptic potential (fEPSP) as a function of presynaptic volley amplitude, and (2) the population spike amplitude as a function of the fEPSP slope. Raising the concentration of K⁺ from the normal level of 3.5 mM to 5 mM caused an average increase of 48% in the population spike evoked by a given presynaptic volley. This was due to an increased electrical excitability of pyramidal cells, as indicated by an increase of the population spike evoked by a given magnitude of fEPSP. Conversely, lowering [K⁺]_o from 3.5 to 2 mM caused a decrease of the population spike relative to a given magnitude of either the presynaptic volley or the fEPSP. Changing [K⁺]_o within these limits caused no significant change of the fEPSP evoked by a given presynaptic volley. Raising [Ca²⁺]_o from 1.2 to 1.8 mM caused a 35% increase in both the fEPSP and the population spike evoked by a given presynaptic volley, and lowering [Ca²⁺]_o to 0.8 mM caused a decrease of both these functions. The amplitude of the population spikes evoked by given fEPSPs changed surprisingly little (but consistently) when [Ca²⁺]_o was varied within these limits. We conclude that moderate changes of [K⁺]_o influence mainly the electric excitability of hippocampal pyramidal cells, with little effect on transmitter release or on the response of the postsynaptic membrane to transmitter, while moderate changes of [Ca²⁺]_o affect the release of excitatory synaptic transmitter more than they affect postsynaptic membrane function.     

Hippocampal excitability and changes in extracellular potassium

Extracellular recordings were made from dendritic and somatic sites in the CA1 region of guinea pig hippocampal slices maintained in vitro. We studied the effects of increasing extracellular K⁺ from 3.25 to 15.25 mm on potentials elicited by synaptic activation of stratum radiatum fibers. Increasing K⁺ from 3.25 to 12.25 mm had little or no detectable effect on the excitability of presynaptic fibers, increased the amplitude of the somatic response (the population spike), and increased the size of the dendritic field EPSP in the range of 3.25 to 9.25 mm. When epileptiform activity induced by penicillin was studied as a function of [K⁺]₀, it was found that elevating K⁺ increased the amplitude and duration of the abnormal population spike. However, significant epileptiform activity was present in 3.25 mm [K⁺]₀. All evoked activity was reversibly abolished by perfusion with 15.25 mm K⁺. These results indicate that [K⁺]₀ has an important role in regulating neuronal excitability and may effect changes both pre- and postsynaptically.     

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/pH_o 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 ‐transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na⁺/ 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.     

Carrier-mediated Cl− transport in cultured mouse oligodendrocytes

We studied the steady state and the regulation of intracellular Cl^? activity (aCl^?_i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl^?-selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl^? above passive distribution (2–3 mM), few cells showed a passive Cl^? distribution. To identify the carriers mediating Cl^? uptake, oligodendrocytes were maintained in a solution with low extracellular Cl^? concentration ([Cl^?]₀) which resulted in a rapid decrease in aCl^?_i. The recovery of aCl^?_i above its passive distribution in normal [Cl^?]₀ was blocked in the absence of Na⁺ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na⁺/K⁺/Cl^? cotransport. We therefore conclude that Cl^? uptake is primarily due to the activity of a Na⁺/K⁺/Cl^? transport system. Cl^? uptake above passive distribution was not affected in HCO₃^?-free solution or in the presence of SITS and DIDS, indicating that Cl^?/HCO₃^? exchange is not involved in Cl^? uptake by oligodendrocytes. Elevation of [K⁺]₀ induced an increase in aCl^?_i and, as shown earlier, intracellular K⁺ activity. This K⁺-induced Cl^? uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K⁺]₀ the combined uptake of K⁺ and Cl^? is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Voltage-dependent Ca influx into identified leech neurones

We determined the relationships between the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential (E_m) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca²⁺]_i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K⁺ concentration ([K⁺]_o) up to 89 mM caused a persistent increase in [Ca²⁺]_i, which was abolished in Ca²⁺-free solution indicating that it was due to Ca²⁺ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca²⁺]_i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca²⁺]_i and E_m were partly due to the cell-specific generation of action potentials. In Na⁺-free solution, the action potentials were suppressed and the [Ca²⁺]_i/E_m relationships were similar. The K⁺-induced [Ca²⁺]_i increase was inhibited by the polyvalent cations Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, and La³⁺, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K⁺-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca²⁺ channels with similar properties.     

Molecular mechanisms of K+ clearance and extracellular space shrinkage—Glia cells as the stars

Neuronal signaling in the central nervous system (CNS) associates with release of K⁺ into the extracellular space resulting in transient increases in [K⁺]_o. This elevated K⁺ is swiftly removed, in part, via uptake by neighboring glia cells. This process occurs in parallel to the [K⁺]_o elevation and glia cells thus act as K⁺ sinks during the neuronal activity, while releasing it at the termination of the pulse. The molecular transport mechanisms governing this glial K⁺ absorption remain a point of debate. Passive distribution of K⁺ via Kir4.1-mediated spatial buffering of K⁺ has become a favorite within the glial field, although evidence for a quantitatively significant contribution from this ion channel to K⁺ clearance from the extracellular space is sparse. The Na⁺/K⁺-ATPase, but not the Na⁺/K⁺/Cl⁻ cotransporter, NKCC1, shapes the activity-evoked K⁺ transient. The different isoform combinations of the Na⁺/K⁺-ATPase expressed in glia cells and neurons display different kinetic characteristics and are thereby distinctly geared toward their temporal and quantitative contribution to K⁺ clearance. The glia cell swelling occurring with the K⁺ transient was long assumed to be directly associated with K⁺ uptake and/or AQP4, although accumulating evidence suggests that they are not. Rather, activation of bicarbonate- and lactate transporters appear to lead to glial cell swelling via the activity-evoked alkaline transient, K⁺-mediated glial depolarization, and metabolic demand. This review covers evidence, or lack thereof, accumulated over the last half century on the molecular mechanisms supporting activity-evoked K⁺ and extracellular space dynamics.     

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex

Maintenance and regulation of intracellular pH (pH_i) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na⁺ gradient by reduction of the extracellular Na⁺ concentration ([Na⁺]_o) resulted in rapid intracellular acidification, inhibited by 5′-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na⁺/H⁺ exchange. In the presence of EIPA and/or 4′,4′-diisothiocyano-stilbene-2′,2′-sulfonic acid (DIDS), an inhibitor of Na⁺-coupled anion exchangers and Na⁺-HCO₃⁻ cotransport, a slow decline of pH_i was seen. Following intracellular acidification imposed by an NH₄Cl prepulse, pH_i recovered at a rapid rate, which was reduced by reduction of [Na⁺]_o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl⁻ gradient by removal of extracellular Cl⁻ significantly increased the rate of pH_i recovery. In HCO₃⁻-free media, the pH_i recovery rate was reduced in control cells and was abolished at zero [Na⁺]_o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pH_i recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl⁻, as well as in the presence of Zn²⁺, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl⁻/HCO₃⁻ and passive H⁺ influx in the recovery of pH_i after alkalinization. J. Neurosci. Res. 51:431–441, 1998. © 1998 Wiley-Liss, Inc.     

INa and IKir are reduced in Type 1 hypokalemic and thyrotoxic periodic paralysis

We evaluated voltage‐gated Na⁺ (I_Na) and inward rectifier K⁺ (I_Kir) currents and Na⁺ conductance (G_Na) in patients with Type 1 hypokalemic (HOPP) and thyrotoxic periodic paralysis (TPP). We studied intercostal muscle fibers from five subjects with HOPP and one with TPP. TPP was studied when the patient was thyrotoxic (T‐toxic) and euthyroid. We measured: (1) I_Kir, (2) action potential thresholds, (3) I_Na, (4) G_Na, (5) intracellular [Ca²⁺], and (6) histochemical fiber type. HOPP fibers had lower I_Na, G_Na, and I_Kir and increased action potential thresholds. Paralytic attack frequency correlated with the action potential threshold, G_Na and I_Na, but not with I_Kir. G_Na, I_Na, and [Ca²⁺] varied with fiber type. HOPP fibers had increased [Ca²⁺]. The subject with TPP had values for G_Na, I_Na, action potential threshold, I_Kir, and [Ca²⁺] that were similar to HOPP when T‐toxic and to controls when euthyroid. HOPP T‐toxic TPP fibers had altered G_Na, I_Na, and I_Kir associated with elevation in [Ca²⁺]. Muscle Nerve, 2010     

Serotonin,norepinephrine, and acetylcholine differentially affect astrocytic potassium clearance to modulate somatosensory signaling in male mice

Changes in extracellular potassium ([K⁺]_e) modulate neuronal networks via changes in membrane potential, voltage-gated channel activity, and alteration to transmission at the synapse. Given the limited extracellular space in the central nervous system, potassium clearance is crucial. As activity-induced potassium transients are rapidly managed by astrocytic Kir4.1 and astrocyte-specific Na⁺/K⁺-ATPase, any neurotransmitter/neuromodulator that can regulate their function may have indirect influence on network activity. Neuromodulators differentially affect cortical/thalamic networks to align sensory processing with differing behavioral states. Given serotonin (5HT), norepinephrine (NE), and acetylcholine (ACh) differentially affect spike frequency adaptation and signal fidelity (“signal-to-noise”) in somatosensory cortex, we hypothesize that [K⁺]_e may be differentially regulated by the different neuromodulators to exert their individual effects on network function. This study aimed to compare effects of individually applied 5HT, NE, and ACh on regulating [K⁺]_e in connection to effects on cortical-evoked response amplitude and adaptation in male mice. Using extracellular field and K⁺ ion-selective recordings of somatosensory stimulation, we found that differential effects of 5HT, NE, and ACh on [K⁺]_e regulation mirrored differential effects on amplitude and adaptation. 5HT effects on transient K⁺ recovery, adaptation, and field post-synaptic potential amplitude were disrupted by barium (200 µM), whereas NE and ACh effects were disrupted by ouabain (1 µM) or iodoacetate (100 µM). Considering the impact [K⁺]_e can have on many network functions; it seems highly efficient that neuromodulators regulate [K⁺]_e to exert their many effects. This study provides functional significance for astrocyte-mediated buffering of [K⁺]_e in neuromodulator-mediated shaping of cortical network activity.     

Effects of pilocarpine on paired pulse inhibition in the CA3 region of the rat hippocampus

Pilocarpine (PILO), a muscarinic agonist, produces status epilepticus when administered to rats in vivo and induces interictal or ictal patterns of epileptiform activity in rat hippocampal slices. We investigated the effects of PILO (10 μM) on paired pulse inhibition (PPI) in the CA3 region of rat hippocampal slices. PPI was assessed by stimulating either the alveus or str. radiatum and recording the extracellular response from str. pyramidale of CA3. The evoked population spike following the second stimulus was compared to the first, PILO was bath applied for 1 h and then washed out to assess acute and long lasting effects. PILO decreased the amplitude of evoked population spikes measured in CA3. PPI following alveus stimulation was not affected by PILO; however, a significant loss of PPI at 15 and 30 ms interpulse intervals occurred following str. radiatum stimulation in the presence of PILO and 5 mM [K⁺]_o artificial cerebrospinal fluid (ACSF). The decrease in PPI at the 15 ms interval persisted following wash-out of PILO. PILO in 7.5 mM [K⁺]_o ACSF produced epileptiform activity and a resultant long lasting loss of PPI that followed str. radiatum stimulation. This effect was not observed following epileptiform activity produced by 7.5 mM [K⁺]_o alone, suggesting that the loss of PPI was due to PILO. Because str. radiatum-evoked PPI was selectively impaired, PILO appears to preferentially decreased feed-forward inhibition. The more dramatic loss of PPI following exposure to PILO and high [K⁺]_o may present the first steps in the development of chronic seizures that results from PILO-induced status epilepticus in rats.