Endogenous rhythm generation in the pre-Bötzinger complex and ionic currents: modelling and in vitro studies

The pre‐Bötzinger complex is a small region in the mammalian brainstem involved in generation of the respiratory rhythm. As shown in vitro, this region, under certain conditions, can generate endogenous rhythmic bursting activity. Our investigation focused on the conditions that may induce this bursting behaviour. A computational model of a population of pacemaker neurons in the pre‐Bötzinger complex was developed and analysed. Each neuron was modelled in the Hodgkin–Huxley style and included persistent sodium and delayed‐rectifier potassium currents. We found that the firing behaviour of the model strongly depended on the expression of these currents. Specifically, bursting in the model could be induced by a suppression of delayed‐rectifier potassium current (either directly or via an increase in extracellular potassium concentration, [K⁺]_o) or by an augmentation of persistent sodium current. To test our modelling predictions, we recorded endogenous population activity of the pre‐Bötzinger complex and activity of the hypoglossal (XII) nerve from in vitro transverse brainstem slices (700 µm) of neonatal rats (P0–P4). Rhythmic activity was absent at 3 mm [K⁺]_o but could be triggered by either the elevation of [K⁺]_o to 5–7 mm or application of potassium current blockers (4‐AP, 50–200 µm , or TEA, 2 or 4 mm ), or by blocking aerobic metabolism with NaCN (2 mm ). This rhythmic activity could be abolished by the persistent sodium current blocker riluzole (25 or 50 µm ). These findings are discussed in the context of the role of endogenous bursting activity in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.     

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

Activation of alpha-2 noradrenergic receptors is critical for the generation of fictive eupnea and fictive gasping inspiratory activities in mammals in vitro

Biogenic amines are not just ‘modulators’, they are often essential for the execution of behaviors. Here, we explored the role of biogenic amines acting on the pre‐Bötzinger complex (pre‐BötC), an area located in the ventrolateral medulla which is critical for the generation of different forms of breathing. Isolated in transverse slices from mice, this region continues to spontaneously generate rhythmic activities that resemble normal (eupneic) inspiratory activity in normoxia and gasping in hypoxia. We refer to these as ‘fictive eupneic’ and ‘fictive gasping’ activity. When exposed to hypoxia, the pre‐BötC transitions from a network state relying on calcium‐activated nonspecific cation currents (I_CAN) and persistent sodium currents (I_Nap) to one that primarily depends on the I_Nap current. Here we show that in inspiratory neurons I_Nap‐dependent bursting, blocked by riluzole, but not I_CAN‐dependent bursting, required endogenously released norepinephrine acting on alpha2‐noradrenergic receptors (α2‐NR). At the network level, fictive eupneic activity persisted while fictive gasping ceased following the blockade of α2‐NR. Blockade of α2‐NR eliminated fictive gasping even in slice preparations as well as in inspiratory island preparations. Blockade of fictive gasping by α2‐NR antagonists was prevented by activation of 5‐hydroxytryptamine type 2A receptors (5‐HT2A). Our data suggest that gasping depends on the converging aminergic activation of 5‐HT2AR and α2‐NR acting on riluzole‐sensitive mechanisms that have been shown to be crucial for gasping.     

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.     

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.     

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.     

Hypoxia induced by Na2S2O4 increases [Na]i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na⁺]_i) and resting potential (E_m) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na⁺-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO₂ 122 Torr), [Na⁺]_i was 12–13 mM corresponding to a Na⁺ equilibrium potential (E_Na) of about 58 mV. Em was about −42 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM (PO₂ 10 Torr), depolarized the cells by about 20 mV, [Na⁺]_i increased by 21 mM and E_Na dropped to about 35 mV. One millimolar of CoCl₂ depressed, or blocked, the effects of Na₂S₂O₄ on [Na⁺]_i but did not affect hypoxic depolarization. Voltage-clamping at −70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba²⁺. All currents were blocked by 1 mM CoCl₂ suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na⁺]_i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na⁺ increases (inflow) probably occurred by disturbing a Na⁺/K⁺ exchange mechanism and not by activation of voltage-gated channels.     

Astrocytes restrict discharge duration and neuronal sodium loads during recurrent network activity

Influx of sodium ions into active neurons is a highly energy‐expensive process which must be strictly limited. Astrocytes could play an important role herein because they take up glutamate and potassium from the extracellular space, thereby dampening neuronal excitation. Here, we performed sodium imaging in mouse hippocampal slices combined with field potential and whole‐cell patch‐clamp recordings and measurement of extracellular potassium ([K⁺]_o). Network activity was induced by Mg²⁺‐free, bicuculline‐containing saline, during which neurons showed recurring epileptiform bursting, accompanied by transient increases in [K⁺]_o and astrocyte depolarizations. During bursts, neurons displayed sodium increases by up to 22 mM. Astrocyte sodium concentration increased by up to 8.5 mM, which could be followed by an undershoot below baseline. Network sodium oscillations were dependent on action potentials and activation of ionotropic glutamate receptors. Inhibition of glutamate uptake caused acceleration, followed by cessation of electrical activity, irreversible sodium increases, and swelling of neurons. The gliotoxin NaFAc (sodium‐fluoroacetate) resulted in elevation of astrocyte sodium concentration and reduced glial uptake of glutamate and potassium uptake through Na⁺/K⁺‐ATPase. Moreover, NaFAc extended epileptiform bursts, caused elevation of neuronal sodium, and dramatically prolonged accompanying sodium signals, most likely because of the decreased clearance of glutamate and potassium by astrocytes. Our experiments establish that recurrent neuronal bursting evokes sodium transients in neurons and astrocytes and confirm the essential role of glutamate transporters for network activity. They suggest that astrocytes restrict discharge duration and show that an intact astrocyte metabolism is critical for the neurons' capacity to recover from sodium loads during synchronized activity. GLIA 2015;63:936–957     

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.     

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     

Blockade of K+ channels induced by AMPA/kainate receptor activation in mouse oligodendrocyte precursor cells is mediated by NA+ entry

AMPA/kainate receptor activation in cultured oligodendrocyte precursor cells from embryonic mouse cortex leads to a blockade of delayed rectifying K⁺ currents. In the present study, we provide evidence using the patch-clamp technique in the whole-cell configuration that the mechanism linking kainate receptor activation and K⁺ conductance blockade is due to the receptor-mediated Na⁺ entry: (1) The blockade was not observed in Na⁺ -free bathing solution nor when intracellular [Na⁺] was elevated by dialzying the cell with a pipette solution containing high [Na⁺]. (2) Elevation of intracellular [Na⁺] alone led to a blockade of outward currents in contrast to cells dialyzed by sucrose. High [Li⁺]_i also reduced the outward currents, and in Li⁺-containing bathing solution the kainate-induced blockade of K⁺ channels was more pronounced. Probably, Li⁺ accumulates intracellularly after permeation through the receptor pore due to slower extrusion mechanisms. Experiments with GTPγS or GDPβS and pertussis toxin indicated that GTP-binding protein-mediated mechanisms were not of importance for the kainate-induced K⁺ conductance blockade. Our data suggest that in glial precursor cells AMPA/kainate receptor activation leads to an intracellular [Na⁺] increase which blocks delayed rectifying K⁺ channels. © 1995 Wiley-Liss, Inc.     

Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

High-frequency fatigue in rat skeletal muscle: Role of extracellular ion concentrations

High-frequency fatigue (HFF), the decline of force during continuous tetanic stimulation (lasting 4–40 s), was studied in isolated bundles of rat skeletal muscle fibers. HFF was slower in slow-twitch soleus fibers than in fast-twitch red or white sternomastoid fibers; denervation accelerated fatigue in soleus. Maximal 200-mmol/L potassium contractures of normal amplitude were induced in fatigued fibers, suggesting that crossbridge cycling and the voltage activation of excitation–contraction coupling could still occur maximally, but that activation by action potentials was impaired. An increase in [Na⁺]_o slowed HFF, while a small increase in [K⁺]_o or reduction in [Cl^?]_o accelerated HFF. Increasing the tetanic stimulation frequency exacerbated fatigue. Recovery from HFF proceeded rapidly since force increased markedly within a few seconds when stimulation ceased. These results support the hypothesis that a redistribution of Na⁺, K⁺, and Cl^? across the transverse tubular membranes during repeated action potential activity induces fatigue by reducing the amplitude and conduction of action potentials. © 1995 John Wiley & Sons, Inc.     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na⁺]_i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na⁺]_i. In dopamine neurons voltage-clamped at a holding potential of ?60 mV elevations of [Na⁺]_i were induced by long-lasting (45–60 s) voltage jumps to more negative membrane potentials (–90 to ?120 mV) but not by corresponding voltage jumps to ?30 mV. These hyperpolarization-induced elevations of [Na⁺]_i were depressed during inhibition of I_h, a hyperpolarization-activated inward current, by Cs⁺. Hyperpolarization-induced elevations in [Na⁺]_i might occur also in other cell types which express a powerful I_h and might signal lack of postsynaptic activity.     

AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes

Cultured mouse cortical astrocytes of the stellate type were studied by using the patch-clamp technique in whole-cell configuration. The astrocytes express at least two types of outwardly rectifying K⁺ channels which mediate a transient and a sustained current. Activation of AMPA receptors by kainate leads to a substantial blockade of both types of K⁺ currents. The blockade is absent when Na⁺ is withdrawn from the external medium, suggesting that it is caused by constant Na⁺ influx through AMPA receptors. The presence of high Na⁺ solutions in the pipette induces a blockade of both K⁺ currents which is very similar to the blockade induced by kainate, supporting thus the view that the mechanism of the blockade of K⁺ channels by kainate involves Na⁺ increases in the submembrane area. The blockade occurs between 20 and 40 mM [Na⁺]_i, which is within the physiological range of [Na⁺]_i in astrocytes. The data may suggest that the blockade of K⁺ channels by high [Na⁺]_i conditions could provide a mechanism to prevent K⁺ leakage from the astrocytes into the extracellular space during periods of intense neuronal activity. GLIA 20:38-50, 1997. © 1997 Wiley-Liss, Inc.     

SHORT COMUNICATION Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons

We investigated the effects of oxygen (O₂)/glucose deprivation on intracellular sodium concentration ([Na⁺]_i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O₂/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na⁺]_i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (d -amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na⁺/Ca²⁺-exchanger, affected these responses to O₂/glucose. The present results demonstrate that, in cortical neurons, O₂/glucose deprivation induces an early rise in [Na⁺]_i which cannot be ascribed to the activity of voltage gated Na⁺-channels, glutamate receptors or of the Na⁺/Ca²⁺-exchanger.     

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.     

Astrocyte sodium signaling and the regulation of neurotransmission

The transmembrane Na⁺ concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non‐excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na⁺‐dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long‐lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na⁺ ([Na⁺]_i) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio‐temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na⁺]_i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na⁺]_i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na⁺‐coupled co‐transporter, can strongly influence, or even reverse, other Na⁺‐dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655–1666     

Comodulation of h- and Na+/K+ Pump Currents Expands the Range of Functional Bursting in a Central Pattern Generator by Navigating between Dysfunctional Regimes

Central pattern generators (CPGs), specialized oscillatory neuronal networks controlling rhythmic motor behaviors such as breathing and locomotion, must adjust their patterns of activity to a variable environment and changing behavioral goals. Neuromodulation adjusts these patterns by orchestrating changes in multiple ionic currents. In the medicinal leech, the endogenous neuromodulator myomodulin speeds up the heartbeat CPG by reducing the electrogenic Na⁺/K⁺ pump current and increasing h-current in pairs of mutually inhibitory leech heart interneurons (HNs), which form half-center oscillators (HN HCOs). Here we investigate whether the comodulation of two currents could have advantages over a single current in the control of functional bursting patterns of a CPG. We use a conductance-based biophysical model of an HN HCO to explain the experimental effects of myomodulin. We demonstrate that, in the model, comodulation of the Na⁺/K⁺ pump current and h-current expands the range of functional bursting activity by avoiding transitions into nonfunctional regimes, such as asymmetric bursting and plateau-containing seizure-like activity. We validate the model by finding parameters that reproduce temporal bursting characteristics matching experimental recordings from HN HCOs under control, three different myomodulin concentrations, and Cs⁺ treated conditions. The matching cases are located along the border of an asymmetric regime away from the border with more dangerous seizure-like activity. We found a simple comodulation mechanism with an inverse relation between the pump and h-currents makes a good fit of the matching cases and comprises a general mechanism for the robust and flexible control of oscillatory neuronal networks.SIGNIFICANCE STATEMENT Rhythm-generating neuronal circuits adjust their oscillatory patterns to accommodate a changing environment through neuromodulation. In different species, chemical messengers participating in such processes may target two or more membrane currents. In medicinal leeches, the neuromodulator myomodulin speeds up the heartbeat central pattern generator by reducing Na⁺/K⁺ pump current and increasing h-current. In a computational model, we show that this comodulation expands the range of central pattern generator''s functional activity by navigating the circuit between dysfunctional regimes resulting in a much wider range of cycle period. This control would not be attainable by modulating only one current, emphasizing the synergy of combined effects. Given the prevalence of h-current and Na⁺/K⁺ pump current in neurons, similar comodulation mechanisms may exist across species.     

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH₄⁺/NH₃). Here, we examined the effects of NH₄⁺/NH₃ on steady‐state intracellular pH (pH_i) and sodium concentration ([Na⁺]_i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH₄⁺/NH₃ on glutamate transporter activity by measuring D ‐aspartate‐induced pH_i and [Na⁺]_i transients. In 20–34 days in vitro (DIV) astrocytes, NH₄⁺/NH₃ decreased steady‐state pH_i by 0.19 pH units and increased [Na⁺]_i by 21 mM. D ‐Aspartate‐induced pH_i and [Na⁺]_i transients were reduced by 80–90% in the presence of NH₄⁺/NH₃, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pH_i and [Na⁺]_i were minimally affected by NH₄⁺/NH₃, and D ‐aspartate‐induced pH_i and [Na⁺]_i transients were reduced by only 30–40%. Next we determined the contribution of Na⁺, K⁺, Cl^?‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH₄⁺/NH₃‐evoked changes in steady‐state pH_i and [Na⁺]_i and attenuated the reduction of D ‐aspartate‐induced pH_i and [Na⁺]_i transients by NH₄⁺/NH₃ to 30% in 20–34 DIV astrocytes. Our results suggest that NH₄⁺/NH₃ decreases steady‐state pH_i and increases steady‐state [Na⁺]_i in astrocytes by an age‐dependent activation of NKCC. These NH₄⁺/NH₃‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE. © 2008 Wiley‐Liss, Inc.