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.     

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.     

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     

Extracellular potassium concentration changes during propagated seizures in neocortex

Cortical surface and intracortical, extracellular K⁺ selective microelectrode recording was carried out in the pericruciate cortex of cats during propagated seizures produced by repetitive stimulation of the surface of the contralateral homotopic neocortex. The K⁺ selective microelectrode consistently recorded a potential change which corresponded to an increase in [K⁺]₀ which was directly related to the seizure amplitude × duration. The interictal [K⁺]₀ in neocortical extracellular fluid was determined to be 4 meq/liter. The maximum increase of [K⁺]₀ during propagated seizures in these experiments was 7 meq/liter which correlates lates well with increases in [K⁺]₀ calculated from neuroglial depolarizations during similar propagated seizures.     

The increase in extracellular potassium concentration in the ischemic brain in relation to the preischemic functional activity and cerebral metabolic rate

The course of ischemic increase of extracellular potassium concentration ([K⁺]_e) was studied in rat cerebral cortex with potassium selective microelectrodes and correlated to the preischemic functional and metabolic state. Complete cerebral ischemia was induced in artificially ventilated rats by cardiac arrest. Seven different functional states including conditions with cerebral hypermetabolism (seizures, amphetamine intoxication, hyperthermia) and hypometabolism (barbiturate anesthesia, hypothermia) were chosen in order to cover a wide range of cerebral metabolic rates (CMR_o2 : 28.7-2.4 ml O₂/(100 g)/min). The ischemic increase of [K⁺]_e was delayed in conditions with low CMR_o2 and accelerated in conditions with high CMR_o2; the time interval to the terminal steep rise in extracellular potassium concentration varied within the extremes of35 ± 5 and 365 ± 12sec (means ± S.E.M.), the control state (N₂O-analgesia) being116 ± 5sec. In groups with high CMR_o2 electrocortical activity ceased within 15 sec and in groups with low CMR_o2 within 22 sec. The rates of the ischemic [K⁺]_e increase, measured as rate of change in the potassium electrode potential (mV/sec), remained high in conditions with high preischemic CMR_o2 and low in conditions with low CMR_o2, indicating a remaining influence of the preischemic metabolism on membrane ion permeability. These results support previous metabolic data indicating that the rate of consumption of high energy phosphates during ischemia mirrors the preischemic cerebral metabolic rate. Phenobarbital anesthesia did not change the initial rate of [K⁺]_e increase but reduced the rate of [K⁺]_e increase later during ischemia, suggesting a special effect of barbiturates on partly depolarized membranes.     

Effects of GABA on Axonal Conduction and Extracellular Potassium Activity in the Neonatal Rat Optic Nerve

GABA depolarizes rat optic nerve axons and modulates axonal conduction through the activation of GABA-A receptors. To address whether an increase of [K⁺]_e plays a major role in GABA actions on the rat optic nerve, we studied the effects of GABA on axonal conduction and [K⁺]_e in the neonatal rat optic nerve in vitro. Double-barrelled K⁺-sensitive microelectrodes were used to record [K⁺]_e. GABA (10^-4-10^-3M) increased [K⁺]_e in the neonatal optic nerve. During prolonged application, the [K⁺]_e slowly recovered. The increase in [K⁺]_e induced by GABA was markedly reduced by the GABA-A receptor blocker bicuculline (10^-4M). Isoguvacine (10^-4M), a GABA-A agonist, mimicked the effect of GABA but produced larger responses at the same concentration. In contrast, baclofen (10^-4M), a GABA-B agonist, had no effect on [K⁺]_e. The changes in the compound action potential induced by GABA correlated only partially with the [K⁺]_e changes. Furthermore, the changes in the compound action potential induced by elevation of K⁺ were far less than those induced by GABA. These results demonstrate that the GABA-evoked accumulation of [K⁺]_e plays a secondary role in GABA actions on the neonatal rat optic nerve.     

Synaptic impairment induced by paroxysmal ionic conditions in neocortex

Purpose: Seizures are associated with a reduction in extracellular Ca²⁺ concentration ([Ca²⁺]_o) and an increase in extracellular K⁺ concentration ([K⁺]_o). The long‐range synchrony observed between distant electrodes during seizures is weak. We hypothesized that changes in extracellular ionic conditions during seizures are sufficient to alter synaptic neuronal responses and synchrony in the neocortex. Methods: We obtained in vivo and in vitro electrophysiologic recordings combined with microstimulation from cat/rat neocortical neurons during seizures and seizure‐like ionic conditions. In vitro the [K⁺]_o was 2.8, 6.25, 8.0, and 12 mm and the [Ca²⁺]_o was 1.2 and 0.6 mm . Key Findings: During seizures recorded in vivo, we observed abolition of evoked synaptic responses. In vitro, the membrane potential of both regular‐spiking and fast‐spiking neurons was depolarized in high [K⁺]_o conditions and hyperpolarized in high [Ca²⁺]_o conditions. During high [K⁺]_o conditions, changes in [Ca²⁺]_o did not affect membrane potential. The synaptic responsiveness of both regular‐spiking and fast‐spiking neurons was reduced during seizure‐like ionic conditions. A reduction in [Ca²⁺]_o to 0.6 mm increased failure rates but did not abolish responses. However, an increase in [K⁺]_o to 12 mm abolished postsynaptic responses, which depended on a blockade in axonal spike propagation. Significance: We conclude that concomitant changes in [K⁺]_o and [Ca²⁺]_o observed during seizures contribute largely to the alterations of synaptic neuronal responses and to the decrease in long‐range synchrony during neocortical seizures.     

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.     

Synchronization of rat hippocampal neurons in the absence of excitatory amino acid-mediated transmission

Extracellular and intracellular recordings and measurements of extracellular K⁺ concentration ([K⁺]_o) were performed in the adult rat hippocampus in an in vitro slice preparation. Excitatory amino acid receptor antagonists, as well as the K⁺-channel blockers 4-aminopyridine (4AP, 50 μM) and/or tetraethylammonium (TEA, 5 mM), were added to the bath. Synchronous, negative-going field potentials were recorded in the CA3 stratum radiatum during application of 4AP and excitatory amino acid receptor antagonists. Each of these events was associated with an intracellular long-lasting depolarization and a concomitant rise in [K⁺]_o that attained peak values of 4.3 + 0.1 mM (mean ± S.E.M., n = 6 slices) and lasted 29 ± 3 s. These field potentials were still recorded in CA3 stratum radiatum after addition of TEA. Under these conditions, prolonged field potentials (40.2 ± 4.5 s, n = 18) characterized by a prominent positive component; discharge of population spikes also occurred. [K⁺]_o, increases associated with these prolonged field-potential discharges had a considerable variability in magnitude (peak value = 3.8–14.1 mM, 6.1 ± 0.7 mM, n = 5) and duration (14–210 s; 48 ± 13 s, n = 5). In 8% of the cases spreading depression-like episodes were observed. [K⁺]_o increases during spreading depression-like episodes attained peak values of 11–27 mM (22.8 ± 0.2 mM, n = 2) and had a duration of 160–396 s (244 ± 29 s, n = 2). All types of synchronous activity were abolished by the GABAA receptor antagonist bicuculline methiodide (t0 μM) ( n= 11). A similar effect was obtained by applying Ca²⁺-free/high-Mg²⁺ medium ( n = 5). Simultaneous field-potential recordings in CA3, CAI, dentate area and subiculum demonstrated that negative-going potentials and prolonged field-potential discharges occurred in all areas in a synchronous fashion. Spreading depression-like episodes were more frequently recorded in the CAI than in the CA3 area and were not seen in the subiculum or dentate area. These experiments indicate that a glutamatergic-independent, synchronous GABA-mediated potential which is elicited by 4AP in the adult rat hippocampus continues to occur in the presence of TEA. In addition, concomitant application of these K⁺-channel blockers induces a novel type of prolonged field-potential discharge as well as spreading depression-like episodes. Since all synchronous potentials (including spreading depression-like episodes) were abolished by bicuculline methiodide, we conclude that their occurrence is presumably dependent upon the post-synaptic activation of GABA_A receptors located on neuronal and glial elements. As excitatory synaptic transmission was nominally blocked under our experimental conditions, we also propose that rises in [K⁺]_o and consequent redistribution processes are per se sufficient to make all types of synchronous activity propagate.     

Potassium activity in rabbit cortex

Measurements of extracellular potassium concentration ([K⁺]₀) were made in rabbit cortex using potassium-sensitive microelectrodes with a rapid response time. Resting levels of [K⁺]₀ averaged about 3 mM and transient increases occurred following direct stimulation of normal cortex, during spreading depression (SD), and during interictal epileptiform discharges.Interictal discharges were associated with increases in [K⁺]₀ to maximum levels of 9–10 mM. These changes in [K⁺]₀ had a laminar distribution through the cortex being maximal in deeper layers. During ictal episodes [K⁺]₀ peaked at 9–10 mM and appeared ‘clamped’ at this level. Neither seizure onset nor termination appeared to occur at particular threshold levels of [K⁺]₀. SDs were associated with increases in [K⁺]₀ to about 40 mM. During SDs, ictal activity might be generated even though local [K⁺]₀ was 25–30 mM. DC shifts in EEG activity recorded adjacent to the K⁺ electrode usually preceded the [K⁺]₀ changes and returned more quickly to baseline levels, but the envelope of DC changes closely mirrored the alterations in [K⁺]₀.     

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.     

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K⁺]₀, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca²⁺]_i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K⁺]₀), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K⁺]₀) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca²⁺]_i induced by 55 mM [K⁺]₀ became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca²⁺]_i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca²⁺]_i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.     

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.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Effects of depolarizing agents on transglutaminase activity,Ca2+ influx,and protein synthesis in superior cervical and nodose ganglia excised from rats

Rapid changes in transglutaminase (TG) activity,⁴⁵Ca²⁺ — influx and [³H]leucine incorporation in superior cervical ganglia (SCG), and nodose ganglia (NG) excised from adult rats were examined following addition of membrane-depolarizing agents veratridine (Ver) or high extracellular [K⁺]_o during aerobic incubation in vitro at 37°C. Addition of KCl (50mM) stimulated TG activity to a maximal extent (four to sixfold) in SCG and NG after 30 min. Ver (0.2 mM) also increased TG activity in both ganglia after 30 min. Kinetic studies showed that the stimulation of TG activity in both ganglia caused by each depolarization condition was associated with a decrease inK _m and an increase inV _max value. The depolarizing agents Ver and high [K⁺]_o also caused significant increases in⁴⁵Ca²⁺ influx into both ganglia. The Ver-induced increases in TG activity and⁴⁵Ca²⁺ accumulation were antagonized by tetrodotoxin (TTX, 1 μM), a sodium channel blocker. The K⁺-induced increase in TG activity was not blocked by tetraethylammonium (TEA, 20 mM), a potassium channel antagonist, although TEA did block the K⁺-induced increase in⁴⁵Ca²⁺ accumulation. The membrane-perturbing, sialic acid-containing compounds, GM1-ganglioside (GM1, 5 nM) and α-sialyl cholesterol (α-SC, 20 μM), were moderate inhibitors of the K⁺-induced effects on TG activity and⁴⁵Ca²⁺ accumulation. The sialyl compounds had little effect on Ver-induced accumulation of⁴⁵Ca²⁺ but enhanced the Ver-evoked stimulation in TG activity. These results suggests that the veratridine-and K⁺-induced increases in TG activity occur via modulation of Ca²⁺ and Na⁺ channel gating mechanisms that are pharmacologically distinct for each depolarizing agent. The veratridine- and K⁺-induced decrease in [³H]leucine incorporation could be a result of stimulation of TG activity as a consequence of degenerative alterations.     

Effects of serotonin on extracellular potassium concentration in the rat hippocampal slice

The effects of serotonin (5-HT) on extracellular potassium concentration ([K⁺]₀) were measured with ion-selective microelectrodes in rat hippocampal slices. Electrical stimulation of an excitatory afferent system, the Schaffer collateral commissural pathway, caused a 2–4 mM rise in [K⁺]₀ in the stratum pyramidale of area CA1. 5-HT caused a 0.6–1.1 mM rise in [K⁺]₀. This rise was associated with hyperpolarization of neurons and cessation of their spontaneous spike discharge. Methysergide, a 5-HT antagonist, reduced the 5-HT effect. The change in [K⁺]₀ was highest in stratum moleculare and lowest in stratum pyramidale, the opposite gradient to that found with excitatory electrical stimulation. The 5-HT-induced [K⁺]₀ changes were maximal in CA1 stratum moleculare, intermediate in the dentate stratum granulare and almost non-existent in the CA3 stratum pyramidale.GABA, but not norepinephrine, produced a small (up to 0.5 mM) rise in [K⁺]₀ in stratum pyramidale. Extracellular calcium concentration measured with a Ca²⁺-sensitive microelectrode was reduced by electrical stimulation but unchanged by 5-HT or norepinephrine. It is suggested that 5-HT hyperpolarizes hippocampal cells by activation of sodium- and calcium-independent potassium channels, which cause a rise in [K⁺]₀.     

Flat and steep terminal negativity in the DC-potential after deprivation of oxygen and glucose in human neocortical slices

The so-called terminal negativity (TN) of the DC-potential is a characteristic reaction of neuronal tissue to hypoxia or ischemia. In a previous study on human neocortical slices, two types of TN with flat and steep slopes of rise (< or >10 mV/min) were found with hypoxia. The aim of the present study was to further investigate causes underlying the occurrence of flat and steep TN. Experiments were performed on 23 human neocortical slices (500 μm) resected from 13 patients (epilepsy and tumour surgery). DC-potential and evoked potentials (white matter stimulation) were recorded in layer III. The extracellular potassium concentration ([K⁺]_o) was measured by K⁺-sensitive microelectrodes. In an interface type chamber, ischemic episodes were induced by oxygen and glucose deprivation. They were terminated when TN had peaked. Both flat and steep TN also existed with ischemic conditions. There was a linear correlation between the slope of rise of TN and the associated slope of rise in [K⁺]_o, respectively, but none regarding latencies of TN or recovery of evoked potentials. Peak levels in [K⁺]_o were 13.9±0.9 mmol/l. Compared to control, the slope of rise and latency of TN were clearly increased by addition of dimethyl sulfoxide (DMSO, 0.4%) to the bath solution, whereas nimodipine (40 μmol/l) in 0.4% DMSO had neither an effect on slope of rise of TN nor on latency of TN. As a whole, our observations suggest, that the actual metabolic state determines the occurrence of flat or steep TN.     

Novel inward rectifier blocked by Cd in crayfish muscle

The characteristics of a voltage- and time-dependent inward rectifying current were examined with voltage clamp techniques in crayfish muscle. The inward current, carried by K⁺, was activated by hyperpolarization. Although this inward current increased with the extracellular K⁺ concentration ([K⁺]_o), the voltage-dependence of the underlying conductance was independent of [K⁺]_o. The current was unaffected by Cs⁺ and Ba²⁺, but was blocked by low concentrations of Cd²⁺. Therefore, this inward rectifier is different than previously described ones.     

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.     

Voltage‐induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation‐secretion coupling is assumed to be dependent only on extracellular calcium ([Ca²⁺]_o). Ryanodine receptor (RyR)‐sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca²⁺]_i) that are voltage‐dependent. We hypothesised that voltage‐elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage‐gated calcium channels and/or from such voltage‐sensitive ryanodine‐mediated calcium stores. Increases in [Ca²⁺]_i upon depolarisation in the presence of voltage‐gated calcium channel blockers, or in the absence of [Ca²⁺]_o, still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca²⁺]_o, there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Furthermore, such depolarisations lead to increases in [Ca²⁺]_i. Pre‐incubation with BAPTA‐AM resulted in > 50% inhibition of peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Nifedipine but not nicardipine inhibited both the high K⁺ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage‐induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation‐induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine‐sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.