Blockade of U50488H on sodium currents in acutely isolated mice hippocampal CA3 pyramidal neurons

The effect of opioid agonist U50488H on Na(+) currents was examined in freshly dissociated hippocampal neurons of mice using the whole-cell patch clamp technique. U50488H (1-100 microM) caused a concentration dependent reversible inhibition of the voltage-activated sodium currents. IC50 of 15.5 microM and Hill constant of 1.4 were calculated respectively. The inhibitory actions of U50488H on I(Na) were still observed in the presence of 30 microM naloxone. Moreover, under the action of U50488H, repetitive stimulation induced further inhibition which was frequency-dependent. The activation curve did not change before and after application of 10 microM U50488H. However, after exposure to 10 microM U50488H and repetitive depolarizing at 10 Hz, frequency-dependent inhibition occurred, and a mean shift of half-activation membrane potential by +20 mV could be induced. The inactivation curve was significantly changed toward negative membrane potential with 10 microM U50488H, and further negative shift was observed after repetitive depolarizing at 10 Hz. Our results indicate that U50488H could directly inhibit neuronal Na(+) currents without involvement in the activation of kappa-opioid receptors.     

Oxidative modulation of the transient potassium current IA by intracellular arachidonic acid in rat CA1 pyramidal neurons

Oxidative stress affects cellular membrane lipids and proteins. Using whole-cell patch-clamp recording we demonstrate differential oxidative inhibition of voltage-gated transient (IA) and delayed rectifier [IK(V)] K+ currents by arachidonic acid (AA) and H2O2 in CA1 neurons in hippocampal slice. We show that intracellular application of 1 pm AA or its non-metabolizable analog eicosatetraynoic acid (100 pm) reduced IA by approximately 42% but did not affect IK(V). AA shifted the voltage dependence of steady-state inactivation of IA by 12 mV to more negative potentials whereas the rate of inactivation was unchanged. Surprisingly, intracellular glutathione (GSH, 20 mm) enhanced the effect of AA on maximal IA (-62%) and with AA slowed inactivation of IA. The combination of GSH and extracellular ascorbate (0.4 mm) prevented reduction of IA by AA. Intracellular Trolox (a vitamin E analog, 10 microm) reduced IA by 61%and IK(V) by 39%. Like AA, intracellular Trolox caused a 10-mV left shift of IA steady-state inactivation but Trolox and AA did not cause a shift when coapplied. Extracellular Trolox (100 microm) had no effects on IA. H2O2 (80 microm) reduced both IA and IK(V) in a GSH- and ascorbate-sensitive manner and slowed the rate of inactivation of IA by a factor of 2. Coapplication of H2O2 with GSH and extracellular ascorbate caused approximately 22 mV negative shifts of both steady-state inactivation and activation. We conclude that AA is extremely potent in affecting IA by oxidative modifications. Antioxidants can augment these effects, probably by catalysis of the underlying reactions between oxidants and IA channel proteins.     

Interaction of phencyclidine with voltage-dependent potassium channels in cultured rat hippocampal neurons: comparison with block of the NMDA receptor-ionophore complex

Whole-cell voltage-clamp recording techniques were used to investigate the blockade of voltage-dependent K+ channels by phencyclidine (PCP) in cultured rat hippocampal neurons. All recordings were carried out in the presence of tetrodotoxin (1-2 microM) to eliminate Na+ currents. Step depolarization from a holding potential of -40 mV activated a slowly rising, minimally inactivating K+ current (IK). PCP (0.5-1000 microM) caused a reduction in the maximum conductance of IK [IC50(+30 mV), 22 microM] without altering its voltage dependency. The PCP block of IK diminished at depolarized potentials. Analysis according to the scheme of Woodhull (1973) suggested that block occurs via binding to an acceptor site (presumably within the channel pore) that senses 40-50% of the transmembrane electrostatic field. PCP had no effect on the kinetic properties of IK and the block failed to show use dependency, suggesting that PCP may bind to the IK channel via a hydrophobic mechanism not requiring open channels. For comparison, we also investigated the effect of PCP on the transient K+ current, IA, activated by step depolarization following a 200 msec prepulse to -90 mV (20 mM tetraethylammonium was present in the bathing solution to reduce IK). In contrast to the potent blocking action of PCP on IK, the drug only affected IA at high concentrations [IC50(+30 mV), 224 microM]. At concentrations causing substantial block (300-500 microM), PCP produced an acceleration in the IA inactivation rate, and, for brief (5-6 msec) depolarizing steps, the suppression of IA was use dependent. These observations suggest that PCP block of IA requires open channels. PCP reduced inward current responses induced by the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) at substantially lower concentrations than those required for its effects on K+ channels [IC50(-60 mV), 0.45 microM]. The PCP-like dioxadrol stereoisomer dexoxadrol (10 microM) blocked NMDA-evoked inward current responses, while its behaviorally inactive enantiomer levoxadrol did not. Dexoxadrol and levoxadrol also blocked IK in a stereoselective fashion (IC50's, 73 and 260 microM, respectively), whereas the sigma ligands (+)- and (-)-SKF 10,047 and (+)-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [(+)-3-PPP] had little effect on the current (IC50's, greater than 300-500 microM). We conclude that PCP causes a selective, voltage-dependent block of IK in hippocampal neurons via a PCP- and not a sigma-type acceptor site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus

The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94+/-4.96 microM and 10.86+/-1.06 microM, Emax of 67.47+/-4.00% and 67.14+/-1.79%, n of 0.77+/-0.08 and 0.96+/-0.07, respectively. Berberine 30 microM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 microM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage.     

Dopamine enhances a voltage-dependent transient K+ current in the MMQ cell, a clonal pituitary line expressing functional D2 dopamine receptors

The influence of dopamine on voltage-dependent K+ current (IK) was studied in cultured MMQ cells using the whole-cell patch-clamp technique. IK in nearly all MMQ cells revealed a transient outward current component and inactivated during maintained depolarization lasting 60 ms. The transient component was inhibited by prepulse potentials more positive than -40 mV or by addition of 4 mM 4-aminopyridine to the bathing solution and was insensitive to the external Ca2+ concentration. Thus, this transient K+ current resembled the A-current (IA) found in other cells. Dopamine at 1 microM increased by 50% (P less than 0.001) the peak of IK evoked by a test potential to +80 mV and the response was prevented by pretreatment with 100 nM haloperidol, a D2 receptor antagonist. These data suggest that MMQ clonal pituitary cells possess a voltage-gated K+ A-current and that this current can be modulated by dopamine via D2 receptors.     

Inhibitory effect of lamotrigine on A-type potassium current in hippocampal neuron-derived H19-7 cells

PURPOSE: We investigated the effects of lamotrigine (LTG) on the rapidly inactivating A-type K+ current (IA) in embryonal hippocampal neurons. METHODS: The whole-cell configuration of the patch-clamp technique was applied to investigate the ion currents in cultured hippocampal neuron-derived H19-7 cells in the presence of LTG. Effects of various related compounds on IA in H19-7 cells were compared. RESULTS: LTG (30 microM-3 mM) caused a reversible reduction in the amplitude of IA. The median inhibitory concentration (IC50) value required for the inhibition of IA by LTG was 160 microM. 4-Aminopyridine (1 mM), quinidine (30 microM), and capsaicin (30 microM) were effective in suppressing the amplitude of IA, whereas tetraethylammonium chloride (1 mM) and gabapentin (100 microM) had no effect on it. The time course for the inactivation of IA was changed to the biexponential process during cell exposure to LTG (100 microM). LTG (300 microM) could shift the steady-state inactivation of IA to a more negative membrane potential by approximately -10 mV, although it had no effect on the slope of the inactivation curve. Moreover, LTG (100 microM) produced a significant prolongation in the recovery of IA inactivation. Therefore in addition to the inhibition of voltage-dependent Na+ channels, LTG could interact with the A-type K+ channels to suppress the amplitude of IA. The blockade of IA by LTG does not simply reduce current magnitude, but alters current kinetics, suggesting a state-dependent blockade. LTG might have a higher affinity to the inactivated state than to the resting state of the IA channel. CONCLUSIONS: This study suggests that in hippocampal neurons, during exposure to LTG, the LTG-mediated inhibition of these K+ channels could be one of the ionic mechanisms underlying the increased neuronal excitability.     

Characterization of the neurons of the mouse hypogastric ganglion: morphology and electrophysiology

The somata of mouse hypogastric ganglion cells injected with Lucifer yellow were ovoid in shape, lacked dendritic processes but gave rise to a single axonal process. Antidromic activation demonstrated that some of the cells contained in this ganglion innervated the vas deferens. The passive and active membrane properties of the ganglion cells were determined in current clamp experiments. Cells fired tetrodotoxin-sensitive action potentials in response to intracellularly-applied depolarizing current. In voltage clamp evidence was obtained for both a persistent inward calcium current at potentials between -30 and -40 mV and a transient calcium current evoked by step depolarizations to around -20 mV. In current clamp, however, cells did not fire calcium action potentials in the presence of tetrodotoxin. Three potassium currents, IM (blocked by 1 mM barium and by 30 microM bethanechol), IA (blocked by 2 mM 4-aminopyridine) and IK(Ca) fast (blocked by 100 microM cadmium and by 5 mM tetraethylammonium) were characterized in these neurons. In addition, IK(Ca) slow was observed in a small proportion of cells. Fast, all-or-nothing, excitatory synaptic potentials were recorded in response to single stimuli applied to the afferent fibres running to the ganglion. In most cells the excitatory synaptic potentials were suprathreshold for action potential initiation and were markedly reduced or abolished by 100 microM mecamylamine, 1 mM hexamethonium and following desensitization to 100 microM nicotine. Excitatory synaptic potentials arose from stimulation of a single presynaptic nerve process and are typical of strong synaptic inputs.     

Galantamine blocks cloned Kv2.1, but not Kv1.5 potassium channels

Galantamine is a cholinesterase inhibitor (AChEI) currently used in treatment of Alzheimer's disease (AD). In the present study, the effects of galantamine on currents of cloned Kv2.1 and Kv1.5 potassium channels were investigated by using patch-clamp whole cell recording techniques. Kv2.1 and Kv1.5 were stably expressed in HEK293 cells. Galantamine blocked Kv2.1 current in a concentration-dependent manner. When depolarizing from -50 to +40 mV, the IC50 of galantamine for inhibition of Kv2.1 was 5.6 microM. Galantamine 10 microM shifted the activation curve of Kv2.1 to negative potential by 4.0 mV. At the same concentration, galantamine shifted the inactivation curve to negative potential by 25.2 mV. While Kv1.5 was not sensitive to galantamine, Kv1.5 current was not changed by galantamine at concentration of 10 microM. Our data suggest that galantamine potently blocks Kv2.1, but not Kv1.5 channels.     

Trans-resveratrol, a red wine ingredient, inhibits voltage-activated potassium currents in rat hippocampal neurons

The red wine ingredient trans-resveratrol was found to exert potent neuroprotective effects in different in vivo and in vitro models. Thus far, the mechanisms underlying the neuroprotection were attributed mainly to its antioxidant properties. The aim of this study was to investigate the actions of trans-resveratrol on voltage-gated K(+) channels, which have been implicated in neuronal apoptosis. Superfusion of trans-resveratrol reversibly inhibited both the delayed rectifier (I(K)) and fast transient K(+) current (I(A)) in rat dissociated hippocampal neurons with IC(50) values of 13.6 +/- 1.0 microM and 45.7 +/- 7.5 microM, respectively. The inhibition on I(K) had a slow onset, was neither voltage dependent nor use dependent. Trans-resveratrol (30 microM) shifted the steady-state inactivation curve of I(K) to the hyperpolarizing direction by 20 mV and slowed down its recovery from inactivation. The inhibition on I(A) was similar to that on I(K), but voltage dependent. Superfusion of trans-resveratrol (30 microM) shifted the steady-state activation curve of I(A) to the depolarizing direction by 17 mV. Intracellular application of trans-resveratrol (30 microM) was ineffective. Based on the comparable effective concentrations, the inhibition of voltage-activated K(+) currents by trans-resveratrol may contribute to its neuroprotective effects.     

Phencyclidine selectively blocks the sustained voltage-dependent potassium conductance in PC12 cells

We investigated the effects of phencyclidine (PCP), a psychotomimetic dissociative anesthetic, and several related drugs on voltage-dependent K+ currents in PC12 cells, a neuron-like clonal cell line derived from a rat pheochromocytoma. Whole-cell voltage clamp recordings demonstrated two kinetically distinct voltage-dependent outward (K+) current components in these cells: a rapidly activating and inactivating component, IA, that was selectively eliminated by 4-aminopyridine (2 mM) and a slowly activating, minimally inactivating (sustained) component, IK, that was specifically blocked by tetraethylammonium (20 mM). PCP (1-100 microM) produced a dose-dependent blockade of both IK and IA, however, at low doses the drug selectively reduced IK with little effect on IA; the IC50s for blockade of IK and IA were 4 and 25 microM, respectively. The blockade of IK was voltage-dependent so that the degree of block decreased with increasing depolarization, indicating that the blocking mechanism is likely one in which the positively charged PCP molecule is drawn into the channel pore. Several PCP related drugs also suppressed IK. Thienyl-PCP (TCP), a drug that is behaviorally more potent than PCP, partially blocked IK at low doses (31% at 1 microM), but even at high doses (25 microM) the degree of block was never as great as that produced by PCP. The optically active PCP congeners (+)-PCMP (1-(1-phenylcyclohexyl)-3-methyl-piperidine) and dexoxadrol were also potent blockers of IK. However, in contrast to the stereospecificity these compounds demonstrate in binding to high-affinity PCP receptors and in eliciting PCP-like behavioral responses, their enantiomers (-)-PCMP and levoxadrol showed similar potencies as the parent compounds in blocking IK. These results demonstrate that PCP and related drugs are powerful, selective blockers of IK in PC12 cells. The structure-activity studies indicate that this effect occurs at a site that is pharmacologically distinct from the behaviorally relevant PCP receptor. Blockade of K+ channels is unlikely to be responsible for the psychotomimetic or anti-convulsant properties of PCP, but could account for the convulsant potential of the drug.     

Hsp70 and Hsp40 improve neurite outgrowth and suppress intracytoplasmic aggregate formation in cultured neuronal cells expressing mutant SOD1

The inhibitory effects of opioids on voltage-dependent calcium channels (VDCCs) were investigated in cultured porcine adrenal chromaffin cells using whole-cell patch clamp technique. The effects of the opioid on [Ca(2+)](i) increase and catecholamine secretion induced by high K(+) were also examined in single cells by fura-2 microfluorimetry and amperometry. A depolarizing pulse to 0 mV (test pulse) from a holding potential of -80 mV evoked an inward barium current (I(Ba)), which was reversibly inhibited by methionine-enkephalin. This inhibitory effect of methionine-enkephalin was abolished by naloxone. Selective agonists of opioid receptor subtypes (DAMGO: mu, DPDPE: delta, U50488: kappa) dose-dependently inhibited I(Ba). In inhibitory potency, the order was DAMGO>U50488>DPDPE. These agonists applied sequentially produced a reversible I(Ba) inhibition in the same cells. The inhibitory effect of DAMGO on I(Ba) almost disappeared in the presence of omega-conotoxin GVIA but not omega-agatoxin IVA plus nifedipine. Application of a conditioning prepulse to +100 mV prior to the test pulse partly retrieved the I(Ba) inhibition by DAMGO, suggesting the involvement of voltage-sensitive components in opioid-induced VDCC inhibition. Intracellular application of GDPbetaS or GTPgammaS as well as pretreatment with pertussis toxin significantly reduced the extent of I(Ba) inhibition induced by DAMGO. DAMGO reversibly inhibited the [Ca(2+)](i) increase and catecholamine release induced by high K(+). RT-PCR revealed the expression of mu-, delta- and kappa-opioid receptor mRNAs in cultured adrenal chromaffin cells. These results suggest that porcine adrenal chromaffin cells possess mu-, delta- and kappa-opioid receptors and activation of opioid receptors mainly inhibits N-type VDCCs via pertussis toxin-sensitive G-proteins.     

Modulatory effects of FMRF-NH2 on outward currents and oscillatory activity in heart interneurons of the medicinal leech.

Using single-electrode voltage clamp, heart interneurons of the medicinal leech were shown to possess both a rapidly inactivating outward current, IA, and a more slowly inactivating outward current, IK. IA and IK could be separated by their voltage sensitivity and kinetic properties. FMRF-NH2 (Phe-Met-Arg-Phe-NH2) modulates IK by shifting both steady state activation and inactivation to more hyperpolarized potentials, but it does not affect the time constants. IA and IK appear to use K+ as a charge carrier; a change in the external [K+] produced a shift in the apparent reversal potential in the direction predicted with potassium as the charge carrier. Both IA and IK are sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), and TEA and 4-AP both interfere with the effects of FMRF-NH2 on IK. The biophysical properties of IA and of IK in the presence and absence of FMRF-NH2 were incorporated into a Hodgkin-Huxley model of these currents that could reproduce voltage-clamp data. FMRF-NH2 produces two apparently dissimilar effects on the heartbeat rhythm--acceleration and disruption. We suggest that both effects could result from the hyperpolarizing shifts in steady state activation and inactivation of IK.     

Interaction between alpha 2- and beta-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for beta-adrenergic receptors.

The interaction between alpha 2- and beta-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [3H]dihydroalprenolol ([3H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 microM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 microM clonidine, the maximum concentration which did not inhibit [3H]DHA binding, inhibition curves of [3H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 microM clonidine decreased a beta-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 microM yohimbine completely prevented the clonidine-induced reduction in the beta-adrenergic high-affinity state. In the presence of 200 microM GTP, the effect of clonidine was not observed. In addition, Kd and Bmax values for [3H]p-aminoclonidine ([3H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [3H]PAC binding. Moreover, isoproterenol inhibition of [3H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 microM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for beta-adrenergic receptors, while isoproterenol did not modulate the binding characteristics of alpha 2-adrenergic receptors. Furthermore, these results suggest that regulation between alpha 2- and beta-adrenergic receptors is not bidirectional, but is instead unidirectional from alpha 2-adrenergic receptors to beta-adrenergic receptors.     

Opioids act at mu-receptors to hyperpolarize arcuate neurons via an inwardly rectifying potassium conductance

Intracellular recordings were made from 48 hypothalamic arcuate (ARC) neurons under current- and voltage-clamp in slices prepared from female guinea pigs which had been ovariectomized and pretreated with estradiol. Twenty ARC neurons were silent (RMP: -62 +/- 2 mV) and 28 cells were spontaneously active (7.3 +/- 1.1 Hz; threshold -57 +/- 1 mV). The input resistance (Rin), determined in the potential range between -60 and -80 mV, was 358 +/- 30 M omega (n = 38) and ARC neurons showed inward rectification at potentials negative to the equilibrium potential for potassium. The selective mu-opioid agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) was applied by pressure pipette application at concentrations of 10 or 20 microM. DAGO decreased spontaneous firing and it hyperpolarized 26 of 31 neurons (9.6 +/- 0.8 mV; range 3-21 mV). Concomitant with the hyperpolarization, DAGO caused a decrease in Rin of 32 +/- 3, and the reversal potential, measured from current-voltage plots, was -94 +/- 2 mV. These effects were mimicked by bath concentrations of 0.5-1.0 microM DAGO. In voltage clamp, DAGO caused an outward current to flow at -60 mV (range 50-185 pA, n = 6). This current reversed at -92 +/- 2 mV (n = 6) and exhibited inward rectification. An additional 6 ARC neurons were tested with DAGO in varying extracellular concentrations of K+ (2.5, 5 and 10 mM) and the reversal potential for the effect of DAGO shifted by 58 mV per decade change in extracellular K+ concentration. DAGO decreased spontaneous postsynaptic potentials in some cells, but TTX (1 microM) had no effect on the ability of DAGO to hyperpolarize the membrane. The hyperpolarization and decrease in Rin induced by DAGO were blocked by the opioid antagonist naloxone (100 nM-1 microM). DAGO responsive cells were unaffected by a kappa-opioid agonist (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulphonate; U50,488H), however, 2 of 5 cells also were hyperpolarized by a selective delta-receptor opioid agonist (Tyr-D-Pen-Gly-Phe-D-Pen; DPDPE). The effects of DPDPE, but not DAGO, were blocked by a delta-antagonist (ICI 174,864; 1 microM). The present results indicate that activation of ARC mu-receptors leads to an increase in an inwardly rectifying potassium conductance and a subsequent hyperpolarization of most ARC neurons. We suggest that this mu-receptor-induced hyperpolarization of ARC neurons may underlie the opioid inhibition of reproductive events in the mammal.     

Differential effects of pentylenetetrazole on ion currents of Aplysia neurones

The effect of the convulsant drug pentylenetetrazole (PTZ) on separated membrane current components has been studied in identified voltage-clamped Aplysia neurones. External PTZ blocks the voltage-dependent Na+, Ca2+ currents and the delayed rectifier current (INa, ICa and IK,V, respectively). The amplitude of the Ca2+-activated K+ current (IK,Ca) is increased. The amplitude of the fast inactivating K+ current (IA) is transiently increased at low concentrations of PTZ but is depressed at higher concentrations or after long-lasting application of the drug. The effect of PTZ on leakage current (IL) seems to depend on the cell type. In some cells (R-15, L-7, LP-1) IL is decreased while it is increased in other cells (L-11, BL-1, BR-1). PTZ accelerates the inactivation of IK,V and IA and shifts the current-voltage relation of ICa to negative voltages by 5-8 mV. Pressure injection of PTZ into the neurone did not affect IK,V or IK,Ca. Thus PTZ seems to act on the outside of the plasma membrane. The effect of external PTZ on INa, ICa, IK,V and IL is also observed if the internal Ca2+ activity is buffered with EGTA suggesting that an increase in the internal Ca2+ activity is not involved. At -40 mV PTZ induces a tetrodotoxin-insensitive inward current carried by Na+ ions. PTZ transforms the beating pacemaker cell L-11 into a bursting pacemaker and the bursting pacemaker cell R-15 exhibits 'square-wave'-like oscillations of the membrane potential.     

Different Types of Potassium Outward Current in Relay Neurons Acutely Isolated from the Rat Lateral Geniculate Nucleus

Different classes of potassium (K+) outward current activated by depolarization were characterized in relay neurons acutely isolated from the rat lateral geniculate nucleus (LGN), using the whole-cell version of the patch-clamp technique. A fast-transient current (IA), activated at around - 70 mV, declined rapidly with a voltage-dependent time constant (tau=6 ms at + 45 mV), was 50% steady-state inactivated at - 70 mV, and rapidly recovered from inactivation with a monoexponential time course (tau=21 ms). IA was blocked by 4-aminopyridine (4-AP, 2 - 8 mM) and was relatively insensitive to tetraethylammonium (TEA, 2 - 10 mM). After elimination of IA by a conditioning prepulse (30 ms to - 50 mV), a slow-transient K+ current could be studied in isolation, and was separated into three components, IKm, IKs and a calcium (Ca2+)-dependent current, IK[Ca]. The slow-transient current was not consistently affected by 4-AP (up to 8 mM), while TEA (2 - 10 mM) predominantly blocked IKs and IK[Ca]. The component IKm persisted in a solution containing TEA and 4-AP, activated at around - 55 mV, declined monoexponentially during maintained depolarization (tau=98 ms at + 45 mV), was 50% inactivated at - 39 mV, and recovered with tau=128 ms from inactivation. IKs activated at a similar threshold, but declined much slower with tau=2662 ms at + 45 mV. Steady-state inactivation of IKs was half-maximal at - 49 mV, and recovery from inactivation occurred relatively fast with tau=116 ms. From these data and additional current-clamp recordings it is concluded that the K+ currents, due to their wide range of kinetics and dependence on membrane voltage or internal Ca2+ concentration, are capable of cooperatively controlling the firing threshold and of shaping the different states of electrophysiological behaviour in LGN relay cells.     

Extracellular calcium ions are required for muscarine-sensitive potassium current in bullfrog sympathetic neurons

Cultured bullfrog sympathetic neurons were voltage-clamped in the whole-cell configuration. The extracellular medium contained tetrodotoxin (3 microM) and cesium (1 mM) to block and inward sodium current and a hyperpolarization-activated cation current Attempts were made to separate the M-current from four other potassium currents. Tetraethylammonium (30 mM) was used to block a classical delayed rectifier current (IK) and a fast calcium-activated current (IC). Apamin (30 nM) was used to block a slow calcium-activated current (IAHP). 4-Aminopyridine (1 mM) was used to reduce the amplitude of a transient current (IA). In these conditions, the maximum M-conductance near 0 mV was reduced by as much as 90% when divalent cations such as cobalt (1 mM) were added to the superfusate. The maximum M-conductance was also reduced by as much as 60% when calcium ions were removed from the superfusate. The half-activation voltage in the steady-state activation curve and the reversal potential of the M-current were not significantly changed in the calcium-free solution. It is suggested that the presence of calcium ions in the extracellular space is required for the M-current activation.     

Effects of glycine on neurons in the rat substantia nigra zona compacta: in vitro electrophysiological study

The effects of bath-applied glycine to substantia nigra zona compacta neurons of rat were investigated by intracellular recording techniques in vitro. Superfusion of glycine (1 mM) in the medium hyperpolarized 53% of the neurons recorded with KCl electrodes, whereas 32% of the cells were depolarized. The remaining 15% of neurons was hyperpolarized and then depolarized by the amino acid. In spite of these membrane changes, the action potential firing was depressed. Both hyperpolarization and depolarization were correlated to an outward and an inward current, respectively, when recording in single-electrode voltage-clamp mode. In response to bath application of glycine, the neurons showed a concentration-dependent conductance increase. Micromolar concentrations of glycine (100-300 microM) in the superfusion medium produced a membrane hyperpolarization (outward current) in most of the tested cells, whereas millimolar concentration of amino acid could cause depolarization (inward current) in the same neurons. When the recording electrodes contained K acetate, only hyperpolarizations (outward current) were produced. The potential and current changes and the increase in membrane conductance produced by glycine showed little desensitization when neurons were recorded with K acetate electrodes. The mean reversal potential for the membrane hyperpolarization was -80 mV with intracellular electrodes containing KCl and -84 mV with electrodes containing K acetate. The mean null potential for the depolarizing effect was -46 mV. The reversal potential for the glycinergic responses was shifted to less negative values upon lowering the extracellular concentration of chloride or increasing the extracellular concentration of potassium. Strychnine (1-10 microM) reversibly antagonized both the conductance increase and the membrane changes produced by glycine. Bath application of bicuculline (100 microM) and picrotoxin (200 microM) did not affect glycine responses, while depressing the actions of GABA and muscimol. It is concluded the glycine exerts an inhibition on substantia nigra zona compacta neurons by acting on strychnine-sensitive receptors. The membrane effects produced by glycine result from the activation of a chloride current. In addition, the simultaneous involvement of potassium ions may contribute to the overall effects of glycine.     

Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants

The body wall muscles in Drosophila larvae are suitable for voltage clamp analysis of changes in membrane excitability caused by mutations. Both inward and outward ionic currents are present in these muscle fibers. The inward current is mediated by voltage-dependent Ca2+ channels. In Ca2+-free saline, the inward current is eliminated. The remaining outward K+ currents consist of two distinct components, an early transient IA and a delayed steady IK, which are separable by differences in the rate and voltage dependence of activation and inactivation. The steady-state and kinetic properties of the activation and inactivation processes of these two currents are analyzed. The results provide a basis for quantitative analysis of altered membrane currents in behavioral mutants of Drosophila. Previous studies indicate that mutations in the Shaker (Sh) locus alter excitability in both nerve and muscle in Drosophila. Our results support the idea that the channels mediating IA are molecularly distinct from those mediating IK. All Sh mutations studied specifically affect IA without changing the properties of the calcium current and IK. In certain alleles (ShKS133, Sh102, and ShM) IA is eliminated, permitting detailed studies of IK in isolation of IA. Studies of the alleles that do not eliminate IA provide additional information of the channels. In one such allele, Sh5, voltage dependence of IA activation is shifted to more positive potentials. This is accompanied by a less pronounced shift in the voltage dependence of inactivation. These results suggest that Sh5 mutation affects the voltage-sensitive mechanism of both activation and inactivation processes and that these two processes are not controlled by independent parts of the channel. Furthermore, the differential effects of these alleles on different excitable membranes imply that other genes take part in the control of IA. The effects of Sh5 on muscle depend on developmental stage. In larval muscle, Sh5 reduces the amplitude of IA because of the shift in the current-voltage (I-V) relation. In contrast, in adult Sh5 muscles, IA is reported to be normal in amplitude but shows abnormally rapid inactivation (Salkoff, L., and R. Wyman (1981) Nature 293: 228-230). A different allele, ShrK0120, causes a clear defect in nerve excitability, but analysis of IA in ShrK0120 larval muscle reveals I-V relations, inactivation, and recovery from inactivation similar to those seen in normal fibers. We suggest a possible mechanism of combinations of multiple interacting genes participating in the control of potassium channels to account for the presence of a variety of potassium channels in different excitable membranes.     

Effects of selective and non-selective -opioid receptor agonists on cutaneous C-fibre-evoked responses of rat dorsal horn neurones

We have studied the effects of 3 putative kappa-opioid receptor agonists, U50488H, ethylketocyclazocine (EKC) and dynorphin A1-13 (DYN) on the processing of nociceptive information in the dorsal horn of the rat under halothane anaesthesia. Extracellular single unit recordings were made from convergent or multireceptive lumbar dorsal horn neurones, which could be excited by impulses in A beta and C fibre afferents following transcutaneous electrical stimulation of their ipsilateral hind paw receptive fields and also by noxious and innocuous natural stimuli. Agonists were applied directly onto the surface of the spinal cord. DYN and U50488H consistently produced both a facilitation and inhibition of the C-fibre evoked nociceptive responses of individual cells, these dual effects being relatively insensitive to naloxone antagonism and cancelled each other for the whole population of cells. A beta fibre-evoked responses were little altered. In contrast, EKC consistently depressed C-fibre transmission in a dose-dependent, naloxone reversible manner, analogous to, but considerably less potent than intrathecal morphine under identical experimental conditions. Agonist-induced effects on neuronal responses to natural stimulation (noxious pinch and innocuous prod) were consistent with the changes observed with the electrically evoked responses. The present results therefore indicate that EKC probably exerts its spinal antinociceptive activity in the rat spinal cord in a manner akin to mu-receptor activation. Results with U50488H and DYN indicate that -opioids can excite and inhibit individual neurones but produce no overall change on the whole population, so differing from effects mediated by the other opiate receptors.