Hydrogen peroxide-induced reduction of delayed rectifier potassium current in hippocampal neurons involves oxidation of sulfhydryl groups.

This study examined the effect of H2O2 on the delayed rectifier potassium current (IKDR) in isolated hippocampal neurons. Whole-cell voltage-clamp experiments were performed on freshly dissociated hippocampal CA1 neurons of SD rats before and after treatment with H2O2. To reveal the mechanism behind H2O2-induced changes in IKDR, cells were treated with different oxidizing and reducing agents. External application of membrane permeable H2O2 reduced the amplitude and voltage-dependence of IKDR in a concentration dependent manner. Desferoxamine (DFO), an iron-chelator that prevents hydroxyl radical (OH) generation, prevented H2O2-induced reduction in IKDR. Application of the sulfhydryl-oxidizing agent 5,5 dithio-bis-nitrobenzoic acid (DTNB) mimicked the effect of H2O2. Sulfhydryl-reducing agents dithiothreitol (DTT) and glutathione (GSH) alone did not affect IKDR; however, DTT and GSH reversed and prevented the H2O2-induced inhibition of IKDR, respectively. Membrane impermeable agents GSH and DTNB showed effects only when added intracellularly identifying intracellular sulfhydryl groups as potential targets for hydroxyl-mediated oxidation. However, the inhibitory effects of DTNB and H2O2 at the positive test potentials were completely and partially abolished by DTT, respectively, suggesting an additional mechanism of action for H2O2, that is not shared by DTNB. In summary, this study provides evidence for the redox modulation of IKDR, identifies hydroxyl radical as an intermediate oxidant responsible for the H2O2-induced decrease in current amplitude and identifies intracellular sulfhydryl groups as an oxidative target.

Oxidative stress alters physiological and morphological neuronal properties.

We investigated the effects of H(2)O(2)-induced oxidative stress on the delayed-rectifier current (IK(DR)), neuronal physiological and morphological properties. Measurements were obtained from hippocampal CA1 neurons in control solution and from the same neurons after exposure to oxidative stress (short- and long-term H(2)O(2) external applications at 0.1, 1, and 10 mM). With short-term (6 min) H(2)O(2) (1 mM) treatment, IK(DR) measured in the H(2)O(2)-containing solution (778 +/- 23 pA, n=20), was smaller than that measured in the control Ca(2+)-free Hepes solution (1,112 +/- 38 pA, n=20). Coenzyme Q(10) (0.1 mM) pretreatment prevented the H(2)O(2)-induced inhibition of IK(DR). With long-term (40, 80 min) H(2)O(2) (0.1, 10 mM) treatment, the neuron lost its distinctive shape (rounded up) and the neurite almost disappeared. These results suggest that oxidative stress, which inhibits IK(DR), can alter neural activity. The morphological changes caused by H(2)O(2) support the idea that oxidative stress causes intracellular damage and compromises neural function.

Vitamins C and E modulate neuronal potassium currents.

We investigated the effects of vitamins C and E on the delayed-rectifier potassium current (IK(DR)), which is important in repolarizing the membrane potential, and on the transient A-type potassium current (IK(A)), which regulates neuronal firing frequency. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The membrane potential was stepped to different voltages between -40 and +60 mV from a holding potential of -80 mV. IK(DR) and IK(A) measured in the vitamin C-containing solution (IK(DR) 305 +/- 16 pA, IK(A) 11 +/- 2 pA) were smaller than those measured in the control solution (488 +/- 21 pA, IK(A )28 +/- 3 pA). By contrast, IK(DR) and IK(A) measured in the vitamin E-containing solution (IK(DR) 561 +/- 21 pA, IK(A )31 +/- 3 pA) were greater than those measured in the control solution (422 +/- 15 pA, 17 +/- 2 pA). These results indicate that vitamins C and E can modulate potassium current amplitudes and possibly lead to altered neuronal excitability.

Modulation of neuronal [Ca2+]i by caffeine is altered with aging.

Voltage-dependent calcium channels play an important role in controlling many neuronal processes such as neuronal excitability and synaptic transmission. Any slight alteration in intracellular calcium concentration ([Ca2+]i) can have a considerable impact on various neuronal functions. The effects of caffeine on [Ca2+]i were studied in CA1 hippocampal neurons of young (2 months) and old (24 months) C57BL mice. Fura 2-AM fluorescence photometry was used to measure [Ca2+]i in the presence and absence of caffeine (100 microM) in response to KCl (26 mM) application. Caffeine enhanced the peak [Ca2+]i as compared to control solution in young mice (control: 325+/-8 nM, caffeine: 402+/-10 nM), but had no effect on the peak [Ca2+]i in old mice (control: 222+/-6 nM, caffeine: 223+/-7 nM). These results indicate that caffeine can impact neuronal functions through the modification of [Ca2+]i. The lack of caffeine-induced modulation of [Ca2+]i in old mice suggests that this role of caffeine has been compromised with aging.

Caffeine modulates potassium currents in Drosophila neurons.

We investigated the effects of caffeine on the delayed-rectifier potassium current (IK(DR)) which is important in repolarizing the membrane potential, and the transient A-type potassium current (IK(A)) which regulates neuronal firing threshold and the rate of repetitive action potentials. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The currents were measured from neurons before and after the application of 1mM caffeine to the external saline of the same neuron. IK(DR) measured in the caffeine-containing solution (470+/-36 pA, n=18), was smaller than that measured in the control 6K/0Ca Tris solution (745+/-51 pA, n=18). IK(A) measured in the caffeine-containing solution (17+/-2 pA, n=16) was smaller than that measured in the control 6K/0Ca Tris solution (35+/-4 pA, n=16). These results indicate that caffeine reduces IK(DR) and IK(A) amplitudes and possibly leads to increased action potential frequency and enhanced neuronal excitability.

Transient K+ current is blocked by lanthanum in Drosophila neurons.

The potassium A-current (IK(A)) is important in regulating the membrane potential between action potentials. The whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IK(A) was measured from neurons before and after application of 0.1 mM lanthanum to the external saline. IK(A) was smaller in the lanthanum-containing saline (7+/-1 pA) than in the control saline (34+/-6 pA). Activation and inactivation of IK(A) were unchanged by lanthanum. These results suggest that lanthanum neurotoxicity may lead to increased neuronal excitability. Moreover, given this inhibition of IK(A), lanthanum should not be used to block calcium current in studies of K+ currents.

Resistance of delayed-rectifier K+ current to cadmium in Drosophila neurons.

The delayed-rectifier potassium current (IKDR) is important in repolarizing the membrane potential and determining the level of neuronal excitability. We investigated the effect of cadmium on this potassium current. The whole-cell patch-clamp technique was used to measure IKDR from cultured Drosophila neurons derived from embryonic neuroblasts. The current was measured from neurons before and after the application of 0.1 mM cadmium to the external saline. IKDR was similar in the cadmium-containing saline (383 +/- 47 pA) and the control saline (401 +/- 60 pA). These results indicate that cadmium neurotoxicity does not specifically affect IKDR in Drosophila neurons.

Pkc-a differentially affects rutabaga and wild-type Drosophila neuronal potassium current.

Learning and memory are defective in the Drosophila mutant rutabaga, which has a low intracellular cyclic adenosine monophosphate (cAMP) concentration. The aim of this study was to compare modulation effects of protein kinase C activator (PKC-A) on the delayed-rectifier potassium current (IKDR) in wild-type and rutabaga neurons. IKDR was measured from cultured (2 days) wild-type and rutabaga neurons. The authors examined the effects of PKC-A on IKDR in wild-type and rutabaga neurons. IKDR was measured from neurons before and after addition of PKC-A to the external solution. IKDR was smaller in rutabaga neurons (380 +/- 25 pA) than in wild-type neurons (529 +/- 44 pA). IKDR was reduced by PKC-A more in wild-type (decreasing 55 +/- 6%) than in rutabaga (decreasing 35 +/- 8%) neurons (single-cell studies). In the presence of PKC-A, there was no difference in IKDR between wild-type (229 +/- 31 pA) and rutabaga (242 +/- 26 pA) neurons (population studies). These results indicate that PKC-A differentially affects the delayed-rectifier channel in wild-type rutabaga.

Blocking effect of lanthanum on delayed-rectifier K+ current in Drosophila neurons.

The delayed-rectifier potassium current (IKDR) is important in regulating neuronal excitability. The authors characterized the neurotoxic effect of lanthanum on IKDR. The conventional whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IKDR was measured from neurons before and after application of 0.1 mM lanthanum to the external saline. IKDR was smaller in the lanthanum-containing saline (441 +/- 57 pA) than in the control saline (680 +/- 35 pA) (p <.001). Activation and inactivation of IKDR were unchanged by lanthanum. Because these results suggest that lanthanum acts as a potent blocker of IKDR, neuronal excitability may be altered during lanthanum neurotoxicity.

Inhibition of transient K+ current by copper in Drosophila neurons.

The transient K+ current (IK(A)) affects the rate of repetitive action potentials. The whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IK(A) was measured from neurons before and after application of 0.1 mM copper to the external saline. IK(A) was smaller in the copper-containing saline (12.0 +/- 1.6 pA) than in the control saline (37 +/- 6.5 pA). Activation and inactivation of IK(A) were unchanged by copper. These results suggest that copper can influence neuronal excitability and may affect neuronal function.

Increased calcium influx through acetylcholine receptors in dunce neurons.

Calcium homeostasis was studied in dunce, a Drosophila mutant that is defective in learning and memory. Fura 2-AM fluorescence photometry was used to measure the intracellular calcium concentration in wild type and dunce cleavage-arrested neurons under resting conditions and in response to neurotransmitters. After acetylcholine application, the peak [Ca2+]i was greater in dunce (693 +/- 125 nM) than in wild type neurons (464 +/- 154 nM), but half decay time was shorter in dunce (66 +/- 15 s) than in wild type neurons (104 +/- 40 s). In contrast, the application of glutamate, NMDA, dopamine, and serotonin had no effect on [Ca2+]i. These results indicate that calcium influx through acetylcholine receptors is increased in dunce, compared to wild type neurons. The results also suggest that calcium extrusion to the outside and/or calcium buffering are enhanced in dunce, compared to wild type neurons. This disturbance in the homeostasis of cytosolic calcium concentration in dunce may be implicated in defective associative learning in Drosophila, and may play a role in acute and chronic neurodegenerative disorders in the mammalian brain.

Drug effects on calcium homeostasis in mouse CA1 hippocampal neurons.

Voltage-dependent Ca2+ channels (VDCC) are important in control of neuronal excitability, synaptic transmission, and many other cellular process. Even the slightest alteration in Ca2+ currents can have a considerable impact on the neuronal function. However, it is still unknown whether Ca2+ currents are affected by neurotoxic drugs such as lead, cobalt, zinc, cadmium, thallium, lanthanum, and aluminum. We have characterized the effects of neurotoxic drugs on Ca2+ homeostasis in CA1 hippocampal C57BL mice. Fura 2-AM fluorescence photometry was used to measure intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of neurotoxic drugs (10 microM) in response to KCl application. The peak [Ca2+]i due to KCl application was reduced in the presence of lead (60%), cobalt (35%), zinc (62%), cadmium (71%), thallium (27%), and lanthanum (66%). By contrast, in the presence of aluminum the peak [Ca2+]i was either increased (46%) or it was not affected. These results indicate that neurotoxic drugs could block the entry of calcium into CA1 neurons via VDCC.

Serotonin reduces potassium current in rutabaga and wild-type Drosophila neurons.

The Drosophila learning mutant rutabaga is defective in short-term memory and has a reduced intracellular cyclic adenosine monophosphate (cAMP) concentration. The delayed-rectifier potassium current (IKDR) was measured from cultured (2 days) wild-type and rutabaga neurons. IKDR was smaller in rutabaga neurons (382 +/- 41 pA) than in wild-type neurons (542 +/- 33 pA). IKDR was measured from neurons before and after addition of serotonin to the external solution. IKDR was reduced by serotonin in wild type (decreasing 37 +/- 7%) and rutabaga (decreasing 33 +/- 6%) neurons (single-cell studies). In the presence of serotonin, IKDR was smaller in rutabaga (218 +/- 24 pA) than in wild-type (426 +/- 35 pA) neurons (population studies). These results indicate that serotonin has affected IKDR so that the inherent difference between the two genotypes was preserved.

Reduced delayed-rectifier K+ current in the learning mutant rutabaga.

In the Drosophila mutant rutabaga, short-term memory is deficient and intracellular cyclic adenosine monophosphate (cAMP) concentration is reduced. We characterized the delayed-rectifier potassium current (IK(DR)) in rutabaga as compared with the wild-type. The conventional whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IK(DR) was smaller in rutabaga (368 +/- 11 pA) than in wild-type (541 +/- 14 pA) neurons, measured in a Ca(2+)-free solution. IK(DR) was clearly activated at approximately 0 mV in the two genotypes. IK(DR) typically reached its peak within 10-20 msec after the start of the pulse (60 mV). There was no difference in inactivation of IK(DR) for wild-type (14 +/- 3%) and rutabaga (19 +/- 3%). After application of 10 mM TEA, in wild-type, IK(DR) was reduced by 46 +/- 5%, whereas in rutabaga, IK(DR) was reduced by 28 +/- 3%. Our results suggest that IK(DR) is carried by two different types of channels, one which is TEA-sensitive, whereas the other is TEA-insensitive. Apparently, the TEA-sensitive channel is less expressed in rutabaga neurons than in wild-type neurons. Conceivably, altered neuronal excitability in the rutabaga mutant could disrupt the processing of neural signals necessary for learning and memory.