6-cyano-7-nitroquinoxaline-2-3-dione and iberiotoxin

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Biophysics; Calcium; Cerebellum; Dendrites; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Mice; Mice, Knockout; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Purkinje Cells; Pyridazines; Shaw Potassium Channels; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin

In adult mice, classical conditioning in which whisker stimulation is paired with an electric shock to the tail results in a decrease in the frequency of head movements, induces expansion of the cortical representation of stimulated vibrissae and enhances inhibitory synaptic interactions within the 'trained' barrels. We investigated whether such a simple associative learning paradigm also induced changes in neuronal excitability. Using whole-cell recordings from ex vivo slices of the barrel cortex we found that layer IV excitatory cells located in the cortical representation of the 'trained' row of vibrissae had a higher frequency of spikes recorded at threshold potential than neurons from the 'untrained' row and than cells from control animals. Additionally, excitatory cells within the 'trained' barrels were characterized by increased gain of the input-output function, lower amplitudes of fast after-hyperpolarization and decreased effect of blocking of BK channels by iberiotoxin. These findings provide new insight into the possible mechanism for enhanced intrinsic excitability of layer IV excitatory neurons. In contrast, the fast spiking inhibitory cells recorded in the same barrels did not change their intrinsic excitability after the conditioning procedure. The increased excitability of excitatory neurons within the 'trained' barrels may represent the counterpart of homeostatic plasticity, which parallels enhanced synaptic inhibition described previously. Together, the two mechanisms would contribute to increase the input selectivity within the conditioned cortical network.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Bicuculline; Conditioning, Classical; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; Large-Conductance Calcium-Activated Potassium Channels; Male; Mice; Mice, Inbred C57BL; Neurons; Patch-Clamp Techniques; Peptides; Piperazines; Potassium Channel Blockers; Somatosensory Cortex

Large-conductance calcium-activated potassium channels (BK channels) have been suggested to play a substantial role in synaptic transmission in the spinal cord dorsal horn. In the present experiments, we attempted to clarify the physiological significance of BK channels in the modulation of synaptic transmission in the superficial dorsal horn where nociceptive information is processed. Spontaneously occurring excitatory postsynaptic currents (sEPSCs) were recorded from the neurons located in the superficial dorsal horn of a mouse spinal cord slice, and the effects of iberiotoxin, a BK channel blocker, on sEPSCs were analyzed. The frequency of sEPSCs was significantly higher in the peripheral nerve-ligated neuropathic mice than in the sham-operated control mice, but the amplitude of sEPSCs was equivalent between the two groups. Iberiotoxin increased the frequency of sEPSCs in the control mice to the same level as that in the neuropathic mice without affecting the amplitude of sEPSCs. In contrast, iberiotoxin did not show any significant effects on the sEPSCs in the neuropathic mice. These findings suggest that the BK channels that are located in presynaptic terminals control synaptic transmission in the superficial dorsal horn, and that functional downregulation of BK channels accompanies the neuropathic pain induced by peripheral nerve injury. This downregulation was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of the BK channel alpha subunit. Taken together, our present results indicate that BK channels play crucial roles in the synaptic transmission of nociceptive information in the superficial dorsal horn.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Down-Regulation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Mice; Mice, Inbred ICR; Patch-Clamp Techniques; Peptides; Peripheral Nervous System Diseases; Posterior Horn Cells; Presynaptic Terminals; Spinal Cord; Synaptic Transmission

The cytosolic redox status modulates ion channels and receptors by oxidizing/reducing their sulfhydryl (SH) groups. We therefore analyzed to what degree SH modulation affects hippocampal susceptibility to hypoxia. In rat hippocampal slices, severe hypoxia caused a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). Oxidizing SH groups by 5,5'-dithiobis 2-nitrobenzoic acid (DTNB, 2 mM) postponed HSD by 30%, whereas their reduction by 1,4-dithio-dl-threitol (DTT, 2 mM) or alkylation by N-ethylmaleimide (500 microM) hastened HSD onset. The DTNB-induced postponement of HSD was not affected by tolbutamide (200 microM), dl-2-amino-5-phosphonovaleric acid (150 microM), or 6-cyano-7-nitroquinoxaline-2,3-dione (25 microM). It was abolished, however, by Ni2+ (2 mM), withdrawal of extracellular Ca2+, charybdotoxin (25 nM), and iberiotoxin (50 nM). In CA1 neurons DTNB induced a moderate hyperpolarization, blocked spontaneous spike discharges and postponed the massive hypoxic depolarization. DTT induced burst firing, depolarized glial cells, and hastened the onset of the massive hypoxic depolarization. Schaffer-collateral/CA1 synapses were blocked by DTT but not by DTNB; axonal conduction remained intact. Mitochondria did not markedly respond to DTNB or DTT. While the targets of DTT are less clear, the postponement of HSD by DTNB indicates that sulfhydryl oxidation increases the tolerance of hippocampal tissue slices against hypoxia. We identified as the underlying mechanism the activation of BK channels in a Ca(2+)-sensitive manner. Accordingly, ionic disregulation and the loss of membrane potential occur later or might even be prevented during short-term insults. Therefore well-directed oxidation of SH groups could mediate neuroprotection.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aniline Compounds; Animals; Calcium; Charybdotoxin; Cortical Spreading Depression; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamine; Hippocampus; Hypoxia; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Neurons; Nickel; Oxidation-Reduction; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Sulfhydryl Reagents; Tolbutamide; Valine; Xanthenes