Ki-Wug Sung

Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia

To determine whether the pathophysiological processes after transient forebrain ischemia are mediated via a signal pathway involving gp130 (a signal transducer for the interleukin‐6 family), we analyzed changes in the expression of gp130 and its downstream transcription factor, signal transducer and activator of transcription factor 3 (STAT3), in the rat hippocampus of a four‐vessel occlusive ischemia model. Expression of gp130 mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Four hours after ischemic injury, astrocytes expressed gp130 mRNA. Expression of gp130 increased preferentially in the CA1 and dentate hilar regions, and was maintained for at least 2 weeks. Increase in gp130 expression was accompanied by the activation of STAT3 following ischemic injury. Four hours after injury, STAT3 and phosphorylated STAT3 (pSTAT3) were observed in the nuclei of the dentate hilar region, and sequentially in the CA1 region at day 1. By day 3, STAT3 immunoreactivity markedly increased in these areas, where small cells with the morphology of astrocytes showed nuclear and cytoplasmic STAT3 and nuclear pSTAT3 immunoreactivities. These patterns were especially maintained in the CA1 area until 14 days of reperfusion. Double‐labeling experiments revealed that the cells expressing STAT3 and pSTAT3 were glial fibrillary acidic protein‐expressing reactive astrocytes. These results show a coordinated and long‐lasting upregulation of gp130 mRNA and STAT3 activation in reactive astrocytes of the postischemic hippocampus, indicating that they may be involved in the astrocytic response to an ischemic insult. GLIA 41:237–246, 2003.

Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfusion

White matter lesions are thought to result from chronic cerebral ischemia and constitute a core pathology of subcortical vascular dementia. This rarefaction has been known to be associated with microglial activation. We investigated whether minocycline, a microglial inhibitor, attenuates the white matter damage induced by chronic cerebral hypoperfusion that is used as a model of vascular dementia. Male Wistar rats were subjected to bilateral, permanent occlusion of the common carotid arteries (BCCAO) to induce chronic cerebral hypoperfusion. Minocycline or saline was injected daily for 2 weeks after BCCAO. In the corpus callosum and the optic tract, white matter damage observed with Klüver‐Barrera staining was significantly attenuated in the minocycline‐treated group compared to saline‐treated controls. In control rats, immunoreactivities of major basic protein (MBP), Ox‐42 as a microglial marker, and matrix metalloproteinase (MMP)‐2 were increased in the corpus callosum. Minocycline significantly reduced these changes. Co‐expression of Ox‐42 and MMP‐2 was confirmed by double immunofluorescence histochemistry. Our results suggest that chronic treatment with minocycline could be protective against at least some ischemic white matter damage, and its mechanism may be related to suppressing microglial activation.

Increased leptin and decreased ghrelin level after smoking cessation

Smoking cessation is associated with transient increases in body weight. Leptin and ghrelin are known to be major mediators of appetite, weight and the reward pathway. Therefore, this study assessed the changes in the plasma leptin and ghrelin level and their relationship with the body weight and appetite after smoking cessation in the Korean population. Eighteen subjects, who had stopped smoking for 2 months were enrolled in this study. The body mass index (BMI), body fat mass (BFM), waist-hip ratio (WHR), weight and appetite were measured before and after smoking cessation. In addition, the plasma leptin and ghrelin levels were measured. The BMI, BFM, WHR, weight and appetite were significantly higher than baseline in those who had gave up smoking for 2 months (p<0.05). The plasma leptin concentration increased and the plasma ghrelin level decreased after smoking cessation. The change in the leptin level was positively correlated with the change in the body mass index and body fat mass. These results do not support the direct mediation of the leptin-ghrelin-neuropeptide Y (NPY) system on weight gain after smoking cessation. It appears that weight and appetite is regulated by a more complicated mechanism after smoking cessation.

Upregulation of haptoglobin in reactive astrocytes after transient forebrain ischemia in rats.

Immunohistochemistry for haptoglobin (Hp) in the postischemic hippocampus demonstrated an immunoreactivity visible one day after reperfusion and continuing to increase until 14 days after ischemia. The immunoreactivity was most prominent in CA1 and the dentate hilar region, especially in cells with astroglial morphology. Double immunofluorescence histochemistry confirmed colocalization of the Hp and glial fibrillary acidic protein. Furthermore, a reverse transcription-polymerase chain reaction study confirmed an elevated Hp mRNA level in the postischemic hippocampus. The Hp gene expression was also upregulated in C6 and A-172 glioblastoma cell lines after H2O2 treatment. These findings suggest that Hp is synthesized in reactive astrocytes in response to ischemia-reperfusion injury.

Inhibition of the cloned delayed rectifier K+ channels, Kv1.5 and Kv3.1, by riluzole

The action of riluzole, a neuroprotective drug, on cloned delayed rectifier K+ channels (Kv1.5 and Kv3.1) was examined using the whole-cell patch-clamp technique. Riluzole reversibly inhibited Kv1.5 currents in a concentration-dependent manner with an IC50 of 39.69+/-2.37 microM. G-protein inhibitors (pertussis toxin and GDPbetaS) did not prevent this inhibition of riluzole on Kv1.5. No voltage-dependent inhibition by riluzole was found over the voltage range in which channels are fully activated. Riluzole shifted the steady-state inactivation curves of Kv1.5 in a hyperpolarizing direction in a concentration-dependent manner. It accelerated the deactivation kinetics of Kv1.5 in a concentration dependent-manner, but had no effect on the steady-state activation curve. Riluzole exhibited a use-independent inhibition of Kv1.5. The effects of riluzole on Kv3.1, the Shaw-type K+ channel were also examined. Riluzole caused a concentration-dependent inhibition of Kv3.1 currents with an IC50 of 120.98+/-9.74 microM and also shifted the steady-state inactivation curve of Kv3.1 in the hyperpolarizing direction. Thus, riluzole inhibits both Kv1.5 and Kv3.1 currents in a concentration-dependent manner and interacts directly with Kv1.5 by preferentially binding to the inactivated and to the closed states of the channel.

Orexin-A increases cell surface expression of AMPA receptors in the striatum

Accumulating evidence suggests that orexin signaling is involved in reward and motivation circuit functions. However, the underlying mechanisms are not yet fully understood. Here, we show that orexin-A potentiates AMPAR-mediated synaptic transmission in the striatum, possibly by regulating the surface expression of AMPARs. Primary culture of striatal neurons revealed increased surface expression of AMPARs following orexin-A treatment. The increase in surface-expressed AMPARs induced by orexin-A treatment was dependent on both ERK activation and the presence of extracellular Ca(2+). In the corticostriatal synapses of rat brain slices, orexin-A bath-application caused a delayed increase in the AMPAR/NMDAR EPSC ratio, suggesting that orexin-A sets in motion a series of events that lead to functional alterations in the striatal circuits. Our findings provide a potential link between the activation of orexin signaling in the striatum in response to addictive substances and neural adaptations in the reward circuitry that may mediate the long-lasting addiction-related behaviors.

Fluoxetine suppresses synaptically induced [Ca2+]i spikes and excitotoxicity in cultured rat hippocampal neurons

Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca(2+) channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca(2+)](i) spikes. We investigated the effects of fluoxetine on [Ca(2+)](i) spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg(2+) in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca(2+)](i) spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5 min inhibited [Ca(2+)](i) increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca(2+)](i) responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca(2+)](i) responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg(2+)](o)-induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca(2+)](i) signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca(2+)](i) spikes and neuronal cell death.

Open Channel Block of A-Type, Kv4.3, and Delayed Rectifier K+ Channels, Kv1.3 and Kv3.1, by Sibutramine

The effects of sibutramine on voltage-gated K+ channel (Kv)4.3, Kv1.3, and Kv3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak Kv4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC50 value for the sibutramine block of Kv4.3 was 17.3 μM. Under control conditions, the inactivation of Kv4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k+1) and dissociation (k–1) rate constants for the sibutramine block of Kv 4.3 were 1.51 μM–1s–1 and 27.35 s–1, respectively. The theoretical KD value, derived from k–1/k+1, yielded a value of 18.11 μM. The block of Kv4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the Kv1.3 and Kv3.1 currents with an IC50 value of 3.7 and 32.7 μM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of Kv1.3 and Kv3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on Kv4.3, Kv1.3, and Kv3.1 as an open channel blocker.

Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia

Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion.

Interaction of Riluzole with the Closed Inactivated State of Kv4.3 Channels

The effect of riluzole on Kv4.3 was examined using the whole-cell patch-clamp technique. Riluzole inhibited the peak amplitude of Kv4.3 in a reversible, concentration-dependent manner with an IC50 of 115.6 μM. Under control conditions, a good fit for the inactivation of Kv4.3 currents to a double exponential function, with the time constants of the fast component (τf) and the slow component (τs), was obtained. τf was not altered by riluzole at concentrations up to 100 μM, but τs became slower with increasing riluzole concentration, resulting in the crossover of the currents. The inhibition increased steeply with increasing channel activation at more positive potentials. In the full activation voltage range positive to +30 mV, however, no voltage-dependent inhibition was found. Riluzole shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. However, the slope factor was not affected by riluzole. The Ki for riluzole for interacting with the inactivated state of Kv4.3 was estimated from the concentration-dependent shift in the steady-state inactivation curve and was determined to be 1.2 μM. Under control conditions, closed state inactivation was fitted to a single exponential function. Riluzole caused a substantial acceleration in the closed state inactivation. In the presence of riluzole, the recovery from inactivation was slower than under control conditions. Riluzole induced a significant use-dependent inhibition of Kv4.3. These results suggest that riluzole inhibits Kv4.3 by binding to the closed inactivated state of the channels and that the unbinding of riluzole occurs from the closed state during depolarization.