Recovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers

The mechanisms through which changes in intracellular Ca2+ concentration ([Ca2+]i) might influence desensitization of neuronal nicotinic receptors (nAChRs) of rat chromaffin cells were investigated by simultaneous patch-clamp recording of membrane currents and confocal microscopy imaging of [Ca2+]i induced by nicotine. Increases in [Ca2+]i that were induced by membrane depolarization or occurred spontaneously did not influence inward currents elicited by focally applied test pulses (10 msec) of nicotine, indicating that raised [Ca2+]i per se did not trigger desensitization of nAChRs. Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B. Conversely, recovery from desensitization was facilitated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the phosphatase 2 B inhibiting complex of cyclosporin A-cyclophilin A, whereas it was impaired by the broad spectrum kinase inhibitor staurosporine. The effects of PMA or staurosporine were prevented by the intracellularly applied Ca2+ chelator BAPTA. The adenylate cyclase activator forskolin accelerated recovery, whereas the selective PKA antagonist Rp-cAMPS had an opposite effect. The action of staurosporine and Rp-cAMPS on recovery from desensitization was additive. It is proposed that when nAChRs are desensitized, they become susceptible to modulation by [Ca2+]i via intracellular second messengers such as serine/threonine kinases and calcineurin. Thus, the phosphorylation state of neuronal nAChRs appears to regulate their rate of recovery from desensitization

PROTECTION OF BRAIN MITOCHONDRIA AGAINST MICRO STROKE-INDUCED INJURY: A STUDY OF ETHYLMETHYLHYDROXYPYRIDINE SUCCINATE IN EXPERIMENTAL MODEL USING TWO PHOTON LASER FLUORESCENT MICROSCOPY

Objective. Mitochondrial injury plays a central role in neuronal death following ischemic stroke. In the present study, we investigated effects of ethylmethylhydroxypyridine succinate on a microstroke-induced mitochondria swelling, a hallmark of mitochondrial injury.Materials and Methods. Ischemic microstroke was induced in Thy1-CFP-MitoS mice expressing cyano fluorescent protein (CFP) in brain mitochondria by impulse infrared laser. Ethylmethylhydroxypyridine succinate 25 mg/kg or saline (control) were administered i.p. at 30 min after the stroke onset. A period of observation was 48 h. Brain images were obtained by two photon laser fluorescent microscopy and analyzed using a software developed in Neurotar Ltd (Finland). Nonparametric Mann-Whitney-Wilcoxon test was used for te statistical analysis of results.Results. Microstroke resulted in mitochondria swelling, i.e. injury, in the zone surrounding the thrombus. The most profound changes of mitochondrial morphology were observed at 2 h from the stroke onset. Ethylmethylhydroxypyridine succinate significantly reduces the stroke-induced swelling at 1 h (p<0.05) and 2 h (p<0.05), as compared to the control.Conclusion. These results suggest that ethylmethylhydroxypyridine succinate significantly protects brain mitochondria against microstroke-induced injury

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl− transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks