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J Neurosci Methods. 2000 Oct 30;102(2):165-76.
Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy.

Ren Y, Ridsdale A, Coderre E, Stys PK.

Loeb Health Research Institute, Division of Neuroscience, 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4K9.

Intracellular Ca(2+) plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca(2+) is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca(2+) dynamics in neurons. In constrast, there is virtually no such information for mammalian central myelinated axons, due in large part to technical difficulty with dye loading and imaging such fine myelinated structures. We developed a technique to allow imaging of ionized Ca(2+) in live rat optic nerve axons with simultaneous electrophysiological recording in vitro at 37 degrees C using confocal microscopy. The K(+) salt of the Ca(2+)-sensitive indicator Oregon Green 488 BAPTA-2 and the Ca(2+)-insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-Ca(2+)/low-Na(+) solution. Axonal profiles, confirmed immunohistochemically by double staining with neurofilament-160 antibodies, were clearly visualized by S101 fluorescence up to 800 microm from the cut ends. The Ca(2+) signal was very low at rest, just above the background fluorescence intensity, indicating healthy tissue, and increased significantly after caffeine (20 mM) exposure designed to release internal Ca(2+) stores. The health of imaged regions was further confirmed by a virtual absence of spectrin breakdown, which is induced by calpain activation in damaged CNS tissue. Red and green fluorescence decayed to no less than 70% of control after 60 min of recording at 37 degrees C, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with confocal images remained largely stable as well.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11040413&dopt=Abstract

Pflugers Arch. 2000 Oct;440(6):809-18.
Activation of the human, intermediate-conductance, Ca2+-activated K+ channel by methylxanthines.

Schroder RL, Jensen BS, Strobaek D, Olesen SP, Christophersen P.

NeuroSearch A/S, Ballerup, Denmark.

This study demonstrated that the methylxanthines, theophylline, IBMX and caffeine, activate the human, intermediate-conductance, Ca2+-activated K+ channel (hIK) stably expressed in HEK-293 cells. Whole-cell voltage-clamp experiments showed that the hIK current increased reversibly and voltage independently after the addition of methylxanthines. In current-clamp experiments, theophylline dose-dependently hyperpolarised the cell membrane from a resting potential of -18 mV to -56 mV. The methylxanthines did not affect large-conductance (BK) or small-conductance (SK2), Ca2+-activated K+ channels, demonstrating that the effects were not secondary to a rise in intracellular Ca2+. However, the activation of hIK by theophylline required an intracellular [Ca2+] above 30 nM. The hIK current was insensitive to 8-bromoadenosine cyclic 3',5'-monophosphate (8-bromo-cAMP), forskolin, 8-bromoguanosine cyclic 3',5'-monophosphate (8-bromo-cGMP) and sodium nitroprusside. Moreover, in the presence of inhibitors of protein kinase A (PKA) or protein kinase G (PKG) theophylline still activated the current. Finally, mutation of the putative PKA/PKG consensus phosphorylation site (Ser334) had no effect on the theophylline-induced activation of hIK. Since the observed activation is independent of changes in PKA/PKG-phosphorylation and of fluctuations in intracellular Ca2+, we suggest that the methylxanthines interact directly with the hIK protein.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11041545&dopt=Abstract

Neuroreport. 2000 Sep 28;11(14):3243-6.
Regulation of nociceptin mRNA expression in the septum by dopamine and adenosine systems.

Dassesse D, Ledent C, Meunier JC, Parmentier M, Schiffmann SN.

Department of Neuroscience, School of Medicine, Universite Libre de Bruxelles, Brussels, Belgium.

Most effects of nociceptin are related to blockade of stress and anxiolytic-like effects. This neuropeptide is highly expressed in septal nuclei, which are involved in response to stressful situations. Dopamine and adenosine may have modulatory effects on stress behaviour by acting on septal neurons. We therefore analysed the regulation of septal nociceptin expression using quantitative in situ hybridization following manipulations of adenosine and dopamine neurotransmission. No difference was observed between wild-type and A2A receptor-deficient mice. In both genotypes, chronic treatments with caffeine, an equipotent A1 and A2A adenosine receptor antagonist, did not significantly modify nociceptin expression. 6-Hydroxydopamine-induced dopamine depletion was also without effect. These results demonstrate that dopamine and adenosine are not involved in the regulation of septal nociceptin expression in spite of the involvement of these three neurotransmitters in stress and anxiety behaviours.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11043556&dopt=Abstract

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