omega-agatoxin-iva and 2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline

R-type calcium channels in postsynaptic spines signal through functional calcium microdomains to regulate a calcium/calmodulin-sensitive potassium channel that in turn regulates postsynaptic hippocampal long-term potentiation (LTP). Here, we ask whether R-type calcium channels in presynaptic terminals also signal through calcium microdomains to control presynaptic LTP. We focus on presynaptic LTP at parallel fiber to Purkinje cell synapses in the cerebellum (PF-LTP), which is mediated by calcium/calmodulin-stimulated adenylyl cyclases. Although most presynaptic calcium influx is through N-type and P/Q-type calcium channels, blocking these channels does not disrupt PF-LTP, but blocking R-type calcium channels does. Moreover, global calcium signaling cannot account for the calcium dependence of PF-LTP because R-type channels contribute modestly to overall calcium entry. These findings indicate that, within presynaptic terminals, R-type calcium channels produce calcium microdomains that evoke presynaptic LTP at moderate frequencies that do not greatly increase global calcium levels.

Topics: Adenosine A1 Receptor Antagonists; Analysis of Variance; Animals; Animals, Newborn; Calcium; Calcium Channel Blockers; Calcium Channels, R-Type; Calcium Signaling; Cerebellum; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Long-Term Potentiation; Membrane Microdomains; Neural Pathways; Nickel; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Phosphinic Acids; Piperidines; Presynaptic Terminals; Propanolamines; Purkinje Cells; Pyrazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spider Venoms; Tetrodotoxin; Xanthines

We investigated the contribution of L-, N- and P/Q-type Ca(2+) channels to the [Ca(2+)](i) changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca(2+) imaging. Selective Ca(2+) channel blockers, namely nitrendipine, omega-Conotoxin GVIA (omega-GVIA) and omega-Agatoxin IVA (omega-AgaIVA) were used. The [Ca(2+)](i) changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-D-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca(2+) channel blocker caused differential inhibitory effects on [Ca(2+)](i) responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by omega-GVIA or omega-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of omega-GVIA or omega-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q-type Ca(2+) channels activated by stimulation of the AMPA/kainate receptors.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Channels, N-Type; Calcium Channels, P-Type; Calcium Channels, Q-Type; Cells, Cultured; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Kainic Acid; Neurons; Nitrendipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Quinoxalines; Rats; Rats, Wistar

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.

Topics: Acetates; Adolescent; Adult; Amines; Anticonvulsants; Calcium; Calcium Channel Blockers; Child; Child, Preschool; Cyclohexanecarboxylic Acids; Excitatory Amino Acid Antagonists; Female; Gabapentin; gamma-Aminobutyric Acid; Humans; Immunoblotting; Male; Middle Aged; Neocortex; Neurons; Norepinephrine; omega-Agatoxin IVA; Potassium; Pregabalin; Quinoxalines; Receptors, AMPA; Synaptosomes

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.

Topics: 2-Amino-5-phosphonovalerate; Acetates; Amines; Animals; Aspartic Acid; Calcium; Cyclohexanecarboxylic Acids; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Gabapentin; gamma-Aminobutyric Acid; Glutamates; Male; Neostriatum; Neurons; Neurotransmitter Agents; Norepinephrine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Potassium; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Synaptosomes; Tritium