omega-agatoxin-iva and nickel-chloride

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

Most crustacean muscle fibers receive double excitatory innervation by functionally different motor neurons termed slow and fast. By using specific omega-toxins we show that the terminals of the slow closer excitor (SCE) and the fast closer excitor (FCE) at a crab muscle are endowed with different sets of presynaptic Ca(2+) channel types. omega-Agatoxin, a blocker of vertebrate P/Q-type channels, reduced the amplitude of EPSCs by decreasing the mean quantal content of transmitter release in both neurons by 70-85%, depending on the concentration. We provide the first evidence that omega-conotoxin-sensitive channels also participate in transmission at crustacean neuromuscular terminals and are colocalized with omega-agatoxin-sensitive channels in an axon-type-specific distribution. omega-Conotoxin, a blocker of vertebrate N-type channels, inhibited release by 20-25% only at FCE, not at SCE endings. Low concentrations of Ni(2+), which block vertebrate R-type channels, inhibited release in endings of the SCE by up to 35%, but had little effects in FCE endings. We found that two neuropeptides, the FMRFamide-like DF(2) and proctolin, which occur in many crustaceans, potentiated evoked transmitter release differentially. Proctolin increased release at SCE and FCE endings, and DF(2) increased release only at FCE endings. Selective blocking of Ca(2+) channels by different omega-toxins in the presence of peptides revealed that the target of proctolin-mediated modulation is the omega-agatoxin-sensitive channel (P/Q-like), that of DF(2) the omega-conotoxin-sensitive channel (N-like). The differential effects of these two peptides allows fine tuning of transmitter release at two functionally different motor neurons innervating the same muscle.

Topics: Animals; Axons; Brachyura; Calcium Channel Blockers; Calcium Channels; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; FMRFamide; In Vitro Techniques; Muscles; Neuromuscular Junction; Neurons; Neuropeptides; Neurotransmitter Agents; Nickel; Oligopeptides; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Synaptic Transmission

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and gamma-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Topics: Action Potentials; Animals; Baclofen; Brain Stem; Cadmium Chloride; Calcium Channel Blockers; Calcium Channels; Electric Stimulation; Evoked Potentials; In Vitro Techniques; Neurons; Nickel; Nifedipine; Nimodipine; omega-Agatoxin IVA; omega-Conotoxins; Patch-Clamp Techniques; Peptides; Rats; Rats, Wistar; Receptors, GABA-B; Receptors, Metabotropic Glutamate; Spider Venoms; Synapses

The involvement of different subtypes of voltage-sensitive (Ca2+ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [3H]acetylcholine ([3H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [3H]ACH were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca(2+)-channel blocker omega-conotoxin GVIA (omega-CgTX, 0.01-1 microM) reduced the stimulation-evoked release of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-type Ca2+ channel antagonist omega-agatoxin IVA (omega-Aga IVA) (0.05 microM) and the inorganic Ca(2+)-channel blocker Ca2+ (0.2 mM) inhibited the outflow of both transmitters, while Ni2+ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) with Ca(2+)-free solution inhibited the evoked-release of ATP and [3H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [3H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+]0 condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca2+ chelators, and dipyridamole (2 microM), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca(2+)-free media. Tetrodotoxin (TTX, 1 microM), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca2+]0 conditions. In summary, we showed that both N- and P-type Ca2+ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [3H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [CA2+]0 conditions an additional release of ATP occurs, which is not associated with action potential propagation.

Topics: Acetylcholine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cadmium Chloride; Calcium; Calcium Channel Blockers; Electric Stimulation; Habenula; In Vitro Techniques; Isotope Labeling; Male; Nickel; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Rats; Rats, Sprague-Dawley; Spider Venoms; Tetrodotoxin; Tritium