Localization of L-type Ca2+ channels at perisynaptic glial cells of the frog neuromuscular junction

Abstract

The presence of L-type Ca2+ channels at the frog neuromuscular junction (nmj) was studied by monitoring changes in intracellular Ca2+ evoked in presynaptic terminals and perisynaptic Schwann cells (PSCs) and by studying the distribution of Ca2+ channels using a monoclonal antibody directed against the alpha 2/delta subunit of L channels. L-type Ca2+ channel agonist and antagonist had no effect on resting level of fluorescence and nerve-evoked Ca2+ responses in presynaptic terminals. However, depolarization of PSCs induced by KCl (25 mM) produced entry of Ca2+, which was prevented by L-type Ca2+ channel blockers, in (+)R Bay K 8644 of nimodipine. Labeling of Ca2+ channels revealed an intracellular epitope with an irregular and spotty distribution along the endplate. Similar results were obtained with a fluorescent phenylalkylamine [(-)DM-BODIPY-PAA], a blocker of L-type Ca2+ channels. Ca2+ channel labeling remained in absence of nerve terminals but was absent after mechanical removal of nerve terminals and PSCs. Most Ca2+ channel spots were distributed in between bands of cholinergic receptors labeled with alpha-bungarotoxin-TRITC. Cross sections of motor endplates revealed that labeling of Ca2+ channels was found only at the level of the synaptic cleft and not all around the PSCs. We conclude that L-type Ca2+ channels are located in perisynaptic glial cells in an appropriate location to sense depolarization induced by neurotransmitters and thus may support possible roles of glial cells on synaptic function.

Expression of voltage‐gated Ca2+ channel subtypes in cultured astrocytes

“…Subsequent pharmacological and physiological investigations further confirmed this finding by reporting L-and T-type Ca 2ϩ currents in cultured astrocytes (MacVicar and Tse, 1988;Barres et al, 1989;Trudeau, 2000;De Pina-Benabou et al, 2001). At the frog neuromuscular junction, L-type voltage-gated Ca 2ϩ channels are strategically localized in perisynaptic glia, suggesting that this location enables them to sense neuronal depolarization and possibly modulate synaptic transmission (Robitaille et al, 1996). Other studies demonstrate that the expression of L-type voltage-gated Ca 2ϩ channels in astrocytes in vivo is increased following diverse neural trauma such as ischemia and anoxia (Westenbroek et al, 1998;Brown et al, 2001).…”

Non-myelin-forming perisynaptic Schwann cells express protein zero and myelin-associated glycoprotein

“…Denervation of muscles was performed in a manner similar to the method of Robitaille et al (1996Robitaille et al ( , 1997. We injected 0.3 mg/g frog body weight of MS-222 (3-aminobenzoic acid ethyl ester, methane sulfonate salt; Sigma Chemical, St. Louis, MO) dissolved in FRS into a dorsal lymphatic sac to anaesthetize frogs.…”

Synapse–Glia Interactions at the Mammalian Neuromuscular Junction

“…The response of PSCs to synaptic activity could be attributable to an increase of extracellular K ϩ ions during high-frequency stimulation that would depolarize the cell and open voltage-gated Ca 2ϩ channels (MacVicar, 1984;Newman, 1986;Barres et al, 1990;Robitaille et al, 1996) or to the release of neurotransmitters (Jahromi et al, 1992;Reist and Smith, 1992;Robitaille, 1995). To distinguish between the two possibilities, the Ca 2ϩ -dependent release of neurotransmitters was prevented by blocking the Ca 2ϩ channels that trigger this process.…”

“…Thus, substances released by the nerve terminal during evoked activity are necessary for the induction of Ca 2ϩ responses in PSCs, suggesting that PSCs are sensitive to transmitters released during synaptic activity. The residual Ca 2ϩ response in PSCs in the presence of -CgTx MVIIC is likely attributable to the depolarization of the cell caused by K ϩ accumulation during synaptic activity (Jahromi et al, 1992;Robitaille et al, 1996).…”