Separation of voltage- and ligand-gated calcium influx by optical imaging

    Thomas G. Oertner and Alexander Borst

    Calcium ions can enter neurons through either ionotropic transmitter receptors or through voltage-gated calcium channels. Thus, an observed rise in intracellular calcium concentration upon synaptic stimulation can be due to either one of these mechanisms or to both of them. There is some controversy in the literature about the permeabilty of insect neuronal acetylcholine receptors for calcium (cf. Goldberg et al., J.Physiol. 514.3 (1999) 759-768; van Eyseren et al., J.Insect Physiol. 44 (1998) 227-230). We analyzed the individual contribution of transmitter- and voltage-gated calcium entry in non-spiking somata, acutely dissociated from thoracic ganglia of the locust Locusta migratoria. By optically recording the calcium signal following different stimulation protocols, we isolated the voltage- and the transmitter-gated component and found that these components indeed summate to the total rise in calcium observed under control conditions.

    minifig1.gif - 11487 Bytes Fig. 1: A typical set of experiments on an isolated nerve cell soma filled with fura2.
    A: Control experiment: Carbachol stimulation in current clamp. Top: Color coded change in internal calcium 5s after stimulation onset, scaled to the maximal value. Curves show the time course of the membrane potential (blue), the injected current (green), the measured calcium concentration (red squares) and the sum of experiments B and C (blue). The gray bar indicates the time of carbachol application.
    B: Voltage replay experiment: The time course of the depolarization recorded in A is used as comand voltage in a voltage clamp experiment without carbachol stimulation. The calcium influx is mediated by voltage-gated channels only.
    C: Membrane potential clamped to -60mV and carbachol stimulation as in A. Only ligand-gated channels are activated.

    minifig2.gif - 1087 Bytes Fig. 2:
    Statistical analysis of all cells measured (n=6). Changes in calcium concentration were normalized to the control experiment (=100%). We evaluated the peak change in calcium concentration (black bars) and the integrated change (light gray bars) during the 10s following the onset of stimulation. For both measures, the voltage- and ligand-gated components summate to about 100%. The calcium influx through voltage-gated channels is about three times as large as the ligand-gated calcium influx.

    minifig3.gif - 3148 Bytes Fig. 3:
    (A) Carbachol stimulation in low calcium saline only slightly reduces the amplitude of depolarization (top panel). The calcium signal, however, is completely and reversibly abolished (bottom panel): Calcium ions are entering the cell from the outside. (B) The addition of 0.1mM d-tubocurarine to the perfusion solution blocks the electrical response upon carbachol stimulation (top panel) as well as the calcium signal (bottom panel). After 10min wash in standard saline, both signals are restored to their original amplitude. This demonstates that the stimulated acetylcholine-receptors are of the nicotinic type.

    This work has been published as:
    T. G. Oertner, S. Single und A. Borst (1999): Separation of voltage- and ligand-gated calcium influx in locust neurons by optical imaging. Neurosci. Lett. 274 (2): 95-98
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