Electrically induced changes in Ca2 in Helisoma neurons: Regional and neuron‐specific differences and implications for neurite outgrowth

Abstract

The present experiments addressed the questions of how electrical stimulation influenced the magnitude, time course, and regional levels of free intracellular calcium of different identified neurons. The calcium concentration in the growth cones, neurites and cell bodies of Helisoma buccal neurons B4 and B19 was measured while somata were electrically stimulated via an intracellular electrode. The findings showed that calcium levels in B4 and B19 increased monotonically with increasing stimulation frequency. However, the range of calcium levels evoked by electrical stimulation differed significantly for each type of neuron. The greater increase in calcium concentration in B4 was correlated with its longer duration action potential compared to B19. The increase in calcium concentration was much smaller in the cell bodies than in the growth cones and neurites. Extending the duration of the B19 action potential produced a sixfold increase in the change in calcium concentration at 2 Hz stimulation. Under conditions where the electrical stimulation produced a calcium concentration of < 160 nM, the elevated level of free intracellular calcium remained constant. When calcium concentration increased above 200 nM in both identified neurons, an initial peak concentration was followed by a decline to a lower concentration suggesting increased calcium buffering occurring above 200 nM. By correlating the calcium concentration data herein with growth data from a previous study, we suggest that specific calcium levels that influence neurite outgrowth may differ widely between neurons.

The role of action potentials in determining neuron‐type‐specific responses to nitric oxide

“…These cell‐specific effects may be attributed to the intrinsic properties of the APs, producing a set point of [Ca 2+ ] i in the growth cone to elicit distinct behaviors. Our findings as well as those of others (Bolsover and Spector, ; Ross et al, ; Torreano and Cohan, ; Kuznetsov et al, ), suggest that the width of an AP may be important in how the electrical activity affects the calcium dynamics and thus the resulting behavior produced by the growth cone.…”

“…We showed that B5 neurons reach a higher [Ca 2+ ] i per defined firing frequency than B19 neurons, which can be attributed to wider APs in B5 neurons. By widening the B19 AP using the K + channel blockers TEA and 4AP, we increased the growth cone [Ca 2+ ] i per AP, similar to what was found previously in Helisoma using long term 45 minute electrical stimulations (Torreano and Cohan, ). Therefore, intracellular signaling events that change the AP waveform are expected to strongly influence cell‐specific calcium dynamics, a finding we needed below to test the relationship between AP parameters, [Ca 2+ ] i , and filopodial elongation.…”

“…Growth cones of Helisoma B5 and B19 neurons produce regenerative APs, which are indistinguishable from those generated in their somata, suggesting the presence of the same ion channels in both cellular compartments (Guthrie et al, ). Measurements of calcium showed a tight temporal relationship between firing frequency and growth cone [Ca 2+ ] i , which has been described previously in studies of both invertebrate (Cohan et al, ; Ross et al, ; Torreano and Cohan, ) and vertebrate (Bolsover and Spector, ; Fields et al, ) neurons. Here, we further develop these findings by showing that deviations from spontaneous activity during depolarizing or hyperpolarizing stimuli can increase and decrease [Ca 2+ ] i , respectively, suggesting for a wider range of fluctuating [Ca 2+ ] i in developing B5 and B19 neurons than previously thought.…”

“…Bolsover et al (1986) first showed that the duration of an AP can shape the magnitude of the resulting [Ca 2+ ] i . Since then a number of studies have supported the notion that AP duration dictates the amount of calcium brought in by an AP (Ross et al, ; Torreano and Cohan, ; Kuznetsov et al, ). We showed that B5 neurons reach a higher [Ca 2+ ] i per defined firing frequency than B19 neurons, which can be attributed to wider APs in B5 neurons.…”

“…The highly regulated intracellular calcium concentration ([Ca 21 ] i ) in neurons determines a variety of cellular processes, such as neurotransmitter release (Schneggenburger et al, 2012), axonal guidance (Zheng and Poo, 2007), synaptic plasticity, and gene regulation (Cohen and Greenberg, 2008). The spiking activity of a neuron is a major factor determining [Ca 21 ] i , and neuromodulators can contribute to changes in [Ca 21 ] i by changing firing frequencies and action potential (AP) parameters within cell bodies, neurites, and growth cones (Bolsover and Spector, 1986;Cohan et al, 1987;Ross et al, 1987;Torreano and Cohan, 1997;Kuznetsov et al, 2012).…”