Action potential modulates Ca2+-dependent and Ca2+-independent secretion in a sensory neuron

Abstract

Neurotransmitter release normally requires calcium triggering. However, the somata of dorsal root ganglion (DRG) neurons possess a calcium-independent but voltage-dependent secretion (CIVDS) in addition to the classic calcium-dependent secretion (CDS). Here, we investigated the physiological role of CIVDS and the contributions of CIVDS and CDS induced by action potentials (APs) in DRG soma. Using membrane capacitance measurements, caged calcium photolysis, and membrane capacitance kinetics analysis, we demonstrated that AP-induced secretion had both CIVDS and CDS components. Following physiological stimuli, the dominant component of AP-induced secretion was either CIVDS for spontaneous firing or CDS for high-intensity stimuli. AP frequency modulates CDS-coupled exocytosis and CIVDS-coupled endocytosis but not CIVDS-coupled exocytosis and CDS-coupled endocytosis. Finally, CIVDS did not contribute to excitatory postsynaptic currents induced by APs in DRG presynaptic terminals in the spinal cord. Thus, CIVDS is probably an essential physiological component of AP-induced secretion in the soma. These findings bring novel insights into primary sensory processes in DRG neurons.

Figure 1

Stimulus-induced secretion consists of CIVDS and CDS components in the somata of DRG neurons. (A) Depolarization- and UV-flash-induced C_m signals in a DRG neuron dialyzed with 5 mM NP-EGTA and 0.2 mM fura-6F. In Ca2+-free extracellular solution (left panel), a step depolarization (200 ms) evoked CIVDS (C_m0). When extracellular Ca²⁺ was added (right panel), UV flashes (arrows) evoked a substantial increase of C_m (C_m(UV)), which was caused by the Ca²⁺ increase in the absence of membrane depolarization. Series conductance (Gs) and membrane current (I_m), and Ca²⁺ trace ([Ca²⁺]i) are also shown. (B) Depolarization-induced C_m signals. In Ca²⁺-free extracellular solution (left panel), a step depolarization (200 ms) evoked a C_m increase (CIVDS) followed by a rapid reversal as in A. C_m1 refers to the entire course. Subsequently, extracellular Ca²⁺ (2.5 mM) was puffed (right panel), and the same stimulus evoked a biphasic response (C_m2). The first Cm increase was followed by a small but rapid reversal and then a slow but substantial increase. C_m2 − C_m1 (dashed line) indicated the CDS component.

Figure 2

Larger AP number increases the fractional contribution by CDS. (A) Different AP numbers induced C_m signals (upper panel) in the presence (solid traces) or absence (dashed traces) of 2.5 mM Ca²⁺. Simultaneous measurements of [Ca²⁺]i are also presented with 0.1 mM fura-2K in the patch pipette. The numbers of APs and their trigger times are shown. (B) Average results from six neurons; each neuron was stimulated by 5 APs at 40 Hz and 20 APs at 40 Hz in Ca²⁺-free and 2.5 mM Ca²⁺ solutions. Empty bars represent Ca²⁺-free bath, and striped bars represent the 2.5 mM Ca²⁺ condition. No significant difference was found when 5 APs at 40 Hz were applied (ΔC_m(0Ca) = 74 ± 10 fF; ΔC_m(2.5Ca) = 75 ± 11 fF); whereas a stronger stimulus, 20 APs at 40 Hz, induced a significant increase under 2.5 mM Ca²⁺ conditions (ΔC_m(0Ca) = 150 ± 28 fF; ΔC_m(2.5Ca) = 293 ± 39 fF; p < 0.01). (C) Fractional contribution of CIVDS to total AP-induced secretion in 2.5 mM Ca²⁺ extracellular solution. This was defined as the ratio of ΔC_m(0Ca)/ΔC_m(2.5Ca). The fractional CIVDS induced by 5, 20, or 40 APs at 40 Hz were 1.03 ± 0.17, 0.56 ± 0.05, and 0.35 ± 0.06, respectively. For 5 APs vs. 20 APs: p < 0.05; 5 APs vs. 40 APs: p < 0.01; 20 APs vs. 40 APs: p < 0.05. (D) Average results of ΔC_m in Ca²⁺-free bath triggered by 5, 20, or 40 APs at 40 Hz were 80 ± 8, 173 ± 19, and 198 ± 28 fF, respectively. Five APs at 40 Hz vs. 20 APs at 40 Hz: p < 0.001; 5 APs at 40 Hz vs. 40 APs at 40 Hz: p < 0.001; 20 APs at 40 Hz vs. 40 APs at 40 Hz: p > 0.1. Numbers in parentheses indicate number of cells.

Figure 3

High AP frequency increases the CDS component in DRG neurons. (A) Profiles of ΔCm induced by different AP frequencies in the presence of 2.5 mM extracellular Ca²⁺ (solid traces) or in Ca²⁺-free bath (dashed traces). (B) Averaged results showing that in Ca²⁺-free bath, 20 APs at 1 Hz induced a similar secretion as in 2.5 mM Ca²⁺ (ΔC_m = 121 ± 22 fF; vs. 153 ± 35; n = 4). On the other hand, 20 APs at 40 Hz induced a greater secretion in 2.5 mM Ca²⁺ (ΔC_m = 318 ± 32 fF) than in Ca²⁺-free solution (ΔC_m = 173 ± 19 fF, p < 0.001; n = 11).

Figure 4

High AP frequency accelerates CIVDS-coupled rapid endocytosis (RE) but not CDS-coupled slow endocytosis (SE). (A, left panel) In Ca²⁺-free extracellular solution, 40 APs at 40 Hz (solid trace) induced faster endocytosis than at 4 Hz (dashed trace). The C_m increases were 185 ± 21 fF and 122 ± 15 fF at 40 Hz and 4 Hz, respectively (n = 7). (Right panel) In 2.5 mM Ca²⁺, 40 Hz or 4 Hz induced similar rates of endocytosis. The C_m increases were 551 ± 44 fF and 519 ± 58 fF at 40 Hz and 4 Hz, respectively (n = 8). (B) Averaged results showing the ratio of endocytosis to exocytosis at low and high frequencies. In the Ca²⁺-free bath (left panel), the ratios at 4 Hz and 40 Hz were 0.37 ± 0.07 and 0.67 ± 0.05, respectively (p < 0.001, n = 7), whereas in 2.5 mM Ca²⁺ (right panel), the ratios were 0.03 ± 0.03 and 0.00 ± 0.02, without significant difference (n = 8).

Figure 5

CIVDS does not contribute to EPSCs of DRG-DH synapses in primary cocultured DRG and DH neurons. (A, upper left, a) DRG and DH neurons were cocultured for at least 6 days before stimulation of DRG neurons with 50 mM KCl. (Right, b) Whole-cell EPSCs recorded in a DH neuron in Ca²⁺-free and 2.5 mM Ca²⁺ bath solution. The bar represents the application of 50 mM KCl. Part of the EPSC trace at 2.5 mM Ca²⁺ was expanded. The slow changes in whole-cell currents during KCl application were probably caused by the voltage-independent K channels responsible for resting membrane potential. Similar results were obtained in four independent experiments. (B) Average results of the peak EPSCs, which were subtractions of total KCl-induced currents in 0 and 2.5 mM Ca²⁺ bath, from DRG-DH coculture was 1.5 ± 0.3 nA (n = 4).