Dependence of transient and residual calcium dynamics on action-potential patterning during neuropeptide secretion

Abstract

Secretion of the neuropeptide arginine vasopressin (AVP) from the neurohypophysis is optimized by short phasic bursts of action potentials with a mean intraburst frequency around 10 Hz. Several hypotheses, most prominently action-potential broadening and buildup of residual calcium, have been proposed to explain this frequency dependence of AVP release. However, how either of these mechanisms would optimize release at any given frequency remains an open question. We have addressed this issue by correlating the frequency-dependence of intraterminal calcium dynamics and AVP release during action-potential stimulation. By monitoring the intraterminal calcium changes with low-affinity indicator dyes and millisecond time resolution, the signal could be dissected into three separate components: rapid Ca(2+) rises (Delta[Ca(2+)](tr)) related to action-potential depolarization, Ca(2+) extrusion and/or uptake, and a gradual increase in residual calcium (Delta[Ca(2+)](res)) throughout the stimulus train. Action-potential stimulation modulated all three components in a manner dependent on both the stimulation frequency and number of stimuli. Overall, the cumulative Delta[Ca(2+)](tr) amplitude initially increased with f(Stim) and then rapidly deteriorated, with a maximum around f(Stim) </= 5 Hz. Residual calcium levels, in contrast, increased monotonically with stimulation frequency. Simultaneously with the calcium measurements we determined the amount of AVP release evoked by each stimulus train. Hormone release increased with f(Stim) beyond the peak in Delta[Ca(2+)](tr) amplitudes, reaching its maximum between 5 and 10 Hz before returning to its 1 Hz level. Thus, AVP release responds to the temporal patterning of stimulation, is sensitive to both Delta[Ca(2+)](tr) and Delta[Ca(2+)](res), and is optimized at a frequency intermediate between the frequency-dependent maxima in Delta[Ca(2+)](tr) and Delta[Ca(2+)](res).

Fig. 1.

Frequency dependence of intraterminal calcium changes. A, Superposition of the fluorescence changes, ΔF/F₀, recorded with the low-affinity calcium indicator dye Mag-Fluo-4/AM (K_{D, Ca} = 22 μm) in the mouse neurohypophysis during trains of 40 action potentials at the indicated frequencies. The fluorescence data were low-pass-filtered at 1 kHz and corrected for dye bleaching by subtraction of a reference trace without stimulation. The data in A were also passed through a seven-point smoothing algorithm, and their final amplitudes were matched to the amplitude of the first Δ[Ca²⁺]_tr of the control trace at 15 Hz. In the inset, the complete time course of the truncated 1 Hz fluorescence trace is shown. B, Initial Δ[Ca²⁺]_tr amplitudes from the data in Figure 1 before matching their peaks. Even without peak matching, ΔF/F₀ values are within 10% of one another. In contrast, the corresponding values for the resting fluorescence F₀ (not in chronological order) changed nearly twofold during the course of these recordings, as shown in C.

Fig. 2.

Amplitude variation of the transient Ca²⁺ rise. Shown are fractional changes in the amplitude of the transient Ca²⁺ rise, Δ[Ca²⁺]_tr, during a train of 40 action potentials at the indicated stimulation frequencies (●, 1 Hz; ○, all other frequencies), normalized to the first transient of each train. Three different patterns of facilitation and depression emerge. At low frequencies (f_Stim ≤ 1 Hz), Δ[Ca²⁺]_tr amplitudes are essentially constant throughout the train (●). At moderate frequencies (1 Hz < f_Stim ≤ 15 Hz), Δ[Ca²⁺]_tr amplitudes facilitate during the initial stimuli but deteriorate later in the train. Finally, at higher frequencies, depression of Δ[Ca²⁺]_tr amplitudes dominates throughout the train, resulting in Δ[Ca²⁺]_tr amplitudes deteriorating to <20% of their initial values at the end of 40 stimuli.

Fig. 3.

Decay of intraterminal calcium levels after stimulation. Shown is the decay of [Ca²⁺]_i at the end of stimulation with 40 action potentials at the indicated f_Stimon linear (A) and logarithmic (B) time scales. The y-axis scale in B is identical to the one in A. Thesolid lines in A represent the raw data points. In B the raw data from A were resampled uniformly on a logarithmic time scale. The open circles in B are the results from double-exponential fits through the data. C, Changes in the shape of the decay phase of the 1st (——), 5th, and 15th (– – –) Δ[Ca²⁺]_i response during action-potential stimulation atf_Stim = 15 Hz. D, Dependence of the amplitudes (open symbols) and time constants (closed symbols) of the double-exponential fits in B on stimulation frequency (⋄, constant background; ■, ▪, first and ○, ●, second fast component).

Fig. 4.

Changes in residual calcium during stimulation. Shown is the increase of residual calcium, Δ[Ca²⁺]_res, in the nerve terminals of the neurohypophysis as a function of (A) time and (B) the stimulus number, n_Stim, during a train of 40 action potentials at the indicatedf_Stim. The open circlesrepresent the values of indicator fluorescence changes (ΔF/F₀) immediately before a transient Ca²⁺ rise. In B, the spacing between data points (○) at differentf_Stim represents an increment in time of Δt = (f_Stim)⁻¹sec, respectively. The solid lines represent single-exponential fits through the data. With increasingf_Stim, the range of data points conforming to this single-exponential rise rapidly decreases.C, Initial rise times (▪) of Δ[Ca²⁺]_res obtained from the single-exponential fits. The open squares represent the values of the plateau amplitudes of Δ[Ca²⁺]_res. Because the single-exponential fits did not reproduce plateau amplitudes properly, their values were instead set equal to Δ[Ca²⁺]_res atn_Stim = 25. The solid lines in C are only guides to the eye.

Fig. 5.

AVP release and cumulative Ca²⁺changes. A, Basal release (○) and changes in AVP secretion (●) from the neurohypophysis in response to a train of 40 action potentials at the indicated f_Stim. The hormone levels were measured with an EIA. Aliquots from the recording chamber were collected for analysis immediately after the measurement of the stimulation-induced changes in [Ca²⁺]_i. B, Relative changes in the cumulative amplitudes of the transient Ca²⁺ rise (ΣΔ[Ca²⁺]_tr, ▪) during the action potential, and the cumulative residual calcium increase (ΣΔ [Ca²⁺]_res, ■) throughout the stimulus train versus f_Stim. Data in B are normalized to their respective peak values. Solid lines in A andB are guides to the eye. The error bars reflect the SDs from six different experiments.

Fig. 6.

Effect of burst stimulation on transient Ca²⁺ rise and AVP release. Shown are Δ[Ca²⁺]_tr amplitudes during stimulation of the neurohypophysis with trains of action-potential bursts at 5 Hz. The bursts (▪) consisted of either (A) 4 × 10, (B) 8 × 5, or (C) 20 × 2 stimuli at 5 Hz, separated by interburst intervals of 4, 2, and 1 sec, respectively. The reference trace (○) in Figure6A–C represents the initial, continuous action-potential train of 40 stimuli at 5 Hz. Thesolid lines through the data are guides to the eye.D, Changes in cumulative Δ[Ca²⁺]_tr amplitudes (white bars) and AVP release (gray bars) for the data in A–C with respect to the values observed with the first reference trace. Values for a second reference trace at the end of the experiments and for the basal release of AVP are shown as well. Results are normalized to the values obtained with the first reference stimulus train.