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. 2004 Feb 4;24(5):1226-35.
doi: 10.1523/JNEUROSCI.4286-03.2004.

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx

Valérie De Crescenzo  1 , Ronghua ZhuGe, Cristina Velázquez-Marrero, Lawrence M Lifshitz, Edward Custer, Jeffrey Carmichael, F Anthony Lai, Richard A Tuft, Kevin E Fogarty, José R Lemos, John V Walsh Jr
Affiliations

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx

Valérie De Crescenzo et al. J Neurosci. .

Abstract

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

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Figures

Figure 1.
Figure 1.
Relationship between measured fluorescence and amount of Ca2+ bound to fluo-3 (CaFl). To determine the total amount of Ca2+ released in a syntilla (CaT), measured fluorescence was determined as a function of the amount of Ca2+ bound to fluo-3 (CaFl). Images were acquired of glass capillaries (internal rectangular cross section 20-μm-deep and 200-μm-wide) loaded with known concentrations of fluo-3 (1, 5, 25 50, 75, 100, and 150 μm for each of the points shown from left to right along the abscissa) and saturating [Ca2+] (1.45 mm). Data were fitted with a straight line (r = 0.85) having a slope of 0.072 intensity units per fluo-3 molecule per msec exposure (14 molecules of bound fluo-3 per count per millisecond).
Figure 2.
Figure 2.
Spontaneous Ca2+ syntillas in isolated nerve terminals in Ca2+-free solution at a membrane potential of –80 mV. A, B, Images of one Ca2+ syntilla in a single isolated terminal in Ca2+-free solution (200 μm EGTA) at a holding membrane potential, Vh =–80 mV. Contour plots (A) show same syntilla as B. C, Time course, corresponding to the images in A and B, of signal mass (black) and its first derivative (red); expanded time scale on right. D, Neither syntilla frequency (N = 13 and 18, with and without Ca2+, respectively) nor Ca2+ signal mass (n = 22 and 27, with and without Ca2+, respectively) were altered by extracellular calcium at Vh = –80 mV. N represents the number of observation periods for 21 nerve terminals used, and n represents the number of syntillas.
Figure 3.
Figure 3.
Ca2+ syntillas are mediated by RyRs. All experiments were performed in Ca2+-free solution at a membrane potential of –80 mV. A, Ryanodine (100 μm in the bathing solution) decreased syntilla frequency (N = 21 and 29, without and with ryanodine, respectively) without affecting signal mass (n = 27 and 72). Caffeine (20 mm in puffer pipette) increased both frequency (N = 21) and signal mass (n = 55) at Vh = –80 mV, but only the frequency increase was attenuated by ryanodine (N = 29) (signal mass data, n = 19). B, Ryanodine (10 μm in puffer pipette) increased syntilla frequency (N = 11) and decreased signal mass (n = 7 and 25, without and with ryanodine, respectively). A total of 57 nerve terminals were used for the experiments in A and B. C, RyR receptor localization in a single terminal (stereo pair). A single terminal has been incubated with ryanodine receptor (RyR) antibody and imaged in 3-D. D, Images showing six different syntillas in one nerve terminal in Ca2+-free solution, recorded over a period of 8 sec. E, Map of locations of all Ca2+ syntillas in the same, single terminal illustrated in D, recorded over 12 sec (Vh = –80 mV). Dots indicate site of one Ca2+ syntilla (E, left panel); diameters of circles are proportional to the signal mass of the syntilla (E, right panel). Red symbols correspond to syntillas in D. Recordings in D and E were made in the presence of 20 mm extracellular caffeine to increase syntilla frequency.
Figure 4.
Figure 4.
Properties of Ca2+ syntillas. (A) Distribution of signal mass for 184 Ca2+ syntillas from 49 nerve terminals; arrow indicates mean of distribution which is fit by a single exponential (r2 = 0.89). Mean is 43 × 10–20 moles of Ca2+ or ∼250,000 Ca ions released per syntilla. B, Averaged derivative of 17 signal mass traces recorded at a rate of one image per 5 msec. These syntillas were recorded in the presence of a low caffeine concentration (1 mm), which increased their frequency but did not affect the mean signal mass. Calibration of current in B does not take account of endogenous buffers (see Materials and Methods).
Figure 5.
Figure 5.
Depolarization from –80 to –40 mV in absence of extracellular Ca2+ increases syntilla frequency. Depolarization from –80 to –40 mV elicits two types of response in different terminals: an increase in syntilla frequency (56% of the terminals) (A), or an increase in global [Ca2+]i (44% of the terminals). A, Syntillas in a nerve terminal, after depolarization from –80 to –40 mV for 3.8 sec. The terminal was depolarized three times (i, ii, iii) with 2 min between depolarizations. B, Effect of depolarization from –80 to –40 mV for those terminals in which there was an increase in syntilla frequency (N = 31 and 29, at –80 and –40 mV, respectively); there was no change in mean syntilla signal mass (n = 27 and 82); ryanodine decreased frequency (N = 29 and 28, in the absence and presence of ryanodine, respectively) without affecting signal mass (n = 40 and 49). A total of 50 nerve terminals were used for the data in the figure.
Figure 6.
Figure 6.
Stronger stimulation causes a rise in global [Ca2+] in absence of extracellular Ca2+. A, Images showing increase in global [Ca2+]i in same terminal after 400 msec depolarization to 0 mV (bottom row) or to –40 mV (top row), and traces showing time course of change in global ΔF/F0 corresponding to the images. B, Percentage change in global ΔF/F0 at end of 400 msec depolarization to –40 mV (N = 23) or 0 mV (N = 22) in the presence and absence of ryanodine (N = 21 and 23, respectively). C, Images showing increase in global [Ca2+]i after 20 mm caffeine perfusion and traces showing time course of change in global ΔF/F0 corresponding to the images. Caffeine perfusion begins at red arrow and continues throughout. D, Percentage increase in global ΔF/F0 after 2 min of perfusion in the presence (N = 3) or absence (N = 3) of ryanodine. A total of 34 nerve terminals were used to acquire the data in the figure.
Figure 7.
Figure 7.
The spatiotemporal profile of [Ca2+] caused by a syntilla. A, The Ca2+ release current waveform for the simulated syntillas. The ICa waveform was taken from the experimentally determined Ca2+ current (see Results and Fig. 4B), shown in black circles and referred to the vertical scale on the left. To avoid the effect of noise in the estimated current, these data were fit with a function of two exponentials, one for the rising and one for the falling phases (red line, see legend for time constants, R2 = 0.92). To take into account the additional Ca2+ taken up by the endogenous buffers, the amplitude of the Ca2+ current was multiplied by the relative buffering capacity) of the total endogenous buffer (see Materials and Methods), and this is reflected in the scale on the right. This fitted, scaled waveform (red line, right-side scale) was used as the syntilla ICa waveform in the simulations shown in B and C. The Ca2+ release was simulated over a period of 150 msec, with the peak of the release occurring at 31 msec. B, Simulation of a syntilla located directly under the plasma membrane. Isoconcentration lines show the values of free [Ca2+] taken along a line through the center of the release site. The vertical axis is distance along the PM away from the release site, and the horizontal axis is time. The observed 2-D symmetry along the vertical axis (distance from release) in fact occurs in 3-D, i.e., in all directions along the PM away from the release. C, Simulation of a syntilla located 400 nm away from the plasma membrane. In this case, the isoconcentration lines show the values of free [Ca2+] taken along a line directly.
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