Article Figures & Data
Figures
- Fig. 1.
Age-dependent motility in GFP-containing neurons.A, P1 cell grown in culture for 6 d expresses considerable spine motility. Two images on the left are successive, taken 5 sec apart, showing several dendrites, and spines in focus on one of them. On the right is a subtracted image, showing in black and white a disparity between the two left images, withgray showing the same position of the dendrite in the two images. Note that most of the spines stain black orwhite and that the parent dendrite is primarilygray. B, GFP-containing cell, grown for 2 weeks in culture, same series of images as in A. Much less motility is detected in the subtraction image on theright. C, Same conditions, 30-d-old culture, showing mature dendritic spines, expressing little or no distinct motility. Below, computer-generated contour frames taken from successive images of the spine shown in C witharrow, to illustrate little or no lateral motility of the spine. Scale bars: A–C, 3 μm; C, bottom, 1 μm.
- Fig. 2.
Lack of correlation between spine motility, calcium variations, and afferent innervation. Cells were loaded individually with a calcium dye, OG-1. At the end of the observation time, cultures were exposed to the membrane dye FM4-64 to identify presynaptic terminals. A, Low-power view of a cell taken from a 2-week-old culture, showing in greenthe dendritic field of the cell, soma on the right edge, and inred, FM4-64 puncta. Scale bar, 10 μm. A1, A2, High-power images of dendritic segments taken fromA, indicated by arrows (1, 2). Each image is a composite picture taken at two time points, at which the dendritic spines are at their farthest spatial positions within the 100 sec of observation. Red indicates one position, green indicates the other position, andyellow denotes overlap in the two positions. The main difference between A1 and A2 is in the presence of FM4-64 dots in A2 and absence of dots in A1. A3, A4, Three-week-old culture, showing different images ontop and the same images counterstained with FM4-64 at the bottom. A large FM particle is seen adjacent to the short spine on the left, and a small dot is seen adjacent to the long spine on the right. The difference image on top shows little movement of the left spine and a large movement of the right spine.A4, High-power image of a medium-sized spine, adjacent to an FM dot to show little disparity in position of the spine in space over time. Scale bars: A1–A4, 1 μm.B1–B4, Correlating movement and calcium transients over time. Spines correspond to those shown in A1–A4, with the notations S for spine, D for dendrite, S1 for spine 1, and S2for spine 2. Overlapped are the calcium transients, incontinuous lines, quantified as ΔF/F, and the movement is indicated with dotted lines. A clear lack of correlation between the calcium transients and local motility is seen. In general, calcium transients in the spine head and the parent dendrites are correlated, with notable exceptions. Larger variations in calcium transients are seen in B2 compared with B1, as well as a large disparity between adjacent spines. In B3, a comparison between the mobile and the immobile spines indicates that although both can express simultaneous calcium transients, the long spine is more independent from the parent dendrite than the short one. B4, At a high temporal resolution, large dendritic calcium events are always seen also in the spine, whereas the opposite is not always true, and independent calcium transients can be seen in the spine. Altogether, the spine expresses little motility compared with the other ones.
- Fig. 3.
Motility, length, and frequency of calcium transients as a function of age in culture. A, Maximal motility over 100 sec of observation, as a function of days in culture;n = 293 spines grouped in single days in vitro. B, Spines are longer in young cells than in older ones. The total population of spines was divided into two age groups, older and younger than 2 weeks in vitro.C, Frequency of calcium transients in 100 sec of observation time in the two age groups. n = 157 spines in the young group and 107 spines in the older group.
- Fig. 4.
Relationship between FM4-64 puncta, calcium transients, and motility. Calcium transients were divided into three categories: low, 0–1 events per 100 sec;medium, 2–6 events per 100 sec; andhigh, >6 calcium transients in 100 sec.A, Both in the absence of FM4-64 (left column) and in its presence (right column), the largest movements are exerted by spines with a moderate number of calcium transients, whereas higher and lower rates of calcium transients are associated with relatively smaller movements of the spines. Note, however, that the presence of FM particles was associated with a reduction in the magnitude of movements by a factor of 10.B, By comparison, the initial length of spine is not correlated with the presence or absence of FM particles, nor with the frequency of calcium transients. n = 62, 23, and 15 spines for the FM−, low, medium, and high calcium transients, respectively. n = 42, 28, and 28 spines for the FM+ group, for the low, medium, and high rates of calcium transients, respectively. C, Same as in A, but when only of the subpopulation of the adult spines is analyzed (>14 din vitro).
- Fig. 5.
The size of the FM particle affects spine motility. The FM particles touching the spines were divided into small (<0.3 μm in diameter) and large (>0.3 μm in diameter). A marked difference was found between the two groups in the percentage of motile spine (A), with far fewer motile spines associated with the large FM particle than with the small one. The magnitude of the movement, however, was not correlated with the size of the particle (B). Altogether, when the spines were divided into motile and stable groups (C), the size of FM particles was larger in the stable spines than in the motile ones.
- Fig. 6.
FM puncta constitute a subgroup of synaptophysin-labeled terminals. EGFP-containing neurons were imaged, followed by staining for FM4-64 (red puncta inA). At the end of the experiment, the same culture was fixed and stained for synaptophysin (Sf) (red puncta in B). As seen in the two images, all of the FM puncta have a corresponding Sf-labeled terminal, but some terminals do not stain for FM. Arrows show an example of spine with Sf, but without FM, and arrowheadsshow an example of a naked spine. Scale bar, 3 μm. C,Summary diagram of 473 spines counted on 58 20 μm dendritic segments. The difference between the number of Sf terminals and FM puncta is highly significant (p < 0.01).
- Fig. 7.
Blockade of actin polymerization reduces spine motility but does not affect spontaneous calcium transients.A, Tracing of spine size by a thresholding procedure and overlapping spine sizes in the two extreme positions across 1 min of observation before (1), 1 min (2), and 20 min (3) after exposure of the cell to 2 μm latrunculin. Note that the disparity between the two positions of the spine disappear at 20 min. Note also the change in spine shape after latrunculin.B, Spines cease to move, but continue to express calcium transients after exposure to latrunculin. Before (B1) and 15 min after (B2) onset of exposure of the culture to latrunculin. Same scheme as in Figure 2; continuousand dotted lines denote calcium transients in the spine and the parent dendrite, and broken line denotes measurement of maximal disparity in spatial location of the spine. Scale bar, 0.5 μm.
- Fig. 8.
Lack of temperature effects on spine motility. Five-week-old GFP-labeled cell was imaged at room temperature (A), warmed to 30°C (B) and to 36°C (C), and imaged for 1–5 min in each of the temperatures. At the end of the imaging session, the cell was counterstained with FM4-64 to identify presynaptic terminals. The two left columns indicate the images that were taken 15 sec apart, and the image column on theright is a subtraction image of the two left ones. Only a minor movement is seen with the top left spine, whereas the other ones were stable. Interestingly, the top left spine is the only one not associated with a presynaptic terminal.
- Fig. 9.
Blockade of spontaneous electrical activity enables spine motility. Three-week-old cell in culture, labeled with calcein was studied herein. A dendrite containing several spines was imaged (A), as before, in control (B), after exposure to TTX for 1 hr (C), and after wash (D). In each of these three cases, a subtraction of two images, 20 sec apart, is shown. No spontaneous motility is seen in the normal conditions, whereas blockade of spontaneous activity enabled this motility as inB. Scale bar, 3 μm.
