Activity-Regulated Dynamic Behavior of Early Dendritic Protrusions: Evidence for Different Types of Dendritic Filopodia

Dendritic filopodia are highly motile protrusions in early postnatal development. Top, Skeleton representation of all 20 frames of the movie shown in Figure 2. Individual protrusions were traced in ImageJ (see Materials and Methods) in all 20 frames of each time-lapse movie. Skeleton versions of movies of filopodia and other protrusions were drawn for all dendrites imaged in this study throughout development from P2 through P12 (a total of 56 dendrite segments and 1008 filopodia), to measure their lengths using custom-written macros in ImageJ. A few representative filopodia are labeled; arrows point to the first and last frame in which those filopodia can be distinguished. Bottom, Graph displaying the lengths of the representative filopodia mentioned above over the same 10-min movie. Most filopodia appear and disappear over the 10-min imaging period (e.g., filopodia #25, #32, and #51). At these early ages, only rare filopodia were present throughout the length of the movie (e.g., filopodium #10).

Dendritic filopodia exhibit a wide range of motile behaviors. The motility of dendritic filopodia at P2-P5 was manifested by a wide range of behaviors. This included extension and retraction (first row), bending along the stem of the filopodium (second row), rotation around an axis at the base of the filopodium (third row), or branching (fourth row). Scale bar, 3 μm (for all panels).

High-resolution two-photon imaging of dendritic filopodia in early postnatal neurons reveals their dynamism: motility is actin dependent. A, Layer 5 pyramidal neuron in visual cortex of a P2 mouse imaged by two-photon microscopy. Inset, Dendrite imaged in B. Scale bar, 25 μm. B, Time-lapse movie of dendrite from cell shown in A (see movie 1 in supplementary data, available at www.jneurosci.org). Numerous filopodia protrude in and out of the dendrite. Note that filopodia seem to be clustered at the dendrite tip (growth cone) compared with the proximal shaft (see also Figs. 6 and 7). Images were acquired every 30 sec, but only half the time points are shown here. Scale bar, 5 μm. C, Collapsed view of all frames of the movies shown above. This displays nicely the exploratory behavior of filopodia throughout the 10-min imaging period. D, Collapsed view of all 20 frames from a 10-min movie of the same dendrite, 5 min after applying cytochalasin D (1 μg/ml), an actin polymerization inhibitor, into the bath (see movie 2 in supplementary data, available at www.jneurosci.org). Cytochalasin D significantly reduced filopodia motility (see Fig. 9A).

Differences between filopodia in axonal and dendritic growth cones and between filopodia in dendritic growth cones and in dendritic shafts. Left, Example of a typical dendritic shaft at P2-P5. Note that filopodia are shorter than in both axonal growth cones and dendritic growth cones, and they are less densely packed. There are also three hotspots of filopodia activity (white arrows). These hotspots were frequently seen in young dendrites but almost never after P6. Middle, Example of a typical dendritic growth cone at P2-P5. Note how individual filopodia are easily identified (no webbing) and that filopodia aim in all directions, including away from the tip of the dendrite. Right, Example of a typical axonal growth cone at P2-P5. Note the long filopodia and the webbing between filopodia. Almost all filopodia are oriented at acute angles toward the tip of the axon. The different rows show different time points in the 10-min time-lapse movies. The last row (below the black arrows) shows collapsed sums of all 20 individual time points from each 10-min movie. Scale bar, 3 μm.

Dendritic shaft filopodia, but not growth cone filopodia, are regulated by neronal activity. A, Bath application of 1 μg/ml cytochalasin D, an inhibitor of actin polymerization (n = 46; four experiments), resulted in an ∼50% decrease in density (p = 0.004), an ∼55% decrease in motility (p = 0.011), and an ∼75% increase in lifetime (p = 0.016) of dendritic protrusions. Shaft and growth cone protrusions were equally affected. An ∼15% decrease in the length of filopodia approached, but did not reach significance (p = 0.074), The effect on density and lifetime was reversible after washing out the drug, because there were no significant differences between control and washout conditions (p > 0.26; n = 90; three experiments). This wash-out effect required several hours (the data come from dendrites imaged on average ≥4 hr after washout of cytochalasin D). B, Blocking neuronal activity with 1 μm TTX (n = 219; five experiments) resulted in an ∼85% increase in the average density of shaft filopodia (p = 0.04) and an ∼75% increase in the average length of dendritic shaft filopodia (p = 0.05). Neither the density nor the length of dendritic growth cone filopodia were significantly affected by TTX (p = 0.70 and p = 0.69, respectively). Motility and lifetime of filopodia were unaffected by TTX in either dendritic shafts (p > 0.21) or growth cones (p > 0.54). C, Blocking neuronal activity with calcium-free ACSF (n = 350; six experiments) also resulted in an ∼125% increase in the average density (p = 0.05) and an ∼45% increase in the average length (p = 0.004) of filopodia in dendritic shafts. Just as with TTX, neither the density nor the length of dendritic growth cone filopodia were significantly affected in the presence of zero-calcium ACSF (p = 0.52 and p = 0.88, respectively). Motility and lifetime of filopodia were similarly unaffected by zero-calcium ACSF in either dendritic shafts (p > 0.23) or growth cones (p > 0.32). The effects of zero-calcium ACSF on dendritic shaft filopodia density and length were reversible ≥30 min after washing in normal ACSF containing 2 mm CaCl₂ (p > 0.28 compared with control; N = 3 successful washouts; n = 59). D, Blocking ionotropic glutamate receptors for 15-30 min with 20 μm CNQX and 40 μm d-APV resulted in an ∼35% decrease in both the density (p = 0.006) and the turnover (p = 0.04) of shaft filopodia (N = 8; n = 319) but had no effect on length, motility, or lifetime of shaft protrusions (p>0.15). CNQX/APV had no effect on growth cone filopodia (p>0.56; N = 5; n = 164). These effects of CNQX/APV on dendritic shaft filopodia were reversed 30-60 min after washing out the drug (p > 0.63 compared with control; N = 4 successful washouts; n = 116).E, PicoSpritzer application of 100-200 μm glutamate resulted in an ∼75% increase in the length of shaft filopodia (p = 0.004; N = 5; n = 251) but did not significantly affect the other parameters (p > 0.25). A trend toward lower density of growth cone filopodia was also observed (p = 0.055; N = 2; n = 76). The histograms in A-E reveal percentage differences compared with control dendrites imaged before the application of the drugs or zero-calcium ACSF. Error bars represent the SEM, using the number of experiments (N), rather than the number of protrusions. ^*p < 0.05; ^**p < 0.01.

Dendritic filopodia are developmentally regulated; dendritic growth cones disappear after P5. The first row (A) shows representative dendritic segments of cortical neurons of mice at P2 through P10 (see movies 3-7 in supplementary data, available at www.jneurosci.org). The second row (B) shows composite views of the first (red) and last (green) frames of the 10-min time-lapse movies of the dendrites. The third row (C) shows a collapsed view of all 20 frames of the 10-min movie at each postnatal age. Scale bar, 5 μm. These panels show four results. First, filopodia are longer and more densely packed in dendritic growth cones than in shafts. Note that dendritic growth cones (bracket in C) are only apparent at P2-P3 and P4-P5. Second, dendritic protrusions become progressively more densely packed throughout early postnatal development and become shorter in dendrite tips. Third, as judged by the overlap between first and last time frames in each movie (second row), a switch from highly motile, transient filopodia to more stable, immobile protrusions occurs throughout early postnatal development. Fourth, spine-like protrusions, with bulbous swellings at their tips (heads), begin to appear at P10-P12 (arrows in A).

Developmental time course of dendritic protrusions: evidence for distinct filopodia in growth cones and shafts. A, Density. The average density of protrusions in dendritic tips decreases steadily throughout postnatal development (from 1.3 per micrometer at P2-P3 to 0.6 per micrometer at P10-P12), whereas the density of protrusions on dendritic shafts increases during the second week of postnatal development (from 0.4 per micrometer to 0.7 per micrometer). The higher density of filopodia in growth cones (as compared with those in shafts) at P2-P3 was statistically significant (large asterisks). Note that values for mice aged P8-P12 consist of pooled data from GFP- and Alexa-labeled neurons for ages >P6, although the majority of analyzed dendrites came from Alexa-loaded neurons. B, Length. The average length of all protrusions on dendritic tips at any given time decreases throughout early development, as growth cones disappear (from 2.2 μm at P2-P3 to 0.95 μm at P10-P12). The average length of shaft protrusions remains relatively stable at 1.5 μm. The longer lengths of filopodia in growth cones (as compared with those in shafts) at P2-P3 and at P4-P5 were statistically significant (black asterisks). The right panel shows the frequency distribution of the average lengths individual protrusions throughout development. C, Motility. The average motility of protrusions in both dendritic tips and shafts decreases throughout early postnatal development (from ∼0.8 μm/30 sec at P2-P3 to 0.25 μm/30 sec at P10-P12). The fastest recorded speeds exceeded 3 μm/30 sec. Motility of protrusions was also significantly higher in dendritic growth cones than in shafts at P2-P3 and at P4-P5, which is when growth cones can be observed (black asterisks). The right panel shows the frequency distribution of protrusion motility throughout development. Note that the frequency of highly motile protrusions (≥0.8 μm/30 sec) decreases from 36% at P2-P3 to 5% at P10-P12. D, Lifetime. The average lifetime of protrusions in both dendritic tips and shafts increases steadily throughout early development (from 3 min at P2-P3 to ∼7 min at P10-P12). Lifetimes were also significantly higher in dendritic tips at P8-P9 and at P10-P12 (black asterisks). The right panel shows the frequency distribution of protrusion lifetimes throughout development. Note that two populations of protrusions can be distinguished on the basis of their lifetimes, one with lifetimes ≥8 min (stable; i.e., protospines) and the other with protrusion lifetimes ≥3 min (transient; i.e., filopodia). For instance, 75% of all protrusions at any age fall into either of these categories. E, Turnover. The average turnover of dendritic tip protrusions (i.e., the number of protrusions added per minute per 10 μm segment of dendrite) decreases from 3.5 at P2-P3 to 0.1 at P10-P12. A proportionally smaller but also statistically significant decrease was observed in the turnover of shaft protrusions, from 1.2 at P2-P3 to 0.5 at P10-P12. The difference in turnover between shafts and tips was also statistically significant at P2-P3, again demonstrating the particularly high dynamism of dendritic growth cones. The reverse was true at P10-P12, indicating that tip protrusions at older ages are the most stable of all protrusions. Number of dendrites and individual protrusions analyzed for all panels: for dendritic tips at P2-P3, P4-P5, P6-P7, P8-P9, and P10-P12, N = 7, 4, 3, 4, and 5, respectively. For dendritic shafts at P2-P3, P4-P5, P6-P7, P8-P9, and P10-P12, N = 8, 7, 4, 6, and 8, respectively, where N = number of dendrites analyzed; for each dendrite, values are averages of 20 time frames, 30 sec apart. In terms of the number of individual protrusions (n), this corresponds to: for dendritic tips at P2-P3, P4-P5, P6-P7, P8-P9, and P10-P12, n = 147, 46, 29, 28, and 13, respectively; for dendritic shafts at P2-P3, P4-P5, P6-P7, P8-P9, and P10-P12, n = 150, 184, 77, 156, 178, respectively. That is a total of 1008 protrusions and a total of 56 dendrite segments. Error bars represent the SEM, which was calculated using the number of dendrites (N) for measurements of density and length and using the number of protrusions (n) for measurements of motility and lifetime. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Blockade of global synaptic activity results in longer, more motile filopodia. A, Dendritic shaft from a control neuron at P3 in control ACSF (2 mm CaCl₂). The left, middle, and right panels in each row show representative snapshots of dendritic filopodia in the beginning, middle, and end of the 10-min movies. Scale bar, 5 μm (for all panels). A′, Same dendritic shaft segment ∼7 min after continuous bath application of 1 μm TTX. Note the dramatic elongation of many filopodia in three different frames of a 10-min time-lapse movie. B, Dendritic growth cone from a control neuron at P4 in control ACSF (2 mm CaCl₂). B′, Same dendritic growth cone ∼30 min after continuous bath application of TTX. Note that there is no change in the density or length of growth cone filopodia. C, Dendritic growth cone from a control neuron at P3 in control ACSF (2 mm CaCl₂). See movie 8 in supplementary data, available at www.jneurosci.org. C′, Same dendritic growth cone ∼25 min after washing in zero-calcium ACSF. Note the dramatic elongation of many filopodia in three different frames of a 10-min time-lapse movie. This effect was reversible because filopodia lengths and motility recovered after washout. See movie 9 in supplementary data, available at www.jneurosci.org.

Focal application of glutamate results in rapid elongation of a subset of filopodia. A, Time-lapse movie of apical dendrite from a layer 5 pyramidal neuron at P4 (see movie 10 in supplementary data, available at www.jneurosci.org). Images were acquired every 60 sec, and representative time points are shown. The white arrows separating certain frames represent the times when three puffs (200 msec, ∼20 psi) of 100 μm glutamate were delivered, corresponding to frames 10, 17, and 28. Note that by the end of the movie, many filopodia have grown to lengths >5 μm. Scale bar, 5 μm. B, In this low magnification view of a pyramidal neuron, the white box delineates the region in which the movie in A was obtained. The arrow points to the location of the pipette tip containing 100 μm glutamate (and Alexa-488). Scale bar, 20 μm. C, Graph displaying the lengths of 45 representative filopodia from the 40-min movie shown in A. Black arrows and gray columns designate the three times when glutamate was puffed (200 msec puffs). Note that during the first 10 frames of the movie filopodia lengths never surpass 4 μm, whereas after just two puffs of glutamate, a subset of filopodia have reached lengths well above 4 μm. The effect of glutamate was quite rapid as shown by the sudden increases (within 1 min) of some filopodia after individual puffs. D, Frequency distribution of filopodia lengths before and after glutamate application from five separate experiments. The gray histograms represent pooled data from 117 filopodia analyzed in the first 10 frames of each movie, whereas the black histograms represent data from 136 filopodia analyzed during 10 consecutive frames after at least two puffs of glutamate. Note that glutamate induced growth to lengths >4 μm in a minority (∼10%) of filopodia, rather than a small increase in the length of all the filopodia. E, F, Another example of the effects of glutamate puffing on a P2 dendrite. The pipette was located ∼50 μm away, up and to the left of the dendrite growth cone. E is a representative frame of the dendrite with a growth cone in normal conditions. F is the same dendrite ∼30 min after three puffs of 200 μm glutamate. Scale bar, 5 μm.

A continuum of dendritic shaft protrusions throughout early postnatal development. This graph plots motility on the x-axis against lifetime on the y-axis for a total of 342 individual shaft protrusions at P2-P5 (dark blue) and 333 individual shaft protrusions at P8-P12 (light orange). The plot again demonstrates that at younger ages shaft protrusions are indeed more motile and have shorter lifetimes than at older ages, as was shown in Figure 5. The distribution of protrusions at either age is best fit by an exponential curve, but the characteristics of the distributions are different. At younger ages, as protrusion motility varies lifetime changes very little. The opposite is true at older ages when, as protrusions shift toward less motility, their lifetime increases considerably (and vice versa). On the basis of this distribution, a continuum of dendritic shaft protrusions can be discerned from the rapidly motile and transient filopodia (bottom right) to the relatively immobile and permanent dendritic spines (top left). We have arbitrarily defined several clusters of filopodia along this continuum. The insets show representative examples of imaged protrusions falling into several arbitrary categories of filopodia along this continuum, as well as an example of dendritic spines at P12. We speculate that neuronal activity regulates early dendritic protrusions such that, as neuronal activity increases throughout development, protrusions change along this continuum from filopodia to spine-like protrusions. Indeed, in more mature dendrites, some filopodia developed heads at their tips, thus resembling dendritic spines (arrowhead). Arrows indicate the position along the dendrite in which filopodia will appear in the next frame. Scale bar, 3 μm. Time stamps in white indicate minutes within a 10-min time-lapse movie.