Figure 3.
Aging neurons can produce multiple novel outgrowths, many of which are associated with mitochondria at the branch site. A–D, In a decrepit class C wild-type animal at day 15 of adult life (20°C culture), the dendrite of ALMR included multiple periodic branches extending inward, away from the cuticle and the primary dendrite. Scale bar, 0.5 μm. A, Anterior portion of the ALM dendrite (yellow overlay) shows a typical cross section view in which the narrow dendrite is virtually filled by a bundle of large-diameter microtubules (Chalfie and Thomson, 1979). The dendrite is enveloped by folding of the extremely thick body cuticle typical of old animals (Herndon et al., 2002), and the nearby extracellular space that surrounds the touch neuron is filled by a “mantle” (white arrow) and a thickened basal lamina. B, Several micrometers further posterior, the same ALM dendrite extends a narrow branch inward (also marked by yellow overlay); the branch is locally swollen to accommodate a mitochondrion (marked as M). Several microtubules in the main bundle (white arrows) seem to extend laterally toward the branch, suggesting that the branch may encompass a smaller microtubule bundle than is connected to the main bundle. C, D, Two more distinct individual branches extend inward from the same ALM dendrite, each spaced several micrometers more posteriorly from the previous branch. Inside each branch, a small mitochondrion is located close to the base of the branch. In portions of the dendrite between each branch, ALM resumes a shape and content similar to A (data not shown). Note that it is unusual to find large branches on axons in aging ALM touch neurons (Fig. 2) and the three small branches depicted in B and D probably correspond to outgrowths too small to have been scored using the dissection microscope as described for Figures 1 and 2. Rather, these branches most likely correspond to the short tiny outgrowths we observe at 600× magnification using a compound microscope. Note also that we identified native developmental branch points in TEM micrographs in 9 young adult animals and found 8/9 for these normal branches are associated with mitochondria at the branch point, suggesting mitochondrial positioning at the branch site may be a common configuration in both normal developmental branches and aberrant novel branches that appear during aging. E–G, GFP-labeled mitochondria distribution in mCherry-labeled touch neuron processes with specific abnormalities. Red marker distributed throughout the cytoplasm is pmec-4mCherry; green mitochondrial marker is pmec-4mitoGFP (extragenic array odEx[Pmec-4::mitoGFP, Pmec-4::mCherry); shown are PLM neurons of 2 d adult. E, Representative process from a young adult. Note that some, but not all, green-labeled mitochondria (green arrows) are associated with beads of concentrated mCherry signal (red arrows). Scale bars: E–I, 10 μm. F, Representative process from a PLM neuron that features multiple beads of concentrated mCherry that appear to protrude from the main dendrite. Note that the dramatic protrusions all include an associated mitochondrion (green arrows), but some mitochondria are not associated with abnormal structures (red arrows). G, A branch site in a 2 d adult PLM neuron from strain with a GFP-labeled mitochondrion situated at the branch point. We find that mitochondrial GPF signals are often, but not always, associated with branch points (at day 10, 90% of new branches were associated with mitochondria, n = 40; at day 15, 68% of new branches were associated with mitochondria, n = 28). We tracked mitochondria in PLM and find the following: (1) for the distal, native branch on which touch neuron synapses are normally made, mitochondria are present at the native synaptic branch site (combined day 2 and day 4 data 87%, n = 39), and do not decline dramatically by day 10 (87% of native PLM branches are associated with mitochondria, n = 32 day 10), although there is a modest decline by day 15 (80% native PLM branches are with mitochondria, n = 36); (2) mitochondria are dynamic and can appear and disappear within a defined region of the process in adult neurons; (3) the average number of mitochondria does not change dramatically with age (mean number of mitochondria on day 2 is 7.3; on day 10, 7.6; on day 15, 6.3; ns). FUDR was not used in quantitative studies of mitochondria summarized here. H, I, Examples of new outgrowths that appear to originate from regions of relative GFP accumulation and dendrite bending. zdIs5 [pmec-4GFP] was used to visualize touch neuron processes; neurons are PLMRs, 6 d old.