Cytoskeletal and Morphological Alterations Underlying Axonal Sprouting after Localized Transection of Cortical Neuron AxonsIn Vitro

Effect of taxol exposure (10 μg/ml) on neurite morphology and cytoarchitecture. A shows a selection of images from a time-lapse series. Times shown on each image indicate minutes after taxol administration. In the presence of taxol, the originally splayed tips of neurites take on a bulbous morphology (arrows). Immunofluorescence labeling for βIII-tubulin demonstrated that neurites elaborated from vehicle-treated cultures (3–5 DIV) were slender and extended radially, often in defined bundles (arrows inB), whereas neurites from taxol-treated cultures (3–5 DIV) were distended, particularly in their distal domains (arrow inC) and often demonstrated a curled morphology (arrowheads in C). D, βIII-Tubulin labeling of vehicle-treated cultures demonstrating examples of growth cones (exhibiting defined filopodia) located at the distal tips of developing axons (arrows). In the distal tips of taxol-treated developing neurites, βIII-tubulin was localized to club-like (arrows) and loop-like (arrowhead) structures (E). Phosphorylated neurofilaments accumulated into club-like (long arrow), ball-like (short arrow), or loop-like (arrowhead) structures (F). Tau was localized throughout distended neurites and in the filopodial-like protrusions emanating from their distal tips (arrow in G), whereas F-actin was most abundant in neurite tips, forming bulb-like accumulations (H). Scanning EM images of growth cones from vehicle-treated (I) and taxol-treated (J, K) neurons (3–5 DIV). Vehicle-treated growth cones had an elaborate three-dimensional structure (H), whereas taxol-treated growth cones were bulbous, with few remaining filopodia (I, J). Double-labeling analysis demonstrated that some neurites regained growth cones after taxol washout (M, F-actin labeling) and underwent more neurite growth than cultures continually exposed to taxol; however, neurites often remained distended (L, βIII-tubulin). Scale bars: A, D–H, L–M, 20 μm; B, C, 60 μm; I–K, 1 μm.

Bar graphs showing the effect of exposing developing cortical cultures to vehicle or taxol for 48 hr between 3 and 5 DIV. A, Proportion of neurites tipped by a growth cone after 48 hr vehicle or taxol treatment. B, Mean neurite length before treatment and after vehicle or taxol treatment. *p < 0.05. Error bars are SEMs.

Effect of nocodazole (100 μg/ml) on neuronal morphology in developing cortical cultures. Vehicle-treated neurons (3–5 DIV, 1.2%DMSO–PBS) had defined cell bodies, axons were slender and tipped by growth cones, and future axonal branch points were indicated by growth cone “remnants” (A). Nocodozole-treated cultures had vastly altered morphology: neurites were stunted and often existed as thick protrusions of the cell, cell bodies were often bordered by a lamellipodial-like fringe (B), and large axonal growth cones often lacked lamellipodia (C). Scale bars, A, 20 μm; B, C, 10 μm.

Confirmation of cortical neuron maturity. BrdU was administered to cultures at 7 (A, B) or 17 (C, D) DIV, for cell proliferation assays, and cultures were grown to 21 DIV. Neuron-specific enolase (NSE) immunopositive neurons (arrows in A andC) lacked BrdU-immunoreactive nuclei (B,D) in double-labeling studies, indicating that they were not proliferating. Double labeling for tau (E) and postmitotic neuronal nuclei (F) at 21 DIV demonstrated that aggregated neurons were not dividing. NSE-labeled neuron (G) in apposition with a profile of synaptophysin-immunoreactive puncta (arrows depict examples inH), demonstrating that neurons had abundant synaptic connections. The presence of synapses was confirmed with electron microscopy (I). J, Electron microscopy demonstrating that axons within axonal bundles were not myelinated (arrows depict examples). Scale bars:A–H, 30 μm; I, 2 μm;J, 1 μm.

Time course of the posttransection response.A, Time-lapse imaging of a cut site immediately after injury, demonstrating retraction away from the injury site (note retraction in relation to the asterisk in each image).B, Time-lapse imaging of a cut site imaged from 2 hr after injury, with sprout growth indicated by arrows at each time point. Time course of postinjury axonal sprouting in cultures immunofluorescence labeled for βIII-tubulin (C), tau (D), and phosphorylated neurofilaments (E). Arrows in each image depict examples of sprouts. By 24 hr after injury, sprouting was extensive and several sprouts had crossed the lesion sites. Times shown on each image indicate postinjury interval in hours. Double labeling for βIII-tubulin and F-actin (F, G, respectively), as well as tau and phosphorylated neurofilaments (H, I, respectively), demonstrated that βIII-tubulin and tau were distributed throughout postinjury sprouts and proximal growth cones, whereas F-actin was most abundant in sprout growth cones and phosphorylated neurofilament were restricted to sprout shafts. Scale bars: A, B, 40 μm;C–E, 60 μm; F–I, 30 μm.

Immunofluorescence labeling for tau, demonstrating the effect of taxol exposure on postinjury axonal sprouting. Times shown on each image indicate hours of vehicle or taxol exposure after injury. Vehicle-treated cultures demonstrated an extensive and progressive sprouting response after axonal transection, with several sprouts crossing the lesion site by 24 hr after injury (A, B). Postinjury sprouts elaborated from vehicle-treated cultures were typically slender with expanded growth cone-like distal domains (arrows in C). Limited postinjury axonal sprouting was demonstrated in taxol-treated cultures up to 24 hr after injury (D, E), and bulb-like structures formed at the tips of sprouts (arrows inF). Arrows in A, B,D, and E depict examples of postinjury sprouts. C and F were captured at 4 and 14 hr after injury, respectively. Taxol washout, after 4 hr of postinjury taxol exposure, resulted in increased sprout growth across injury sites, and some sprouts gained growth cone-like structures at their tips (arrows in G). Scale bars: A,B, D, E, 60 μm;C, F, 20 μm; G, 10 μm.

Bar graphs demonstrating the effect of exposing cultures to taxol for 4 hr after axonal transection. A, Mean sprout length. B, Mean number of sprouts per 100 μm of injury site length. *p < 0.05. Error bars are SEMs.