Growth cones are not required for initial establishment of polarity or differential axon branch growth in cultured hippocampal neurons

Abstract

Hippocampal neurons developing in culture exhibit two types of differential, seemingly competitive, process outgrowth in the absence of external cues. During the initial acquisition of polarity, one of several equivalent undifferentiated minor neurites preferentially grows to become the axon. Once the axon has formed, it typically branches, and the branches grow differentially rather than concurrently. In axons with only two branches, growth alternates between branches. In both axon establishment and branch growth alternation, growth among sibling processes or branches must be differentially regulated. We found that elaborate and dynamic growth cones were associated with growth, whereas diminished growth cones were associated with nongrowing processes or branches. To test whether growth cones were necessary for differential growth, growth cone motility was eliminated by application of cytochalasin E. Although cytochalasin treatment before axon formation yielded longer processes overall, a similar percentage of both treated and untreated neurons had one process that grew more rapidly and became much longer than its sibling processes. Immunostaining to visualize dephospho-tau, an axonal marker, demonstrated that these single dominant processes were axons. Axons that formed in cytochalasin were thicker and showed more intense anti-tubulin staining than their sibling processes. Branched axons deprived of growth cones retained a pattern of differential growth and often included alternation. These results indicate that neither formation of a single axon nor differential growth of branches are dependent on growth cone motility and suggest that the neuron can regulate neurite elongation at sites other than at the growth cone.

Fig. 1.

Axon determination: the transition of a neuron from stage 2 to stage 3. The undifferentiated minor processes of a stage 2 neuron exhibit net growth over time as each process undergoes cycles of growth and retraction. Growth cone size reflects the dynamic changes in the growth states of the different processes, becoming large in actively growing processes (arrows). Eventually, one process grows longer than the others and continues to grow rapidly, differentiating into an axon. Relative times are shown in minutes, beginning at ∼8 hr in culture. Scale bar, 20 μm.

Fig. 2.

Formation of an axon in the presence of 0.2 μg/ml cytochalasin E. A stage 2 neuron is shown before addition of CE and at time intervals thereafter. Growth cones are eliminated by treatment with CE, but processes nonetheless elongate. The elimination of growth cone function is clearly demonstrated by occasional lifting of the process tip off the substrate. Although substantial growth is seen in several of the processes, one process becomes significantly longer and also takes on an apparently greater thickness compared with the other processes. Branching in the absence of growth cone formation was also noted to occur. Times are shown in minutes relative to the time of CE addition. Scale bar, 20 μm.

Fig. 3.

Tau-1 and Texas Red phalloidin staining of an axon that formed in the presence of cytochalasin E. A, An axon forms from a stage 1 neuron treated with 0.17 μg/ml CE. The cell exhibits the typical actin-based lamellipodial fringe characteristic of stage 1 neurons before addition of CE. A long process grows from the remnant of the lamellipodial fringe after CE is applied. The image taken at 235 min after addition of CE shows the process tip lifting off the substrate, underscoring the effectiveness of the CE application in eliminating growth cones. Times are shown in minutes relative to the time of CE addition. B, The same cell shown inA was fixed ∼16 hr after CE addition and double-stained with tau-1 antibody (C) and Texas Red phalloidin (D). Tau-1 specifically stained the longest process in a distal to proximal gradient. Phalloidin staining shows aggregates of F-actin in the cell body and along the processes, confirming the effectiveness of the CE. Scale bars, 20 μm.

Fig. 4.

DM1A anti-tubulin and Texas Red phalloidin staining of an axon that formed in the presence of cytochalasin E.A, A stage 1 neuron is shown before and after addition of 0.2 μg/ml CE. After CE is added, lamellipodial activity ceases, leaving behind a membranous remnant. Processes grow out of the lamellipodial remnant. Times are shown in minutes relative to the time of CE addition. B, By ∼16 hr in CE, the cell has extended multiple long processes, with one of these processes at least twice as long as any of the other processes and also thicker.C, The cell was fixed and stained with DM1A, an antibody to tubulin. A greater degree of staining in the longest process (the presumptive axon) suggests that the apparently greater thickness of this process is at least in part attributable to a higher amount of tubulin. D, Phalloidin staining shows a staining pattern indicative of F-actin aggregates in the cell body and along the processes. Scale bars, 20 μm.

Fig. 5.

Alternation of axon branch growth.A, Phase contrast images of a neuron with a branched axon at selected time points. The two branches alternate their growth state such that only one branch grows at a time. Growth cone size reflects the growth state of the branch, with the growing branch exhibiting an elaborate and highly motile growth cone, whereas the growth cone of the nongrowing branch is relatively small and inactive. Branches are labeled as 1 and 2 in reference to the graph in B. Relative times are shown in minutes and refer to the times in the graph in B. Scale bar, 20 μm. B, Branch length (measured from the branch point) is graphed as a function of time for the cell shown inA. Branch 1 was growing at the onset of recording, branch 2 was not. Soon after recording commenced, the branches switched growth states. Growth alternated once more at ∼200 min of recording.Asterisks mark times that correspond to the images shown in A.

Fig. 6.

Differential axon branch growth in the presence of cytochalasin E. A, The branched axon of a neuron is shown before and after addition of 0.17 μg/ml CE. Growth cones are eliminated by the treatment with CE, but growth of the branches nonetheless proceeds. Growth of the branches continues to occur one branch at a time. Branches are marked as 1 and2 in reference to the graph in B. Times are shown in minutes relative to the time of CE addition. Scale bar, 20 μm. B, Branch length (measured from the branch point) is graphed as a function of time. Before cytochalasin (open bar at top), the branches show differential growth that alternates at approximately −50 min. After addition of CE (dotted line, time = 0), both branches cease growing for ∼2 hr. In the continued presence of CE (filled bar at top), branch 2 begins to grow after ∼150 min in CE, whereas branch 1 remains nongrowing. At ∼380 min in CE, the branches alternate their growth state such that growth ceases in branch 2 and begins in branch 1.Asterisks mark times that correspond to phase contrast images in A.