A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth

Abstract

Although maturing neurons undergo a precipitous decline in the expression of genes associated with developmental axon growth, structural changes in axon arbors occur in the adult nervous system under both normal and pathological conditions. Furthermore, some neurons support extensive regrowth of long axons after nerve injury. Analysis of adult dorsal root ganglion (DRG) neurons in culture now shows that competence for distinct types of axon growth depends on different patterns of gene expression. In the absence of ongoing transcription, newly isolated neurons can extend compact, highly branched arbors during the first day in culture. Neurons subjected to peripheral axon injury 2-7 d before plating support a distinct mode of growth characterized by rapid extension of long, sparsely branched axons. A transition from "arborizing" to "elongating" growth occurs in naive adult neurons after approximately 24 hr in culture but requires a discrete period of new transcription after removal of the ganglia from the intact animal. Thus, peripheral axotomy-by nerve crush or during removal of DRGs--induces a transcription-dependent change that alters the type of axon growth that can be executed by these adult neurons. This transition appears to be triggered, in large part, by interruption of retrogradely transported signals, because blocking axonal transport in vivo can elicit competence for elongating growth in many DRG neurons. In contrast to peripheral axotomy, interruption of the centrally projecting axons of DRG neurons in vivo leads to subsequent growth in vitro that is intermediate between "arborizing" and "elongating" growth. This suggests that the transition between these two modes of growth is a multistep process and that individual steps may be regulated separately. These observations together suggest that structural remodeling in the adult nervous system need not involve the same molecular apparatus as long axon growth during development and regeneration.

Fig. 6.

In vitro transition from arborizing to elongating growth by naive neurons. The percentage of naive and pre-axotomized neurons with elongating (A) or arborizing (B) neurites was determined at various times after plating, using visual inspection of all neurons in each culture (circles) or by quantitative analysis of neurite length and branching frequency for randomly selected neurons, as described in Figure 4D (squares). Filled symbols represent cultures from naive ganglia, and open symbols are from pre-axotomized ganglia. Each visually derived time point (circles) represents the mean ± SD of three to seven separate experiments, with duplicate cultures in each experiment. Time points derived from quantitative analysis include 480 randomly selected naive neurons pooled from three separate experiments (squares). The onset of elongating growth is delayed for ∼24 hr in naive cultures, but the subsequent emergence of elongating axons parallels the initial outgrowth from pre-axotomized neurons. Arborizing growth plateaus near 20–25% and then declines as more neurons begin to exhibit the elongating morphology.

Fig. 7.

Onset of elongation from naive neurons requires a finite period of new gene transcription after plating.A, Naive DRG cultures were exposed to DRB, a reversible inhibitor of mRNA synthesis for the indicated intervals. Neurons in both DRB-treated and untreated cultures were fixed 40 and 66 hr after plating and visually scored as arborizing (gray bars) and elongating (black bars). Early DRB treatment dramatically reduced the percentage elongating at 40 hr, but not at 66 hr. When transcription was blocked between 12 and 24 hr after plating, the effect was less pronounced, and by 24 hr after plating, neurons were no longer dependent on ongoing transcription to initiate and sustain neurite elongation. Bars represent the mean ± SD from three separate experiments with duplicate cultures in each experiment.B, Estimate of delay produced by transient DRB treatment. The percentage of neurons with elongating neurites was rescored by quantitation of length and branching frequency.Filled circles represent neurons treated with DRB for the first 12 hr; open circles represent control neurons in untreated cultures. Each data point represents the mean ± SD for three separate experiments (30 individual neurons). These data were then compared to the previously determined time course for the onset of elongation under standard conditions (solid curve, from Fig. 6B). To estimate the length of delay produced by transient DRB treatment, we displaced this curve to the right until it intersected with the data points for DRB-treated cultures. The result indicates that an initial exposure to DRB for 12 hr produces a delay in the onset of elongating growth of ∼16 hr.C, The delay was not attributable to an overall decrease in protein synthesis, because transient DRB exposure for the first 12 hr after plating did not grossly alter protein synthesis measured 12 hr later, when elongating neurites begin to emerge in control but not in DRB-treated cultures.

Fig. 4.

Neurites of naive and pre-axotomized neurons define two distinct modes of axon growth. Previous axotomy in vivo influenced the growth characteristics of DRG neurons, prompting the extension of longer neurites with fewer branches during the first day in culture. The length and number of branch points were determined for the longest neurite extended by randomly selected DRG neurons from three separate experiments in which cultures had been fixed 16 hr after plating. Open symbols represent individual neurons from naive ganglia; filled symbolsthose from pre-axotomized ganglia. A, Naive cells plated under standard conditions (circles) or at high density, which promotes outgrowth from a majority of neurons (diamonds). B, Naive neurons cultured in the presence of schwannoma-conditioned medium (triangles) or NGF (squares).C, Pre-axotomized neurons plated under standard conditions. D, Inclusion of the transcription inhibitor DRB during dissociation and plating had no effect on length and branching. E, Data fromA–C plotted on a single graph. All neurites extended by naive neurons, regardless of culture conditions, exhibit a branching frequency > 1.5 branches per 100 μm (dotted line). This line, therefore, serves as a simple objective criterion for categorizing neurons. For subsequent quantitative analysis, data points that fall above this line will be scored as “arborizing” and those that fall below the line will be scored as “elongating.”

Fig. 1.

Distinct time courses of neurite outgrowth from naive and previously axotomized neurons. Neurons from L4 and L5 DRG of adult rats were dissociated and plated under standard conditions, as described in Materials and Methods. Naive neurons from uninjured animals (N) and pre-axotomized neurons the peripheral axons of which had been injured in vivo 1 week earlier (AX) were observed over time in culture. A, Neurons and neurites in typical cultures photographed 6, 16, and 40 hr after plating. Immunoperoxidase staining for GAP-43 labels all neurons and their neurites to varying degrees. Scale bar, 100 μm. B, Time course showing the percentage of neurons in naive cultures (solid line,filled circles) and pre-axotomized cultures (dashed line, open circles) that had extended neurites by the indicated times after plating. Each time point represents the mean ± SD of at least three to seven separate experiments, with duplicate cultures in each experiment.

Fig. 2.

Genes commonly expressed in intact adult neurons are sufficient to support robust axon outgrowth. A, The percentage of neurons with neurites 16 hr after plating was determined in transcriptionally active cultures (black bars) and for cultures in which mRNA synthesis was blocked by 80 μmDRB, a potent inhibitor of RNA polymerase II (gray bars). Only 20–25% of naive neurons extended neurites by 16 hr under standard conditions (STD), but the addition ofNGF to the medium or use of medium condition by schwannoma cells (not shown) stimulated growth from more neurons during the same interval. Plating at 4× the standard density (HD) had a more pronounced effect. Neuritic growth stimulated by these conditions was not inhibited by continuous exposure to DRB. Neurite extension from neurons axotomized 1 week earlier by sciatic nerve crush in vivo [STD (Ax)] was also unaffected by DRB. Data represent the mean ± SD for four separate experiments with duplicate cultures in each experiment. B, Representative neurite-bearing neurons at 16 hr after plating in the absence (top row) or presence (bottom row) of DRB. C, DRB effectively inhibits mRNA synthesis in DRG cultures. At the doses used in these experiments (80 μm), uptake of [H³]uridine is inhibited >90%.

Fig. 3.

Naive and pre-axotomized neurons extend different types of neurites in acute cultures. A, Naive adult DRG neurons after 16 hr under standard plating conditions extend neurites that are highly branched and extend over a limited radius, terminating in large, palmate growth cones with numerous filopodia.B, Neurites in 16 hr cultures from previously axotomized rats (1, 2, 4,5) or in 40 hr cultures from naive rats (3) branch much less frequently, often exceed 1–2 mm in length, and tend to fasciculate with other processes. Growth cones are typically tapered with few filopodia (5). Immunoperoxidase labeling with antibodies to major neuronal proteins stained all neurons to varying degrees and were often used to facilitate counting: GAP-43 (A: 1,2, 5, 6; B:1–3), NF-H (A:4; B: 4), or MAP-2 (A: 3). Scale bar, 75 μm.

Fig. 5.

Neurites observed at later times in cultures from naive animals resemble those extended early by previously axotomized neurons. Randomly selected naive neurons from three separate naive cultures at different times were analyzed for length and number of branch points of their longest neurite. High frequency of branching and limited length persisted through 16 and 22 hr (A,B), but by 48 hr (C) neurites were typically longer with fewer branches, resembling neurites in 16 hr cultures from pre-axotomized ganglia. When plotted together (D), the data segregate on either side of the same line used in Figure 4, with neurites from 16 and 22 hr cultures falling above the line and the majority of those from 48 hr cultures falling below the line.

Fig. 8.

Disruption of axonal transport induces competence for elongating growth. Pliable plastic cuffs impregnated with colchicine or vehicle control were placed around sciatic nerves of anesthetized rats. Assays were performed after 48 hr. A, Electron microscopy confirmed that axons in the nerve segment surrounded by the plastic cuff remained intact. Colchicine produces an increase in irregular profiles of myelinated axons compared to vehicle controls, consistent with its disruption of microtubules. In contrast, sciatic nerve crush produces dramatic degeneration of axons just proximal to the crush site.B, The percentage of elongating (black bars) and arborizing cells (gray bars) after 16 hr in culture. Colchicine did not block the induction of elongating growth after peripheral axotomy (Ax./Colch.), but in the absence of sciatic nerve crush, the drug induced competence for elongating growth in otherwise uninjured neurons (Colch.). Data from naive (N.) and pre-axotomized (Ax.) untreated animals are provided for comparison. Application of cuffs containing vehicle alone (Veh.) had no effect on the onset of axon elongation (compare to 16 hr time points in Fig.5A,B). Each bar represents the mean ± SD of four separate experiments with duplicate cultures in each experiment. C, Quantitative analysis of neurite branching and length shows that neurites extended in three separate experiments by neurons from colchicine-treated animals (filled squares) resemble the elongating axons of pre-axotomized neurons and are clearly segregated from the arborizing neurites extended after application of vehicle-containing cuff (open circles).

Fig. 9.

Competence for elongating growth is regulated by a retrograde signaling pathway(s) activated by contact with peripheral targets. A, The length of the proximal nerve stump affects the timing, but not the extent, of the transition to elongating growth competence. Sciatic nerve crush was performed either 1.0–1.5 cm (black bars) or 5.0–5.5 cm (gray bars) from neuronal cell bodies, and neurons were removed and transferred to tissue culture 24, 48, or 72 hr later. By 48 hr after injury, both proximal and distal lesions produced a large increase in the number of neurons able to extend elongating axons by 16 hr after plating (fewer neurons are injured by the distal lesion, so the maximal response is lower than after proximal injury). The response to proximal crush reaches 45% of the maximum response by 24 hr after injury. In contrast, the response to a more distal lesion by 24 hr is only 12% of the maximum. This may reflect time required for the depletion of retrogradely transported inhibitory molecules in longer proximal nerve segments. Each bar represents the mean ± SD of three separate experiments with duplicate cultures in each experiment.B, Sciatic nerves were subjected to a crush lesion, which permits effective regeneration of axons, or complete transection or resection of a 0.5 cm nerve segment, both of which delay or prevent reinnervation of peripheral targets. DRG neurons were removed 1 week (shaded bars) or 8 weeks (black bars) after injury, and plated for 16 hr in vitro. By 1 week after injury, all three types of lesion had stimulated competence for elongating growth. By 8 weeks after injury, competence for elongation had declined in neurons with crush lesion but remained elevated after nerve transection or resection. Each bar represents the mean ± SD of three separate experiments with duplicate cultures in each experiment.

Fig. 10.

Central and peripheral axotomy affect the growth characteristics of DRG neurons differently. DRG neurons subjected to central axotomy displayed growth characteristics intermediate between naive and peripherally axotomized neurons. A, Fewer neurons were able to elongate (black bars) 2 or 7 d after a central root crush than 2 d after a peripheral crush. In addition, more neurons extended arborizing neurites (gray bars) after the central root lesions. Each bar represents the mean ± SD of three separate experiments with duplicate cultures in each experiment. B, Analysis of neurite length and branching revealed that the neurites in these cultures did not fall into distinct groups but, instead, covered a wide range of intermediate morphologies. Filled circles, Neurites measured in 16 hr cultures from ganglia removed 2 d after dorsal root crushes were performed in vivo. Open circles, Neurites measured in 16 hr cultures from ganglia removed 7 d after dorsal root crushes were performed.