Peripheral and central target requirements for survival of embryonic rat dorsal root ganglion neurons in slice cultures

Abstract

Developmental cell death in the nervous system usually is controlled by the availability of target-derived trophic factors. It is well established that dorsal root ganglia (DRG) neurons require the presence of their peripheral target for survival, but because of their central projections, it is possible that the spinal cord also may be required. Before examining this possibility in rat embryos, we first used terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to determine that thoracic DRG cell death occurred from embryonic day 15 (E15) to E18. To determine the target requirements of DRG neurons, we used organotypic slice cultures of E15 thoracic trunk segments. After peripheral target removal, essentially all DRG neurons disappeared within 5 d. In contrast, after removal of the spinal cord, approximately half of the DRG neurons survived for at least 8 d. Hence, some E15 DRG neurons could survive without the spinal cord. However, those DRG neurons that died after spinal cord ablation apparently required trophic factors from both central and peripheral targets, because the presence of only one of these tissues was not adequate by itself to support this cell group. Addition of neurotrophin-3 (NT-3) to the culture medium rescued some DRG neurons after CNS removal, suggesting a possible role for NT-3 in vivo. In other experiments, cultures were established from older (E16) embryos, and essentially all neurons survived after spinal cord ablation, even without added factors. These and other experiments indicated that approximately 65% of DRG neurons are transiently dependent on the CNS early in development.

Fig. 1.

TUNEL-positive profiles in developing DRGs. TUNEL appears in these micrographs as black,small, mostly round profiles.A, At E14, very few TUNEL profiles were present (arrowheads). B, Only 1 d later at E15, many positive profiles were scattered throughout the DRGs.C, Beginning at E16, the labeling was greatly reduced compared with E15, but it was still noticeably more than on E14. Dorsal is top, and medial is right. Scale bar, 50 μm.

Fig. 2.

Mean number of TUNEL-positive profiles per section of DRG at various ages. The height of each barrepresents the mean value for one to four animals at each age analyzed (no animals were examined on E20 or E22). Error bars indicate SEM. There were statistically significant differences among these ages (p < 0.0001, Kruskal–Wallis test). Specifically, the ages marked with asterisks had significantly greater numbers of TUNEL profiles than the other ages (p < 0.01).

Fig. 3.

E15 rat thoracic spinal cord and DRG, stained with diaphorase histochemistry. This developmental stage represents the time of peak TUNEL in the DRG and the age at which slice culture experiments were initiated. At this age, all DRG neurons appeared to be diaphorase-positive (Wetts and Vaughn, 1993). Their central processes were visible extending into the dorsolateral funiculus of the spinal cord (arrow), and the peripheral processes were visible branching in the peripheral target tissue (arrowheads). The autonomic motor neurons (AMNs) (Wetts et al., 1995) also were diaphorase-positive, as were the endothelial cells of blood vessels. Scale bar, 100 μm.

Fig. 4.

Peripheral target dependency of DRG neurons in organotypic slice cultures of E15 thoracic trunk. A, A freshly prepared transverse slice consisted of the spinal cord, DRGs, and adjacent peripheral tissues such as the body wall.B, After 5 d of culture in serum-free medium, slices were fixed, sectioned, and processed for combined ChAT immunocytochemistry and diaphorase histochemistry. Numerous diaphorase-positive DRG neurons were clearly visible in these intact cultures (neuron counts are presented in Table 1). C, For periphery-removed cultures, most of the peripheral tissue was surgically removed at the beginning of the culture period.D, After 5 d without peripheral tissue and without added growth factors, only a small remnant of the DRG is visible. No surviving cells can be discerned in this section, a result that occurred in most sections of periphery-removed cultures. Dorsal istop (A–D), and medial isright (B, D). Scale bars:A, C, 100 μm; B,D, 20 μm.

Fig. 5.

Rescue of periphery-deprived DRG neurons by NGF treatment. A, After removal of the peripheral tissue (as in Fig. 4C) and NGF treatment for the entire 8 DIV, many diaphorase-positive neurons were present (Table 1). These results confirm that NGF can prevent the death of DRG neurons, even in the absence of peripheral tissues. B, Delayed-NGF cultures were maintained in factor-free medium for the first 4 d after periphery removal, and then the medium was supplemented with NGF for the last 4 d. Only a few DRG neurons were present in these cultures (Table 1), indicating that most DRG neurons were dead after 4 d of peripheral target deprivation. In the time lines below each micrograph, the thin line indicates the days with NGF present, and the thick line indicates the days without NGF. Dorsal is top, and medial is left. Scale bar, 20 μm.

Fig. 6.

Independence of some DRG neurons from their central target tissue. A, A CNS-ablated culture was prepared by excising the spinal cord from a freshly prepared E15 slice.B, After 8 d in vitro, many diaphorase-positive DRG neurons were clearly present in such CNS-ablated cultures, indicating that some DRG neurons survived in the absence of the spinal cord. Quantitative data are presented in Tables 1and 2. Dorsal is top. Scale bar, 100 μm.