Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2613-2617, April 1993 Neurobiology

Neurotrophin 3 supports the survival of developing muscle sensory neurons in culture FREYA HORY-LEE*, MICHELLE RUSSELLt, RONALD M. LINDSAYt, AND ERIC FRANK*t *Department of Neurobiology, Anatomy, and Cell Science, University of Pittsburgh School of Medicine, 814 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15213; and tRegeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591 Communicated by Gerald D. Fischbach, October 26, 1992 (receivedfor review August 11, 1992)

ABSTRACT Target-dependent cell death of different sub- vitro, have been identified within the past few years. Brain- populations of sensory neurons may be regulated by different derived neurotrophic factor (BDNF) promotes survival of trophic factors. To investigate this possibility, we have taken subpopulations of all sensory neurons known to project into advantage of the fact that the fractions of muscle sensory and the CNS (from which this factor was initially isolated) (see cutaneous sensory neurons in chicken dorsal root ganglia ref. 15 for review). Neurotrophin 3 (NT-3), identified and (DRG) are probably different at different segmental levels, and cloned through its sequence homology to NGF and BDNF, we have compared the responses of chicken DRG from levels has been reported to promote the survival of up to 60% of that do and do not innervate limb tissue to various growth DRG neurons in culture (7). Unlike NGF, however, NT-3 has factors in vitro. Nerve growth factor (NGF) and brain-derived been reported to save >90% of the proprioceptive neurons neurotrophic factor (BDNF) both supported neurite outgrowth from the trigeminal mesencephalic nucleus (6). Also, unlike from DRG explanted from all segmental levels. In contrast, NGF mRNA, NT-3 mRNA is present in significant amounts neurotrophin 3 (NT-3) supported robust neurite growth only in adult skeletal muscle (7, 8). These observations led us to from DRG explanted from the cervical or lumbar levels, which examine whether NT-3, rather than NGF, might be the factor innervate limb muscles. Similarly, NGF and BDNF both pro- involved in development of muscle sensory afferents. moted survival of dissociated neurons from limb and nonlimb The following study was designed to examine whether the segmental levels, whereas NT-3 promoted survival of more survival ofmuscle sensory afferents from developing chicken neurons from limb compared to nonlimb levels. This suggests DRG might be specifically promoted by NT-3 and not NGF. that muscle sensory neurons, which are probably more prev- The density of muscle spindles is generally higher in limb alent at the cervical and lumbar levels, may be specifically muscles than in axial muscles (16), so it is likely that sensory affected by NT-3. To evaluate this possibility directly, we ganglia supplying limbs have a higher proportion of muscle compared the survival of retrogradely labeled muscle and sensory afferents. We therefore compared the response of cutaneous neurons in NGF, BDNF, and NT-3. Identified both explanted ganglia and dissociated neurons from differ- muscle sensory neurons survived best in vitro in the presence of ent segmental levels to NGF, BDNF, and NT-3 in vitro. To NT-3, while the survival of identified cutaneous sensory neu- test the effects of the three neurotrophins on identified rons was greatest in NGF. This work provides direct evidence populations of sensory neurons directly, we compared the for a potential role of NT-3 versus NGF in the survival of a survival of retrogradely labeled muscle and cutaneous neu- specific subpopulation of DRG neurons. rons in NGF, BDNF, and NT-3. METHODS Due to its abundance in the male mouse salivary gland and its relative ease of purification, the best characterized of the Explant Cultures. DRG were removed asceptically from neurotrophic factors is nerve growth factor (NGF). If a chicken embryos at embryonic day 6, 8, or 10 (E6, E8, or E10) chicken embryo is treated with NGF during the period of and were cultured as explants in collagen gel as described (9). naturally occurring cell death, the dorsal root ganglia (DRG) Briefly, single right and left pairs of midsacral, midlumbar, are larger than normal and contain fewer degenerating neu- midthoracic, and midcervical ganglia were removed from rons (1). Evidence that NGF does not act uniformly on all embryos at each age and pooled separately in tissue culture classes of sensory neurons, however, comes from studies of medium such that explant cultures of six to eight sacral, the lumbar, thoracic, or cervical ganglia were established in 1 ml trigeminal ganglion. The portion of this ganglion that lies of collagen gel in Nunc 35-mm tissue culture dishes. Ganglia outside the central nervous system (CNS), whose neurons were grown project extensively to cutaneous targets in the face, is re- either in the absence of neurotrophic factor or in sponsive to NGF in culture. As well, the epithelial target cells the presence of NGF, BDNF, or NT-3 over a dose range of of this ganglion begin to express NGF mRNA just coincident 20 pg to 100 ng/ml. NGF was purified mouse submaxillary with the arrival of trigeminal cutaneous sensory axons (2). In gland NGF, and BDNF and NT-3 were both the human contrast, the portion of the ganglion that lies within the CNS, recombinant proteins, generously provided by James Miller the trigeminal mesencephalic nucleus, which consists of (Amgen Biologicals), produced and purified as recently de- that scribed (10). After 24 h, the extent of neurite outgrowth from proprioceptive neurons innervate skeletal muscle, is not each explant was scored as described (9) on an arbitrary scale responsive to NGF in culture (3, 4). Trigeminal mesenceph- of0-5+, with 0 being almost no fiber outgrowth and 5+ being alic nucleus neurons do survive and produce neurites when a dense halo of fibers, the maximum fiber outgrowth seen cultured in extracts of skeletal muscle, which suggests that with a saturating dose of NGF (5-10 ng/ml). the survival of these proprioceptive neurons may be target Dissociated Neuron-Enriched Cultures. For a more quanti- dependent but regulated by some factor other than NGF. tative comparison of the survival and Two NGF-related neurotrophic factors, both of which are neurite-outgrowth pro- capable of influencing the survival of sensory neurons in Abbreviations: NGF, nerve growth factor; DRG, dorsal root ganglia; CNS, central nervous system; BDNF, brain-derived neurotrophic The publication costs of this article were defrayed in part by page charge factor; NT-3, neurotrophin 3; E6, etc., embryonic day 6, etc.; diI, payment. This article must therefore be hereby marked "advertisement" 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. fTo whom reprint requests should be addressed. 2613 Downloaded by guest on September 24, 2021 2614 Neurobiology: Hory-Lee et al. Proc. Natl. Acad. Sci. USA 90 (1993) moting effects ofthe neurotrophins on lumbar versus thoracic DRG neurons, separate cultures of dissociated neuron- enriched lumbar and thoracic DRG neurons were prepared from E8 chicken embryos. Cultures were established as de- scribed (5) except that separate pools of midlumbar (two pair of right/left ganglia from each embryo) and midthoracic gan- glia were collected and used to prepare lumbar or thoracic DRG cultures. Neuron-enriched cell suspensions were pre- pared by differential preplating to remove nonneuronal cells, and neurons were plated at a density of 10,000 phase bright cells per 35-mm dish. After 48 h in the presence or absence of NGF, BDNF, or NT-3, the cultures were fixed in 4% para- formaldehyde and the number of surviving neurite-bearing neurons was determined in microscope fields covering 11% of each dish. All conditions were established in triplicate. Retrogradely Labeled Dissociated Cultures. Sensory neu- rons from E7 embryos were labeled in ovo as follows: muscle sensory neurons were retrogradely labeled with an aqueous suspension of green fluorescent microspheres (Lumafluor, New City, NY) pressure injected into the leg muscles through a micropipette (tip diameter, -100 ,um). Cutaneous sensory neurons were retrogradely labeled by pressure injection of an ethanol solution (12.5 mg/ml) of the fluorescent dye 1,1'- dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlo- rate (diI) (Molecular Probes) onto the surface ofthe skin ofthe hindlimb. In the latter case, care was taken not to break the skin of the embryo or to allow excess dye to escape into the amniotic fluid. The eggs were then sealed and returned to the incubator. After 22-24 h, lumbar ganglia corresponding to the labeled limb were removed, and cultures were established as described above, except that droplets of the single cell sus- FIG. 1. Transverse sections of chicken E8 spinal cord and DRG pension (7-8 ,ul, each containing -12,000 cells) were preplated following peripheral labeling of sensory neurons. (A) Green fluores- onto the bottoms of collagen-coated Nunc 1-cm-well multi- cent latex beads injected into limb muscle retrogradely label motoneu- dishes. Medium containing NuSerum (Collaborative Re- rons in the lateral motor column (LMC) and sensory neurons in DRG. search) plus one or more of the growth factors was added to (B) DiI applied to the skin labels sensory neurons in the DRG only. the wells after 1 h. In each experiment, each condition was replicated six to eight times. The cultures were left for 48 h, motoneurons, whereas the injection of diI onto the skin after which they were switched to serum-free medium (11) labeled only sensory neurons. containing one or two growth factors (see Results) and, thereafter, fed every 2-3 days for the duration of the experi- RESULTS ment. Serum-free medium was used because it does not Explant Cultures. In a previous study (R.M.L., unpub- support the survival of nonneuronal cell types. Labeled cells lished data), it was observed that the maximum response of were counted in living cultures with fluorescein or rhodamine E8 DRG explants to NGF was quantitatively similar at all optics. The entire colony was scanned to check for a uniform segmental levels. As shown in Fig. 2 (Middle), this is also true density of labeled cells. Subsequently, a representative field for BDNF, although the extent offiber outgrowth in response from the center ofthe culture was selected, and the number of to BDNF was seen to be uniformly less than NGF at all fluorescent cells within that field was recorded. Growth factor segmental levels. In contrast, the response of E8 ganglia to concentrations were 100 ng of NT-3, 100 ng of BDNF, 50 ng NT-3 showed clear segmental differences. Lumbar and cer- of NGF, and 25 ,ug of muscle extract per ml. Muscle extract vical ganglia exhibited robust responses to NT-3, although was prepared from E18 chicken hindlimb according to Davies somewhat less than NGF. (3). Control cultures were grown in medium without the Sacral and thoracic ganglia showed addition of any growth factors. weak responses to NT-3, which were only marginally above For sensory neurons labeled by injection of diI onto the scores for control ganglia (range, 0-0.5). The results in Fig. skin, the specificity of labeling was determined by checking 2 are in each case the maximum responses to each factor for the absence of retrogradely labeled motoneurons. The taken from dose-response experiments covering the range of segments of the spinal hemisection innervating the injected 20 pg to 100 ng/ml. No greater effects of NT-3 on sacral or limb were removed, and the spinal neurons were dissociated. thoracic ganglia were observed at concentrations up to 1 If the proportion of labeled cells to total cells in the spinal ,ug/ml. To determine whether the differential response of E8 cord cell suspensions was >10% that in the corresponding DRG at different segmental levels to NT-3 might represent a DRG single cell suspensions, the cells from that preparation temporal rather than a strict spatial difference, we also were discarded. In contrast, after muscle injections, a higher compared the effects of NGF, BDNF, and NT-3 on explants proportion oflabeled to total cells could usually be seen in the of E6 and E10 ganglia. As shown in Fig. 2 (Top and Bottom), spinal cell suspensions as compared to those from the asso- the patterns of effects of NGF, BDNF, and NT-3 are very ciated DRGs. similar at all three ages, suggesting that the subpopulations of The specificity of these labeling procedures is demon- sensory neurons that respond to either NGF, BDNF, or NT-3 strated in Fig. 1. The lumbosacral spinal column was re- are already established at E6. Furthermore, it is clear that the moved 24 h after labeling and fixed overnight in 4% parafor- proportion of NT-3-responsive neurons is greater in ganglia maldehyde. Transverse 50-gm frozen sections were cut and ofthe lumbar and cervical enlargements ofthe body trunk, as viewed with fluorescein or rhodamine optics. The injection of compared to ganglia that contain sensory neurons innervating microspheres into the limb labeled both sensory neurons and sacral and thoracic regions. Downloaded by guest on September 24, 2021 Neurobiology: Hory-Lee et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2615

E6 60

-4 50 Q 0 40 42\ C~ 30

o X 20

10 S LT C S LTC SLT C z 0 E8 0 001 .01 10 100 60- BDNF u 50- N zZ CD NGF 40- BDNF Ol m 30- * NT3 _ 20- 61 Ol IN1KNWKNJ [.:..l..1.:111 S LTC S LTC S LT C 10 E10 0- 5_ O .001 01 1 10 100

X 60 - NT3 I- 0XD 50- 0 P:EC Z 40 -

X 30 - E: m ;s 0- E 20 - S LTCt S L TC S LTC8L - SEGMENTAL LEVEL 10" FIG. 2. Bar graph showing maximum response of explants of E6, e O E8, and E10 chicken embryo sacral (S), lumbar (L), thoracic (T), and 0 .001 .01 .1 10 100 cervical (C) DRG to NGF, BDNF, and NT-3, respectively. Data represent maximum fiber outgrowth observed on dose-response CONCENTRATION (ng/ml) curves established for each growth factor across the range 20 pg to FIG. 3. Dose-response curves ofthe effects ofNGF, BDNF, and 100 ng/ml as described. Results are means ± SEM (n = 6-8) of NT-3 on dissociated neuron-enriched cultures ofeither lumbar (-) or scores offiber outgrowth taken at 24 h on an arbitrary scale of0-5+. thoracic (o) level DRG. (Top) Maximal effects of NGF are similar at Values for neurite growth in NT-3 at lumbar and cervical levels are both segmental levels. (Middle) BDNF shows a similar pattern at different from those at the sacral level at P < 0.001 for all three ages, both levels but a lower maximal response than NGF. (Bottom) Effect except at E10, where the response at the cervical level is different of NT-3 on lumbar neurons is almost 2-fold higher than on thoracic from the sacral level only at P < 0.075. neurons of the same age. Results are means ± SEM of triplicate The data in Fig. 2 were obtained from single pairs of cultures and are representative of several experiments. right/left ganglia dissected at midsacral, lumbar, thoracic, or Taken together, the above data suggest that NT-3 affects a cervical levels. When all intermediate segmental levels were subpopulation of spinal sensory neurons that have a greater examined separately, the maximum effects of NT-3 showed in the and a repeating wave-like pattern along the neural axis, with representation DRG of lumbar cervical enlarge- peaks at the midcervical and midlumbar ganglia and troughs ments. On the basis that these DRG probably contain more at midthoracic and midsacral levels (data not shown). proprioceptive/muscle afferents than sacral or thoracic gan- Dissociated Neuron-Enriched Cultures. For a more quanti- glia, we proceeded to studies to obtain direct evidence that tative evaluation ofthe greater effect ofNT-3 on lumbar DRG NT-3 may be a specific neurotrophic factor for sensory neurons as compared to thoracic DRG neurons, we used neurons that predominantly innervate muscle. dissociated neuron-enriched cultures prepared from E8 Retrogradely Labeled Dissociated Cultures. Muscle and chicken embryos. As shown in Fig. 3 the percentage of DRG cutaneous sensory neurons, identified by retrograde labeling neurons that survived in the presence of NGF was similar in ovo, responded very differently from each other when (40-50%) in 48-h cultures derived from either lumbar or cultured with each of the neurotrophins. Muscle sensory thoracic neurons. Similarly, the effect of BDNF on promot- neurons survived well in medium containing NT-3 or muscle ing neuronal survival was essentially the same in neuron- extract but not NGF, whereas the converse was true for enriched cultures derived from either segmental level, al- cutaneous sensory neurons. Typical experiments are shown though the maximal effect of BDNF was less than that of in Fig. 4. Labeled neurons were followed over time in NGF as seen before (5). In contrast, NT-3 promoted the serum-free medium containing one or two of the growth survival of almost twice as many lumbar DRG neurons as factors. Within 2 days, the differences in survival became thoracic neurons. No greater or lesser effect was observed apparent and they persisted for as long as the labeled cells when any of the neurotrophins was tested between 5 and 500 could be identified. In most experiments, the number of ng/ml. labeled muscle sensory neurons in medium containing NT-3 Downloaded by guest on September 24, 2021 2616 Neurobiology: Hory-Lee et al. Proc. Natl. Acad. Sci. USA 90 (1993) A C Z 80- 00

Z 60- OmX,zZI. CJ2X 9z4r1

;P 20-

0O 3 4 5 DAYS IN CULTURE DAYS IN CULll)RE

SERUM + SELECTED SERUM + AU FACTOR I SELECTED FACTOR ONLY FACrORS _ I| SELECTED FACTOR ONLY B 80- 0 Z < NGF NT-3+NGF

I-<< r:k 40- =,^ BDNF NT-3 ME B1Y2z 20- CONTROL

0 1 2 3 4 5 6 7 DAYS IN CULTURE DAYS IN CULTURE

FIG. 4. Survival of labeled dissociated sensory neurons retrogradely labeled as in Fig. 1. Surviving labeled neurons were counted at -2-day intervals after plating. Each point represents the mean SEM of six to eight separate wells. (A and B) Muscle-labeled (A) or skin-labeled (B) neurons grown for 2 days in medium enriched with NuSerum plus one or two growth factors or muscle extract (ME) and subsequently changed to serum-free medium containing the same growth factor(s). (C and D) Muscle-labeled (C) or skin-labeled (D) neurons grown for 2 days in medium enriched with NuSerum plus all growth factors and subsequently changed to serum-free medium containing only one or two growth factors. Muscle-labeled neurons survive best with NT-3 or muscle extract, while skin-labeled neurons survive best with NGF.

or muscle extract did not decrease significantly between 1 in individual experiments (Fig. 4), on average they were and 6 days in culture. The same was true for the number of intermediate between NT-3 and NGF. labeled cutaneous sensory neurons in medium containing Medium containing muscle extract was just as effective in NGF. This suggests that these factors may be necessary and saving muscle sensory neurons as NT-3. Moreover, the effect sufficient for the short-term survival of these neurons. of a combination of muscle extract with NT-3 was no larger In about half the experiments, the DRG cultures were than the effect of either one alone. A simple interpretation of initially grown in medium containing serum plus a single this result is that NT-3 may be the major component of factor, or a combination of two factors, and then deprived of muscle extract responsible for promoting the survival of serum after 48 h (Fig. 4 A and B). In this type of experiment, sensory neurons. Consistent with this, NT-3 mRNA has been we could not exclude the possibility that the different factors detected within skeletal muscle (8). might alter the initial adhesion of the different types of Trophic effects ofthe growth factors might be mediated by neurons to the substrate rather than their subsequent sur- nonneuronal cells. We believe this is unlikely. Muscle sen- vival. For the remainder of the experiments, we therefore sory neurons can survive for at least 18 days in serum-free grew both types of labeled DRG cultures in medium that medium (data not shown), but NT-3 is still necessary to contained multiple factors for the initial 48 h (Fig. 4 C and D). maintain muscle sensory neurons in culture after the first 7 After 48 h, at the same time that the cultures were deprived days, after which time virtually all the nonneuronal cells have of serum, they were also deprived of all but one or two ofthe disappeared. Cutaneous afferents labeled with diI cannot be factors. The results from the two procedures were indistin- followed for longer than 1 week in culture because the label guishable. The combined results of seven independent ex- gradually fades during this time. periments are presented in Fig. 5, where cell counts are An unexplained result is that the survival of muscle sensory shown after a total of 6 days in culture. For each of the two neurons in NGF and of cutaneous sensory neurons in NT-3 is types of sensory neuron, counts have been normalized to significantly greater than in control medium. Several factors 100% for the best factor for each cell type (NT-3 for muscle may contribute to this result. Although the control experiments sensory neurons and NGF for cutaneous sensory neurons). described in Methods make it unlikely that appreciable numbers Over all seven experiments, NT-3 promoted the survival of of muscle sensory neurons were labeled by diI applied to the approximately twice as many muscle sensory neurons as did skin, corresponding controls could not be made to ensure that NGF, while twice as many cutaneous neurons survived in cutaneous afferents were not labeled by latex beads injected NGF as in NT-3. Although the effects ofBDNF were variable into the limb. In fact, this seems likely. Another possible Downloaded by guest on September 24, 2021 Neurobiology: Hory-Lee et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2617 120- ulation by retrograde labeling from muscle or skin in ovo i SENSORY NEURONS suggests that NT-3 may be a specific growth factor for a * MUSCLE population of muscle sensory afferents. g 100- Our results are consistent with those from recent experi- 5 CUTANEOUS 14 ments in which neonatal rats were treated with a highly T specific antibody to NGF (13). Small caliber sensory neurons, ;> 80- whose central processes ramify only in the dorsal laminae of the spinal cord, are destroyed by anti-NGF. In contrast, large ) muscle spindle proprioceptive neurons, whose central collat- 60- eral axons project deep into the spinal gray matter (where they N make monosynaptic connections with motoneurons) survive < 40- anti-NGF treatment. This work suggests that NGF is the trophic factor regulating target-dependent cell death for small 0 sensory DRG neurons, which provide a rich innervation of the Z 20r_v -- skin, but that large muscle sensory neurons are regulated in some other manner. Our work suggests that NT-3 may be involved. In support of NT-3 being specific for the largest 0 NT3 NGF MUSCLE BDNF CONTROL diameter sensory neurons, DiStefano etal. (10) have recently EXTRACT shown in a comparative study of the retrograde transport of radiolabeled NGF, BDNF, and NT-3 in adult rat sciatic nerve FACTORS that 125I-labeled NT-3 is predominantly transported to the FIG. 5. Summary of survival of labeled sensory neurons in largest diameter DRG neurons. Furthermore, it appears that culture medium enriched with different growth factors. Each bar the recently identified NT-3-specific receptor trkC (14) is value (±SEM) represents average of seven separate experiments, expressed in the largest DRG neurons of adult rat lumbar such as those shown in Fig. 4. Labeled neurons were counted 6 days ganglia (unpublished data). after plating. Counts of muscle-labeled sensory neurons have been The work presented here shows that NT-3 is capable of normalized to 10%to for the value in NT-3. Counts of skin-labeled a of identified muscle sensory neurons sensory neurons have been normalized to 100% for the value in NGF. supporting population Each bar value (except that of muscle sensory neurons in BDNF) is from DRG in culture. It does this in a manner indistinguish- significantly different (P < 0.005) from its matched control (NT-3 for able from extracts made from skeletal muscle, the target muscle sensory neurons, NGF for cutaneous sensory neurons) using tissue of these cells. Moreover, it does this better than NGF, the two-tailed t test for paired samples. which we have shown to be better at supporting cutaneous sensory neurons. These experiments are one step in deter- explanation stems from evidence that DRG neurons in vivo may mining whether NT-3 may be an authentic trophic factor also require trophic support from their central targets (12). regulating the survival of muscle sensory neurons in vivo, and NGF, BDNF, and NT-3 mRNAs are present at high levels in our results are consistent with this idea. Further steps will many regions of the developing CNS (8). We used these factors require an evaluation of the role of this factor in vivo. NT-3 at much greater concentrations than would be likely in vivo. If must be demonstrated to be present in the targets of muscle there is a subpopulation of sensory neurons that would normally sensory neurons during the appropriate stages of develop- need two distinct factors, one each from the periphery and the ment. Furthermore, these neurons must be demonstrated to CNS, we may have kept this subpopulation alive in culture with undergo a greater than normal degree of cell death if the a high dose of one factor or the other. Finally, perhaps the most amount of NT-3 available from these targets is reduced by obvious explanation is that some subpopulation of muscle blocking antibodies during development. sensory afferents (for example, the smaller diameter muscle 1. Hamburger, V., Bruno-Bechtold, J. K. & Yip, J. W. (1981) J. sensory afferents) is responsive to NGF and not NT-3. Neurosci. 1, 60-71. 2. Davies, A. M., Bandtlow, C., Heumann, R., Korsching, S., Rohrer, DISCUSSION H. & Thoenen, H. (1987) Nature (London) 326, 353-358. In vitro studies have shown that a number of neurotrophic 3. Davies, A. M. (1986) Dev. Biol. 115, 56-67. factors, including ciliary neurotrophic factor and all members 4. Wayne, D. B. & Heaton, M. B. (1990) Dev. Biol. 138, 473-483. 5. Lindsay, R. M., Thoenen, H. & Barde, Y.-A. (1985) Dev. Biol. 112, of the neurotrophin family (mammalian NGF, BDNF, NT-3, 319-328. and Xenopus NT-4) promote survival and neurite outgrowth 6. Hohn, A., Leibrock, J., Bailey, K. & Barde, Y.-A. (1990) Nature of chicken embryo DRG neurons. NGF seems to affect the (London) 344, 339-341. greatest percentage of E8 DRG neurons (50-60%), whereas 7. Maisonpierre, P. C., Belluscio, L., Squinto, S., Ip, N. Y., Furth, BDNF and NT-3 support the survival of 20-40% of these M. E., Lindsay, R. M. & Yancopoulos, G. D. (1990) Science 247, in dissociated cultures derived from ganglia pooled 1447-1451. neurons 8. Maisonpierre, P. C., Belluscio, L., Friedman, B., Alderson, R. F., from various segmental levels. It has previously been shown Wiegard, S. J., Furth, M. E., Lindsay, R. M. & Yancopoulos, that the effects of BDNF are additive with those of NGF in G. D. (1990) Neuron 5, 501-509. neuron-enriched cultures of E6, E8, or E10 DRG, suggesting 9. Lindsay, R. M. & Peters, C. (1984) Neuroscience 12, 45-51. that these two neurotrophins act on distinct subpopulations 10. DiStefano, P. S., Friedman, B., Radziejewski, C., Alexander, C., of DRG neurons (5). Boland, P., Schick, C., Lindsay, R. M. & Wiegand, S. J. (1992) To date we know of no study that has specifically examined Neuron 8, 983-993. 11. Bottenstein, J. E., Skaper, S. D., Varon, S. S. & Sato, G. H. (1980) possible differential effects of neurotrophic factors on the Exp. Cell Res. 125, 183-190. survival of DRG neurons at different segmental levels. In the 12. Yip, H. K. & Johnson, E. M., Jr. (1984) Proc. Natl. Acad. Sci. USA present study, we have shown that the effects of NGF and 81, 6245-6249. BDNF are essentially the same on DRG neurons taken from 13. Ruit, K. G., Elliott, J. L., Osborne, P. A., Yan, Q. & Snider, W. D. all segmental levels, suggesting that NGF- and BDNF- (1992) Neuron 8, 573-587. 14. Lamballe, F., Klein, R. & Barbacid, M. (1991) Cell 66, 967-979. responsive neurons are relatively constant along the neural 15. Lindsay, R. M. (1993) in Neurotrophic Factors, eds. Loughlin, axis. In contrast, many more NT-3-responsive neurons were S. E. & Fallon, J. (Academic, London), in press. found in lumbar and cervical ganglia than in either thoracic or 16. Bolterman, B. R., Bindu, M. D. & Stuart, D. G. (1978) Am. Zool. sacral ganglia. Characterization of this NT-3-responsive pop- 18, 135-152. Downloaded by guest on September 24, 2021