Neuron,

Vol. 8, 573-587, March,

1992, Copyright

0 1992 by Cell Press

Selective Dependence of Mammalian Dorsal Root Ganglion Neurons on Nerve G rowth Factor during Embryonic Development Kenneth C. Ruit,*+* Jeffrey 1. Elliott,* Patricia A. Osborne,+ Qiao Yan,§ and William D. Snider* *Department of Neurology +Department of Molecular Biology and Pharmacology Washington University School of Medicine St. Louis, Missouri 63110 §Amgen Inc. Thousand Oaks, California 91320

Summary We have investigated the NCF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer antiNCF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NCF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3. Introduction The different sensory modalities are known to be subserved by distinct classes of neurons in dorsal root ganglia (DRGs). In cats, at least eight classes have been described on the basisof axonal conduction velocities and peripheral projections to different sensory receptors (for reviews see Brown, 1981; Willis and Coggeshall, 1991). Each class of DRG neurons has a characteristic central projection within the spinal cord. Other species have been less well studied, although most of these classes of DRG neurons are known to be present in rat (Smith, 1983; Fitzgerald, 1987; Woolf, 1987). Because of their accessibility and ease of maintenance in vitro, DRC neurons are frequently utilized in studies of neuronal cell biology. Nerve growth factor (NGF), the prototypical neuronal growth factor, was characterized on the basis of its effects on neurite outgrowth in DRG explants. Despite the fact that the * Present address: Department of Anatomy and Cell Biology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202.

influences of NGF on DRG neurons have been known for almost 40 years, which of the several classes of DRG neurons require NGF for survival during development has not been clearly determined. Problems in identifying the NGF-dependentclassesof DRG neurons in vivo have included the following: DRG neurons are most dependent on NGF during embryonic life, when experimental manipulations are difficult; the specificity of anti-NGFantibodies used in previous experiments is unknown in relation to the newly described members of the NGF neurotrophin family; and noconvenient method has been availableto identify functional classes of DRG neurons. There is general agreement that small, substance P-containing neurons, which presumably mediate nocic:eption, require NGF (Otten et al., 1980; Johnson et al.,, 1980; Ross et al., 1981; Goedert et al., 1981,1984; Yip et al., 1984). Whether other classes of DRG neurons require NGF to the same extent, to a lesser extent, or are NGF independent is unclear. On one hand, NGF appears to be required for the survival and growth of most embryonic DRG neurons. In vitro, the dense neuriteoutgrowth from embryonic DRG explants observed when NGF is added to the medium suggests that most DRG neurons respond to this factor (Levi-Montalcini and Angeletti, 1968). In vivo, exogenous NGF administration prevents naturally occurring neuronal death in DRGs of embryonic chicks, sparing both the “large” ventrolateral and the “small” dorsomedial classes of DRG neurons (Hamburger et al., 1981). Furthermore, administration of exogenous NGF can almost completely prevent the substantial DRG neuronal death that accompanies axotomy during the neonatal period (for a review, see Johnson et al., 1986), and virtually all DRG neurons can transport the protein from the periphery (Yip and Johnson, 1986). Finally, exposure of embryos to circulating maternal or exogenous anti-NGF antibodies results in the lossof upto85% of DRG neurons(Johnson et al., 1980). On the other hand, some DRG neurons invariably survive prenatal exposure to antibodies in the immunological NGF deprivation paradigms employed to date (Johnson et al., 1980; Aloe at al., 1981; Goedert et al., 1984). Furthermore, only 50%-60% of DRG neurons bind NGF with high affinity (Verge et al., 1989a, 1989b). Physiological studies in neonatal animals have shown that NGF is required for the appropriate phenotypic development of cutaneous high threshold mechanoreceptors, whereas development of class D hair follicle afferents appears to be NGF independent (Ritter et al., 1991; Ritter and Mendell, 1991, Sot. Neurosci., abstract; Lewin et al., 1991, Sot. Neurosci., abstract). One study of the effects of NGF on axotomized DRG neuronsdemonstrated that NGFdoes not rescue large, functionally identified proprioceptive neurons, whereas small neurons are saved (Miyata et al., 1986).

Table 1. Determination

of Antibody

Specificity

Neurotrophic Factor

Antiserum Dilution

Neurite Outgrowth”

NGF (1 q/ml)

1:1,000 1:10,000 Control I:100 1:1,000 1:10,000 Control I:100 1:1,000 1:10,000 Control

0, 0, 3, 3, 3, 3, 3, 4, 4, 4 4,

BDNF (1 q/ml)

NT-3 (1 q/ml)

0, 0, 3, 3, 3, 3, 3, 4, 4, 4, 4,

0, 0, 3, 2, 2, 3, 3, 4, 4, 4, 4,

0, 0.5 0, 1 4, 4 2, 2 2 2.5, 2.5 2.5, 2 3, 3 4, 3 4, 4 3

a Score on a O-5 scale (0 = no neurite outgrowth; 5 = maximum neurite outgrowth). Neurite outgrowth was assessed in 4-5 E8 DRGs for each antiserum dilution. Control assays contained no antiserum.

All of these results suggest that different requirements for NGF may exist among populations of DRG neurons. In addition to NGF, three recently described members of the NGF family of neurotrophins, brainderived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), have demonstrated effects on DRG neurons in vitro (Lindsay et al., 1985; Davies et al., 1986; Leibrock et al., 1989; Hohn et al., 1990; Maisonpierre et al., 1990; Hallbijtik et al., 1991). BDNF also diminishes naturally occurring neuronal death in DRGs in vivo (Kalcheim et al., 1987; Hofer and Barde, 1988). However, whether these neurotrophins affect the same, overlapping, or completely different classes of DRG neurons is unknown. The fact that NGF and BDNF have additive effects on the survival of embryonic DRG neurons provides some evidence that different classes of DRC neurons may be supported by each factor (Lindsay et al., 1985; Davies et al., 1986). An observation critical to the interpretation of previous NCF deprivation experiments is that there is substantial sequence homology among all of these neurotrophins (Leibrock et al., 1989; Hohn et al., 1990; Maisonpierre et al., 1990; Hallb66k et al., 1991). This finding raises the issue of whether polyclonal antibodies raised against NGFand used in numerous previous experiments may have cross-reacted with BDNF or NT-3. It has been suggested that such cross-reactivity may have led to misinterpretation in these experiments, in which antibodies of unknown specificity were utilized (Acheson et al., 1991; but see also Barde et al., 1982). In a recent major advance, the putative high affinity receptors (trk, trkB, and trkC) that mediate the physiological effects of these neuronal growth factors have been identified (Hempstead et al., 1991; Kaplan et al., 1991; Klein et al., 1991; Lamballe et al., 1991; Soppet et al., 1991; Squint0 et al., 1991). Although receptor localization promises to identify DRG neurons that are responsive to the different neurotrophins at the level of the ganglion (Carroll et al., submitted), neuro-

trophin receptor expression has not yet been directly correlated with the physiological and morphological characteristics of particular populations of DRG neurons. In the present study, we have deprived embryonic rats of NGF by injecting individual rat embryos at precise developmental time points with a specific, high titer anti-NGF antiserum. We have identified different classes of DRG neurons by visualizing their characteristic central projections utilizing the intenselyfluorescent, lipid-soluble carbocyanine dye, Dil (see Honig and Hume, 1986; Godement et al., 1987; Snider and Palavali, 1990). These techniques have allowed us to describe the consequences of prenatal NGF deprivation for particular populations of DRG neurons. Results Administration of Anti-NCF Antiserum to Embryonic Animals The antiserum used in these experiments (generously provided by E. M. Johnson, Jr.) was raised in a goat immunized with 2.5s NGF. This anti-NGF antiserum is exceedingly useful for studies in vivo because of its high titer of antibodies directed against NGF. To determine the specificity of this antiserum for NGF, we tested its ability to block neurite outgrowth induced by neurotrophins in conventional in vitro bioassays employing embryonic chick DRG explants (Lindsay et al., 1985). The results of one series of experiments are given in Table 1. The antiserum prevented NGFinduced neurite outgrowth from the DRG explants at dilutions of up to 1:10,000, confirming its high level of activity against NGF. Conversely, the antiserum had no effect on BDNF- or NT-3-,induced neurite outgrowth, even at dilutions as low as 1:lOO. A second series of experiments was carried out in order to confirm this specificity, and the results were identical (data not shown). Therefore, we conclude that this antiserum is absolutely specific for NGF. A significant problem with previous paradigms of immune deprivation is that the precise point at which animals were deprived of NGF during embryonic development is unknown. For example, in pregnant mothers rendered autoimmune, when embryonicanimals were first exposed to NGF antibodies was unclear. How long NGF antibody titers were maintained in experiments utilizing single injection paradigms was also unknown. We therefore utilized the chick DRG explant assay to assess the anti-NGF activity in sera from injected fetuses. We determined how long demonstrable antibody titers persisted in embryos after injections of anti-NGF antiserum in utero at precise developmental time points. The titer is defined as the reciprocal of the highest serum dilution that blocked neurite outgrowth. Even with the limited amount of sera that could be obtained from embryonic animals, it was possible to demonstrate NGF antibody activity (titer >2) after a single in utero injection of our anti-NGF antiserum at embryonic day 15 (E15).

Selective NCF Dependence 575

Table 2. Number Antibody Titers

After

of Prenatal Sensory Neurons

of Embryos

Single Injection

with

Serum

% OF TOTAL CELLS 30

NCF After Double Injection (El5 and EIB)

(E15)

El7

El8

El9

E21

3/4 (75%)

6/9 (67%)

317 (43%)

23/24 (96%)

Embryoswereconsidered to havedemonstrable serum anti-NGF activity if serum blocked the outgrowth of neurites in the chick DRG bioassay at a dilution of at least 1:2. At E21, when more serum was available for analysis, anti-NCF activity was present at dilutions of I:32 or greater. Only embryos with demonstrable anti-NGF titers were chosen from litters for cell counting and Dil staining.

However, over the E15-El9 period, the number of animals exhibiting serum antibody titers after a single injection decreased (Table 2). For example, 75% of the embryos had demonstrable titers at E17, whereas only 43% of the embryos had titers at E19. Therefore, we determined that a second injection of anti-NCF antiserum was necessary to maintain antibody titers in embryonic animals up to the day of birth (E21). El8 was chosen as the day for the second injection because a majority of the embryos had detectable titers on El8 after a single injection (Table 2). After receiving a second injection of antiserum, 96% of the embryos had very significant serum anti-NGF antibody titers (>32) on E21. Therefore, by using the double injection paradigm, we were confident that embryos were deprived of NCF during the entire E15-E21 period. Effects of Prenatal Exposure to Antibody on DRC Neuronal Survival and Size In utero exposure to anti-NGF antibodies significantly reduced the number of neurons within the C5 DRG. Cell counts revealed that in control animals injected with preimmune goat serum, the left C5 DRG contained 3872 (k 232 SEM) neurons on the day of birth (Table 3). After continuous exposure to anti-NGF anti-

Table 3. Effects of In Utero Neuronal Survival Group Control (n = 6)

NGF-deprived (n = 4)

NCF Deprivation

on DRG

Raw Count

Correction Factor

Corrected Count

Corrected Mean + SEM

8931 7197 7911 6330 8457 6414 2436 1470 2388 2331

0.52 0.50 0.52 0.51 0.51 0.52 0.54 0.53 0.54 0.58

4644 3599 4114 3228 4313 3335 1315 779 1290 1352

3872 f 232

1184 f 136

Correction factors were calculated by the Abercrombie (1946) formula (section thickness/average nuclear diameter + section thickness). n represents the number of ganglia.

I

I I

25

n Control 0 NGF deprived

m

:: 100

150

200

250

300

350

400

450

Figure 1. Effects of In Utero NGF Deprivation Sectional Areas of DRC Neuron Somata

500

550

600

on the Cross-

Histogram illustrating the distribution of DRC neuron crosssectional areas from control and NGFdeprived anirnals.Animals immunologically deprived of NCF in utero lost the population of the smallest DRG neurons, whereas the larger neurons were spared. Therefore, the mean cross-sectional area of neurons present within the ganglia of NCF-deprived animals was increased, and the larger cells represented a greater percentage of thecelIswithintheganglia.Atleast250celI profilesineachgroup were used to generate these histograms.

body over the E15-E21 period, the number of neurons in the ganglion was reduced to 1184 (k 136 SEM). This representsa70% decrease in neuronal number.A caveat in interpreting this result is that, in the presence of the severe atrophy accompaning trophic deprivation, nonneuronal satellite cells may be difficult to distinguish from neurons. Thus, the degree of cell death may have been slightly overestimated. However, these results clearly demonstrate that a majority of DRG neurons require NGF for survival during development. Some indication about the classes of DRG neurons most affected by NGF deprivation could be gained by measuring neuronal cross-sectional areas taken from semi-thin (1 urn) plastic sections. A histogram illustrating the distribution of DRG neuronal soma areas in control and NGF-deprived animals is shown in Figure 1. The average cross-sectional area of neurons present withintheC5DRGoftheratonthedayofbirthwas275 pm2 (k 9 SEM). After prenatal exposure to anti-NGF antibody over the E15-E21 period, the distribution of cross-sectional areas of neurons still present within the ganglion shifted substantially toward larger values, and the average area increased to 394 urn* (f 10 SEM). This increase was due predominantly to the the virtual elimination of the population of small neurons ( 0.05). Finally, it was possible to quantify the branching of type la muscle afferents in an even more direct way. These afferents defasciculate and elaborate terminal arborizations as they near motor neurons in the ventral horn (Figures 5A and 5B, large open arrows). We therefore quantified the type I muscle afferent projection by measuring the accumulated total length of the la projection in laminae VII-IX within a 70 pm section of the C5 segment. In doing so, we chose a section from the middle of the C5 segment that we felt adequately represented the projection and drew the projection in detail from photoconverted material by using camera lucida (Figures 5A and 5B). Quantitative analysis showed that the accumulated length of the type la muscle afferent projection to the ventral horn in embryos exposed in utero to anti-NGF antibodies (14,320 pm f 1,402 SEM)was not significantly different (p > 0.05) from that measured in control animals (14,572 pm + 848 SEM) (Table5). Therefore, NGF deprivation did not have any demonstrable effect on the

N‘.ZU”3” 580

Figure 5. Summary

of Primary

Afferent

Distribution

at E21 in Control

and NCF-Deprived

Embryos

(A) Camera lucida drawing of the primary afferent projection to the C5 spinal cord segment of an E21 control embryo. The primary afferents projecting to the superficial dorsal horn (nociceptors and thermoreceptors, long black arrows), the deeper laminae of the dorsal horn (mechanoreceptors, large black arrow), and the ventral horn (type la muscle afferents, large open arrow) are well developed at this age, and their patterns of innervation within appropriate target fields are very complex. cc, central canal. Bar, 100 Lrn. (B) Camera lucida drawing of the primary afferent projection to the C5 spinal cord segment of an E21 NGF-deprived embryo. Although the superficial dorsal horn (long black arrows) essentially lacks primary afferent input, the afferents innervating the deeper laminae of the dorsal horn (large black arrow) appear unaffected and sprout into the dorsal uninnervated regions. The complexity of the afferent arborizations within the ventral horn (large open arrow) also appears unaffected when compared with that of controls. cc, central canal. Bar, 100 pm. (C) Photomicrograph of a transverse C5 spinal cord section showing the Dil-labeled, photoconverted primary afferent projections within the spinal cord of an E21 NCF-deprived embryo. Illustrated in the photomicrograph are the regions of interest (1 and 2) used in the optical densitometric analysis. Region 1 corresponds to laminae I and II, and region 2 corresponds to laminae Ill and IV. Bar, 100 pm.

branching of the central processes of DRG neurons that project to muscle spindles in the periphery.

Discussion Experimental

Paradigms of Immune Deprivation

The objective of the present study was to determine whetherdifferentclassesof mammalian DRG neurons differ in their dependence on NGF during embryonic development. Previous studies have described cell loss in DRGs as well as biochemical alterations associated with prenatal NGF deprivation (Johnson et al., 1980; Aloe et al., 1981; Goedert et al., 1984). We now show, by using an antibody that is strictly specific for NGF and an anatomical technique that can identify DRG neurons by their characteristic central processes, that the dependence of mammalian DRG neurons on NGF during embryonic development is strikingly selective. Since mammalian DRG neurons are most sensitive to NGF during embryonic development, an experimental approach was utilized to deprive embryos of NGF in utero. In this study we have demonstrated the feasibility of direct in utero injections of a specific antiserum as a means of depriving developing DRG

neurons of trophic support (see also Goedert et al., 1984; Johnson et al., 1989). Double injections of antiNGF antiserum directly into developing rat embryos effectively maintained elevated serum NGF antibody titers from E15-E21. In contrast to previous studies, we demonstrated that our embryos were actually deprived of NGF throughout this entire period. A potential problem associated with these experiments is cross-reactivity of the polyclonal antibody with BDNF, NT-3,0rother membersoftheNGFneurotrophin family that share considerable sequence homology with NGF (Leibrock et al., 1989; Hohn et al., 1990). Other authors have recently demonstrated that some polyclonal NGF antibodies significantly crossreact with BDNF and have questioned the validity of previous studies that utilized anti-NGF antibodies of unproven specificity(Acheson etal., 1991). In the present study, the ability of our antiserum to prevent neurite outgrowth induced by BDNF and NT-3 was specifically tested. In dilutions as low as 1:100, our antiserum failed to block BDNF- or NT-3-induced neurite outgrowth from embryonic chick DRG explants. Therefore, it is unlikely that our results are compromised because of the cross-reactivity of our antibody with other neurotrophins.

Selective NGF Dependence 581

Table 4. Mean Values

of Prenatal Sensory Neurons

for Area and Optical

Group Region I (laminae I and II) Control (n = 3)

NCF-deprived

(n = 4)

Region 2 (laminae III and IV) Control (n = 3)

NGF-deprived

(n = 4)

Density

of Dil-Labeled

Regions

Area of Region (# of Pixels)

Mean Area f SEM (# of Pixels)

3394 2958 3216 3203 2582 2217 2110

3189 f 127

2765 3099 3173 3063 2600 2949 2762

3012 f 125

2528 + 247'

2844 + 102a

of the Dorsal

Pixel Gray Value 57 51 58 213 222 111 155

+ + f i f + *

0.5 0.5 0.7 0.6 0.9 1.0 1.0

59 50 76 112 133 67 76

f f * + * f f

0.5 0.5 0.8 1.0 1.2 0.6 0.6

Range of pixel gray values (pixel values): 0 (black) to 256 (white). n represents the number from each animal. a Values not significantly different from controls Lp > 0.05 by Student’s t test). b Value significantly different from control tp < 0.02 by Student’s t test).

Identification of DRG Neurons That Require NGF for Survival In agreement with the results of experimental autoimmune approaches to NGF deprivation (Johnson et al., 1980,1982, 1983), our results show that in utero NGF deprivation results in the loss of a majority (70%) of mammalian DRG neurons. However, some neurons clearly survived the treatment with anti-NGF antiserum. A clue to the functional class of these neurons was obtained by examining the distribution of DRG neuron cross-sectional areas in experimental animals and comparing it with that of controls. Analysis of these data by frequency histograms revealed that NGF deprivation had marked effects on the neurons with the smallest diameters, whereas the neurons with the largest diameters were unaffected. It was difficult to draw firm conclusions about neurons of intermediate diameter using this technique. Although previous studies agree that the small diameter DRG neurons are most affected by NGF deprivation (Johnson et al., 1980; Yip et al., 1984; see also Goedert et al., 1984), the extent to which the larger DRG neurons also require NGF has been unclear. To identify the classes of DRG neurons affected by

Table 5. Average Cumulative Muscle Afferent Projections

Lengths

of Type la

Group

Length

Control (n = 4) NGF-deprived (n = 4)

14,572 f 848 14,320 f 1,402

n represents determined.

the number

of animals

(pm) f SEM

from

which

values

were

Horn

of separate

Mean Pixel Gray Value f- SEM 55 f 2

175 k 26b

62 f 8

97 + 15"

images; one image was generated

NGF deprivation, we have labeled the characteristic central projections of DRG cells with Dil. We and others havedescribed thetimecourseof prirnaryafferent ingrowth, as well as the specific laminar distribution and characteristic morphology of DRG neuronal projections within the embryonic rat spinal cord (Smith, 1983; Fitzgerald, 1987; Snider et al., submitted). Because the different classes of DRG cells project characteristically to distinct regions of spinal gray matter, the consequences of peripheral manipulations on particular populations of DRG neurons can be readily determined. The ability of the neuroanatomical tracer Dil to label central DRG projections in remarkable detail has allowed us to describe easily and confidently the effects of NGF deprivation on these projections. Our results show that NGF is responsible for the survival and terminal arborization of the primary sensory neurons that innervate laminae I and II of the dorsal horn. These are the primary afferents that are generally associated with nociception and thermoreception (for a review see Willis and Coggeshall, 1991). In contrast, the central projections of other classes of DRG neurons appeared unaffected; fibers that project to laminae III and IV (hair follicle afferents and other low threshold mechanoreceptors) and the fibers projecting to the ventral horn (muscle afferents) appeared normal. Interestingly, in NGF-deprived animals, the central arborizationsof lowthreshold mechanoreceptors normally confined to laminae III and IV sprouted into laminae I and II, invading the region of dorsal horn uninnervated as a result of NGF deprivation. Video-based optical densitometry of bulk-labeled, photoconverted material demonstrated quantitatively the dependence of the superficial dorsal horn projection on NGF. The striking depletion of fibers in lami-

nae I and II is consistent with the widespread loss of small neurons within the individual DRG. In contrast, we could find no evidence for effects of NGF deprivation on other populations of DRG neurons. Densitometric analyses performed on deeper laminae of the dorsal horn showed no significant differences between control and NGF-deprived animals. Furthermore, quantitation of the distal branching of the group la muscle afferents showed no significant difference between control and NGF-deprived animals. These results demonstrate that different requirements for NGF exist among the various populations of DRG neurons. It is not possible to state with certainty that DRG neurons projecting to deeper spinal laminae are completely unaffected by NGFdeprivation. Possibly, some DRG neurons in other classes were lost, but the central projections of surviving neurons sprouted, yielding a spinal projection quantitatively similar to that of controls. Indeed, such a phenomenon was hypothesized to explain the normal appearance of the highly ordered pattern of projections to primary sensory cortex (the “barrel” fields) after massive destruction of trigeminal ganglion cells in NGF-deprived guinea pigs (Sikich et al., 1986). We can, however, conclude that in utero NGF deprivation results in the massive depletion of the central projection to a specific region of the spinal cord while resulting in no qualitative or quantitative changes in projections to other regions. Thus, even in the most conservative interpretation of our results, striking differences are seen in the degree of NGF dependence of DRG neurons projecting to these different regions. Relationship of the Present Results to Previous Investigations of NGF Dependence Many previous studies have either directly or indirectly addressed the issue of the NGF dependence of various classes of DRG neurons. However, most of these studies have been performed during the postnatal period, when dependence on NGF is less acute. Furthermore, the specificity of polyclonal NGF antibodies employed in these studies is uncertain. Finally, many of these studies have utilized methods less convenient or direct than those employed here to identify various classes of DRG neurons. Several previous studies have classified DRG neurons by the expression of different neuropeptides. All of these studies agree that substance P-containing DRG neurons are particularly vulnerable to NGF deprivation (Ottten et al., 1980; Ross et al., 1981; Schwartz et al., 1982; Goedert et al., 1984; Lindsay and Harmer, 1989; see also Kessler and Black, 1980). However, the extent to which other types of peptide-expressing neurons require NGF is controversial. For example, studies have produced conflicting results about whether DRG neurons expressing somatostatin require NGF (Ross et al., 1981; Goedert et al., 1984; see also Verge et al., 198Va). Thus, classifying DRG neu-

rons by peptide expression has not yielded a clear conclusion about whether NGF dependence is selective. A previous finding that strongly supports the idea that DRG neurons may have differing requirements for NGF is that not all DRG neurons are capable of binding NGF with high affinity. Indeed, studies have shown that only 50%-60% of DRG neurons bind NGF with high affir;ity during the postnatal period (Verge et al., 198Vb). Assuming that the biological effects of NGF are mediated via high affinity binding sites, the results of Verge et al. argue that only .50%-60% of DRG neurons .may require NGFfor survival. However, many more DRG neurons are capable of binding NGF with lower affinity (Yan and johnson, 1988). Whether these neurons require NGF for survival or whether they can exhibit biological responses to NGF is uncertain. Interestingly, both “large” and “small” DRG neurons bind NGF with high affinity. The functional identity of these neurons is not clear, except for the fact that many of the small DRG neurons express the neuropeptide substance P (Verge et al., 198Va). Several studies reporting effects of exogenously administered NGF have suggested that NGF may influence avariety of classes of DRG neurons. In the chick, NGF prevents naturally occurring cell death in populations of both large ventrolaterai and small dorsomedial DRG neurons (Hamburger et al., 1981). In addition, axotomy-induced cell death in the neonatal period can be largely prevented by NGF administration (for a review see johnson et al., 1986). These studies utilizing exogenous NGF suggest rather generalized effects of NGF on many classes of DRG neurons. A possible explanation for these results is that NGF administered in supra-physiological doses exerts its effects nonspecifically. For example, exogenous NGF might act via effects transduced by the low affinity NGF receptor, which is expressed by most, if not all, DRG neurons (Yan and Johnson, 1988), or by binding to the high affinity receptor of another neurotrophin, such as BDNF, and exerting an agonistic effect (Rodriguez-Tkbar et al., 1990). However, exogenously administered NGF does not rescue specifically identified proprioceptive DRG neurons after neonatal axotomy (Miyata et al., 1986). This result suggests that even the effects of exogenously administered NGF may be more specific than previously realized. Dependence of DRG Neurons on Other Neurotrophins Our results are consistent with work in vitro suggesting that distinct populations of neurons within peripheral sensory ganglia depend preferentially on different neurotrophins. Using NGF and the other members of the NGF neurotrophin family, BDNF and NT-3, these studies have shown that peripheral neurons which respond to these molecules appear to be largely distinct (Davies et al., 1986; Lindsay et al., 1985; Maisonpierre et al., 1990; Hohn et al., 1990). For example, many DRG neurons, as well as neural crest-de-

Selective 583

NGF

Dependence

of Prenatal

Sensory

Neurons

rived trigeminal neurons, respond to NGF in culture with neurite outgrowth. Conversely, placode-derived trigeminal neurons and neurons from other peripheral ganglia (e.g., nodose) respond to BDNF and NT-3 rather than to NGF. Interestingly, within DRGs, a clear overlap exists in the responses of the various classes of neurons to the different neurotrophins. For example, both NGF and BDNF appear capable of supporting the majority of DRC neurons in vitro (Lindsay et al., 1985; Davies et al., 1986). Nevertheless, more DRG neurons survive in the presence of both NGF and BDNF than survive with either agent alone (Lindsay et al., 1985; Davies et al., 1986). This result supports the hypothesis that classes of DRG neurons specifically require one or the other of these neurotrophic molecules and, as such, is consistent with the findings of the present study. An interesting issue to consider is whether some DRG neurons in our study may have survived NGF deprivation by acquisition of trophic support via their central processes. To what extent our systemically administered antibody crossed the blood-brain barrier and exerted its effects within the central nervous system of rat embryos is unknown. Sensory neurons have the ability to transport NCF retrogradely over their central as well as their peripheral processes (Richardson and Riopelle, 1984; Yip and Johnson, 1984). Indeed, the substantial cell death that occurs after transection of the central processes of DRG neurons suggests that theexpression of neurotrophic factors by target populations of spinal cord neurons may be important to the survival of DRG neurons during development (Yip and Johnson, 1984; see also Davies and Lumsden, 1984). Furthermore, one neurotrophin, NT-3, is now known to be synthesized by motor neurons in embryonic rat spinal cord (Ernfors and Persson, 1991). This finding suggests that the large DRG neurons which innervate muscle spindles and synapse on motor neurons in the ventral horn may be NT-3 dependent and may acquire this factor by virtue of their spinal connections as well as from the periphery. However, in the present study we chose El5 as the starting date for injections of anti-NGF antiserum because this is 24-48 hr before any DRG neurons establish connections within the spinal cord (Smith, 1983; Kudo and Yamada, 1987; Snider et al., submitted). Therefore, the results presented here are not influenced by potential acquisition of trophic support via the central connections of DRG neurons. Physiological Significance The potential physiological consequences of prenatal NGF deprivation are worth emphasizing. Since small diameter, substance P-containing primary sensory neurons projecting to laminae I and II of the dorsal horn may be specifically related to mechanisms of pain (for a review see Willis and Coggeshall, 1991), a consequential reduced sensitivity to noxious stimuli would be expected in animals deprived of NGF during

development. Indeed, in previous studies of embryonic autoimmune NGF deprivation, offspring deprived of NGF were unable to feel pain and died in the early neonatal period (Johnson et al., 1980, 1983; see also Aloe et al., 1981). Even postnatal NGF deprivation, which results in far less extensive loss of DRG neurons, significantly diminishes response to nociceptive stimuli (Urschel et al., 1991). Interestingly, systemic injections of the chemical irritant capsaicin to neonatal rats also selectively lesion small diameter, substance P-containing nociceptive DRG neurons that innervate the superficial dorsal horn (Jancso et al., 1977; Nagy et al., 1981, 1982; Russell and Burchiel, 1984). Moreover, as we find in this study, the cutaneous mechanoreceptive fibers in the deeper laminae of the dorsal horn sprout into the denervated superficial laminae in response to capsaicin treatment (Nagy and Hunt, 1983; Rethelyi et al., 1986; Beal and Knight, 1987; Shortland et al., 1990). NGF partially prevents the effects of capsaicin, leading to the hypothesis that capsaicin exerts its effects on these neurons by blocking the retrograde transport of NGF and, thus, its biological action (Otten et al., 1983). However, whether the populations of DRG neurons lesioned by NGF deprivation and capsaicin administration are identical remains to be demonstrated. Fibers subserving thermal sensation also terminate largely in laminae I and II of the dorsal horn (see Willis and Coggeshall, 1991). An implication of our work, then, is that NGF may be important in the development of pathways subserving both nociception and thermoreception. Results showing that NGF mRNA is expressed in skin, that NGF mRNA is expressed at higher concentrations in more superficial (epidermis) as opposed to deeper layers (dermis), and that NGF is responsible for the normal development of afferents which terminate superficially (Davies et al., 1987) all support the idea that skin-derived NGF may regulate the development of pain- and temperature-responsive pathways. Reasoning by analogy, low threshold mechanoreceptors, which subserve tactile sensation and innervate deeper cutaneous and subcutaneous targets, and proprioceptive afferents, which innervate muscle spindles and Golgi tendon organs, may be organized around specific dependence on other neurotrophins. Classification of DRC Neurons by Expression of High Affinity Neurotrophin Receptors In a recent advance with major implications for the results of the present study, the putative high affinity receptor responsible for the biological action of NGF was identified as an integral membrane glycoprotein identical to the product encoded by the trk protooncogene (Hempstead et al., 1991; Kaplan et al., 1991; Klein et al., 1991). Similarly, the putative high affinity receptors mediating the biological effects of BDNF and NT-3 have also been identified as the products of related genes, trkB (Soppet et al., 1991; Squint0 et al.,

Neuron 504

1991) and t&C (Lamballe et al., 1991), respectively. The results presented here suggest that these different high affinity neurotrophin receptors are expressed by nonoverlapping populations of DRC neurons and, hence, that only one population would be lesioned by NGF deprivation. In a separate study we have shown that this is indeed the case; NGF deprivation has remarkably selective effects, totally lesioning the trk while largely population of neurons expressing sparing the populations of neurons expressing trkB and trkC (Carroll et al., submitted). The results of Carroll et al., taken together with those presented here, imply that trkB- and trkC-expressing neurons may be the mechanoreceptive and proprioceptive DRG neurons spared in the current study. Conclusion In summary, we have shown that the NGF dependence of embryonic rat DRG neurons is selective. Although a majority of the neurons within the ganglion are lost in response to prenatal NGF deprivation, these neurons are specifically those that project to the superficial dorsal horn. The central projections of neurons remaining within the ganglion appear remarkably normal, suggesting that these neurons are unaffected by NCF deprivation. By using a similar experimental approach, it should be possible to determine which neurotrophins are responsible for supporting these other populations of DRG neurons in vivo. Experimental

Procedures

Antibody Specificity A high antibody titer, unfractionated goat anti-mouse 2.55 NGF serum was utilized in these experiments. The serum antibody titer was >lOO,OOO (