Nerve growth factor receptor trkA is down-regulated during postnatal development by a subset of dorsal root ganglion neuronsкод для вставкиСкачать
THE JOURNAL OF COMPARATIVE NEUROLOGY 381:428–438 (1997) Nerve Growth Factor Receptor TrkA Is Down-Regulated During Postnatal Development by a Subset of Dorsal Root Ganglion Neurons DEREK C. MOLLIVER AND WILLIAM D. SNIDER* Center for the Study of Nervous System Injury, Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110 ABSTRACT Nerve growth factor (NGF), signaling through its receptor tyrosine kinase, TrkA, is required for the survival of all small and many intermediate-sized murine dorsal root ganglion (DRG) neurons during development, accounting for 80% of the total DRG population. Surprisingly, NGF/TrkA-dependent neurons include a large population that does not express TrkA in adult mice (Silos-Santiago et al., 1995). This finding suggests two hypotheses: Neurons lacking TrkA in the adult may express TrkA during development, or they may be maintained through a paracrine mechanism by TrkA-expressing neurons. To determine whether TrkA is expressed transiently by DRG neurons that lack the receptor in adulthood, we examined the distribution of TrkA protein during development. We show here that TrkA expression is strikingly developmentally regulated. Eighty percent of DRG neurons expressed TrkA during embryogenesis and early postnatal life, whereas only 43% expressed TrkA at postnatal day (P) 21. Because the period of TrkA down-regulation corresponds with a critical period during which nociceptive phenotype can be altered by NGF (see Lewin and Mendell  Trends Neurosci. 16:353–359), we examined whether NGF modulates the downregulation of TrkA. Surprisingly, neither NGF deprivation nor augmentation altered the extent of TrkA down-regulation. Our results demonstrate a novel form of regulation of neurotrophin receptor expression that occurs late in development. All DRG neurons that require NGF for survival express TrkA during embryogenesis, and many continue to express TrkA during a postnatal period when neuronal phenotype is regulated by NGF. The subsequent down-regulation of TrkA is likely to be importantly related to functional distinctions among nociceptive neurons in maturity. J. Comp. Neurol. 381:428–438, 1997. r 1997 Wiley-Liss, Inc. Indexing terms: nerve growth factor; nociceptors; dorsal horn; regulation of phenotype; neurotrophins Early investigations into the actions of nerve growth factor (NGF) determined that NGF supports the survival of sympathetic neurons and neural crest-derived sensory neurons during development (for review, see Thoenen and Barde, 1980). Unlike sympathetic neurons, dorsal root ganglion (DRG) neurons lose their dependence upon NGF for survival during the first few days of postnatal life (Gorin and Johnson, 1980; Lewin et al., 1992). However, in the first 2 weeks of postnatal life, the specificity of unmyelinated and thinly myelinated DRG neurons for nociceptive stimuli is regulated by NGF (for review, see Lewin and Mendell, 1993). In adulthood, approximately 40% of mature DRG neurons express the NGF receptor tyrosine kinase, TrkA, suggesting that NGF continues to act upon a r 1997 WILEY-LISS, INC. subset of sensory neurons in maturity (Verge et al., 1992; Averill et al., 1995; Molliver et al., 1995). Indeed, in adult DRG neurons, NGF regulates the levels of expression of substance P and calcitonin gene-related peptide (CGRP), two neuropeptides associated with nociceptive transmis- Contract grant sponsor: NINDS; Contract grant numbers: NS31768, P01-NS17763. *Correspondence to: W.D. Snider, M.D., Center for the Study of Nervous System Injury, Department of Neurology, Box 8111, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail: firstname.lastname@example.org Received 24 July 1996; Revised 18 October 1996; Accepted 27 October 1996 DOWN-REGULATION OF TrkA BY DRG NEURONS sion (Lindsay and Harmar, 1989; Verge et al., 1995; for review, see Hökfelt, 1991) that are highly colocalized with TrkA (Averill et al., 1995; see also Verge et al., 1989, 1995). Furthermore, administration of NGF causes thermal and mechanical hyperalgesia, whereas depletion of NGF with blocking antibodies results in a reduced sensitivity to painful stimuli (see Lewin and Mendell, 1993). Thus, in addition to regulating neuronal survival during embryonic development and determining modality specificity in early postnatal life, NGF regulates functional properties of nociceptive sensory neurons in adulthood. We have recently characterized a subset of small DRG neurons that have morphological features associated with nociceptive neurons but that do not express TrkA in adulthood (Molliver et al., 1995; see also Averill et al., 1995). Similar to TrkA-expressing neurons, these TrkA2 neurons are small in diameter, project to the superficial dorsal horn, and presumably have cutaneous peripheral axons (see Silverman and Kruger, 1988). However, the small TrkA2 neurons differ from TrkA-expressing neurons in that they lack substance P and CGRP, bind the Bandeiraea simplicifolia lectin I-B4 and are immunoreactive for the D subunit of protein kinase C and the neurofilament protein a-internexin (Molliver et al., 1995). These TrkA2 neurons presumably correspond to the 25–35% of DRG neurons that do not express any member of the Trk family, or the low-affinity neurotrophin receptor, p75 (McMahon et al., 1994; Wright and Snider, 1995). Furthermore, small TrkA1 and TrkA2 neurons project to different targets in the superficial dorsal horn. Whereas TrkA-expressing neurons project to spinal cord lamina I and the dorsal portion of lamina II (lamina IIo), small neurons lacking TrkA project to the interior of lamina II (lamina IIi; Molliver et al., 1995). Thus, several neurochemical and morphological characteristics distinguish TrkA1 neurons from small neurons lacking TrkA. Despite the restriction of TrkA expression to one subset of small DRG neurons in adulthood, the elimination of TrkA through gene targeting results in the loss of virtually all small neurons (Silos-Santiago et al., 1995). This finding suggests one of two possibilities: that all small DRG neurons initially express TrkA, and the I-B4-binding neurons subsequently down-regulate TrkA expression, or that I-B4 neurons never express TrkA but are dependent for their survival on support from TrkA-expressing neurons via a paracrine mechanism. Potential for paracrine interactions in the developing DRG is supported by findings that brain-derived neurotrophin factor (BDNF) and neurotrophin 3 (NT-3) are expressed by many DRG neurons during development (Ernfors et al., 1992; Schecterson and Bothwell, 1992; Elkabes et al., 1994). To address this issue, we examined the distribution of TrkA protein at selected time points during embryonic and postnatal development. In addition, we examined the ontogeny of CGRP and p75, which are expressed by most or all mature TrkA1 neurons (Verge et al., 1989, 1992; McMahon et al., 1994; Averill et al., 1995; Wright and Snider, 1995), and binding of the lectin I-B4, which selectively labels small TrkA2 neurons (Averill et al., 1995; Molliver et al., 1995). Finally, in order to investigate the possibility that NGF is involved in the down-regulation of TrkA and in the differentiation of small DRG neurons into I-B4-binding and CGRP-expressing neuronal populations, we examined whether systemic injections of NGF or antibodies to NGF during the first 2 429 TABLE 1. Primary Antisera1 Type Rabbit anti-TrkA Rabbit anti-p75 Rabbit anti-CGRP Mouse antineurofilament I-B4-biotin I-B4-HRP Dilution Source 1:8,000 1:750 1:1,000 1:40 1:200 1:200 Louis Reichardt Barbara Hempstead Peninsula Sigma Sigma Sigma 1The antisera and lectins used in this study are listed here with the working dilution used and the source from which they were obtained. CGRP, calcitonin gene-related peptide; I-B4-biotin, a biotinylated form of the lectin I-B4 from Bandeiraea simplicifolia; I-B4-HRP, I-B4 conjugated to horseradish peroxidase (HRP). weeks of postnatal life altered expression of TrkA, CGRP, or I-B4 binding sites. We show here that TrkA is expressed by 80% of DRG neurons during embryogenesis and into postnatal development and is down-regulated in a subset of small neurons during the 3 weeks after birth, well after the time at which DRG neurons lose their dependence on NGF for survival. We conclude that essentially all DRG neurons dependent upon NGF/TrkA signaling for survival express TrkA during embryonic development. In contrast to TrkA, CGRP exhibited a mature expression pattern by postnatal day (P) 1. Manipulation of postnatal NGF levels, which regulate the differentiation of nociceptive phenotype, did not alter the down-regulation of TrkA, nor did it alter the distribution of CGRP or I-B4 binding. The discrepancy between TrkA and CGRP expression during development suggests that these neurochemical features are regulated by different mechanisms. Finally, we suggest that the downregulation of TrkA contributes to the emergence of functionally distinct subsets of small DRG neurons. MATERIALS AND METHODS Tissue preparation TrkA immunocytochemistry was performed in CF-1 mice (Charles River, Wilmington, MA) n 5 3 for each developmental stage examined. Embryos at embryonic day (E) 13 and E15 were immersion fixed in 3% paraformaldehyde, 15% saturated picric acid in 0.1 M phosphate buffer, pH 7.4, for 2 hours. Postnatal day (P) 1, P7, P14, P21, and adult mice were deeply anesthetized, transcardially perfused first with phosphate buffer and then with the fixative described above, and postfixed for 2 hours. P1 animals were anesthetized on ice; older animals were anesthetized by sodium pentobarbital injection. In postnatal animals, the spinal column was dissected out for cutting; embryos were cut whole. All tissue was suspended overnight in 30% sucrose after fixation. Tissue was then frozen in Optimal Cutting Temperature (OCT) embedding medium (Baxter, McGaw Park, IL), and 12 µm sections were cut on a cryostat and stored at 220°C. All animal handling and protocols were approved by the Animal Studies Committee of Washington University. Immunocytochemistry Mounted sections were encircled with a Teflon coating by using a Pap pen (Kyota International, Elk Grove Village, IL) and were incubated for 1 hour in a blocking solution consisting of Superblock buffer, a commercial blocking buffer purchased from Pierce (Rockford, IL), 0.3% Triton X-100 (Electron Microscope Sciences, Ft. Washington, PA), 1.0% porcine gelatin (Sigma), and 1.5% normal serum 430 D.C. MOLLIVER AND W.D. SNIDER (Vector, Burlingame, CA). Table 1 lists the primary reagents used in this report. Primary and secondary antibodies were diluted in the same solution, diluted 1:1 with Superblock buffer/1.5% normal serum. Sections were incubated in primary antibody overnight. They were then washed three times for 5 minutes each with phosphatebuffered saline (PBS) and were placed in secondary antibody solution (donkey anti-rabbit IgG conjugated to the indocarbocyanine dye Cy3; Jackson Immunoresearch Laboratories, West Grove, PA), washed three times for 5 minutes each in PBS, coverslipped in PBS, and then examined under epifluorescence. For horseradish peroxidase-diaminobenzidine (HRP-DAB) visualization of antibody labeling, sections were removed from primary antibody, washed in PBS as above, processed with a Vector Vectastain kit according to instructions in the kit, then dehydrated, and coverslipped with DPX. For a negative control, representative sections were processed without a primary antibody. Thirty-five millimeter slides of representative sections for all procedures were taken on a Nikon Microphot FXA microscope and digitized by using a Polaroid Sprintscan connected to a Power Macintosh 9500. After creation of the figures and adjustment for color, tone, and contrast, images were printed on a Tektronics dye-sublimation printer. Lectin histochemistry Sections were processed for lectin histochemistry by substituting lectins for primary antibodies, as described above, with the following modifications. Sections were incubated overnight in I-B4 (Sigma) diluted to 10 µg/ml in modified PBS containing 0.01 M MgCl2 and CaCl2, pH 6.8. Slides received an additional 5 minute wash in 50 mM Tris/1.8% NaCl after the primary incubation. Avidinylated Cy3 (1:2,500) was diluted in the same buffer, and sections were incubated for 1 hour. The high salt concentration reduced background staining during avidin-biotin binding (L. Slomianka, personal communication). After incubation in the avidinylated reagent, the slides were washed in PBS and incubated in the secondary antibody, as described above. I-B4 conjugated to HRP was used for quantitation of I-B4 histochemistry, which was followed by visualization with the avidin biotin complex (ABC)-DAB procedure, as described above. NGF perturbations P1 mice were separated into three groups for injections. The first group received 5 µg/g NGF in L15 medium (purchased from Washington University Tissue Culture Support Center, St. Louis, MO) once daily from P2 until P14. The second group received 5 µl/g anti-NGF once daily from P3 to P14, and the third group was given daily injection of vehicle. All animals were killed by perfusion, as described above, on P21. This procedure was performed twice, for a total number of five mice for each group. NGF was purified from mouse salivary glands (Boccini and Angeletti, 1969). The titer of the goat anti-mouse NGF antiserum was 80,000, which was determined as the reciprocal of the highest dilution that blocked DRG neurite outgrowth in vitro in the presence of 5 ng/ml NGF (Fenton, 1970; Ruit et al., 1992). Unlike DRG neurons, sympathetic neurons do not lose dependence upon NGF for survival after birth (Gorin and Johnson, 1980). Therefore, superior cervical ganglia (SCGs) from two control, two NGFtreated, and two anti-NGF-treated P14 mice were com- pared for a gross indication of the effectiveness of the treatment protocol. SCGs from NGF-injected mice were clearly larger than control SCGs, whereas SCGs from anti-NGF-injected mice were drastically smaller (not shown). Quantification To provide an estimate of the proportion of DRG neurons expressing TrkA at each age examined, we calculated the percentage of TrkA-IR neurons in selected sections of DRG. Sections of lower lumbar ganglia used for quantification of TrkA immunoreactivity (IR) were counterstained with cresyl violet prior to coverslipping and examined under brightfield microscopy. Sections from E13, E15, and P1 mice were examined with a 3100 water-immersion objective; for older animals, a 350 water-immersion objective was used. Cells with clearly defined nuclei were counted in multiple representative sections through the ganglia and were considered positive if they contained a clear deposition of cytoplasmic DAB reaction product compared with background. For examination of the developmental time course of TrkA distribution, at least 1,000 cells were scored from each of three animals for each age group: E15, P1, P7, P14, P21, and adult. The mean percentage of positive neurons for each age group was calculated, and the means were compared between groups. An issue that must be addressed is whether our results are accurate without stereological analysis. For example, could the observed reduction in the percentage of TrkA-IR neurons be the result of a differential increase in the size of the nuclei of TrkA2 neurons over the first 3 postnatal weeks? We consider this to be a highly unlikely possibility for three reasons. First, at P1, TrkA-IR is excluded only from large-diameter neurons, whereas, at P21, TrkA-IR is excluded from a subset of small-diameter neurons as well. Second, the selective loss of TrkA-IR from afferents in the interior of lamina II is independent corroboration of the down-regulation of TrkA in a specific subset of small neurons (see Molliver et al., 1995). Finally, the percentage of TrkA-IR neurons at P1 corresponds to the percentage of TrkA-IR neurons in the adult plus the percentage of neurons binding I-B4, providing further confirmation that an identified subset of DRG neurons down-regulate TrkA. Cross-sectional areas of TrkA-IR neurons in NGF- and anti-NGF-treated animals were analyzed as a measure of treatment effectiveness (see Rich et al., 1984). Sizefrequency histograms of cross-sectional areas of TrkA-IR neurons from P21 NGF- and anti-NGF-treated animals were compared to determine whether the treatments had affected cell size. More than 300 neurons per treatment group were counted in sections taken from each of the animals treated. Quantification of TrkA-IR, CGRP-IR, and I-B4-binding in treated animals was performed at P21, as described above. The statistical significance of differences between NGF and anti-NGF treated animals was determined by using a Student’s t-test. RESULTS TrkA is down-regulated by a subset of DRG neurons after birth TrkA immunocytochemistry resulted in dense cytoplasmic staining of DRG neurons (Fig. 1) as well as both central and peripheral processes. At E13, the earliest time DOWN-REGULATION OF TrkA BY DRG NEURONS 431 Fig. 1. TrkA protein is down-regulated during postnatal development. Shown here are representative sections from mice of different ages stained for TrkA and counterstained to visualize negative neurons. Scale bars 5 40 µm in top and middle sections. Bottom two sections are high-magnification micrographs of postnatal day (P) 1 and P21 dorsal root ganglia (DRGs). Scale bars 5 20 µm in bottom sections. At P1, the great majority of neurons (80%) are intensely TrkA- immunoreactive (-IR). There is a progressive decrease in the number of TrkA-IR neurons between P1 and P21, at which point the percentage of TrkA-containing neurons is similar to that in the adult. High-magnification micrographs illustrate the large size of negative neurons both at P1 and at P21. However, in contrast to P1, negative neurons at P21 include many small-diameter neurons (arrows). point examined, the majority of neurons in the DRG were immunoreactive for TrkA (not shown). The widespread distribution of TrkA staining in the DRG was maintained between E13 and the day of birth. Quantification of TrkA-IR neurons in lumbar ganglia at E15 revealed that 82% of all neurons in the DRG were immunopositive (Fig. 1). At this age, there was a clearly discernible range of soma sizes, and the largest neurons were consistently immunonegative for TrkA. After birth, there was a gradual, progressive decrease in the number of DRG neurons labeled (Figs. 1, 2). A distribution of TrkA-IR approximating that found in the adult was not seen until P21. Because CGRP is highly colocalized with TrkA in adult DRG (Averill et al., 1995), we determined whether the extent of colocalization was similar during development. Whereas the percentage of TrkA-IR neurons between E15 432 D.C. MOLLIVER AND W.D. SNIDER Fig. 2. The percentage of TrkA-IR DRG neurons diminishes during postnatal development. The percentage of TrkA-IR DRG neurons was plotted against age to illustrate the gradual decrease in the proportion of neurons expressing TrkA between embryonic day (E) 15 and adulthood. Approximately 80% of DRG neurons are TrkA-IR between E15 and P1, a proportion that decreases between P1 and P21 to adult levels of roughly 40%. Vertical bars indicate S.E.M. and P1 was twice that seen in the adult, DRG neuronal CGRP-IR was visible in very few cells at E15 (not shown) and increased to approximately adult numbers of neurons by P1. Figure 3 shows that many small neurons were clearly CGRP2 at P1. Thus, CGRP expression is restricted to a subset of small and medium-sized neurons at an age when TrkA is expressed by the great majority of DRG neurons. In the adult rat DRG, all neurons expressing TrkA also express p75 (Verge et al., 1992; Wright and Snider, 1995). We examined the distribution of p75 to determine whether the low-affinity neurotrophin receptor showed an enhanced distribution during embryonic development similar to that of TrkA. Immunocytochemistry revealed that, like CGRP-IR, p75-IR was more restricted than TrkA-IR at P1, in that many small neurons did not contain p75-IR (Fig. 3). In contrast to CGRP, p75 was also seen in large neurons, consistent with its reported colocalization in the adult with TrkA, TrkB, and partial colocalization with TrkC (Wright and Snider, 1995). In addition to examining markers of TrkA1 neurons, we analyzed the development of I-B4 binding sites to determine whether they are expressed prior to the downregulation of TrkA. DRG neuronal binding by I-B4 was first seen at P1, at which point it was very faint (not shown). However, staining was sufficient to determine that I-B4-binding labeled a subset of small neurons and that the pattern was qualitatively similar to that seen in the adult. Staining was more evident at P7 and, at P14, Fig. 3. Calcitonin gene-related peptide (CGRP) and low-affinity neurotrophin receptor (p75) are not expressed by all TrkA neurons at P1. DRG sections from P1 mice were processed for fluorescence immunohistochemistry for p75, TrkA, or CGRP, which are highly colocalized in adulthood. This figure displays the clear discrepancy between the large percentage of TrkA-IR neurons and the smaller populations of CGRP-IR and p75-IR neurons. In addition to TrkAexpressing neurons, p75 labels a subset of large-diameter TrkCexpressing and TrkB-expressing neurons. Scale bars 5 40 µm. DOWN-REGULATION OF TrkA BY DRG NEURONS 433 Fig. 4. Neurons down-regulating TrkA project to the interior of lamina II. Sections of lumbar spinal cord stained for TrkA, neurofilament (NF), or p75 reveal the central target field of neurons downregulating TrkA. NF and p75 staining was used to identify the boundaries of lamina II, the major target field of unmyelinated DRG afferents. The white dots in the NF and p75 sections trace the lamina II-III border. At P1, TrkA staining covers the entirety of laminae I and II of the dorsal horn. TrkA staining in the dorsal horn retreats from the ventral edge of lamina II as the percentage of TrkA1 neurons in the ganglion decreases. At P14 , extensive TrkA label is seen only in lamina I and at the dorsal edge of lamina II. Note that p75-IR avoids the interior of lamina II, indicating that neurons projecting to this region do not express p75. The comparison of TrkA-IR with neurofilament-IR and p75-IR supports our interpretation that the major population of neurons down-regulating TrkA is the Bandeiraea simplicifolia lectin (I-B4)-binding population, which innervates the interior of lamina II (see Molliver et al., 1995). Scale bar 5 40 µm. appeared similar in intensity to the adult (not shown). The presence of I-B4 binding at P1 suggests that these neurons initially express both TrkA and lectin binding sites, as essentially all small neurons express TrkA at this age. While the percentage of DRG neurons expressing TrkA decreased, there was a selective reduction in the ventral extent of TrkA staining in lamina II (see Fig. 4). Between P1 and P21, TrkA staining retreated from the ventral border of lamina II until only afferents innervating lamina I and the dorsalmost portion of lamina II were immunopositive. In addition, the overall density of staining for TrkA in the dorsal horn decreased between birth and adulthood, suggesting a reduction in the concentration of TrkA protein in central afferents after birth. To verify that the intensely stained band of TrkA-IR afferents seen in the dorsal horn at P1 was restricted to lamina II, we determined the location of the lamina II–III border histochemically. Antibodies to phosphorylated neurofilament triplet proteins label the large-caliber axons destined to become myelinated but not the fine-caliber unmyelinated C-fiber afferents (see Lawson, 1992). Because lamina II is primarily innervated by unmyelinated axons in adult animals, whereas lamina III and IV are innervated by large-caliber, low-threshold mechanoreceptors (for review, see Light, 1992), we used neurofilament-IR to discern the boundary of laminae II and III. Comparison of TrkA and neurofilament staining at P1, as Central projections of neurons down-regulating TrkA Both central and peripheral processes of DRG neurons were robustly labeled by TrkA immunocytochemistry during embryonic and early postnatal development. The intense staining made it possible to examine developmental changes in the innervation of the spinal cord by TrkA-IR processes and, thus, identify the laminar target fields of sensory neurons that down-regulate TrkA (Fig. 4). At P1, TrkA-IR axons in the lumbar dorsal horn extended throughout the entire superficial dorsal horn but stopped abruptly at the border of laminae II and III, with some staining in lamina V. Occasional axons were visible projecting into the lower laminae of the dorsal horn. These axons may be collaterals projecting to lamina V or may indicate a small population of TrkA-IR neurons with targets in laminae III–IV. Because these axons are also visible at P21, they probably do not arise from neurons down-regulating TrkA. 434 D.C. MOLLIVER AND W.D. SNIDER TABLE 2. Neuronal Populations in NGF vs. Anti-NGF-Treated Mice1 Treatment TrkA NGF Anti-NGF CGRP NGF Anti-NGF I-B4 NGF Anti-NGF % Labeled 43 6 4.20 41 6 2.44 38 6 3.80 36 6 2.90 39 6 1.04 39 6 1.99 1Data from the quantification of TrkA-IR, CGRP-IR, and I-B4 binding in nerve growth factor (NGF) and anti-NGF-treated animals at postnatal day (P) 21. % Labeled, percentage of positive neurons 6 S.E.M. There was no significant difference in the distribution of TrkA, CGRP, or I-B4 between NGF and anti-NGF-treated mice (TrkA: P 5 0.56; CGRP: P 5 0.36; I-B4: P 5 0.65). NGF, nerve growth factor; CGRP, calcitonin gene-related peptide; I-B4, the lectin I-B4 from Bandeiraeia simplicifolia. shown in Figure 4, indicates that the major band of intense TrkA-IR in lamina II stops abruptly at the border of laminae II and III. CGRP-IR and p75-IR were examined in the spinal cord to distinguish axons that express TrkA in the adult from those expressing TrkA only during development (Fig. 4). The adult pattern of CGRP-IR in dorsal horn laminae I, IIo, and V was clearly established by P1 (not shown) and did not extend into lamina IIi, as seen with TrkA staining at that age. At P1, p75 staining revealed intensely labeled afferents in laminae I, IIo, and III–IV (Fig. 4). However, consistent with our hypothesis that the subset of small neurons lacking p75 is identical to the I-B4-binding neuronal population, little or no p75 staining was seen in lamina IIi, the primary target field of I-B4-binding neurons. Comparison of p75 and TrkA staining in the dorsal horn provides additional evidence that the majority of TrkA-IR afferents do not penetrate ventral to lamina IIi at P1. Furthermore, analysis of CGRP (not shown) and p75 staining in the dorsal horn indicated that TrkA-IR afferents projecting to lamina IIi at P1 contain neither CGRP nor p75. Perturbation of NGF levels does not alter down-regulation of TrkA To determine whether NGF is involved in the downregulation of TrkA expression, mice that had been injected with either NGF or anti-NGF from P3 to P14 were examined at P21 for changes in the distribution of TrkA-IR in the DRG and dorsal horn. CGRP-IR and IB-4 binding were also examined in these animals to evaluate the effect of treatment on different subsets of neurons that express TrkA early in postnatal life. The treatments, as expected, had significant effects on the size of TrkA-IR neurons. The average TrkA-IR neuronal area in NGF-treated animals was 242.9 µm2 compared with 194.1 µm2 in anti-NGFtreated animals, a significant difference (P , 0.001; Students t-test). Size-frequency histograms revealed a shift to larger areas of TrkA-IR neurons from animals treated with NGF compared with anti-NGF (not shown). Thus, perturbation of NGF supplies had a significant biological effect on DRG neurons in treated animals. Quantification of the percentage of TrkA-IR DRG neurons in NGF- and anti-NGF-treated animals showed no significant difference in the extent of TrkA staining between treatment groups (see Table 2), indicating that manipulation of postnatal NGF levels does not alter the extent of TrkA down-regulation. Likewise, the percentage of neurons staining for CGRP or IB-4 binding was not affected (Fig. 5). Furthermore, examination of the patterns of staining in the dorsal horn for TrkA, CGRP, and I-B4 revealed that the laminar organization of central projections to the superficial dorsal horn was not altered by systemic injections of NGF or anti-NGF (not shown). Although we did not find an effect of NGF on any of the parameters examined, we cannot absolutely exclude the possibility of a transient effect of NGF on TrkA expression that disappeared between P14 and P21. DISCUSSION Developmental regulation of TrkA expression Previous studies have demonstrated that NGF and TrkA null mutant mice lose approximately 80% of the normal complement of DRG neurons (Crowley et al., 1994; Smeyne et al., 1994; Silos-Santiago et al., 1995). Thus, sensory neuron loss in these animals is far more extensive than the 40% of DRG neurons that normally express TrkA in maturity (Verge et al., 1992; Averill et al., 1995; Molliver et al., 1995). We show that this apparent discrepancy is due to the developmental down-regulation of TrkA expression. Approximately 80% of DRG neurons express TrkA between E15 and P1. The number of TrkA1 neurons diminishes gradually, dropping to adult levels during the first 3 weeks of postnatal life. Thus, our results suggest that 50% of TrkA1 neurons down-regulate TrkA during postnatal development. The reduction in the percentage of TrkA-expressing neurons occurs for the most part well after P2, the age at which DRG neurons lose their dependence upon NGF for survival (Lewin et al., 1992), indicating that the TrkA population is reduced through the down-regulation of TrkA rather than through selective cell death. The population of neurons that down-regulates TrkA consists of small neurons that bind the lectin I-B4, do not express CGRP or p75, and project to lamina IIi of the dorsal horn. This conclusion is supported by the progressive loss of TrkA-IR from afferent axons in lamina IIi between P1 and P21. Our results should be compared with previous in situ hybridization studies of TrkA expression in the DRG during development that yielded conflicting results. Several investigators have suggested, based on visual analysis, that most or all DRG neurons express TrkA during early embryonic life (Ernfors et al., 1992; Schecterson and Bothwell, 1992). However, percentages were not computed in these studies. When we examined the percentage of DRG neurons expressing TrkA during development by using in situ hybridization in paraffin-embedded tissues and excluded cells with low grain counts, we found that a lower percentage (40–45%) of neurons expressed TrkA than was reported in these earlier studies (Mu et al., 1993). Our principal goal in that study was to achieve optimal morphology in order to examine the relative sizes of neurons expressing the different Trks. Paraffin-embedded tissue, as we pointed out, is thought to exhibit a weaker signal in RNA hybridization studies than freshfrozen tissue (Tecott et al., 1987), and we considered it plausible that we had failed to detect many TrkAexpressing neurons. We concluded that the determination of absolute numbers and percentages of cells expressing TrkA in embryonic life would require further investigation (Mu et al., 1993). Antibodies against TrkA are now available, and we consider immunocytochemical analysis to be more accurate in the quantification of embryonic neurons DOWN-REGULATION OF TrkA BY DRG NEURONS 435 Fig. 5. The availability of nerve growth factor (NGF) does not modulate the down-regulation of TrkA or neurochemical phenotype. Representative sections of lumbar DRGs taken from P21 mice treated with NGF or anti-NGF and reacted for TrkA immunocytochemistry or lectin histochemistry. There was no apparent change in the percentages of TrkA or I-B4-binding neurons as a result of treatment. Arrows indicate representative I-B41 neurons, and arrowheads indicate several large negative neurons. Scale bars 5 40 µm. than autoradiographic in situ hybridization, because most neurons in the embryonic DRG are very small and have little cytoplasm, making it difficult to assign silver grains to specific cells. In contrast, immunocytochemical staining for TrkA filled positive cell bodies, allowing a more definitive identification of TrkA1 neurons. For these reasons, we chose to use immunocytochemistry for the purpose of quantification. An important implication of these results is that virtually all neurons lost in the NGF and trkA null mutant mice express TrkA during development. Thus, the expression of TrkA by 80% of DRG neurons at P1 corresponds to the 80% cell loss in the trkA(2/2) mouse. Furthermore, both major populations of small neurons expressing TrkA during development, CGRP-IR neurons and I-B4-binding neurons, are lost in the trkA null mutant (Silos-Santiago et al., 1995). One might have expected that sensory neuron loss in the trkA null mutant would have included more than the TrkA population because of the potential for TrkA neurons to provide a local source of neurotrophins within the DRG. Both NT-3 and BDNF are widely expressed within the ganglion during development (Ernfors et al., 1992; Schecterson and Bothwell, 1992; Elkabes et al., 1994), and TrkA-expressing neurons are known to express BDNF in adulthood (Apfel et al., 1996). Apparently, however, NGF-unresponsive neurons do not require paracrine support from TrkA-expressing neurons for survival during embryogenesis. It is not surprising that NGF supports the survival of both I-B41 and CGRP-IR neuronal populations during embryonic life, because both populations extensively innervate cutaneous tissues that express NGF as early as E11.5 (Davies et al., 1987; Silverman and Kruger, 1988; Wheeler and Bothwell, 1992; White et al., 1996). What is surprising is that I-B41 neurons cease expression of TrkA, whereas CGRP-IR neurons continue to express TrkA throughout life. Several other examples of developmental regulation of neurotrophin receptor expression have been documented. In the peripheral nervous system (PNS), sensory neurons switch Trk expression and neurotrophin dependence between the times of neurogenesis and target innervation (Buchman and Davies, 1993; Gaese et al., 1994; White et al., 1996; Williams and Ebendal, 1995). This switch may allow local sources of neurotrophins to support neurons before they gain access to target-derived factors. In most neuronal populations, expression of Trks persists into adulthood, suggesting that neurotrophins continue to function in maturity (Merlio et al., 1992; Yan et al., 1993). 436 D.C. MOLLIVER AND W.D. SNIDER However, expression of truncated TrkB and TrkC receptors increases after birth, and these noncatalytic isoforms may restrict the signaling potential of neurotrophins in adulthood in a region-specific manner (Ernfors et al., 1993; Escandon et al., 1994; see also Biffo et al., 1995). Here, we describe a novel form of developmental regulation of Trk expression in which a population of neurotrophin-dependent neurons ceases expression of neurotrophin receptors altogether after central and peripheral projections are formed. As a result, many DRG neurons that are NGF responsive during development are not subject to regulation by NGF in adulthood. Functional implications of TrkA down-regulation The reduction in the percentage of TrkA1 neurons occurs gradually over a period of 3 weeks after birth. The persistence of widespread TrkA expression after the dependence on NGF for survival has passed suggests that there may be a transient role for TrkA during early postnatal life. Notably, the postnatal time course of TrkA downregulation coincides roughly with the critical period described by Mendell and colleagues during which injections of NGF or anti-NGF can permanently alter the physiology of DRG neurons with unmyelinated (C-fiber) and thinly myelinated axons (Lewin et al., 1992; Lewin and Mendell, 1994). Neonatal NGF treatment recruits nonnociceptive thermoreceptors and ‘‘mechanically insensitive’’ afferents to a nociceptive phenotype, producing twice the control number of C-fiber polymodal nociceptors (Lewin and Mendell, 1994). Conversely, treatment with anti-NGF altered more than half of C-fiber polymodal nociceptors to an abnormal low-threshold mechanoreceptor phenotype. The gradual down-regulation of TrkA suggests that not just the peptidergic neurons but also the I-B41 neurons are subject to phenotypic regulation by NGF. Thus, during early postnatal life, NGF may determine the stimulus specificity of afferents that subsequently lose their NGF responsiveness. Although differences in projection patterns and histochemical profiles between I-B4-binding neurons and CGRPexpressing neurons have been known for some time, there has been debate about whether there are functional differences between these two populations (Silverman and Kruger, 1988; Averill et al., 1995; Molliver et al., 1995; see Hunt and Rossi, 1985; Hunt et al., 1992; Light, 1992). Indeed, the action potential characteristics of C-fibers are largely homogenous (Lawson et al., 1996), and at least 73% of C-fibers in the rat DRG are polymodal nociceptors, too large a population to be contained within the CGRPexpressing subset alone (Lynn and Carpenter, 1982). However, the ability to respond to NGF in the adult almost certainly leads to functional distinctions between CGRP and I-B4 populations. Up-regulation of expression of substance P and CGRP by NGF in the TrkA population is likely to underlie central sensitization in hyperalgesia; these peptides are also involved in the generation of the peripheral inflammatory response (for reviews, see Basbaum and Levine, 1991; Hunt et al., 1992; Lewin and Mendell, 1993). Furthermore, inflammation results in increased levels of NGF both in the skin and in nerves innervating the inflamed area (Woolf et al., 1994). Finally, the ability to respond to certain noxious stimuli, such as acid and the chemical irritant capsaicin, is regulated by NGF (Bevan and Winter, 1995; McMahon et al., 1995), suggesting a response mediated in the adult exclusively by the TrkA population. Given these regulatory actions of NGF, it is highly probable that the down-regulation of TrkA by I-B4 neurons results in important differences in the responses of I-B4-binding and peptidergic neurons to certain classes of nociceptive stimuli. It is important to acknowledge the possibility that I-B41 neurons are capable of responding to NGF through TrkA expressed at levels below the limit of immunocytochemical detection or via p75. However, several lines of evidence suggest that these neurons are unresponsive to NGF. First, in the adult, the distribution of TrkA mRNA (Verge et al., 1992; McMahon et al., 1994; Wright and Snider, 1995) and the distribution of high-affinity NGF binding sites (Verge et al., 1989) support the immunocytochemical data in both percentages and cell sizes. Furthermore, in situ hybridization analysis failed to detect mRNA for any Trk family member or p75 in approximately 30% of adult DRG neurons, a population that most likely corresponds to the I-B4-binding neurons (McMahon et al., 1994; Wright and Snider, 1995). The lack of p75-IR at P1 in many TrkA-IR neurons or in lamina IIi supports the notion that I-B4-binding neurons do not express p75 either in adulthood or during development. Finally, studies of the effects of NGF on neuropeptide (Verge et al., 1995) and neurofilament (Verge et al., 1990) expression after injury in the adult DRG found that administration of NGF produced phenotypic changes only in neurons with high-affinity NGF binding sites. Developmental changes In TrkA expression are not regulated by NGF In view of the importance of NGF in the determination of nociceptive phenotypes during postnatal development (Lewin and Mendell, 1993; Mendell, 1995), it was of interest to determine whether the postnatal downregulation of TrkA is also modulated by NGF. Therefore, we examined the effects of postnatal NGF or anti-NGF administration during the period of TrkA down-regulation by DRG neurons. Perhaps surprisingly, the perturbation of endogenous NGF supplies did not alter the extent of TrkA down-regulation, indicating that the availability of NGF does not determine which neurons down-regulate TrkA. We also show that CGRP, I-B4, and p75, which discriminate between TrkA1 and TrkA2 neurons in the adult, distinguish subsets of TrkA-IR neurons in neonates. The essentially mature distribution of these markers at P1 indicates that their expression does not mature in concert with TrkA expression. These results are in agreement with a recent study indicating that the CGRP phenotype in rat DRG neurons is determined before E15 (Robertson and Hall, 1995). Furthermore, administration or depletion of NGF did not alter the distribution of I-B4 binding or CGRP IR, confirming previous findings that perturbation of postnatal NGF supplies does not change the distribution of CGRP (Lewin and Mendell, 1994). Thus, despite its importance in regulating physiological properties, NGF does not appear to determine the neurochemical phenotype of TrkAexpressing neurons, as defined by NGF receptors, neuropeptides, and lectin binding sites. Our results showing that NGF levels do not affect expression of TrkA are consistent with other studies of TrkA regulation in the peripheral nervous system, both at early developmental stages and in adulthood. Studies of the effects of NGF administration on sympathetic and DOWN-REGULATION OF TrkA BY DRG NEURONS DRG neurons both in vitro and in vivo provide evidence that TrkA expression is not normally regulated by NGF (Verge et al., 1992; Wyatt and Davies, 1993; Miller et al., 1994), although NGF can partially rescue normal expression levels reduced by axotomy (Verge et al., 1992). Furthermore, in embryonic trigeminal ganglion neurons, the marked increase in TrkA expression seen after target innervation occurs normally in NGF null mutant mice (Davies et al., 1995). Notably, however, several studies have shown that NGF can transcriptionally up-regulate TrkA in the central nervous system (CNS; Holtzman et al., 1992; Gibbs and Pfaff, 1994). These findings suggest that there are important differences in the regulation of TrkA expression between the PNS and the CNS, although it is not clear what the role of this differential regulation might be. CONCLUSIONS Our findings have established that the great majority of DRG neurons express TrkA throughout embryonic development. NGF/TrkA signaling acts at this time to support neuronal survival (Crowley et al., 1994; Silos-Santiago et al., 1995). Persistent widespread expression of TrkA in the neonate after P2, when DRG neurons no longer require NGF for survival, is consistent with the idea that NGF regulates DRG neuronal phenotype during postnatal development (see Lewin and Mendell, 1993; Mendell, 1995). However, the actions of NGF appear to be specific for physiological aspects of phenotype, such as modality specificity, whereas neurochemical aspects of phenotype, such as neuropeptide and neurotrophin receptor expression, are not determined by NGF. 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