Neurotrophins Cause a New Sensation
Authors: W.D Snider, D.E Wright
Journal: Neuron (1996)

An important organizational principle of the developing peripheral nervous system is the reliance of neurons on specific members of the NGF family of neurotrophins for survival. The primary sensory neurons of the dorsal root ganglion (DRG) have become a favored group for exploring neurotrophin function owing to both their accessibility for in vivo and in vitro studies and the potential importance of neurotrophins in regulating sensory functions, particularly pain. Neurotrophin studies have benefited from the wealth of information available about the physiological properties, peptide expression, cytoskeletal markers, and connectivity of DRG neurons (see20Scott S.A Sensory Neurons. Oxford University Press, New York1992Google Scholar). This information has allowed an initial characterization of sensory neuron loss in settings where neurotrophin or neurotrophin receptor function has been blocked by antibodies or gene targeting (for review, see23Snider W.D Cell. 1994; 77: 627-638Abstract Full Text PDF PubMed Scopus (1310) Google Scholar). To date, however, the neurotrophin dependence of several classes of DRG neurons has remained obscure. In this issue of Neuron, Airaksinen et al. characterize the neurotrophin requirements of two classes of cutaneous mechanoreceptive neurons. Further, these investigators present evidence for previously unappreciated complexities of neurotrophin regulation, even in this relatively simple region of the nervous system. Recent studies of neurotrophin receptor (Trk) localization in association with known DRG neuron histochemical markers have led to a rough classification of DRG neurons according to neurotrophin receptor expression (Figure 1). The localization of the nerve growth factor (NGF) receptor, TrkA, to small- and medium-sized neurons and the neurotrophin-3 (NT-3) receptor, TrkC, to large neurons suggested that NGF- and NT-3-dependent populations in the ganglion were functionally distinct classes (17Mu X Silos-Santiago I Carroll S.L Snider W.D J. Neurosci. 1993; 13: 4029-4041Crossref PubMed Google Scholar, 15McMahon S.B Armanini M.P Ling L.H Phillips H.S Neuron. 1994; 12: 1161-1171Abstract Full Text PDF PubMed Scopus (495) Google Scholar). Further studies in wild-type and null mutant mice have characterized TrkA neurons as predominantly unmyelinated and small myelinated neurons expressing calcitonin gene-related peptide (CGRP) and innervating the superficial laminae of the spinal cord (see21Silos-Santiago I Molliver D.C Ozaki S Smeyne R.J Fagan A.M Barbacid M Snider W.D J. Neurosci. 1995; 15: 5929-5942PubMed Google Scholarreferences therein). In the periphery, most TrkA-positive sensory axons terminate in the superficial layers of skin in proximity to sites known to synthesize NGF mRNA and protein from early developmental stages (6Davies A.M Bandtlow C Heumann R Korshing S Rohrer H Thoenen H Nature. 1987; 326: 353-358Crossref PubMed Scopus (446) Google Scholar, 19Schecterson L.C Bothwell M Neuron. 1992; 9: 449-463Abstract Full Text PDF PubMed Scopus (481) Google Scholar, 24Snider W.D Silos-Santiago I Phil. Trans. Roy. Soc. (Lond.) B, in press. 1996; Google Scholar). The lack of behavioral responses to nociceptive stimuli in NGF and trkA null mutant mice is consistent with the hypothesis that most TrkA neurons convey nociceptive information (5Crowley C Spencer S.D Nishimura M.C Chen K.S Pitts-Meek S Armanini M.P Ling L.H McMahon S.B Shelton D.L Levinson A.D Phillips H.S Cell. 1994; 76: 1001-1012Abstract Full Text PDF PubMed Scopus (885) Google Scholar, 22Smeyne R.J Klein R Schnapp A Long L.K Bryant S Lewin A Lira S.A Barbacid M Nature. 1994; 368: 246-249Crossref PubMed Scopus (835) Google Scholar). In contrast, TrkC neurons have medium to large soma areas, contribute the largest myelinated axons to the dorsal roots, and many express the calcium-binding proteins parvalbumin and calretinin (17Mu X Silos-Santiago I Carroll S.L Snider W.D J. Neurosci. 1993; 13: 4029-4041Crossref PubMed Google Scholar, 4Copray J.C.V.M Brouwer N Neuroscience. 1994; 63: 1125-1135Crossref PubMed Scopus (97) Google Scholar, 9Ernfors P Lee K.-F Kucera J Jaenisch R Cell. 1994; 77: 503-512Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 12Klein R Silos-Santiago I Smeyne R.J Lira S.A Brambilla R Bryant S Zhang L Snider W.D Barbacid M Nature. 1994; 368: 249-251Crossref PubMed Scopus (534) Google Scholar, 15McMahon S.B Armanini M.P Ling L.H Phillips H.S Neuron. 1994; 12: 1161-1171Abstract Full Text PDF PubMed Scopus (495) Google Scholar). NT-3 and trkC null mutant mice display loss of large diameter parvalbumin-positive neurons, loss of spinal projections from muscle afferents, and severe movement abnormalities. These features are consistent with the hypothesis that TrkC neurons convey proprioceptive information (9Ernfors P Lee K.-F Kucera J Jaenisch R Cell. 1994; 77: 503-512Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 10Fariñas I Jones K.R Backus C Wang X.-Y Reichardt L.F Nature. 1994; 369: 658-661Crossref PubMed Scopus (559) Google Scholar, 12Klein R Silos-Santiago I Smeyne R.J Lira S.A Brambilla R Bryant S Zhang L Snider W.D Barbacid M Nature. 1994; 368: 249-251Crossref PubMed Scopus (534) Google Scholar). In the periphery, proprioceptive axons convey information from stretch and tension receptors (muscle spindles and Golgi tendon organs) in skeletal muscle. NT-3 mRNA is diffusely expressed in muscle during development but becomes restricted to muscle spindles, a target end organ of proprioceptive afferents, postnatally (19Schecterson L.C Bothwell M Neuron. 1992; 9: 449-463Abstract Full Text PDF PubMed Scopus (481) Google Scholar, 4Copray J.C.V.M Brouwer N Neuroscience. 1994; 63: 1125-1135Crossref PubMed Scopus (97) Google Scholar). The most puzzling groups of DRG neurons related to neurotrophin dependence have been the low threshold cutaneous mechanoreceptive neurons that convey information from receptor organs in the dermis such as hair follicles, Pacinian corpuscles, Ruffini corpuscles, Meissners corpuscles, and Merkel cells. These DRG neurons have small- and medium-sized somas with myelinated axons and spinal projections that terminate in the deep dorsal horn (see2Brown A.G Organization of the Spinal Cord. Springer Verlag, Berlin1981Crossref Google Scholar). To date, however, no good histochemical markers have been described that specifically identify these populations. It was predicted that low threshold mechanoreceptive neurons would require brain-derived neurotrophic factor (BDNF) for survival since the BDNF receptor, TrkB, is expressed by a subpopulation of small- to medium-sized DRG neurons (7Ernfors P Merlio J.-P Persson H Eur. J. Neurosci. 1992; 4: 1140-1158Crossref PubMed Scopus (449) Google Scholar, 19Schecterson L.C Bothwell M Neuron. 1992; 9: 449-463Abstract Full Text PDF PubMed Scopus (481) Google Scholar, 17Mu X Silos-Santiago I Carroll S.L Snider W.D J. Neurosci. 1993; 13: 4029-4041Crossref PubMed Google Scholar). Also, BDNF mRNA is synthesized in dermal mesenchyme during development in regions sensitive to touch, including the digits, lips, tongue, and mandible (19Schecterson L.C Bothwell M Neuron. 1992; 9: 449-463Abstract Full Text PDF PubMed Scopus (481) Google Scholar). However, the survival effects of BDNF on DRG neurons have been difficult to discern. Neuronal counts of DRGs in BDNF and trkB null mutant mice have been confusing. In 2-week-old BDNF (−/−) mice, approximately 30% of lumbar DRG neurons are lost (8Ernfors P Lee K.-F Jaenisch R Nature. 1994; 368: 147-150Crossref PubMed Scopus (899) Google Scholar, 11Jones K.R Fariñas I Backus C Reichardt L.F Cell. 1994; 76: 989-1000Abstract Full Text PDF PubMed Scopus (901) Google Scholar). However, in neonatal trkB (−/−) mice, the neuronal loss seems to be minimal (21Silos-Santiago I Molliver D.C Ozaki S Smeyne R.J Fagan A.M Barbacid M Snider W.D J. Neurosci. 1995; 15: 5929-5942PubMed Google ScholarSoc. Neurosci., abstract). Furthermore, DRG afferents innervating the deep dorsal horn appear relatively normal in the trkB (−/−) mice (24Snider W.D Silos-Santiago I Phil. Trans. Roy. Soc. (Lond.) B, in press. 1996; Google Scholar). Thus, it is clear that not all classes of low threshold cutaneous mechanoreceptors require BDNF for survival as previously predicted. Interestingly, NT-3, like NGF and BDNF, is also expressed in the skin. In contrast with NGF, NT-3 mRNA is expressed primarily in deep regions of dermal mesenchyme, although there is considerable overlap in the two patterns (19Schecterson L.C Bothwell M Neuron. 1992; 9: 449-463Abstract Full Text PDF PubMed Scopus (481) Google Scholar). The function of this NT-3 expression in skin has not been apparent. Sensory neurons have been separated into distinct groups according to different physiological criteria (for review, see18Perl, E. (1992). In Sensory Neurons (New York: Oxford University Press), pp. 3–23.Google Scholar). The conduction velocities of sensory axons in peripheral nerve differ markedly, resulting in the grouping of axons into either myelinated A-fibers (subdivided further into Aα, Aβ, and Aδ) or unmyelinated C-fibers. Furthermore, sensory neurons respond to precise stimuli with very different thresholds and adaptation responses, properties that further delineate functional classes. Thus, some DRG neurons (nociceptors) respond to various combinations of noxious thermal and mechanical stimuli and have C or Aδ conduction velocities. In contrast, other DRG neurons (cutaneous mechanoreceptors) exhibit low threshold responses to tactile and vibration stimuli and have Aδ and Aβ conduction velocities. The low threshold mechanoreceptors are further divided into populations that rapidly adapt to stimuli and populations that slowly adapt to stimuli. One reason the neurotrophin requirements of low threshold mechanoreceptive populations have remained unclear is that the physiological properties of DRG neurons have not been examined in animals deficient in neurotrophins other than NGF (for a review of work pertaining to NGF, see14Lewin G.R Mendell L.M Trends Neurosci. 1993; 16: 353-359Abstract Full Text PDF PubMed Scopus (472) Google Scholar). Lewin and colleagues (1Airaksinen M.S Koltzenburg M Lewin G.R Masu Y Helbig C Wolf E Brem G Toyka K.V Thoenen H Meyer M Neuron. 1996; 16 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) have now employed a straightforward electrophysiological analysis in NT-3 null mutant mice to dissect the NT-3 dependence of various classes of cutaneous mechanoreceptors. These investigators developed an in vitro preparation of the cutaneous saphenous nerve and attached skin to record from single sensory axons in the desheathed nerve. For each unit, the conduction velocity was determined by electrically stimulating its receptive field, the mechanical threshold was determined with calibrated von Frey monofilaments, and the adaptation properties were analyzed with constant force stimuli via a computer-controlled probe placed on the receptive field. Four different classes of myelinated cutaneous mechanoreceptors were readily recognized using this preparation: high threshold A-fiber mechanonociceptors that tonically respond to suprathreshold mechanical stimuli, low threshold D-hair afferents that respond dynamically to skin stimulation, low threshold rapidly adapting mechanoreceptors (RA neurons) that respond to movement of the skin, and low threshold slowly adapting mechanoreceptors that respond tonically to skin indentation (SA1 neurons). Because NT-3 (−/−) mice die shortly after birth, measurements were performed on NT-3 (+/−) mice to determine the identity of surviving cutaneous afferents. In NT-3 (+/−) animals, the percentage of SA1 afferents was reduced by almost 80%. Lewin and colleagues further showed that the NT-3 dependence is not restricted to the SA1 population. The percentage of D-hair afferents was also reduced by 50% in NT-3 (+/−) animals. It is important to point out that not all cutaneous low threshold mechanoreceptors require NT-3. In NT-3 (+/−) mice, percentages of axons exhibiting responses consistent with RAs increased in proportion to the SA1 loss, suggesting that RAs do not require NT-3 for survival. A surprising feature of these studies was the extensive afferent loss in (+/−) animals. In contrast, NGF-dependent DRG populations exhibit little loss in NGF and trkA (+/−) mice (5Crowley C Spencer S.D Nishimura M.C Chen K.S Pitts-Meek S Armanini M.P Ling L.H McMahon S.B Shelton D.L Levinson A.D Phillips H.S Cell. 1994; 76: 1001-1012Abstract Full Text PDF PubMed Scopus (885) Google Scholar, 21Silos-Santiago I Molliver D.C Ozaki S Smeyne R.J Fagan A.M Barbacid M Snider W.D J. Neurosci. 1995; 15: 5929-5942PubMed Google Scholar). The reason for these apparent differences in dependence on neurotrophin levels is unclear. As pointed out by Lewin and colleagues, a caveat with regards to their findings is that neuron loss was not actually demonstrated and that some more complex explanation such as a phenotype switch remains a formal possibility. An alternative method for analyzing the loss of cutaneous afferents in NT-3-deficient animals is to examine the target end organs for these classes of sensory neurons. Merkel cells are specialized receptors found in specific regions of hairy skin called touch domes. These cells are the end organs of SA1 axons (for review, see18Perl, E. (1992). In Sensory Neurons (New York: Oxford University Press), pp. 3–23.Google Scholar). Lewin and colleagues demonstrate that at birth, both NT-3 (+/−) and (−/−) mice have normal numbers of Merkel cells and a normal complement of innervating sensory axons. However, by 2 weeks of age, the number of Merkel cells per touch dome was reduced by nearly 50% in NT-3 (+/−) mice; Merkel cells were absent in (−/−) mice. These findings strongly argue that SA1 neurons do not require NT-3 for survival prior to birth, but rather acquire NT-3 dependence postnatally. It is tempting to speculate that another neurotrophin can support SA1s in the embryonic period and is either the predominant factor during that time or at least can compensate for NT-3 loss in the prenatal period (see below). Another important implication of this study is that the number of receptor organs of the low threshold mechanoreceptors appear to be regulated by NT-3 levels. NT-3 (+/−) and (−/−) mice have reduced numbers of Merkel cells. Conversely, elevating levels of NT-3 in the skin substantially increases the number of Merkel cells (Albers et al., 1995, Soc. Neurosci., abstract). These results seem analogous to previous findings that levels of NT-3 regulate the number of muscle spindles and Golgi tendon organs in muscle. In NT-3 (+/−) mice, the number of surviving spindles is 50% of wild-type animals; NT-3 (−/−) mice have no muscle spindles or Golgi tendon organs (9Ernfors P Lee K.-F Kucera J Jaenisch R Cell. 1994; 77: 503-512Abstract Full Text PDF PubMed Scopus (686) Google Scholar). These peripheral end organs are presumably not regulated by NT-3 directly. Rather, it is known that muscle spindles form only after contact by sensory fibers, and the survival of muscle spindles requires sustained sensory innervation. In fact, the number of spindles is directly correlated with the number of surviving proprioceptive neurons (13Kucera J Fan G Jaenisch R Linnarsson S Ernfors P J. Comp. Neurol. 1995; 363: 307-320Crossref PubMed Scopus (87) Google Scholar). These results suggest that NT-3 indirectly regulates target end organs by supporting the survival of specific classes of sensory neurons. Axons of surviving neurons then induce differentiation of target end organs in an as yet uncharacterized manner. The finding by Lewin and colleagues that SA1 and D-hair mechanoreceptors do not become dependent on NT-3 until the postnatal period is important since most neuronal loss in neurotrophin-deficient mice has been detected much earlier in development (13Kucera J Fan G Jaenisch R Linnarsson S Ernfors P J. Comp. Neurol. 1995; 363: 307-320Crossref PubMed Scopus (87) Google Scholar, 21Silos-Santiago I Molliver D.C Ozaki S Smeyne R.J Fagan A.M Barbacid M Snider W.D J. Neurosci. 1995; 15: 5929-5942PubMed Google Scholar, 24Snider W.D Silos-Santiago I Phil. Trans. Roy. Soc. (Lond.) B, in press. 1996; Google Scholar). Presumably, these neurons require another neurotrophin (or other neuronal growth factor) for embryonic survival and then switch to survival dependence on NT-3 after birth. In support of this view, a number of examples of changes in neurotrophin receptor expression and neurotrophin dependence have now been reported. Some of these that are operative in DRG are outlined schematically in Figure 2. First, most DRG neurons (or neuronal precursors) express TrkC and appear to require locally synthesized NT-3 at early developmental stages (7Ernfors P Merlio J.-P Persson H Eur. J. Neurosci. 1992; 4: 1140-1158Crossref PubMed Scopus (449) Google Scholar; Buchman and Davis, 1993; 24Snider W.D Silos-Santiago I Phil. Trans. Roy. Soc. (Lond.) B, in press. 1996; Google Scholarreferences therein). By E13.5 in mouse, most DRG neurons have down-regulated TrkC and can be shown to be dependent on TrkA signaling for survival (24Snider W.D Silos-Santiago I Phil. Trans. Roy. Soc. (Lond.) B, in press. 1996; Google Scholar). The proprioceptive population expresses TrkC throughout development, and a small, as yet unidentified, subpopulation expresses both TrkA and TrkC into adulthood (25Wright D.E Snider W.D J. Comp. Neurol. 1995; 13: 4029-4041Google Scholar). After birth, 50% of TrkA neurons down-regulate TrkA, suggesting that this population loses its capacity for regulation by NGF postnatally (24Snider W.D Silos-Santiago I Phil. Trans. Roy. Soc. (Lond.) B, in press. 1996; Google Scholar). The findings of Lewin and colleagues suggest additional postnatal plasticity in that a subpopulation of cutaneous low threshold mechanoreceptors acquires a new neurotrophin dependence in the postnatal period. It is tempting to speculate that SA1s might require NGF or BDNF during embryonic life. Although there is apparently no loss of SA1 afferents in BDNF (−/−) mice (Koltzenburg et al., 1995, Soc. Neurosci., abstract), it is important to point out that some type of compensation may occur in neurotrophin-deficient animals. Thus, SA1s might survive into postnatal life if either BDNF or NT-3 alone is deficient but might not survive in the absence of both of these molecules. Despite the fact that NGF was discovered more than 40 years ago, an understanding of neurotrophin regulation of sensory functions is only now emerging. Strong evidence links NGF to regulation of pain transmission, both in mediating acute responses to inflammation and other noxious stimuli and in regulating gene expression that may lead to chronically enhanced nociceptor transmission (14Lewin G.R Mendell L.M Trends Neurosci. 1993; 16: 353-359Abstract Full Text PDF PubMed Scopus (472) Google Scholar, 16McMahon S.B Bennett D.L.H Priestley J.V Shelton D.L Nature Med. 1995; 1: 774-780Crossref PubMed Scopus (372) Google Scholar). From a clinical standpoint, this powerful regulation of nociception is likely to become the most important pharmacological action of this family of molecules. Studies of neurotrophin regulation of other sensory modalities are only beginning. The continued expression of NT-3 by muscle spindles in adult animals suggests the potential for regulation of proprioceptive function by NT-3. The study of Airaksinen et al. now suggests tactile sensation as an additional target for NT-3 regulation. We will look forward to future studies that will further clarify the roles of neurotrophins in sensory physiology.