reviews Nervegrowthfactorandnociception G a r y R. L e w i n a n d Lorne M . M e n d e l l Nerve growth factor (NGF) is thought of as a targetderived factor responsible for the survival and maintaining the phenoOrpe of specific sets of peripheral and central neurons during development and maturation. Recently, using physiological techniques, we have shown that specific functional b'pes of nociceptive sensory neurons require NGF, first for survival during development in utero and then for their normal phenob,pic development (but not survival) in the early postnatal period. In adulthood, the physiological role of NGF changes dramatically and here it may serve as a link between inflammation and hyperalgesia. Despite apparent changes in NGF's mode of action as the animal matures, it always interacts specifically with nociceptive sensory neurons. The neurotrophic hypothesis states that, during development, neurons are critically dependent for survival on target-derived factors. The presence of limiting amounts of such factors ensures that only a proportion of neurons survive naturally occurring cell death and that the appropriate innervation density of the target is attained. Nerve growth factor (NGF) is the prototypical neurotrophic factor and is a survival factor for both sympathetic and sensory neurons during development1. Many sympathetic neurons continue to depend on NGF for survival into adulthood, whereas sensory neurons cease to rely on this molecule in the postnatal period2~. Furthermore, it appears that at no point during development are all sensory neurons dependent on NGF for survival. Indeed, the number of neurons that need NGF for survival gets smaller as development proceeds (see Box 1). This change in dependence on NGF for survival occurs despite the fact that many smalldiameter peptide-containing sensory neurons in the adult have high-affinity NGF receptors and retrogradely transport NGF from their target tissues 5'6. This suggests that NGF has a physiological role in the adult that is distinct from that in development. Several lines of evidence have indicated that the action of NGF on sensory neurons might be directed to specific functional groups of nociceptive neurons. For example, small sensory neurons containing neuropeptides, such as substance P, do not survive NGF deprivation during development (see Box 1); and in adults, the levels of these peptides are downregulated after NGF deprivation 7'8. High-affinity NGF receptors are present primarily on this same population of neuropeptide-containing neurons6. Conversely, large cells devoid of peptides do not require NGF for survival and do not have high-affinity NGF receptors 6'9'1°. It is widely assumed that peptidecontaining neurons are nociceptive sensory neurons with unmyelinated or thinly myelinated axons 13 (see Box 2). The fact that they are susceptible to NGF deprivation suggests that NGF regulates a functional group of sensory neurons. In addition, the sensitivity of adult sensory neurons to capsaicin, a compound TINS, Vol. 16, No. 9, 1993 that elicits pain in humans and excites C-mechanoheat fibers (see Box 2) in vivo n, can be regulated in culture by the supply of NGF (Ref. 12). Animals subjected to pre- or postnatal anti-NGF treatments appear to suffer from a decreased sensitivity to noxious mechanical and heat stimuli14'~5. In these cases, the death of sensory neurons (perhaps specifically nociceptive neurons) has been proposed to be responsible for the observed hypoalgesia 14'15. However, nociceptive neurons are very heterogeneous in their functional properties (see Box 2), and not all small peptidergic neurons can be associated with physiologically identified nociceptors 16. Thus the relationship between NGF and nociceptors can be determined in a definitive fashion only by using physiological analysis rather than by inference from anatomical, behavioral and immunocytochemical studies. GaryR. Lewinand LomeM. Mendel/are at the Deptof NeurobioloD, and Behavior,SUNYat StonyBrook, StonyBrook, New York, NY 11794, USA. Role of NGF in the postnatal development of nociceptors Anti-NGF treatment. The innervation of hindlimb skin by sensory neurons in adult rats subjected to anti-NGF treatments has been analysed by recording from the axons of single afferents in dorsal root filaments and characterizing their receptive fields4'17. By using an electrical search stimulus on the sural nerve it is possible to obtain an unbiased estimate of the types of primary afferents innervating skin. After treatment with anti-NGF in the first two weeks after birth we observed a loss of just one myelinated afferent type, the thinly myelinated high-threshold mechanoreceptor (A6-HTMR) 4' 17. This selective loss might have been due to the anti-NGF-induced death of these fibers. However, this is unlikely for two reasons. First, the selective depletion of HTMRs can be obtained even when anti-NGF is administered too late to cause cell death (after postnatal day 2; see Box 1). Second, one would have expected the proportions of the two other afferent types present within the A6 range (low-threshold D-hair afferents, and afferents innervating subcutaneous structures, deep afferents) to increase as HTMRs are lost. However, only D-hairs exhibited this proportional increase (see Fig. 1). By using more circumscribed anti-NGF treatments in the postnatal period, we determined a critical period for the effects of antiNGF on A6 nociceptors. This period, from postnatal days 4-11, coincides with a period of a normally occurring rearrangement of afferent terminals in skin (see below). We have proposed that after neonatal anti-NGF treatment AS-HTMRs are forced to assume the phenotype of D-hair afferents4'17'18. This would account for the reciprocal changes in the proportions of D-hairs and HTMRs described above. It also implies that sensory neurons may be subject to respecification of their phenotype by environmental cues well into the postnatal period. © 1993,ElsevierSciencePublishersLtd,(UK) 353 Box 1. How and when can you kill sensory neurons? One can kill sensory neurons in vivo by depriving them of neurotrophic support. This can be achieved in two ways: first, by systemically treating animals with high titre antibodies raised against NGF; second, by making auto-immunized animals that will produce antibodies to their own NGF. Auto-immunized females can produce litters of rat pups that have been deprived of NGF in utero by the transfer of maternal anti-NGF to the foetus a. The effect of neurotrophin deprivation on the survival of sensory neurons varies according to the time at which it is implemented. Deprivation of NGF in utero During development up to 80% of sensory neurons can be killed by depriving them of NGF in utero a'b. The cells that are lost after these treatments all appear to be small cells that probably expressed the trk A receptorc (high-affinity NGF receptor). Many contain neuropeptides and most project to the superficial dorsal horn c-e. The large cells that remain are probably the precursors of mature low-threshold mechanoreceptors innervating skin and muscle targets f. The proportion of neurons in the mature dorsal root ganglion with large cell bodies and thickly myelinated axons is around 20% of the total in the lumbar ganglia g. Interestingly, administration of anti-NGF after postnatal day 2 fails to produce any cell death g. Post-natal Control Anti-NGF Adult NGF deprivation Both methods of NGF deprivation (autoimmune and systemic antibodies) fail to result in the death of any sensory neurons in the adult or after postnatal day 2 (Refs g, j). Adult Control Anti-NGF In utero Control Anti-NGF Perinatal deprivation of NGF In the early postnatal period, studies have shown that 20-35% of sensory neurons could be killed after administration of anti-NGF g-~. The higher percentage was obtained in thoracic ganglia where the proportion of afferents innervating cutaneous targets is greater. The cells that are killed are all small; however, appreciable numbers of these small neurons still remain. This more resilient population might represent a functional group or neurons that are more mature. The development of sensory innervation provides certain important clues as to how this might occur. Although peripheral afferent fibers have innervated the skin by the time that the rat is born, this innervafion undergoes fundamental changes during the immediate postnatal period 19'2°. From around embryonic day 17 (El7) to around postnatal day 5, the sensory innervation of the skin appears to be largely epidermal with virtually no fibers terminating in the dermis. The innervafion takes on the adult pattern in the hindlimb with the appearance of hair follicles in the dermis from around postnatal days 5-10, and so some fibers must move fl'om epidermis to dermis. In the adult animal, the terminations of A6--HTMR afferents appear to be in the epidermis 21. This suggests that when NGF is eliminated by anti-NGF, HTMRs are unable to sustain their normal epidermal terminals and 'drop down' into the dermis to innervate receptors characteristic of D-hairs. 354 References a Johnson, E. M., Jr, Rich, K. M. and Yip, H. K. (1986) Trends NeuroscL 9, 33-37 b Johnson, E. M., Jr, Gorin, P. D., Brandeis, L. D. and Pearson, J. (1980) Science 210, 916-918 c Carroll, S. L. etal. (1992) Neuron 9, 779-788 d Otten, U., GoederL M., Mayer, N. and Lernbeck, F. (1980) Nature 287, 158--159 e GoederL M. et aL (1984) Proc. Natl Acad. Sci. USA 81, 1580-1584 f Ruit, K. G., Elliot-[, J. L., Osborne, P. A., Yah, Q. and Snider, W. D. (1992) Neuron 8, 573-587 g Lewin, G. R., Ritter, A. M. and Mendell, L. M. (1992) J. Neurosci. 12, 1896-1905 h Yip, H. K., Rich, K. M., Larnpe, P. A. and Johnson, E. M., Jr (1984) J. Neurosci. 4, 2986-2992 i Hulsebosch, C. E., Perez-Polo,J. R. and Coggeshall, R. E. (1987) J. Comp. Neurol. 259, 445-451 j Gorin, P. D. and Johnson, E. M., Jr (1980) Brain Res. 198, 27-42 The phenotype of A6 afferents after anti-NGF treatment was determined by characterizing their adequate stimulus. It is possible that the D-hair afferents recorded had only partially changed so that their cell bodies still expressed phenotypes more characteristic of HTMRs. However, an important characteristic difference between D-hair and HTMR afferents is the configuration of their somatic spikes recorded in the dorsal root ganglion. D-Hairs have a very brief spike with a smooth falling phase, whereas HTMRs have a much broader spike with a characteristic inflection on the falling phase 22. In antiNGF-treated animals no D-hairs are observed with broad HTMR-like spikes a3, suggesting that the conversion occurs not only in the skin terminals, but also in the cell body. This finding argues more convincingly for a true phenotypic conversion as a result of the withdrawal of NGF during the postnatal critical period. TINS, Vol. 16, No. 9, 1993 Among the population of unmyelinated C-fibers, at least five different types of nociceptor have been described (see Box 2) z4'25. After neonatal anti-NGF treatment (postnatal days 2-14) it was found that the numbers of C-mechanoheat fibers (see Box 2) in adult animals had been severely reduced (by about 60%). These lost fibers appeared to be replaced by a new population of C-fibers that responded exclusively to mechanical stimuli. The new population of mechanoreceptors was unusual in that their mechanical thresholds were intermediate between those of nociceptors and low-threshold mechanoreceptors. This change in threshold is unlikely to be due to physical changes in the skin, since the mechanical sensitivity of the few remaining C-mechanoheat fibers was normal. That the increase in the fraction of C-mechanoheat afferents seen after excess NGF is given neonatally2s is additional evidence that the fibers conferring noxious heat sensitivity are selectively susceptible to the supply of NGF. The ability of A6 and C-fiber afferents to elicit antidromic vasodilation was found to be impaired in neonatal anti-NGF-treated animals26. This result suggests that the sensory neurons lost (i.e. converted) after anti-NGF treatment (A6--HTMRs and C-mechanoheat fibers) probably mediated the antidromic vasodilation, and this is in agreement with data from other studies 27'2s. NGF treatment. We have administered excess amounts of NGF to neonatal animals in the first two weeks after birth. It is possible that large amounts of systemic NGF might lead to changes that do not reflect this factor's normal function. However, for the most part, the physiological consequences were specific for the same types of fibers affected by lack of NGF (Refs 18, 29). In some ways the results of NGF administration were less dramatic than anti-NGF treatments. For instance, the relative proportions of Ab-HTMRs, D-hair and deep afferents in the sural nerve were unchanged. If our interpretation of the phenotypic switch is correct, this implies that the naturally occurring amount of NGF present in skin is sufficient to ensure that all the A6 nociceptors develop normally. Excess NGF does not apzpear to force more sensorv neurons to become ASs~L The physiological properties of the AS-HTMRs in neonatally NGF-treated animals were different from those of controls. A6-HTMRs in NGF-treated fibers were about twice as sensitive to mechanical stimulation as normal control fibers. This increased sensitivity after neonatal treatment with NGF is not permanent, and wears off when the animal is about nine weeks of age 29. However, if animals were treated with NGF after the second postnatal week, this change in AS-HTMR receptor sensitivity was never observed 29. This suggests that the mechanical thresholds of the A6 fibers are in part regulated by NGF during the postnatal critical period. Further evidence for this suggestion comes from the anti-NGF experiments in which HTMRs not converted by treatment with anti-NGF during the critical period have a decreased mechanical sensitivity (thresholds are about double those of control units) 4. Box 2. Physiological heterogeneity of sensory neurons Sensory neurons are normally classified physiologically using two main criteria, their axonal conduction vel- ocities and their adequate stimuli. Sensory neurons with thickly myelinated axons (A~-fibers) almost all respond to very low-threshold stimuli, such as hair movement in skin or length changes in skeletal muscle. Most of the slower conducting thinly myelinated sensory neurons (A6-fibers) innervating skin are highthreshold mechanoreceptors (HTMRs) as they respond exclusively to noxious stimuli such as pinching the skin. However, a significant proportion of Ai5 sensory neurons respond to very-low-threshold stimuli such as hair movement; these are often called D-hairs1'2. The majority of sensory neurons have unmyelinated axons (C-fibers) and the majority of these neurons in rodents, monkeys and man respond to noxious stimulia-c. However, this does not mean that C-fibers are all functionally equivalent. These neurons can respond to noxious mechanical, heat, cold and chemical stimuli, either exclusively or in combinationd. Thus the most common type has been termed C-mechanoheat (C-MH) as it responds to both these modalities. Other types include C-mechanoreceptors (C-M), C-mechanocold (C-MC) and C-Cold (C-C) d. Finally, some C-fibers innervating skin, viscera and joints appear to be unresponsive to any of these stimuli and these have been termed silent or mechanically insensitive fiberse. For both A6 and C-fibers, no clear correspondence between cytochemical markers and the physiological phenotype of the sensory neurons has been foundf. References a Burgess, P. R. and Perl, E. R. (1973) in Handbook of Sensory Physiology, Vol. 2 (Iggo, A., ed.), pp. 29-78, Springer b Lynn, B. and Carpenter, S. E. (1982) Brain Res. 238, 29-43 c Treede, R. D., Meyer, R. A., Raja, S. N. and Campbell, J. N. (1992) Prog. Neurobiol. 38, 397-421 d Kress,M., Koltzenburg, M., Reeh, P, W. and Handwerker, H. O. (1992) J. Neurophysiol. 68, 581-595 e McMahon, S. B. and Kolt.zenburg, M. (1990) Trends Neurosci. 13, 199-201 f Leah, J. D., Cameron, A. A. and Snow, P. J, (1987) Neurosci. Lett. 56, 257-263 the physiology of A6 fibers. However, since adult sensory neurons possess functional high-affinity NGF receptors s'6, NGF must have some functional role. Behavioral experiments where the effects of excess NGF were tested on adult animals may provide some insights. After just one systemic injection of NGF (1 ~tg/g, intraperitoneal), adult rats develop a profound hypersensitivity to noxious heat and mechanical stimuli29, a so-called hyperalgesia. The mechanical hyperalgesia that develops is particularly profound as the mechanical threshold needed to elicit a flexion reflex drops from around 150 g in controls to as low as 20 g in NGF-treated animals29. This hyperalgesia does not appear to be accompanied by any ongoing pain, or pain elicited by stimuli sufficient to excite lowthreshold AI5 afferents (the latter is often called allodynia). The mechanical hyperalgesia takes around six hours to develop and there are no visible signs of skin inflammation. We have recorded from A6R o l e of N G F in a d u l t a n i m a l s Under normal circumstances, treatment of adult HTMRs in animals displaying this NGF-induced animals with anti-NGF does not lead to any change in hyperalgesia and they are not sensitized to mechanical TINS, Vol. 16, No. 9, 1993 355 A Control Anti-NGF Cell death? or Phenotypic conversion? OI mice displaying behavioral changes (Johnson, E. M., Davis, B. M. and Albers, K., unpublished observations). In adult rats, the same injection of NGF gave rise to a heat hyperalgesia in addition to the mechanical hyperalgesia. The heat hyperalgesia develops within minutes of the injection and thus may be due to the sensitization of C-mechanoheat fibers to noxious heat. However, as yet we have no direct evidence for this. Is NGF t h e h y p e r a l g e s i a - i n f l a m m a t i o n linchpin? The hyperalgesia that follows an NGF injection is qualitatively very similar to that which often accompanies inflammatory tissue injury 31. Could over• HTMR • D-Hair (~ Deep expression of NGF in inflamed tissue be a critical link between tissue injury and the hyperalgesia that B ensues? There is, in fact, abundant evidence supporting a critical role for NGF in this process. The O HTM Rs 8o levels of NGF in damaged or inflamed tissue have (t) [] D-Hairs p,,,, been shown to be increased many fold above normal Q. ~ 60 levels32-34, and this effect can be seen within hours E of the initiation of inflammation 3z. An enhanced retrograde transport of NGF by the sciatic nerve occurs in ~ 40 conjunction with this peripheral increase 35 (Fig. 2). Many cytokines and growth factors, levels of which • 20 are probably elevated at the site of inflammation, can cpotently upregulate the production of NGF by skinI I i I | I I | i and nerve-derived fibroblasts36'37. These mediators £ 0 Cont. 2 - 5 7 - 1 4 2 - 9 4-11 2 - 1 4 0 - 1 4 0 - 5 0-5* include acidic and basic fibroblast growth factor wks days days days days days wks wait (FGF), interleukin 113 (IL-I[3), tumor necrosis factor Duration of treatment (TNF-00, platelet-derived growth factor (PDGF), transforming growth factor (TGF-~t and TGF-[31), and Fig. 1. (A) AAodel of two possible scenarios to account for epidermal growth factor (EGF) 36'37. Skin keratinodepletion of high-threshold mechanoreceptors (HTAARs) cytes appear to be major sources of NGF in viv03°'38; after administration of anti-NGF antibodies over the however, it is not known if the above cytokines can neonatal critical period (postnatal days 4-11). Each of regulate NGF expression in these cells. In recent the four dorsal root ganglia shown contains three types of A6 sensory neurons with axons in the sural nerve: years it has become increasingly clear that NGF may HTAARs, D-hairs and deep (subcutaneous) receptors4,17,18. have an important immunomodulatory role and it Upper panel shows selective loss of HTAARs; lower panel might potently affect the responsiveness of immune shows conversion of HTAARs to D-hairs. (B) Relative competent cells during inflammation39'4°; in short, proportions of the three types of A6 neurons after NGF may be involved in the process of inflammation. different times and durations of postnatal anti-NGF We will not review this large literature here, but it is treatment. Critical period is from postnatal days 4-11, shown by the fact that HTAARs are converted to D-hairs only when anti-NGF is administered over this period. (Reproduced, with permission, from Ref. 4.) Note that the actual changes in the proportion of each type observed conforms to the predictions of the conversion model shown schematically above in (A). 5 4 E stimuli. Stimuli sufficient to excite low-threshold A[3 afferents did not lead to any behaviors indicative of pain, i.e. there was no allodynia present. Recently, Albers et al. 3° described a transgenic mouse where an overproduction of NGF in skin was achieved by attaching the K14 keratin promoter to the NGF gene. These mice exhibited a mechanical hyperalgesia very similar to that found in rats after a systemic NGF injection (Davis, B. M., Lewin, G. R., Mendell, L. M., Johnson, M. and Albers, K., unpubfished observations); this result suggests that an increased level of NGF restricted to the skin is sufficient to produce mechanical hyperalgesia. Preliminary data indicate that biologically active NGF is not present in the systemic circulation of many of the 356 Q. LL 3 2 1 I I I I 1 2 3 4 Time (days) Fig. 2. Levels of NGF in sciatic nerve after inflammation induced by injection of 0.15ml of complete Freunds adjuvant. Note the increase in the NGF content in the ipsilateral sciatic nerve (filled squares) compared to contralateral sciatic nerve (open squares). Statistical significance is indicated by * (p <0.05). (Reproduced, with permission, from Ref. 35.) ~NS, Vo1.16, No. 9,1993 Periphery Cell body Spinal cord Sensory consequence clear that NGF seems to be particularly important for the action of histamine-secreting mast cells and circulating basophils 4v-44. An increase in NGF might have I Injury t Neuropeptides two major effects on the sensory e.g. SP + CGRP neurons innervating inflamed 1 tissue. The first effect may be ¢ indirect as NGF is a potent stimuCentral ~ Mechanical Mechanical lator of mast cells and can cause sensitization [ I hyperatgesi~ hyperatgesia their degranulation 42'45. Rat peritoneal mast cells have recently NGF been found to possess the putadependent tive high-affinity NGF receptor trk \ A (Ref. 46), and they require NGF for survival in vitro 47. Substances Heat L hyperalgesia released by stimulated mast cells could directly sensitize the peripheral endings of C-mechanoheat fibers, which might explain the ~ ] [ LTMR-mediated heat hyperalgesia that immediately ' [ sensptiz~ion I'--[-~"1 pain I ] H l I=io~y~l I NGF follows treatment with exogenous independent NGF. In the rat, mast cells release 5-hydroxytryptamine (5-HT) as well as histamine upon stimulation48. However, neither of these substances can directly sensitize C- Fig. 3. Schematic diagram of possible mechanisms for NGF-dependent (above) and NGFmechanoheat fibers to heat 49. In independent (below) sensory consequences of tissue damage and inflammation. Increased levels of preliminary studies we have found NGF can lead to both mechanical and heat hyperalgesia29. Two major pathways are proposed to be that animals with degranulated involved in these two types of hyperalgesia. The long-lasting heat and mechanical hyperalgesia may come about via the upregulation of neuropeptide levels in the dorsal root ganglion; the central mast cells do not display heat release of these neuropeptides might lead to central sensitization 51. We have obtained evidence hyperalgesia in the first three that the long-lasting heat hyperalgesia may be dependent on an increase in NMDA-mediated hours following treatment with neurotransmission. The rapid heat hyperalgesia after treatment with exogenous NGF might result exogenous NGF (Ref. 50); thus from the sensitization of C-fibers to heat in the periphery. This may be initiated by the NGF-induced the nature of the peripheral mech- release of agents from mast cells5~. Low-threshold mechanoreceptor (LTMR)-mediated pain (allodynia) and spontaneous pain do not appear to be produced by the administration of anism requires further study. The second effect of increased exogenous NGF. These sensory consequences may be produced via other mechanisms of central NGF on sensory neurons in in- sensitization or by the continuous activation of nociceptors or the recruitment of silent fibers (see flamed tissue is more direct. NGF Box 2). Note that where the boxes are tinted, the associated processes have not been definitively might bind to its receptor on sen- proven to be involved in the mechanism indicated. Abbreviations: SP, substance P; CGRP, calcitonin gene-related peptide. sory neurons and be retrogradely transported to the cell body where it can lead to a rapid and large increase in the the inflammation, can be abolished by concurrent production of neuropeptides 5 such as substance P and administration of anti-NGF (Ref. 57). Unfortunately, calcitonin gene-related peptide (CGRP) at the level of the use of antibodies presents problems as very large gene expression 5L52. Increases in the levels of these amounts of these molecules are required to have any two peptides in sensory neurons and their peripheral effect. In future, the development of non-pepride axons innervating inflamed tissue have been reported antagonists to the high-affinity NGF receptor may In • many models of inflammatory pain 3 5 ' 5 3 - 5 6 . For provide a better tool to address this question 58. example, increases in these peptides accompany skin Hyperalgesia following tissue injury has in recent inflammation induced by complete Freunds adjuvant years been increasingly attributed to the sensitization (CFA) or carrageenin 35'54, as well as damage to the of spinal cord circuits in response to an afferent skin by ultraviolet light (sunburn) 56 and joint inflam- barrage from the injured tissue al. The increased mation or experimental arthritis 5a'55. In one of these synthesis of neuropeptides that follows tissue inflammodels, where CFA is used to inflame the foot pad of marion may well lead to an increased release of these a rat, there is now direct evidence that NGF is the substances from the central terminals of primary cause of these changes in peptide levels. Donnerer et afferents, and indeed there is evidence to support this aL 35 demonstrated that the increases in the levels of contention 54'55'59. Neuropeptides released in the substance P and CGRP in the skin and dorsal root dorsal horn may sensitize central neurons, perhaps by ganglion after CFA-induced inflammation of the skin enhancing neurotransmission via the N-methyl-Dcould be blocked with the exogenous administration of aspartate (NMDA) receptor by producing long-lasting anti-NGF. Unfortunately, these workers did not test depolarizations 6° or by directly modulating the recepto see if this blockade of increases in the peptides also tor complex61'62. The NMDA receptor has been blocked hyperalgesia associated with the inflam- shown to be necessary for the generation and marion. Preliminary experiments carded out in this maintenance of many central sensitization events 63. In laboratory using the same CFA model have shown recent experiments we have obtained direct evidence that heat hyperalgesia, which normally accompanies that the long-lasting heat hyperalgesia that follows mS m TINS, VoL 16, No. 9, 1993 / 357 Hyperalgesia Mechanical (central?) Heat (peripheral?) Maintenance of HTMR phenotype I I Survival of small peptidergic cells '" I i I i i } }' I Adult Neonate Fetus Fig. 4. Actions of NGF depend on the developmental stage. Up to postnatal day 2, NGF acts as a survival factor. Between postnatal days 2-14, NGF influences the phenotype of the sensory neuron. In the adult, NGF can lead to hyperalgesia. NGF with a delay of around seven hours is dependent on activation of the NMDA receptor, since administration of a noncompetitive NMDA antagonist blocks the hyperalgesias°. However, the mechanical hyperalgesia that is induced by NGF appears to be independent of NMDA receptor activation~°. Whatever the central mechanism of sensitization, there is direct evidence that artificially increased levels of NGF in a peripheral target can sensitize central neurons to afferent barrages from afferents innervating that target c~. The long latency of NGF-induced mechanical hyperalgesia is consistent with the initiation of increased synthesis of peptides in sensory neurons and their transport to the central terminals in the spinal cord (Fig. 3). Changes in the functional connectivity of these afferents in the spinal cord may then underlie the mechanical hyperalgesia. Neuropepfides that are upregulated by NGF are also transported in large amounts to the periphery 11'3S. Their release in skin may contribute to the inflammationl~; however, it is unlikely that sensory neurons would be further sensitized since neurogenic extravasation does not sensitize peripheral fibers to heat 65. This model suggests that inflammation-induced increases in NGF in the periphery may be the linchpin that connects tissue damage to the accompanying hyperalgesia. The hyperalgesia that follows NGF injections in normal adult rats is not accompanied by detectable inflammation29,34, and this suggests that NGF has in this case short-circuited the normal physiological process. The fact that abnormal sensations such as spontaneous pain and allodynia do not result from exogenous NGF suggests that these sensations arise via a pathway that is independent of NGF (see Fig. 3). More experiments designed to test this model specifically will be needed before NGF's role in generating the hyperalgesia that follows tissue injury is more clearly defined. Nevertheless, the existing evidence suggests that NGF may be an important target of drug therapies aimed at alleviating hyperalgesia following tissue injury. 358 In summary, it appears that the physiological role of NGF in sensory neurons is highly dependent on their developmental context (see Fig. 4). The presence of NGF is critically important for the normal development of important subsets of nociceptive primary afferent neurons. The developmental mechanisms by which NGF regulates nociceptor development in some cases do not include a role as a survival factor, but rather as a molecule that maintains certain neuronal phenotypes. In the adult, NGF may act through entirely different mechanisms to serve as a link between the physiological state of nociceptors and the health of the tissue they innervate. The growing complexity of NGF's physiological role may serve as a model by which the other largely uncharacterized neurotrophins comprising the NGF family can be studied. Selected references 1 Barde, Y-A. (1989) Neuron 2, 1525-1534 2 Gorin, P. D. and Johnson, E. M., Jr (1980) Brain Res. 198, 27-42 3 Ruit, K. G., Osborne, P. A., Schmidt, R. E., Johnson, E. M., Jr and Snider, W. D. (1990) ]. NeuroscL I0, 2412-2419 4 Lewin, G. R., Ritter, A. M. and Mendell, L. M. (1992) J. Neurosci. 12, 1896-1905 5 Goedert, M., Stoeckel, K. and Otten, U. (1981) Proc. Natl Acad. 5ci. USA 78, 5895-5898 6 Verge, V. M., Richardson, P. M., Benoit, R. and Riopelle, R. J. (1989) J. Neurocytol. 18, 583-591 7 Goedert, M. eta/. (1984) Proc. Natl Acad. ScL USA 81, 1580-1584 8 0 t t e n , U. (1984) Trends Pharmacol. Sci. 5, 307-310 9 Johnson, E. M., Jr, Rich, K. M. and Yip, H. K. (1986) Trends Neurosci. 9, 33-37 10 Carroll, S. L. eta/. (1992) Neuron 9, 779-788 11 Holzer, P. (1991) Pharmacol. Rev. 43, 143-201 12 Winter, J., Forbes, C. A., Sternberg, J. and Lindsay, R. M. (1988) Neuron 1,973-981 13 Hunt, S. P., Mantyh, P. W. and Priestley, J. V. (1992) in Sensory Neurons: Diversity, Development and Plasticity (Scott, S. A., ed.), pp. 60-76, Oxford University Press 14 Aloe, L., Cozzari, P., Calissano, P. and Levi-Montalcini, R. (1981) Nature 291,413-415 15 Urschel, B. A., Brown, P. N. and Hulsebosch, C. E. (1991) Exp. Neurol. 114, 44-52 16 Leah, J. D., Cameron, A. A. and Snow, P. J. (1987) Neurosci. Lett. 56, 257-263 17 Ritter, A. M., Lewin, G. R., Kremer, N. E. and Mendell, L. M. (1991) Nature 350, 500-502 18 Ritter, A. M., Lewin, G. R. and Mendell, L. M. (1993) Brain Res. Bull. 30, 245-249 19 Fitzgerald, M. J. T. (1965) J. Comp. Neurol. 126, 37-42 20 Reynolds, M. L., Fitzgerald, M. and Benowitz, L I. (1991) Neuroscience 41,200-211 21 Kruger, L., Perl, E. R. and Sedivec, M. J. (1981) J. Comp. Neurol. 198, 137-154 22 Koerber, H. R. and Mendell, L. M. 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(1992) Ann. NYAcad. Sci. 657, Acknowledgements Thisresearch was supported by a subproject of NIH Program Project Grant PC)1 N514899. Additional support was derived from NIH R01 N516966 to L. M. M. (Javits NeuroscienceA ward). 505-506 60 Thompson, S. W. N., King, A. E. and Woolf, C. J. (1990) Eur. J. Neurosci. 2, 638-649 61 Randic, M., Hecimovic, H. and Ryu, P. D. (1990) Neurosci. Lett 117, 74-80 62 Rusin, K. I., Ryu, P. D. and Randic, M. (1992) J. Neurophysiol. 68, 265-286 63 Dubner, R. and Ruda, M. A. (1992) Trends Neurosci. 15, 96-103 64 Lewin, G. R., Winter, J. and McMahon, S. B. (1992) Eur. J. Neurosci. 4, 700-707 65 Reeh, P., Kocher, L. and Jung, S. (1986) Brain Res. 384, 42-50 TheTINS/TiPSLecture Themolecularbiologyof mammalianglutamatereceptor channels Peter H. Seeburg At the 1992 ENA meeting, held in Munich, the plenary lecture given by Peter Seeburg was sponsored jointly by TINS and TIPS. The following article is adapted from this lecture. In native brain membranes the principal excitatory neurotransmitter L-glutamate activates cation-conducting channels with distinct biophysical and pharmacological properties. Molecular cloning has revealed the existence of 16 channel subunits that can assemble in homomeric or heteromeric configurations in vitro to form receptor channels with disparate functional properties. This review describes the different channel types obtained by recombinant means and the genetic mechanisms controlling the expression of functionally important channel structures. Many actions of the neurotransmitter L-glutamate, ranging from fast excitatory synaptic transmission to the regulation of developmental processes, are mediated by ionotropic receptors 1-3. Biophysical analysis using membrane patches taken from the soma of neurones indicates that different glutamate-sensitive channel types coexist in many cells4. These channels differ with respect to activation and deactivation timecourses, display distinct desensitization kinetics, and have differing ion permeabilities and conductance properties. Selective agonists and antagonists permit a distinction between ionotropic glutamate receptor (GluR) classes 5'6. One selective agonist is c~-amino-3TINS, Vol. 16, No. 9, 1993 hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), which activates channels with fast kinetics 7-9 (onset, offset and desensitization time-courses are in the order of ms); in most neurones these channels are characterized by very low Ca2+ permeability 1°. NMethyl-i)-aspartate (NMDA) selectively gates a channel with comparatively much slower kinetics 4'11 and high Ca2+ permeability 12. These unique properties, together with a voltage-dependent block by Mg2+ (Refs 13, 14), a requirement for glycine as coagonist 15, and a large single channel conductance 14, collectively determine the functional aspects of NMDA receptors. The potent neurotoxin kainate generates currents with different properties in different receptor channels: through AMPA receptors kainate activates a current that persists in the continued presence of this agonist16'17; in another receptor type, called high-affinity kainate receptors (referred to here as kainate receptors) kainate activates fast desensitizing currents 18. The neurophysiological functions of these three channel classes (AMPA, NMDA, kainate) can be summarized as follows. AMPA receptors, found in the majority of excitatory synapses, mediate the majority of all fast excitatory neurotransmission. Their rapid kinetics render them ideally suited for this purpose. The generally very low Ca 2+ permeability of AMPA receptor channels means that glutamate-activated ionic currents mediated by these channels do not carry sufficient Ca 2÷ ions into cells to initiate © 1993,ElsevierScience Publishers Ltd,(UK) Peter H. Seeburgis at the Universityof Heidelberg, Centre of Molecular Biology (ZMBH), Laboratory of Molecular Neuroendocrinology, POBox 106249, Im NeuenheimerFeld 282, 69052 Heidelberg, Germany. 359