Trophic factors and neuronal survival

Neuron,

Vol. 2, 1525-1534,

June, 1989, Copyright

Q 1989 by Cell Press

Trophic Factors and Neuronal Survival Yves-Alain

Barde

Max-Planck Institute for Psychiatry Department of Neurochemistry D-8033 Martinsried Munich Federal Republic of Germany

Introduction A recurrent observation in the development of the nervous system is the generation of neurons followed by their large scale elimination. This phenomenon, usually referred to as naturally occurring neuronal death, takes place at a particularly important time during the development of embryonic neurons-the period immediately followirig the arrival of their axons in the target fields. The extent of naturally occurring neuronal death varies considerably from region to region. In their seminal study of 1949, Viktor Hamburger and Rita Levi-Montalcini noted that the number of pyknotic (dying) neurons in the chick dorsal root ganglia that innervate a large peripheral field, such as the limb, is much smaller than the number of pyknotic neurons in ganglia innervating a small peripheral field, such as the neck and thorax. In addition, the degree of neuronal death can be dramatically increased by removing a prospective target early in development. In the absence of target, the number of neurons initially present is not changed, indicating that the target does not influence the number of neurons generated. Some of these target deprivation experiments were performed at the turn of the century, and their consequences on the development of neurons clearly reported. However, it took about 50 years to realize that, in fact, experimentally induced neuronal cell death merely exaggerates a phenomenon that is taking place during normal development (for a review of the slow maturation of this concept, see Oppenheim, 1981). Conversely, the degree of naturally occurring neuronal death can be decreased by adding an extra target or by blocking neurotransmission. For example, essentially all motoneurons can be rescued in chick embryos when paralyzed by applications of a-bungarotoxin or d-tubocurarine (Laing and Prestige, 1978; Pittman and Oppenheim, 1978). These observations demonstrate that the neurons normally eliminated are not programed to die, but can be rescued provided the conditions in which they grow are changed. In vertebrates then, it seems that the fate of the neurons at the time of target innervation can be markedly influenced by epigenetic factors, with the target tissue apparently playing a major role in this regulation. Although neuronal death has been observed during development in a variety of invertebrates, the role played by the target is unclear. For example, in grasshopper embryos, many motoneurons die after sending their axons to the developing musculature. However, the early removal of the target limb bud in these embryos does not

Review

prevent the survival and differentiation of the motoneurons (Whitington et al., 1982). This review focuses on the idea that cells which act as targets for developing neurons produce limiting amounts of specific molecules required for further survival of neurons, and for which the nerve terminals appear to compete (Figure 1). The properties of three molecules, which are now known to affect neuronal survival in vivo, are discussed: nerve growth factor, brain-derived neurotrophic factor, and fibroblast growth factor.

Nerve Growth

Factor

The providential discovery of remarkably high quantities of nerve growth factor (NGF) in the adult male mouse submandibular gland is the main reason why we have been able to learn so much about the physiology and molecular biology of this protein (for a recent historical account, see Levi-Montalcini, 1987). For over two decades, NGF provided the only basis for the concept that trophic molecules are necessary for neuronal survival. Indeed, in his 1960 publication, Stanley Cohen not only described the purification of NGF from the mouse submandibular gland, but also provided the best single piece of evidence that a neurotrophic protein is required during normal development: the observation that anti-NGF antibodies injected into newborn rodents specifically destroy the peripheral sympathetic nervous system (Cohen, 1960). In the last decade, considerable progress has been made toward a better understanding of the role of NGF during development, and some of these results are reviewed here.

NCF and Sympathetic

Neurons

Although peripheral sympathetic neurons have long been known to respond to NGF (see Levi-Montalcini and Angeletti, 1968, for a review of the early work), the unambiguous demonstration that exogenous NGF decreases naturally occurring neuronal death in sympathetic ganglia is relatively recent. Fewer pyknotic neurons are seen after administration of NGF (Oppenheim et al., 1982), suggesting that the amounts of endogenous NGF are limiting. Importantly, it has now also been established that NGF (Korsching and Thoenen, 1983) and its mRNA (Heumann et al., 1984; Shelton and Reichardt, 1984) are present in target tissues of the sympathetic nervous system. Indeed for many years, there were no reliable measurements of NGF in tissues likely to be relevant to nervous system development (see Thoenen and Barde, 1980, for a review). With the introduction of sensitive and reliable techniques, it was found that the levels of NGF in the target tissues of sympathetic neurons are very low, consistent with the view that these amounts are limiting. Indeed, it was possible to establish a correlation between the levels of NGF (both in the protein and its mRNA) and the density of sympathetic innervation

A

(Korsching and Thoenen, 1983; Heumann et al., 1984; Shelton and Reichardt, 1984). Thus, the conclusion that NGF is a target-derived molecule that controls neuronal survival at the time of target innervation has been established by the observation of the degeneration of neurons after anti-NGF antibody treatment, the examination of the tissue distribution of NGF and its mRNA, the reduction of naturally occurring neuronal death after NGF administration, and the fact that interruption of retrograde transport has the same consequences as antibody treatment (Hendry, 1975; Johnson. 1978). Beyond its general role in the control of cell number in vivo, NGF is also likely to regulate the degree of axonal branching locally. In tissue culture, where the cell bodies of sympathetic neurons and their terminal neurites can be analyzed separately (Campenot, 1977), increasing the NGF concentration above a threshold level necessary for the survival of all neurons increases the number of neurites. The increase in neurite number is entirely controlled by the local concentrations to which the neurites are exposed and is independent of the concentrations of NGF to which the cell bodies are exposed (Campenot, 1982a, 198213). Moreover, the administration of NGF in vivo produces profuse axonal branching and dendritic arborization (see Purves et al., 1988, for a recent review). Sympathetic neurons do not respond to NGF very early in development, and, presumably, NGF is not the agent responsible for the initiation of fiber outgrowth, since this can readily be observed in the absence of NGF (Coughlin and Collins, 1985). In the adult, NGF is not necessary for the survival of most sympathetic neurons. However, the levels of proteins such as tyrosine hydroxylase, which are crucial to the function of sympathetic neurons, are elevated after administration of NGF and are markedly decreased after antibody treatment (see Thoenen and Barde, 1980, for a review). NGF and Sensory Neurons As it does with sympathetic neurons, NGF can reduce naturally occurring death of sensory neurons. In dorsal root ganglia (DRG), fewer pyknotic neurons are found in vivo after administration of NGF to chick embryos (Hamburger et al., 1981). The role of NGF during normal development of the peripheral sensory system has been

fully appreclatrd only recently (see Johnson et J., 1986, for a review). Exposure of fetuses to anti-NGF antibodies reduces the number of dorsal root and trigeminal ganglion neurons by up to 80% (Johnson et al., 1980; Pearson et al., 1983: Johnson et al., 1986). The survival of peripheral sensory neurons ib affected at earlier stages by anti-NGF antibodies, primarily during the intrauterine period, than the survival of sympathetic neurons. Moreover, not all primary sensory neurons are affected by the antibodies: in particular, neurons derived from ectodermal placodes, such as those forming the nodose ganglia, are not affected by this treatment (Pearson et al., 1983: Rohrer et al., 1988b). Also, proprioceptive and other largediameter sensory neurons are less affected by anti-NGF antibodies. After injection of anti-NGF antibodies into rat embryos, there is a 90% decrease in nonmyelinated sensory axons, with no reduction in the number of large myelinated axons (Goedert et al., 1984; see also Laing et al., 1988). Conversely, after nerve section in newborn rats, injections of NGF do not rescue identified proprioceptive neurons, although the injections rescue many (mostly) small-didmeter neurons (Miyata et al., 1986). Finally, the chick trigeminal mesencephalic neurons (consisting entirely of proprioceptive neurons, of neural crest origin) cannot be rescued by NGF in vivo (Straznicky and Rush, 1985). In this context, it is interesting to note that whereas NGF receptors have been found on most (if not all) neurons of the sympathetic ganglia, in the DRG, many sensory neurons. including in particular the largest ones, do not display NGF binding in either chick or rat (Raivich et al., 1985; Richardson et al., 1986). In clarifying the role of NGF during development, studies of the developing mouse trigeminal ganglion have proven to be of particular interest, since the precise time at which trigeminal axons encounter their targets, as well as the onset of the NGF responsiveness, is known (Davies and Lumsden, 1984). Trigeminal axons terminate just under the epidermis (later forming the Merkel disks), with the basal lamina of the nerve fusing with that underlying the epidermis, and on the sides of the whisker follicles (Iggo and Andres, 1982). NGF immunoassays and Northern blot analysis have demonstrated that NGF and its mRNA appear in the target only after the trigeminal axons have arrived (Davies et al., 1987). In situ hybridization has directly demonstrated the presence of

Review: 1527

Trophic

Factors

and Neuronal

Survival

NGF mRNA in the hair follicles and the epidermis (Davies et al., 1987; Bandtlow et al., 1987). Furthermore, analysis of theinitial outgrowth from trigeminal explants, previously shown to be NGF-independent (Lumsden and Davies, 1983), revealed that the axons lacked NGF receptors (Davies et al., 19871, ruling out a long-range chemotropic role for NGF during the initial development of this system. Currently, this is the most direct demonstration of the presence of NGF in the target fields of NGFresponsive neurons during normal development. The factors that determine the initiation of transcription of the NGF gene are still not known. The tight correlation between the time of target innervation and the detection of NGF mRNA in the target suggests that the incoming nerves mght release a signal necessary to initiate transcription. However, early and complete denervation of the skin in chick embryos in vivo does not affect the steady-state level of NGF mRNA measured in the skin (Rohrer et al., 1988a). As with sympathetic neurons, the effects of NGF and its deprivation are not limited to the regulation of survival during development. For example, after postnatal administration of anti-NGF antibodies in the rat, there is a profound reduction in the levels of peptides such as substance P (Otten et al., 1980). Furthermore, NGF can regulate important pharmacological characteristics of adult sensory neurons: the sensitivity of many mammalian sensory neurons to the excitotoxin capsaicin is seen in vitro only when NGF is present (Winter et al., 1988).

NCF and Central

Cholinergic

Neurons

A third neuronal population affected by NGF is composed of the central cholinergic neurons (for a review see Thoenen et al., 1987). These neurons are predominantly, but not exclusively, located in the cholinergic nuclei of the basal forebrain that project to the hippocampus and cortex. In much the same way that embryonic neurons die after interruption of contact with their targets, the basal forebrain cholinergic neurons die after axotomy, and it is striking that these axotomized neurons can be rescued by the intraventricular application of NGF (Hefti, 1986; Kromer, 1987; Montero and Hefti, 1988; Williams et al., 1986). The distribution of the NGF protein and its mRNA supports the general model thought to operate in the PNS (Korsching et al., 1985; Shelton and Reichardt, 1986; Whittemore et al., 1986): NGF is synthesized by the target cells, in this case the pyramidal cells of the hippocampal neurons of the dentate gyrus (Ayer-Lelievre et al., 1988; Whittemore et al., 1988). NGF is then transported retrogradely from the hippocampus back to the cholinergic septal neurons (Schwab et al., 19791, which do not synthesize NGF (Korsching, et al., 1985). However, convincing data demonstrating a role for NGF during the development of this central cholinergic system are lacking. In particular, it is unclear whether cholinergic cell numbers can be affected by administration of NGF or its antibodies in vivo, though it has been shown

that the intraventricular administration of NGF, both in the newborn and in the adult, increases the levels of choline acetyltransferase (Gnahn et al., 1983). In addition, recent studies using dissociated septal cultures have demonstrated that NGF can increase the number of surviving cholinergic neurons (Hartikka and Hefti, 1988; Hatanaka et al., 1988). New experimental approaches seem necessary to clarify the exact physiological role of NGF in the CNS; in particular, methods to regulate experimentally the expression of this gene in the CNS must be developed. It seems unlikely that the approach used so successfully in the PNS-the neutralization of endogenous NGF by antibodies-will work satisfactorily in the CNS, due to the poor penetration of injected antibodies into the brain (Springer and Loy, 1985).

NGF: Biosynthesis Much of the protein chemistry done on NGF has used the mouse protein. NGF exists in solution as a homodimer (Bothwell and Shooter, 1977), each monomer consisting of 118 amino acids, with 6 cysteines and 3 disulfide bridges (Angeletti and Bradshaw, 1971). Mouse NGF cDNA clones were first isolated in 1983 by Scott et al. and Ullrich et al. (1983) using cDNA libraries from the adult male mouse submandibular gland and oligonucleotide probes based on the amino acid sequence of NGF (Angeletti and Bradshaw, 1971). The mouse gene spans 45 kb and has several small 5 exons (Selby et al., 1987). At least four different mRNA transcripts can be produced by differential splicing, although the functional significance of these various transcripts is not yet fully understood. Most of the pre-pro NGF coding sequence, including the full sequence of the biologically active, processed NGF, is located in one exon at the 3’ end of the gene. Gene transfer studies in various cell lines have established that the two primary translation products (27 or 34 kd corresponding to the two major NGF mRNA transcripts) give rise to a glycosylated precursor of 35 kd that has little, if any, biological activity (Edwards et al., 1988a, 1988b). Interestingly, a variety of cells capable of processing NGF to its biologically active form were found, indicating that the trypsin-like enzymes necessary for the correct processing of NGF are found in many cells (Edwards et al., 198813). This is in accord with in vivo data indicating that NGF is made by a variety of cells, such as epidermal cells, smooth muscle cells, or hippocampal neurons. It appears that, depending on the tissue or the cell transfected, NGF can be stored in vesicles and released either by a calcium-dependent mechanism or via the’constitutive” pathway (see Barth et al., 1984; Edwards et al., 1988b; Wallace and Partlow, 1976). The primary structure of NGF (deduced from cDNA clones) is now also available for human, bovine, rat, chick, and snake (Naja naja samiensis) NGFs (see Whittemore et al., 1988, for sequences). The comparison of the sequences shows a high degree of conservation among the different species. Only limited information is avail-

Neuron 1528

able on the residues participating in the binding to its receptors, and no data are yet available three-dimensional structureofany NGF. In spite attempts, there has been no successful synthesis tides displaying NCF biological activity. Brain-Derived Brain-derived extremely only from

Neurotrophic

of NGF on the of many of pep-

Factor

neurotrophic factor (BDNF) is a protein of low abundance that has so far been purified pig brain. Over I@-fold purification was

necessary to achieve homogeneity (Barde et al., 1982; Hofer and Barde, 1988). The relative resistance of BDNF’s biological activity toward denaturing agents allowed its molecular weight, 12,300, to be determined by SDS gel electrophoresis. Additionally, BDNF was found to have a pl of about 10; thus it resembles the monomer of NGF in terms of both size and isoelectric point. Although the determination of the primary structure of BDNF has not yet been completed, sequence comparison with NCF reveals a significant number of amino acid identities, indicating that these proteins belong to the same family (Barde and Lottspeich, unpublished data). BDNF induces sprouting from embryonic peripheral sensory ganglia cultured in vitro in a manner that is indistinguishable from that of NGF (Davies et al., 198613). Neurons originating from ectodermal placode5 are also responsive to BDNF (in contrast to NGF), whereas sympathetic and parasympathetic neurons are not (Lindsay et al., 1985; Davies et al., 1986b). When the response of DRG neurons is examined during development, a curious shift in responsiveness is observed. At early stages, a considerable overlap is seen between NCF- and BDNFresponsive neurons (Lindsay et al., 1985; Ernsberger and Rohrer, 1988). This is also the case in vivo, since administration of either NGF or BDNF can prevent naturally occurring neuronal death in the DRG to the same extent (Hofer and Barde, 1988). After EY, however, when the period of naturally occurring cell death is over and presumably all neurons have established stable connections with their respective targets, the neuronal populations supported by either BDNF or NGF seem to be complementary, the effects of NCF and BDNF com-

A

B

bined being additive. Among the primary rons supported by BDNF are large-diameter tive neurons (Davies et al., 1986a). At very early stages, BDNF will support

sensory neupropriocepthe survival

oi

DRG neuron5 In culture only in the presence of laminin (Lindsdy et dl., 19851, as first found for NGF with sympathetic neurons (see Edgar, 1985). In vivo, BDNF and laminin are also both necessary components for the early phase of DRG development (Figure 2). Removal ot the neural tuhr from chick embryos just after the neural crest cells have started their migration results in the disappearance of the cells aggregating to form the DRG anlage, and consequently no DRG are formed (Teillet and Le Douarin, 1983). This finding has led to the hypothesis that neural tub+derived signals might be required by the DRG vtry early in development (Le Douarin. 1986). In support of this, interposition of rl silastic membrane, between the nrural tube and the DRG anlage resulted in the disapprdrdnce of the DRG anlage (Kalcheim and Le Doudrin, 1986). When the hilastic membrane is coated with a neural tube extract, many cells distal to the membrane could bc rescued (Kalcheim and Le Douarin, 1986). The effect of the extract or neuronal survival could be mimicked by c-eating the membrane with laminin and BDNF, hut not with either laminin or BDNF alone (KalL cheim et cil., 1987) (Figurr 2). Lamlnln dnd NGF together were also inactive, a finding explained by the absenct> of NGF rrcrptors at this stage of development (Bernd, 1985). In the DRG, BDNf~ responsivene55 heem to be extremely tightly regulated. In 30.somitr stage embryos, BDNF supports the survival of very large numbers of HNK-1-immunoredctive cells in rostra1 segments, many of which also contain the sensory neuronal marker sub stance l? tHowevc>r, the less mature cells in more caudal segment5 are c-onsiderably less re5ponslve to BDNF (Kalcheim and Gendreau, 1988). Quantitative analyses performed in VIVO in the quail have shown that exogenous BDNF, like NCF, prevents naturally occurring neuronal death in DRGs. d5 demonstrated not only by increased neuronal numbers after the period of neuronal death, but also by reduced numbers of pyknotic neurons (Hofer and Bardr, 1988). BDNF also increases the number ot

C

Review: 1529

Trophic

Factors

and Neuronal

Survival

neurons in the nodose ganglion, whereas NGF out effect (Hofer and Barde, 1988), consistent vitro studies (Lindsay and Rohrer, 1985; Lindsay

is withwith in et al.,

1985). These data on BDNF and primary sensory neurons can be rationalized by assuming that BDNF is synthesized in the CNS target fields of primary sensory neurons and transported back to the cell bodies of BDNFdependent neurons by their central axons (see Lindsay et al., 1985; Davies et al., 1986a). However, information on the site of synthesis of BDNF is still lacking. Retinal ganglion cells are another target for BDNF. The majority of El7 rat ganglion cells can be rescued in vitro by the addition of BDNF (Johnson et al., 1986), without affecting the total number of cells or the total number of neurons (the ganglion cells representing only a small fraction). The effects of BDNF are not limited to the prenatal period: in adult rat retinal explants, BDNF can support the survival of rat retinal ganglion cells and increase their rate of axonal elongation (Thanos et al., 1989). Also, like NGF, BDNF increases the rate of neurite elongation from isolated, adult rat DRG neurons (Lindsay, 1988). In many respects it therefore seems that BDNF belongs to the same category of agents as NGF. It prevents the death of neurons during development, at a time when these embryonic neurons are crucially dependent on interactions with their targets in vivo. As with NGF the action of BDNF seems not to be limited to a role affecting embryonic neurons, since effects can also be observed on adult neurons.

NCF and BDNF Receptors Both NGF and BDNF show strikingly similar binding to neurons. Target neurons display both highand lowaffinity receptors (10-r’ M and 10mg M, respectively), the only difference being that in the case of BDNF, the kinetics of binding to and dissociation from the lowaffinity receptors are considerably slower than those of NGF (Sutter et al., 1979; Rodriguez-Tebar and Barde, 1988). The low-affinity NGF receptor gene has been cloned (Johnson et al., 1986; Radeke et al., 1987), and examination of its deduced amino acid sequence shows that in contrast to that of several receptors mediating mitogenic signals, the cytoplasmic domain of this receptor does not possess an ATP binding site. It is possible that the key to the mechanism of action of NGF (and, by analogy, of BDNF) will be the elucidation of the mechanisms involved in the transition from the low- to the high-affinity state. This could result from the intracellular binding of a cytoplasmic protein and/or from posttranslational modifications. It seems that only one NGF receptor mRNA exists for both types of receptors, since gene transfer into mutated cells (unable to bind NGF) with a construct coding for the low-affinity NGF receptor restores in thesecells both low-and high-affinity NGF binding (Hempstead et al., 1989). At present, it is still unclear how the NGF response is transduced following the binding of NGF to its receptors. One current speculation is that a ras or a ras-like

protein might be involved in the transduction pathway. This was first suggested by experiments of Bar-Sagi and Feramisco (1985): direct microinjections of the Ha-ras ~21 protein into PC12 cells result in the formation of fibers by these cells. In addition, injections of antibodies to ras inhibit the effects of NGF (Hagag et al., 1986). Moreover, introduction into dissociated embryonic neurons of the oncogene protein T24-ras mimics the effects of NGF on survival and fiber outgrowth, as well as those of BDNF, suggesting that, irrespective of the ligand, identical transducing mechanisms might be operating to promote neuronal survival and fiber outgrowth (Borasio et al., 1989).

Fibroblast

Growth

Factor

Basic and acidic fibroblast growth factors (bFGF and aFGF), usually isolated from pituitary and brain, respectively, are proteins of about 16,000 daltons. They have distinct isoelectric points but are structurally related, sharing about 55% amino acid sequence identities (Esch et al., 1985; Gimenez-Gallego et al., 1985). Both have a strong affinity for heparin (Shing et al., 1984), a finding that was of considerable help in their final purification. Both are found in embryonic brain (Risau et al., 1988) and in adult nervous tissues in relatively large amounts (about 500 pg/kg for aFGF and bFGF from brain and pituitary, respectively; Gospodarowicz, 1987), when compared with NGF or BDNF (which are roughly 100.fold less abundant). In normal brain, bFGF is localized primarily in neurons (Pettmann et al., 1986; Finkelstein et al., 1988), although cultured astrocytes also synthesize (mostly basic) FGF (Ferrara et al., 1988; Hatten et al., 1988). Originally, both FGFs were identified as mitogens for fibroblasts and myoblasts (see Gospodarowicz, 1988, for a review). Later, they were found to induce the division of a variety of cells, in particular endothelial cells, and to stimulate both the differentiation and the migration of these cells, suggesting a role for FGFs in the formation of blood vessels (Folkman and Klagsbrun, 1987). In addition, FGFlike molecules are thought to be involved in mesodermal induction in amphibian blastulae (Slack et al., 1987; Kimelman et al., 1988). Recently, a number of reports have indicated that FGF (both acidic and basic in most cases) can promote the in vitro survival of embryonic neurons, including those from the hippocampus (Walicke et al., 1986), the cerebral cortex (Morrison et al., 1986; Walicke, 1988), the striatum, septum, and thalamus of El8 rats (Walicke, 1988), the early postnatal mouse cerebellum (Hatten et al., 1988), and the chick ciliary ganglia and spinal cord (Unsicker et al., 1987). The acidic or basic forms of FGF have also been shown to induce fiber outgrowth from a variety of cells, including PC12 cells (Togari et al., 1983; Rydel and Greene, 1987; Neufeld et al., 1987), newborn rat adrenal chromaffin cells (Stemple et al., 1988; Claude et al., 1988), and postnatal rat retinal ganglion cells (Lipton et al., 1988). Importantly, effects have also been noted on neuronal survival after lesion in vivo. Thus, Sievers et al. (1987)

NellKNl 1530

have shown that the loss of retinal ganglion cells after section of the adult rat optic nerve can be partially prevented by application of FGF to the cut nerve stump. Furthermore, after lesions of the fimbria-fornix, cholinergic neurons can be rescued in the adult rat forebrain by administration of FGF (Anderson et al., 1988). Thus, in the adult, the FGFs might act as general “rescue” factors released by damaged cells after lesioning, eliminating the problem of how they are released by intact cells (see below). The exact role played by the FGFs in the development of the nervous system is more difficult to evaluate. As discussed recently (Barde, 1988), the relatively large amounts of FGF found in the nervous system are not consistent with the idea of limiting amounts of factors regulating naturally occurring neuronal death. However, both FGFs do not behave like typical secretory proteins (see e.g., Blam et al., 1988), and this might restrict their availability. Although the mechanism by which FGFs are released is unclear, there is evidence that FGF cross-reactive material is librated by astrocytes in vitro. bFGF permits the survival and outgrowth of processes of cerebellar granule cells, mimicking the effects of co-cultured astrocytes on these cells (Hatten et al., 1988). Importantly, addition of polyclonal antibodies raised against bFGF prevents fiber extension by the granule cells when co-cultured with astrocytes, though, curiously, survival is not affected (Hatten et al., 1988). This suggests that some, but not all, of the effects observed in astrocyte-granule cell co-cultures may derive from FGF released by astrocytes. An interesting feature of the FGFs is that they apparently act on a wide variety of neurons. In many studies, it has been difficult to assess whether the effects of FGFs on neurons are direct or indirect via cell types known to respond to the FGFs, which include astrocytes and oligodendrocytes (Eccleston and Silberberg, 1985; Pettmann et al., 1985), as well as fibroblasts and endothelial cells. However, with the neuron-enriched fraction obtained from chick ciliary ganglia and the El8 rat hippocampal neurons (in which no correlation between the survival effects of the FGFs and the number of nonneuronal cells present has been found), a direct action of FGF on neurons appears likely (Unsicker et al., 1987; Walicke and Baird, 1988). It is important to note that the commonly used basic and acidic FGFs are, probably, not the only heparin binding mitogens. Evidence is accumulating for the existence of other FGF-like genes, for example the int-2 (Dickson and Peters, 1987), hst/KS3/K-fgf (Yoshida et al., 1987; DelliBovi et al., 1988), and FGF-5 genes (Zhan et al., 1988). The int-2 gene is transcribed during gastrulation and neurulation in mouse tissues, as demonstrated by in situ hybridization (Wilkinson et al., 1988). These gene products may be of particular interest in neural development, since some of them have the sequence characteristics of typical secretory and heparin binding proteins. In fact, K-fgf has already been demonstrated to be glycosylated and secreted by COS cells after cDNA transfection and to interact with heparin (Delli-Bovi et al., 1988). As with acidic and basic FGF, K-fgf and FGF-5 have been shown

to be mitogenic for a able work remains to velopmental and the new FGFs, as well as

variety of cells. Clearly, considerbe done to determine both the detissue-specific expression of these their role during neurogenesis.

Conclusion Work on NGF, BDNF, and FGF has established that these small proteins can influence the survival of vertebrate neurons in \‘ivo. The admlnistration of NGF or BDNF during development, and of NGF or FGF after lesions, can rescue neurons that would otherwise be eliminated. Although the exact role of FGF during the development ofthe nervous bystem is difficult to evaluate, the data obtained with NGF suggest that the target cell synthesizes and releases the factor and thereby participates in the regulation of its own innervation. This model seems also to apply to BDNF and supports the speculation that the survival of yet other neurons is regulated by the limited availability of additional as yet unidentified trophic factors. Among these, the factor thought to regulate the surviva1 of motoneurons during development is of special interest (see Oppenheim dnd Heverkamp, 1988; Henderson, 1988, for reviews), in particular because the nerve-driven activity ofthe skeletal musculature appears to regulate the production of this factor. As mentioned in the introduction, paralysis ofthe neuromuscular junction during development almost completely prevents naturally occurring neuronal death, suggesting that electrical activity might down-regulate the levels of this factor. The activity-dependent expression of this factor and, by analogy, of others could play a considerable role in the regulation of naturally occurring cell death, as well as in collateral retraction during normal development and sprouting after various experimental manipulations. While the contribution of trophic factors to the regulation of neuronal survival is the major topic of this review, there are likely to be other important regulatory mechanisms. Given the integrated nature of the nervous system, trophic mechanisms may be meaningful only when complemented by other control systems. These include the number and appropriateness of the synaptic contacts made by the embryonic neurons, interactions of neurons and their axons with components of the extracellular matrix, direct cell-cell contacts, and hormonal influences. Elucidating these combined regulatory influences and the molecular mechanisms underlying the actions of trophic factors is likely to represent the focus of this field in the coming few years. Acknowledgments I thank Dawd Edgar, Tom Jessell, Werner for their comments and suggestions.

RIUU,

and

tians

Thoenen

References Anderson, K. I., Dam, D., Lee. S, and Cotman. C. W. (1988). fibroblast growth factor prevents death of lesloned cholinergic 10”s in \‘1\1> Nature 332, 360-361. Angelettl,

R

II

Rand Hrad
R A. llYill

Nerve

growth

Baste neufactor

Review: 1531

Trophic

Factors

and Neuronal

from mouse submaxillary gland: Acad. Sci. USA 68, 2417-2420.

Survival

amino

acid sequence.

Proc.

Natl.

Ayer-Lelievre, C., Olson, L., Ebendal, T., Seiger, A., and Persson, H. (1988). Expression of the B-nerve growth factor gene in hippocampal neurons. Science 240, 1339-1441. Bandtlow, C. E., Heumann, R., Schwab, M. E., and Thoenen, H. (1987). Cellular localization of nerve growth factor synthesis by in situ hybridization. EMBO J. 6, 891-899. Barde, Trends

Y.-A. (1988). What, if anything, Neurosci. 1 I, 343-346.

is a neurotrophic

factor?

Barde, Y.-A., Edgar, D., and Thoenen, H. (1982). Purification new neurotrophic factor from mammalian brain. EMBO 549-553. Bar-Sagi, D., and Feramisco, J. R. (1985). oncogene protein into PC12 cells induces ation. Cell 42, 841-848.

Microinjection morphological

j.

of a I,

of the ras differenti-

Barth, E.-M., Korsching, S., and Thoenen, H. (1984). Regulation of nerve growth factor synthesis and release in organ cultures of rat iris. J. Cell Biol. 99, 839-843. Bernd, P (1985). Appearance of nerve growth factor cultured neural crest cells. Dev. Biol. J12, 145-156.

receptors

on

Blam, S. B., Michell, R., Tischer, E., Rubin, J. S., Silva, M., Silver, S., Fiddes, J. C., Abraham, J. A., and Aaronson, S. A. (1988). Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein. Oncogene 3, 129-136. Borasio, C. D., John, J., Wittinghofer, A., Barde, Y.-A., Sendtner, M., and Heumann, R. (1989). ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons. Neuron 2, 10871096. Bothwell, M. A., and Shooter, E. M. (1977). Dissociation equilibrium constant of B-nerve growth factor. J. Biol. Chem. 252, 85328536. Campenot, R. B. (1977). Local control nerve growth factor. Proc. Natl. Acad.

of neurite development Sci. USA 74, 4516-4519.

by

Delli-Bovi, P, Curatola, A. M., Newman, K. M., Sato, Y., Moscatelli, D., Hewick, R. M., Rifkin, D. B., and Basilica, C. (1988). Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential. Mol. Cell, Biol. 8, 2933-2941. Dickson, C., and Peters, related to growth factors.

C. (1987). Potential Nature 326, 883.

oncogene

product

Eccleston, P A., and Silberberg, D. H. (1985). Fibroblast growth factor is a mitogen for oligodendrocytes in vitro. Dev. Brain Res. 27, 315-318. Edgar, D. (1985). tracellular matrix 107-113.

Nerve growth factors and molecules of the exin neuronal development. J. Cell Sci. (Suppl.) 3,

Edwards, R. H., Selby, M. J., Garcia, P D., and Rutter. W. J. (1988a). Processing of the native nerve growth factor precursor to form biologically active nerve growth factor. J. Biol. Chem. 263,6810-6815. Edwards, R. H., Selby, M. J., Mobley, W. C., Weinrich, S. L., Hruby, D. E., and Rutter, W. J. (198813). Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector. Mol. Cell. Biol. 8, 2456-2464. Ernsberger, U., and Rohrer, H. (1988). chick dorsal root ganglia: differentiation Biol. 126, 420-432.

Neuronal precursor cells in and survival in vitro. Dev.

Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen l?, and Guillemin, R. (1985). Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino terminal sequence of bovine brain acidic FGF. Proc. Natl. Acad. Sci. USA 82, 6507-6511. Ferrara, N., Ousley, F., and Gospodarowicz, D. (1988). Bovine astrocytes express basic fibroblast growth factor, a neurotropic angiogenic mitogen. Brain Res. 462, 223-232.

brain and

Finkelstein, S. P, Apostolides, P J., Caday, C. G., Prosser, J., Philips, M. F., and Klagsbrun, M. (1988). Increased basic fibroblast growth factor (bFGF) imunoreactivity at the site of local brain wounds. Brain Res. 460, 253-259.

Campenot, R. B. (1982a). Development of sympathetic compartmentalized cultures. I. Local control of neurite nerve growth factor. Dev. Biol. 93, 1-12.

neurons growth

in by

Folkman, J., and Klagsbrun, 235, 442-447.

Campenot, R. B. (198213). Development of sympathetic compartmentalized cultures. II. Local control of neurite nerve growth factor. Dev. Biol. 93, 13-21.

neurons survival

in by

Gimenez-Gallego, G., Rodkey, J., Bennet, C., Rios-Candelore, M., DiSalvo, J., and Thomas, K. A. (1985). Brain-derived acidic fibroblast growth factor: complete amino acid sequence and homologies. Science 230, 1385-1388.

K. A., D’Amore, t? A., and growth factor stimulates adreto extend neurites, but is not 1, 783-790.

Gnahn, H., Hefti, F., Heumann, R., Schwab, M., and Thoenen, H. (1983). NGF-mediated increase of choline acetyltransferase (ChAT) in the neonatal forebrain: evidence for a physiological role of NGF in the brain. Dev. Brain Res. 9, 45-52.

Claude, t?, Parade, I. M., Gordon, Wagner, J. A. (1988). Acidic fibroblast nal chromaffin cells to proliferate and a long-term survival factor. Neuron

Cohen, S. (1960). Purification of a nerve-growth promoting protein from the mouse salivary gland and its neurocytotoxic antiserum. Proc. Natl. Acad. Sci. USA 46, 302-311. Coughlin, M. D , and Collins, M. B. (1985). Nerve growth independent development of embryonic mouse sympathetic rons in dissociated cell culture. Dev. Biol. 110, 392-401.

factorneu-

Davies, A. M., and Lindsay, R. M. (1985). The cranial sensory ganglia in culture: differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor. Dev. Biol. 1 I I, 62-72. Davies, A. M., and Lumsden, A. C. S. (1984). Relation of target encounter and neuronal death to nerve growth factor responsiveness in the developing mouse trigeminal ganglion. J. Comp. Neurol. 223, 124-137. Davies, A. M., Thoenen, H., and Barde, Y.-A. (1986a). Different factors from the central nervous system and periphery regulate the survival of sensory neurons. Nature 319, 497-499. Davies, A. M., Thoenen, H., and Barde, Y.-A. (1986b). The response of chick sensory neurons to brain-derived neurotrophic factor. J. Neurosci. 6, 1897-1904. Davies, A. M., Bandtlow, C., H., and Thoenen, H. (1987). synthesis in developing skin sion of the receptor. Nature

Heumann, R., Korsching, S., Rohrer, Timing and site of nerve growth factor in relation to innervation and expres326, 353-358.

M. (1987).

Angiogenic

factors.

Science

Goedert, M., Otten, U., Hunt, S. P, Bond, A., Chapman, D., Schlumpf, M., and Lichtensteiger, W. (1984). Biochemical and anatomical effects of antibodies against nerve growth factor on developing rat sensory ganglia. Proc. Natl. Acad. Sci. USA 81, 1580-1584. Gospodarowicz, D. (1987). and basic fibroblast growth

Isolation and characterization of acidic factor. Meth. Enzymol. 147, 107-119.

Gospodarowicz, D. (1988). Molecular and developmental biology aspects of fibroblast growth factor. In Biology of Growth Factors, V. 234, J. E. Kudlow, D. H. Maclennan, A. Bernstein, and A. I. Gotlieb, eds. (New York: Plenum Publishing Corp.), pp. 23-39. Hagag, N., Halegoua, S., and Viola, M. (1986). Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras ~21. Nature 319, 680-682. Hamburger, V., and Levi-Montalcini, R. (1949). Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. Zool. I J 1, 457-502. Hamburger, V, Brunso-Bechtold, 1. K., and Yip, 1. W. (1981). Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J. Neurosci. I, 60-71. Hartikka, neurons of NGF

J., and Hefti, F. (1988). Development of septal cholinergic in culture: plating density and glial cells modulate effects on survival, fiber growth and expression of transmitter-

NellrOll 1532

speck

enzymes.

j

Neurosc~.

8, 2967.2985.

Hatanaka, H., Tsukui, H., and Nlhonmatsu, I. (lY88). Devrlopmt’ntal change in the nerve growth factor action from induction of c holine acetyitransferase to promotion ofcell \urv~vai in cultured hd\al forebrain chollnergic neurons from postnatal rat\. Dev. Br,lln Res. iY, 85-95. Hatten, M. E., Lynch, M., Rydel. R. E., Sanchcxr. I., Joseph-Sllvrrstein, J., Moscatelli, D., and Rifkin, D. B. (1988). In vitro nrurltc’ c’xtension by granule neuron\ 15dependent upon ,I~troglial-drrlv~~rl ilbroblast

growth

factor.

Hefti, F. (1986). septal cholinergic 215.5-2162.

Drv

Biol.

125.

280-289.

Nerve growth factor neuron\ afterfimbrral

(NCFJ promotes \ur~ival transection. 1. Ncuro\c~.

ot 0,

Hempstead, B. L.. SC hlrlter, L. S., and Chao, M. I! 119891. tkl)rt+ sron of functional nerve growth factor receptor\ atter gene tran\ft,r. Science 243, 373-375. Henderson, C. E (1988). The role of must lc II, the drvc~l~~lumc~nt and differentratIon of spinal motoneurons in \jltro \tutile\. In Plasticity of the Neuromuscular System, Ciba Foundation Symposium / 38, D. Evered and I Whelan, edi. (Chic hr5tc.r: W~ley~, pp 172.191. Hendry, I A. (1975). The response of adrenerglc neuron\ otomy and nerve growth tactor. Brain Re\. 9-I 87-97. Heumann, Relatronship

R., Korsching, j., Scott, J., and between level5 of nerve growth

messenger RNA in sympathetic \ues. EMBO J. 3, 318% 3189. tiofrr, factor

M. M., and garde. prevents neuronal

ganglia

and

Thoenen, H factor lNCFi ptxrlpheral

structure

D., Lanahan, E., Bothwell, of the

A.. Buck, C. M , and Chao,

human

NCF

receptor.

Johnson, E. M., Jr. (1978). Destruction system in neonatal rat> and hamsters concomitant admlnrstratlon of nerve 105-l 18.

Gil

J’.

545-554.

to nerve

antibody

growth

and Pearson, by exposure

tartor.

Sc~rnt

J I,,

(2 2 10,

K. M.. and YIP, H. K. (1986). The r& in viva. Trends Neurosc-1. 4, 33- 17

lohn>on. I. E., Barde, Y.-A, Schwab, M., ~ntl Thuenen. Brarn-derrved neurotrophlc factor supports the \urv~val rat retinal ganglion cells. 1. Neurosci. 6, 3033038. IGllcheim, C., trophlc factor cultured avlan

rt’( <‘,I-

of the \ympathetlc norvou\ by vlnbla\tlne: prc’vc’ntlon bv growth factor. Brain Res Iii.

utero to 916-918.

jr., Rich, neuron\

ti\-

R.. SehgJ A., ~Morg<~ll, ( M. 119861 Expre\\~on rind

I’ M., Brandeli. L. [I.. neurons are destroyed

Johnson, E. M., NCF I” sensory

target

01 c utaneou’.

Johrxons, E. M., Jr., Corln. (1980). Dorsal root ganglion maternal

II984). and it\

Y.-A. (1988). Brarn-derlveti nc‘ttrotrophlc death in VIVO. Nature i i I, Lh I Lh.!

Iggo, A., and Andres, K. H. (1982). Morphology tars. Annu Rev. Neurosrl i l-31. Johnson, Mercer,

IO ax-

ot

ti 11986~ ot c ulturcxl

and Gendreau, M. (1988). Br,un-derived ncxurostimulate5 iurvrval and neurona dlfferrntlatlon tn neural crest. Dev. BraIn Re\. -ii. 79-86.

Kalchelm, C., and Le Douarin. N. M. (1986). RequIremrnt ral tube slgnal for the differentiation of neura crest cells root ganglia. Dev. Biol. I fh, 451-466. Kalcheim, C., Barde. Y.-A, Thoenen, 11987). In VIVO effect of br,aln-derived survival of developing dorsal root 2871-2873

ot a ntu into tioru

H , and Lta Douarln. \1 M neurotrophlc factor on the ganglion ceI\. EMBO I, 6

Klmelmdn. D., Abraham, J. A., HaaparantJ, 1 P,~~IsI, T. M., antI Klrschner, M. W. (1988) The presence of fihroblast growth factor in the frog egg: its role as a natural mesoderm ~nduccr. Science 232. 1053~1056. Korschlng, S.. and Thoenen, H. (1983). Nerve growth factor in \yrnpathetic gangha and corresponding target organ5 of the rat: c orreIdtion with density of sympathetic innervation Pror Natl. Acad. Sci USA 80, 3513-3516. Korschlng, S., Auhurger, C Heumann, R., Scott, I., and Thoenen. H. (1985). Levels of nerve growth factordnd Its mRNA in the central nervous system of the rat correlate with chollnerglc innervation. EMBO 1. 4, 1389-1393.

Review: 1533

Trophic

Factors

and Neuronal

Survival

Pettmann, B., Weibel, M., Sensenbrenner, M., and Labourdette, C. (1985). Purification of two astroglial growth factors from bovine

antiserum sprouting.

brain.

Stemple, D. L., Mahanthappa, N. K., and Anderson, D. 1. (1988). Basic FGF induces neuronal differentiation, cell division, and NGF dependence rn chromaffin cells: a sequence of events in sympathetic development. Neuron I, 517-525.

FEBS Lett.

Pettmann,

189,

102-108.

B., Labourdette,

C., Weibel.

(1986). The brain fibroblast Neurosci. Lett. 68, 175-180. Pittman,

growth

R., and Oppenheim,

M., and

factor

Sensenbrenner,

is localized

R. W. (1978).

M.

in neurons.

Neuromuscular

ade increases motoneurone survival during chick embryo. Nature 271. 364-366.

normal

block-

cell death

Straznicky,

in the

Purves, D., Snider, W. D., and Voyvodic. I. T. (1988). Trophic regulation of nerve cell morphology and innervation in the autonomrc nervous system. Nature 336, 123-128.

Raivich, C.. Zimmermann, A., and Sutter, A. (1985). The spatial and temporal pattern of BNGF receptor expression in the developing chick embryo. EM60 J. 4, 637-644.

Risau, growth man

P M., Verge Issa, V. M. K.. and Riopelle, R. J. (1986). of neuronal receptors for nervegrowth factor in the rat. 6. 2312-2321.

W., Gautschi-Sova, factors in embryontc acidic

fibroblast

Rodrrguez-Tebar. tics of brain-derived from

the

chick

P., and Bohlen, P (1988). Endothelial cell and adult chrck brain are related to hu-

growth

factor.

A., and Barde, neurotrophic embryo.

EMBO

J. 7, 959-962.

Y.-A. (1988). Binding factor to its receptors

I. Neurosci.

H.,

Hofer,

M.,

Hellweg,

charactenson neurons

8, 3337-3342.

R., Korsching,

S., Stehle,

A. D.,

Saadat, S.. and Thoenen, H. (1988b3. Antib.odies against mouse nerve growth factor interfere rn viva with the development of avran sensory and sympathetic neurones. Development 103, 545-552. Rydel. growth

R. E., and Greene, L. A. (1987). Acidic and basic fibroblast factors promote stable neurite outgrowth and neuronal dif-

ferentiation

in cultures

of PC112

cells.

J. Neurosci.

7, 3639-3653.

Schwab, M., Otten, U., Agid, V., and Thoenen, H. (1979). Nerve growth factor (NGF) rn the rat CNS: absence of specrfic retrograde axonal transport and tyrosine hydroxylase rnduction in locus coeruleus and substantia nigra. Brain Res. 168, 473-482. Scott, J., Selby, M., Urdea, M., Quiroga, M., Bell, G. I., and Rutter, W. 1. (1983). Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor. Nature 302, 538-540. Selby,

M. J., Edwards,

nerve growth 7. 3057-3064. Shelton, growth vation

factor

R.. Sharp, gene:

F., and

structure

D. L., and Reichardt,

Rutter,

W. J. (1987).

and expressron.

L. F. (1984).

Mol.

Expression

Mouse

Cell.

Brol.

D. L., and

Reichardt,

L. F. (1986).

Studies

on the expression

of the B nerve growth factor (NGF) gene in the central nervous system: level and regional distribution of NGF mRNA suggest that NGF functions as a trophic factor for several distinct populations of neurons. Proc. Natl. Acad. Sci. USA 83, 2714-2718. Shing, Y., Folkman, j., Sullivan, Klagsbrun, M. (1984). Heparm derived capillary endothelial

R., Butterfield, C., Murray, I., and affinity: purification of a tumorcell growth factor. Science 223,

1296-1299. Sievers, 1,. Hausmann, B., Unsicker, K., and Berry, M. (1987). Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neurosci. Lett. 76, 157-162. Slack, J. M. W., Darlington, B. G., Heath, J. K., and Godsave, S. F. (1987). Mesoderm induction in early Xenopus embryo by heparinbinding growth factors. Nature 326, 197-200. Springer,

J. E., and

Loy,

R. (1985).

tntrahippocampal

injections

R. A. (1985). sensory Embryol.

The

effect

of nerve

neurons of the 171, 91-95.

growth

trigeminal

Teillet, M.-A., and Le Douarin, N. M. (1983). Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo. Dev. Biol. 98, 192-211. Thanos, S., Bahr, M., Barde, Y.-A., and Vanselow, J. (1989). Survival and axonal elongation of adult rat retinal ganglion cells: in vrtro effects of lesioned sciatic nerve and brain-derived neurotrophic factor (BDNF). Eur. 1. Neurosci. I, 19-26. Thoenen, H., and Barde, Y.-A. (1980). factor. Physiol. Rev. 60, 1284-1335. Thoenen,

H.,

Bandtlow,

C., and

Physiology

Heumann,

Togari, A., Baker, neurite-promoting Biochem. Biophys.

of nerve

R. (1987).

The

growth physro-

D., Dickens, G., and Guroff, C. (1983). The effect of fibroblast growth factor on PC12 cells. Res. Commun. 114, 1189-1193.

Ullrich, A., Gray, A., Berman, C., and Dull, T. 1. (1983). nerve growth factor gene sequence highly homologous mouse. Nature 303, 821-825.

of

Human to that

Bof

Unsicker, K., Reichert-Preibsch, H., Schmidt, R., Pettmann, B., Labourdette, G., and Sensenbrenner, M. (1987). Astroglial and fibroblast growth factor have neurotrophic functions for cultured peripheral and central nervous system neurons. Proc. Natl. Acad. Sci. USA 84, 5459-5463. Walicke, P A. (1988). Basic and acidic fibroblast growth have trophic effects on neurons from multiple CNS regions. rosci. 8, 2618-2627.

factors J. Neu-

Walicke, P A., and Baird, A. (1988). Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells. Dev. Brain Res. 40, 71-79. Walicke, F!, Cowan, W. M., Ueno, N., Baird, A., and Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. USA 83, 3012-3016. Wallace, L. J., and Partlow, L. M. (1976). secretion of mouse saliva rich in nerve Acad. Sci. USA 73, 4210-4214.

of the B-nerve

factor gene correlates with the density of sympathetic innerin effectororgans. Proc. Natl. Acad. Sci. USA81, 7951-7955.

Shelton,

Rush,

logical function of nerve growth factor in the central nervous system: comparison with the periphery. Rev. Physiol. Biochem. Pharmacol. 109, 145-178.

Rohrer, H., Heumann, R., and Thoenen, H. (1988a). The synthesis of nerve growth factor (NGF) in developing skin is independent of innervation. Dev. 6101. 128, 240-244. Rohrer,

C., and

on developing primary in chick embryos. Anat.

sympathohippocampal

Sutter, A., Riopelle, R. J., Harris-Warrick, R. M., and Shooter, E. M. (1979). Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J. Biol. Chem. 254, 5972-5982.

Radeke, M. I., Misko, T. P., Hsu, C., Herzenberg, L. A., and Shooter, E. M. (1987). Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 32.5, 593-597.

Richardson, Distribution I. Neurosci.

factor nerve

to nerve growth factor inhibit Brain Res. Bull 15, 629-634.

a-Adrenergic regulation of growth factor. Proc. Natl.

Whitington, i? M., Bate, M., Seifert, E., Ridge, K., and Goodman, C. S. (1982). Survival and differentiation of identified embryonic neurons in the absence of their target muscles. Science 215, 973975. Whittemore, S. R., Ebendal, T., LBirkfors, L., Olson, L., Seiger, A., Stromberg, I., and Persson, H. (1986). Developmental and regional expression of B-nerve growth factor messenger RNA and protein in the rat central nervous system. Proc. Natl. Acad. Sci. USA 83, 817-821. Whittemore, S. R., Friedman, P L., Larhammar, D., Persson, H., Gonzalez-Carvajal, M., and Holets, V. R. (1988). Rat B-nerve growth factor sequence and site of synthesis in the adult hippocampus. 1. Neurosci. Res. 20, 403-410. Wilkinson, D. G., Peters, G., Dickson, C., and McMahon, A. D. (1988). Expression of the FGFrelated proto-oncogene int-2 during gastrulation and neurulation in the mouse. EM60 1. 7, 691-695. Williams, L. R., Varon, W., Bjorklund, A., and

S., Peterson, G. M., Wictorin, Gage, F. H. (1986). Continuous

K., Fischer, infusion of

nerve growth factor prevents flmbria fornix transection.

basal forebraIn neuronal death after Proc. Natl. Acad. Sri. USA 83,

9231-9235. Winter, I., Forbe,, C. A., Sternberg. 1.. and Lindsay, R. M 11988). Nerve growth factor (NCFJ regulates adult rat cultured dor\d/ root ganglion neuron responses to the excitotoxrn rapwcjn. Neuron I. 973-981. Yoshida, T.. Mlyagawa, K., Odaglrr, H., Sakamoto. H , LIttIe. P F K.. Terada, M., and Sugimura. T. (1987). Cenomlc sequence of hrt. J transforming gene encoding a protein homologous to irhrohlast growth factors and the Int-l-encoded protein. Proc. N&l. Acad SCI USA 84, 7305-7309. Zhan. FGF-5 growth

X., Bates, B., Hu, X , and Coldfarb, M (19881. The human oncogene encodes a novel protein related to flbrohla,t factors. Mol. Cell. Bio. 8. 3487-3495