Download - Further characterization of the effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on axotomized neonatal and adult mammalian motor neurons

THE JOURNAL OF COMPARATIVE NEUROLOGY 342~45-56 (1994)

Further Characterization of the Effects of Brain-Derived Neurotrophic Factor and

Ciliary Neurotrophic Factor on Axotomized Neonatal and Adult

Mammalian Motor Neurons

RICHARD E. CLATTERBUCK, DONALD L. PRICE, AND VASSILIS E. KOLIATSOS Departments of Pathology, Neurology (D.L.P., V.E.K.), and Neuroscience and the

Neuropathology Laboratory (R.E.C., D.L.P., V.E.K.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196

ABSTRACT Neurotrophins and neural cytokines are two broad classes of neurotrophic factors. It has

been reported that ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) prevent the degeneration of axotomized neonatal motor neurons. In addition, BDNF is transported retrogradely to a-motor neurons following injection into the muscle, and patterns of BDNF expressed in spinal cord and muscle suggest a physiological role for this factor in motor neurons. In the present study, we characterize the effects of BDNF on axotomized neonatal facial motor neurons and extend these observations to adult models of motor neuron injury (axotomy-induced phenotypic injury of lumbar motor neurons). BDNF reduces axotomy- induced degeneration of neonatal neurons by 55% as determined by Nissl staining (percentage of surviving neurons in vehicle-treated cases, 25%; in BDNF-treated cases, 80%). Rescued neurons have an intact organelle structure but appear smaller and slightly chromatolytic on electron microscopic analysis. As demonstrated by intense retrograde labeling with horseradish peroxidase (HRP) applied to the proximal stump of the facial nerve, neurons rescued by BDNF have intact mechanisms of fast axonal transport. CNTF did not appear to have significant effects on neonatal motor neurons, but the lack of efficacy of this factor may be caused by its rapid degradation at the application site. BDNF is not capable of reversing the axotomy-induced reduction in transmitter markers [i.e., the acetylcholine-synthesizing enzyme choline acetyltransferase ( C U T ) or the degrading enzyme acetylcholinesterase (AChE)] in neonatal or adult animals or the axotomy-induced up-regulation of the low-affinity neurotrophin receptor p75NGFR (nerve growth factor receptor) in adult motor neurons. However, BDNF appears to promote the expression of p7ijNGFR in injured neonatal motor neurons. In concert, the findings of the present study suggest that BDNF can significantly prevent cell death in injured motor neurons. However, this neurotrophin may not be a retrograde signal associated with the induction and/or maintenance of some mature features of motor neurons, particularly their transmitter phenotype.

Key words: cell death, cytokines, facial nucleus, nerve growth factor receptor, neurotrophins

D 1994 Wiley-Liss, Inc.

Neurotrophic factors are classified broadly into neurotrophins and neural cytokines. Neurotrophins, a family of peptides with > 60% amino acid homology, are transported retrogradely from the central nervous system or peripheral targets to support the survival and induce or maintain the mature phenotype of certain populations of neurons express- ing specific receptors. Nerve growth factor (NGF) is the prototypical member of this family (Barde et al., 1982; Levi-Montalcini, 1987; Lo, 1992) that includes also brain-

derived neurotrophic factor (BDNF) (Leibrock et al., 1989; Rosenthal et al., 1991), neurotrophin-3 (NT-3; Ernfors et al., 1990; Hohn et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990), and NT-415 (Berkemeier et al.,

Accepted October 12,1993. Address reprint requests to Vassilis E. Koliatsos, MD, Nenropathology

Laboratory, The Johns Hopkins University School of Medicine, 558 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196.

0 1994 WILEY-LISS, INC.

46 R.E. CLATTERBUCK ET AL.

1991; Hallbook et al., 1991; Ip et al., 1992). Neural cytokines are a group of heterogeneous pleiotropic peptides (fibroblast growth factors, interleukins, transforming growth factors-fi, leukemia inhibitory factor, and CNTF), many of which (e.g., fibroblast growth factors) have been known previously for their effects on nonneural cells (Hil- ton, 1992; Unsicker et al., 1992). Neural cytokines and some of their receptors are expressed ubiquitously and may share some of the properties of neurotrophins in preventing neuronal cell death (Sendtner et al., 1990; Clatterbuck et al., 1993) or maintaining phenotype, but their functions are still poorly understood.

Various trophic factors and muscle preparations, puta- tively enriched in the appropriate factors, have been used to promote the survival of motor neurons in vivo and in vitro. Skeletal muscle extracts enhance the survival of motor neurons in dissociated cultures from the embryonic chick and rat and in spinal cord explants (Dohrmann et al., 1986, 1987; Smith et al., 1986; Appel et al., 1989). These effects are augmented when extracts are prepared from denervated muscle (Nurcombe et al., 1984). The neural cytokine CNTF has been shown to prevent axotomy-induced cell death in rat neonatal motor neurons (Sendtner et al., 1990) and developmental cell death in avian motor neurons (Oppen- heim et al., 1991). However, its effects on axotomized neonatal motor neurons have not been replicated (Snider et al., 1992; Unsicker et al., 19921, and its observed effects in a mouse mutant with progressive motor axonopathy have not been characterized adequately (Sendtner et al., 1992a).

Earlier studies on the role of NGF in the normal development and maintenance of motor neurons were inconclusive (Wayne and Heaton, 1988; Yan et al., 1988). Although motor neurons of early chick embryos and neonatal rats were shown to transport NGF from their target fields (Yan et al., 1988), NGF did not prevent developmental motor neuron death in the chick (Oppenheim et al., 1982) or axotomy-induced death of mammalian neonatal motor neurons (Miyata et al., 1986; Yan et a]., 1988). However, very recent investigations employing non-NGF neurotrophins have had a very positive outcome. First, it has been shown that the neurotrophins BDNF and NT-3 are transported to lumbar motor neurons following injections into the dam- aged sciatic nerve of the adult (DiStefano et al., 1992a) and the hindlimb or footpad of the neonatal rat (Yan et al., 1992; Koliatsos et al., 1993). Second, four independent groups have shown that BDNF prevents retrograde cell death of neonatal motor neurons in the facial nucleus and lumbar cord (Yan et al., 1992; Sendtner et al., 199213; Koliatsos et al., 1993) as well as developmental cell death of lumbar motor neurons in the chick (Oppenheim et al., 1992).

The present study was designed to characterize the effects of BDNF on degenerating neonatal motor neurons at the ultrastructural and functional levels and to compare these effects with those of CNTF. We also assessed whether BDNF can prevent axotomy-induced phenotypic injury in adult motor neurons by employing transections of the sciatic nerve. Our findings indicate that BDNF, but not CNTF, prevents retrograde degeneration of motor neurons; motor neurons rescued by BDNF have near-normal ultrastructure and maintain active retrograde transport.

MATERIALS AND METHODS Neonatal facial nerve transection:

Histological studies PO male Sprague-Dawley rats were anesthetized by hypo-

thermia. Using aseptic procedures, the left facial nerve,

including its posterior auricular branch, was transected 1-1.5 mm distal to the stylomastoid foramen. A small piece of gelfoam (2 x 2 mm), immersed in human recombinant BDNF (10-20 pg, n = 5) or human recombinant CNTF (10-20 pg, n = 5) in phosphate-buffered saline (PBS), was apposed to the proximal stump, and the overlying skin was sutured with 5-0 silk. Control pups were treated with vehicle solution (n = 3). Wounds were inspected daily for signs of dehiscence or infection.

One week after surgery, animals were deeply anesthetized with chloral hydrate (400 mg/kg) and perfused transcardially with 0.1 M PBS (1-2 minute flush), followed by 100-200 ml of 4% freshly depolymerized paraformaldehyde. The brains were removed, blocked, and postfixed in 4% paraformaldehyde for at least 48 hours. Blocks containing the caudal pons/medulla were dehydrated in graded alcohols and embedded in paraffin. Serial coronal sections (7 pm) were stained with cresyl violet.

Three sections through the facial nucleus of each animal were taken for neuronal counts (BDNF and CNTF, five cases each; vehicle, three cases). One section corresponded to the middle of the facial nucleus, and the two other sections corresponded to planes at equal anterior and posterior distances from the middle level on each side of the brain. Nucleolated profiles belonging to a-motor neurons were counted at x 40 magnification, using a computerized image analysis system; counts were corrected for split cells with the Abercrombie formula (Abercrombie, 1946). Cell numbers from the lesioned facial nuclei were expressed as percentages of cell numbers in the contralateral unlesioned nuclei. For statistical evaluation of the effects of various treatments on axotomized motor neurons, an analysis of variance was computed by using the average percentage value for each of the three groups. The Newman-Keuls multiple range posthoc test was used to test for simple effects.

Ultrastructural studies For electron microscopic analysis of axotomized, BDNF-

treated neurons of the neonatal facial nucleus, perfusion fixation followed a protocol adapted for developing neural tissues (n = 3). Blood was cleared with a brief rinse with PBS (30 seconds, 37°C) that was bubbled with a mixture of 02/C02 (95%:5%) for 30 minutes prior to the perfusion. Perfusion fixation was performed using a neutral-buffered solution of 1% paraformaldehyde and 1% glutaraldehyde for 5-8 minutes (37"C), followed by 4% glutaraldehyde for 20 minutes (4°C). Brains were removed and postfixed in the former fixative overnight (4°C). Small tissue slabs at the pontomedullary junction were subdissected from brainstem blocks, stained with 2% osmium tetroxide for 1 hour, and dehydrated in graded alcohols. Blocks were then embedded in an Araldite-based plastic for thin sectioning. Thin sections counterstained with uranyl acetateilead citrate were studied with a Hitachi 600 electron microscope.

Studies of axonal transport Animals were treated with BDNF (n = 3) or vehicle (n =

31, as described above (under Neonatal Facial Nerve Tran- section). On day 6, gelfoam pieces immersed in BDNF or vehicle were replaced by identical pieces immersed in 10% horseradish peroxidase (HRPI-wheat germ agglutinin, following visualization of the nerve stump. On day 7, animals were perfused essentially as in the histology group (see above), with 3% neutral-buffered paraformaldehyde. Brains were postfixed overnight in 2% neutral-buffered paraform-

BDNF EFFECTS ON MOTOR NEURONS 47

aldehyde, immersed for 24 hours in 20% glycerol in 0.1 M phosphate buffer, frozen in isopentane, and sectioned on a sliding microtome (40 pm). Sections through the pons/ medulla were processed in series for cresyl violet and HRP histochemistry, using the tetramethylbenzidine (TMB) pro- cedure according to standard protocols (Mesulam, 1982).

CNTF bioactivity studies Gelfoam pieces containing 10 pg CNTF (n = 26) or PBS

(n = 2) were implanted subcutaneously in the postauricular region in PO male Sprague-Dawley rats. These gelfoams were recovered at days 1 (n = 6), 3 (n = 6 ) , and 7 (n = 6) postimplantation frozen on dry ice, and stored at -70°C. Some gelfoam pieces were immersed in CNTF (n = 8), and all gelfoams containing PBS were frozen immediately following impregnation with the appropriate solution. Subse- quently, gelfoams were thawed and incubated in culture media for 2-3 hours. Media were then assayed for the ability to promote ciliary ganglion neuron survival, as described previously (Gupta et al., 1992).

Phenotypic studies in neonatal and adult animals: Immunocytochemistry for ChAT and

p75NGFR and histochemistry for AChE PO pups were treated with BDNF (n = 2) or vehicle (n =

21, as reported above. One week later, animals were perfused transcardially with 3% paraformaldehyde (see above). Tissue blocks that contained the pontomedullary region were postfixed overnight in 2% paraformaldehyde and then immersed in 20% glycerol in 0.1 M phosphate buffer for 24 hours and frozen in isopentane. Transverse sections were cut on a sliding microtome (40 pm) and processed in series for Nissl, AChE histochemistry, and p75NGFR immunocytochemistry using the monoclonal antibody 192-IgG.

Six adult male Sprague-Dawley rats (250-350 g) were subjected to a unilateral transection of the left sciatic nerve at a level corresponding to the middle of the sacroiliac joint ( - 50 mm from the origin of motor axons) in the L4 and L5 spinal segments, as reported previously (Koliatsos et al., 1991a). Immediately following the transection, the catheter of a ventricular access device (Model 44100; Connell Neuro- surgical, Exton, PA) was lowered at the transection through a passage between the sacral and iliac bones constructed by removing a small piece of bone at symmetrical points across the sacroiliac joint. The reservoir was placed on the superfi- cial fascia of the gluteus maximus, and the reservoir/ cannula was secured in place with sutures that passed through the flange of the reservoir and the fascia; other sutures were used to affix the genu of the cannula to the muscle. The gluteus medius and maximus were sutured in layers with Dexon, and the skin was sutured with silk (vehicle, n = 3; BDNF, n = 3). Subjects treated with BDNF were injected immediately after surgery and then every second day for a total of four applications with 10 pg of BDNF in 300 pl of acidified PBS, followed by washing of the reservoir with an additional 300 p1 of PBS. Controls were treated with 300 p1 of acidified PBS. On the day following the last treatment, all animals were perfused with 3% paraformaldehyde, and L4-L5 tissue blocks were frozen and processed in series for Nissl, C U T , and ~ 7 5 ~ ~ ~ ~ . ChAT and p75NGFR immunocytochemistries were performed on floating sections. Sections were incubated for 30 minutes in 0.4% Triton-X 100 in 0.05 M Tris-buffered saline (TBS); for 1 hour in 3% normal goat serum (NGS), including 0.1% Triton in TBS; and for 48 hours in primary antibodies (the monoclonal antibody 192-IgG for p75NGFR and a commer-

TABLE 1. Effects of Trophic Factors on Numbers of Facial Motor Neurons

Average number of neurons in facial Treatment nucleus ir S.E.M. as percentage of aouu n contralateral. unlesioned side

BDNF 5 CNTF 5 Vehicle 3

79.02 2 5.85* 30.38 ? 3.83 24.14 i 2.44

*One-way ANOVA revealed a significant variance among the three groups [F(2,10) = 39.82, P < 0.0011. A Newman-Keuls multiple range test found the BDNF group to differ significantly (P < 0.05) from the other two groups. There were no other significant differences between groups.

cially available monoclonal antibody against ChAT; Boeh- ringer-Mannheim, Indianapolis, IN). The 192-IgG was used at a concentration of 1:100, and the ChAT antibody was used at 1:150, both in a TBS solution containing 0.1% Triton and 2% NGS. After rinsing in TBS, sections were incubated with affinity-purified goat antimouse IgG (Cap- pell, West Chester, PA) at 1 : l O O concentration in a TBS solution containing 0.1% Triton-X and 2% NGS for 1 hour and rinsed again. Sections were then incubated for 1 hour in a mouse monoclonal peroxidase-antiperoxidase (Stern- berger-Meyer, Baltimore, MD) 1:200 and rinsed for a final time. Sections were developed for 8 minutes in a 0.5 mgiml solution of diaminobenzidine containing 100 p1 of 1% hydrogen peroxide per 10 ml of diaminobenzidine solution.

RESULTS Effects of BDNF on degenerating neonatal

motor neurons: Light and electron microscopy One week postaxotomy, there was a 75% loss of motor

neurons in the facial nucleus ipsilateral to lesions in vehicle-treated animals. In CNTF-treated animals, there was a trend for higher numbers of surviving motor neurons than in vehicle-treated animals, but this trend did not reach statistical significance (Table 1). In view of the fact that retained CNTF bioactivity could be consistently recovered only at day 1 postimplantation (data not shown), the absence of a biological effect of CNTF on axotomized neonatal facial motor neurons may be caused by the rapid degradation of this factor at the delivery site. In BDNF- treated animals, cell death in the axotomized facial nucleus was reduced to - 20% (Table 1). The Nissl pattern appeared normal in most rescued neurons, but some cells showed mild chromatolytic changes. Neuronal size was slightly smaller than on the control side (Fig. 1).

At the ultrastructural level, BDNF-treated facial motor neurons had overall a normal appearance. However, nuclei demonstrated some degree of eccentricity and had indentations in many cells. Many nucleoli of BDNF-treated, axotomized facial motor neurons showed prominent coiled bodies (Fig. 2). Some dissociation of ribosomes and endoplasmic cisternae was observed, leading to a slight increase in free polysomes. Cisternae of the rough endoplasmic reticulum (RER) tended to be stacked in parallel arrays, but, in some neurons, parallel orientation was disrupted. Although, in some cells, the RER was clustered in multiple locations throughout the cytoplasm (Nissl bodies), clustering was not clear in many cells, and there was a tendency for peri- nuclear or submembranous RER accumulation (Fig. 2). Reactive microglia with phagosomes in their cytoplasm were abundant in the facial nucleus of vehicle-treated animals but were observed only occasionally in BDNF- treated subjects.


Fig. 1. Light photomicrographs of cresyl violet-stained sections through the facial nucleus of neonatal rats that underwent facial axotomy at PO followed by trophic factor treatment. E-H represent magnifications of areas indicated by curved arrows in A-D. A,E: Motor neurons in a facial nucleus contralateral to axotomy (control). B,F: Remaining motor neurons in an axotomized facial nucleus treated with

vehicle solution. C , G Remaining motor neurons in an axotomized facial nucleus treated with ciliary neurotrophic factor (CNTF). D,H Rescued motor neurons in an axotomized facial nucleus treated with brain-derived neurotrophic factor (BDNFiScale bars = 100 pm in A-D, 50 Fm in E-H.

BDNFEFFECTSONMOTORNEURONS 49

Fig. 2. Electron micrograph of a representative motor neuron from an axotomized facial nucleus treated with BDNF. Note the somewhat eccentric position of the nucleus and the prominent indentations (arrowheads) but the good preservation of cell organelles and rough endoplasmic reticulum (RER). The framed area containing a submembranous Nissl body is magnified in inset B. Note the parallel organiza- tion of RER stacks and the adherence of ribosomes (arrowheads); some

free polyribosomes are also present (arrows). In inset A, a control motor neuron from an intact facial nucleus has been photographed for comparison with the BDNF-treated axotomized facial motor neuron. Asterisks mark the prominent Nissl bodies. mac, fragment of an activated microglial cell displaying phagocytic activity; oli, perineuronal oligodendrocyte. Main panel and inset A, X2,500; inset B, X 15,000.


Fig. 3. Light- and darkfield photomicrographs of BDNF-treated (A-C) and vehicle-treated (D-F) neonatal facial motor neurons following application of horseradish peroxidase (HRP) to the proximal stump of the transected facial nerve. A Cresyl violet-stained facial nucleus demonstrates the rescue of axotomized neonatal facial motor neurons by BDNF in a section adjacent to that processed for tetramethylbenzidine (TMB1 histochemistry. B: In this darkfield micrograph, the majority of rescued motor neurons are shown to transport HRP retrogradely. C: Darkfield micrograph through the level of the genu of

Motor neurons rescued by BDNF demonstrated normal retrograde transport, as shown by the excellent visualization of TMB reaction product in their cell bodies following HRP application to the proximal nerve stump at day 6 following axotomy (Fig. 3). In vehicle-treated animals, only a few facial motor neurons were found to contain HRP reaction product under the same conditions; the pattern of retrograde labeling was apparently associated with the small number of facial motor neurons that survived after facial nerve transection.

the facial nerve from a BDNF-treated animal shows that most motor axons are filled with HRP. D: Cresyl violet-stained facial nucleus from a vehicle-treated animal. E: Darkfield micrograph of a section adjacent to that in D processed for TMB histochemistry shows reaction product in perikarya of a few surviving facial motor neurons. F: Darkfield micrograph through the level of the genu of the facial nerve from a vehicle-treated animal shows the extremely poor labeling of the facial nerve with HRP because of the degeneration of the vast majority of motor axons. Scale bars = 100 pm.

'

Effects of BDNF on phenotype of injured neonatal and adult motor neurons

In normal neonatal rats (PO-P7), motor neurons of the facial nucleus express both AChE and ~ 7 5 ~ ~ ~ ~ . Following facial nerve axotomy, motor neurons show dramatic reduc- tions in both AChE and p75NGFR immunoreactivity, an alteration consistent with the retrograde death of a majority of motor neurons. Treatment with BDNF restored

BDNF EFFECTS ON MOTOR NEURONS 51

Fig. 4. Light micrographs of sections through the facial nucleus of P7 rats following facial nerve transection at PO and either vehicle (A,B) or BDNF (C,D) treatment. A and C represent sections processed for acetylcholinesterase (AChE) histochemistry, whereas sections in B and D have been stained with p7EiNGFR immunocytochemistry. Asterisks indicate the control facial nucleus (contralateral to the transection

side). Arrowheads mark the position of the axotomized facial nucleus. In the vehicle-treated animal, note the profound reduction in both AChE histochemical activity (A) and p75NGFR immunoreactivity (B) in the axotomized facial nucleus. Following BDNF treatment, p7!jNGFR immunoreactivity is restored in rescued neurons (D); however, there are no effects on AChE staining (C). Scale bars = 500 km.

p75NGFR in a large number of facial motor neurons but did not have an apparent effect on AChE (Fig. 4).

In vehicle-treated adult animals with L4-L5 nerve axotomies, reactive gliosis was observed in the ventral horn on the side of the lesion at 1 week postaxotomy. Levels of ChAT immunoreactivity in motor neurons were decreased, whereas many motor neurons appeared to be immunoreactive for p75NGFR (Fig. 5). Treatment with BDNF had no apparent effect on any of the three responses described above (Fig. 6).

DISCUSSION The results of the present study indicate that BDNF

prevents experimental degeneration of motor neurons in vivo. The neural cytokine CNTF does not demonstrate statistically significant effects under the same experimental conditions, Except for slight chromatolytic changes, motor neurons rescued by BDNF have near normal morphology and exhibit fast axonal transport. Despite its effects on the survival of degenerating motor neurons, BDNF cannot reverse the phenotypic alterations that occur as part of the axonal reaction of adult motor neurons.

Axotomy paradigms involving neonatal motor neurons represent the best available model to study in vivo effects of

trophic factors on motor neuron survival (Kreutzberg, 1986; Snider et al., 1992). Neonatal motor neurons are extremely vulnerable to this type of injury, which induces retrograde cell death in a large number of motor neurons in the brainstem and spinal cord (Snider et al., 1992). This axotomy-induced cell death is reduced significantly when axotomies are performed in the second postnatal week and becomes undetectable when lesions are performed in 1-month-old animals (Schmalbruch, 1984; Snider and Thanedar, 1989).

In the facial nucleus of animals treated with BDNF, the morphology of motor neurons is characterized by some degree of disassociation of ribosomes from the RER cisternae, a phenomenon compatible with slight chromatolysis (Barron et al., 1967; Lieberman, 1971; Price and Porter, 1972; Johnson and Sears, 1989). The size of motor neurons rescued by BDNF is also smaller than normal. In addition, AChE is reduced greatly in axotomized BDNF-treated motor neurons. The persistence of slight chromatolysis, reduction in size, and decrease in the expression of AChE in BDNF-treated animals are consistent with the view that, although BDNF rescues these neurons from retrograde degeneration, it does not interfere with the mechanisms of axonal reaction in neonatal motor neurons. It is interesting, however, that in this context BDNF promotes the expres-


Figui

Figs. 5, 6. Nomarski photomicrographs of Nissl and immunocyto- chemical preparations from the ventral horn of L5 of adult animals following sciatic transections and vehicle treatment (Fig. 5) or sciatic transection and treatment with BDNF (Fig. 6). Upper panels represent sections stained with cresyl violet, middle panels are from sections immunostained with choline acetyltransferase (ChAT), and bottom panels represent ~ 7 5 ~ ~ ~ ~ - i m m u n o s t a i n e d sections. Left panels represent the ventral horn contralateral to the side of the transections, whereas right panels are photographs of axotomized motor neurons (asterisks). A: Axotomized motor neurons appear with near-normal basophilia after both vehicle (Fig. 5A) and BDNF (Fig. 6A) treatment.

-e 5

Reactive gliosis, evident by numerous microglial nuclei, is present in both cases. B: ChAT immunocytochemistry reveals marked reduction in cholinergic markers in axotomized motor neurons in the vehicle- treated animal (Fig. 5B). This reduction is not prevented by treatment with BDNF (Fig. 6B). C: Immunocytochemistry for p7ijNGFR shows that axotomized motor neurons are induced to express high levels of p75NCFR, which are normally not present in adult motor neurons (Fig. 5C). BDNF does not induce a reduction of p7FjNGFR expression in axotomized motor neurons (Fig. 6C); indeed, BDNF-treated motor neurons appear to be more intensely immunoreactive for p75NGFR. Scale bars = 100 km.

BDNFEFFECTSONMOTORNEURONS

Figure 6

53

sion of p7EiNGFR. This apparent effect of BDNF on neonatal motor neurons may be caused either by the persistence of a developmentally appropriate phenotype in surviving neurons (“passive” phenomenon) or by a specific up-regulation of p7EiNGFR by BDNF (“active” phenomenon). The p7EiNGFR is expressed normally in motor neurons during the first postnatal week (Yan and Johnson, 1988; Ernfors et al., 1989), but it is unknown whether it is down-regulated by axotomy, because this type of injury results in retrograde

cell death. On the other hand, as has been indicated primarily by experiments utilizing NGF, p7EiNGFR mRNA is up-regulated by neurotrophins up to fourfold (Cavicchioli et al., 1989). BDNF appears to have similar effects on p7EjNGFR expression in neurons of the central nervous system, albeit not as intense as those of NGF (V.E. Koliat- sos, personal observation).

The responses of adult motor neurons to axotomy are quite different from those of neonatal motor neurons.


Although adult motor neurons exhibit characteristic phenotypic changes following axotomy, they do not degenerate (Lowrie and Vrbova, 1992; Snider et al., 1992). These characteristic retrograde changes include down-regulation of the expression of ChAT and up-regulation of the expression of p75NGFR (Ernfors et al., 1989; Wood et al., 1990; Armstrong et al., 1991; Koliatsos et al., 1991a). Our data suggest that the delivery of BDNF to transected axons of the sciatic nerve does not affect the previous responses. The lack of any effect of BDNF on axotomy-induced phenotypic changes in adult motor neurons is consistent with the absence of effects on axotomy-induced reduction in cholinergic markers in neonatal motor neurons. Both findings suggest that BDNF does not interfere with the mechanisms of axon reaction in motor neurons. Although the molecular mechanisms involved in the axon reaction are poorly understood, the timing of the reappearance of cholinergic markers on motor neurons and the down-regulation of p75NGFR coincide with the reinnervation of target muscles by adult motor neurons following crush injury (Koliatsos et al., 1991a). These observations suggest that target-derived signals may be involved in the elaboration of the responses of motor neurons to axotomy. Based on our present findings, it is likely that these target-derived signals are distinct from BDNF. The effects of BDNF on degenerating adult motor neurons should await the development of appropriate experimental models of death of adult motor neurons. Observations from our laboratory suggest that transection of motor axons proximal to cell bodies in the lumbar spinal cord induces rapid retrograde degeneration of motor neurons 1-2 weeks posttransection (Koliatsos et al., 1994). These lesions (proximal ventral rhizotomies) may be useful in evaluating the usefulness of BDNF in adult motor neuron degeneration, including motor neuron disease.

The physiological relevance of BDNF as a trophic factor for motor neurons is supported further by recent findings from other lines of investigation (Koliatsos et al., 1993). First, BDNF is expressed in the local environment of motor neurons throughout their postnatal life. In targets of motor neurons (hindlimb muscle), BDNF expression is main- tained throughout adult life. Significantly, BDNF expression in muscle is up-regulated by denervation. The axotomy- induced increase in BDNF expression in muscle represents a pattern common in retrogradely transported trophic factors (Gasser et al., 1986) and is consistent with the enhanced trophic efficacy of denervated muscle on spinal cord neurons (Nurcombe et al., 1984). On the other hand, the local expression of BDNF in the ventral horn may explain why separation of adult motor neurons from their muscle targets following axotomy does not result in cell death. The spinal expression of BDNF may also explain why spinal cord extracts added to muscle extracts augment the trophic effects of the latter on cultured motor neurons (Dohrmann et al., 1986, 1987; Smith et al., 1986; Appel et al., 1989).

Second, a-motor neurons express trkB, a receptor involved in BDNF signal transduction and already considered identical to the high-affinity receptor for BDNF (Klein et al., 1991; Soppet et al., 1991; Squinto et al., 1991; Meakin and Shooter, 1992). The expression of trkB in motor neurons of the brainstem and spinal cord is present throughout their postnatal life. Although in situ hybridization experiments using full-length trkB riboprobes may detect

noncatalytic isoforms of trkB that lack the tyrosine kinase domain, recent observations from our group suggest that full-length products of trkB (p14PkB) as well as a very abundant truncated isoform ( ~ 9 5 ~ ' ~ ~ ) are both expressed in adult mammalian ventral horn (M.D. Ehlers, D. Kaplan, and V.E. Koliatsos, personal observations).

Third, BDNF is transported rather selectively to motor neurons from skeletal muscles (Yan et al., 1992; Koliatsos et al., 1993). The retrograde transport of other neurotrophins, such as NGF or NT-3, is not consistent or has not been characterized adequately. [12511NT-3, although transported to ventral horn following muscle injection, accumulates in areas not clearly associated with the cell bodies of a-motor neurons (DiStefano et al., 1992a; Koliatsos et al., 1993). Although some investigators have seen intense retrograde labeling of neonatal motor neurons with [lz5I1NGF, others have not seen consistent labeling under the same conditions (Koliatsos et al., 1993) or have failed to see retrograde transport following injections of [1251]NGF into the sciatic nerve (DiStefano et al., 1992a). Because the retrograde transport of neurotrophins appears to require high-affinity binding-a condition also sufficient for the mediation of the biological effects of neurotrophins in vivo (Koliatsos and Price, 1993)-it can serve as an independent marker of responsivity of motor neurons to BDNF. For example, the transport of [1251]NGF to specific populations of neurons in the basal forebrain predicted novel in vivo effects of NGF in the nervous system (Schwab et al., 1979; Hefti, 1986). On the other hand, the consistency and intensity of the retrograde transport of [1251]BDNF as compared to that of iodinated L1251]NT-3 and [1z511NGF point towards a selective role of BDNF in postnatal motor neurons.

Significantly, a subset of motor neurons that corresponds to the sexually dimorphic motor neuron groups of the lumbar sacral cord express the gene encoding p75NGFR (Koliatsos et al., 1991b). These neurons represent the animal homologue of Onufs nucleus (Onuf, 1899). Al- though p75NGFR is a low-affinity neurotrophin receptor, cotransfection of the gene encoding p75NGFR and trkA in COS cells generates NGF receptors with higher affinity than transfection with either p75NGFR or trkA alone (Hemp- stead et al., 1991). In addition, the small cytoplasmic domain of p75NGFR can activate signaling mechanisms that lead to the differentiation of PC12 cells (Yan et al., 1991). Therefore, BDNF signal transduction may be more efficient in sexually dimorphic motor neurons that express both trkB and p75NGFR, a condition that may explain why these neurons are spared in several types of spinal cord degeneration (Mannen et al., 1977, 1982; Iwata and Hirano, 1978). In addition, the up-regulation of p75NGFR in motor neurons following injury (Ernfors et al., 1989; Koliatsos et al., 1991a) may enhance the binding of BDNF expressed (and perhaps released) in the ventral horn of injured motor neurons, a function that may facilitate BDNF transduction via ~ 1 4 5 ~ ' ~ ~ or combined p145trkB-p75NGFR (Meakin and Shooter, 1992).

The neural cytokine CNTF has been reported to have effects on degenerating motor neurons in a variety of settings. CNTF purified from peripheral nerve has been shown to prevent cell death in axotomized facial motor neurons of neonates (Sendtner et al., 19901, whereas recombinant CNTF has been shown to ameliorate the developmental cell death of motor neurons in the chick (Oppenheim et al., 1991). Moreover, although somewhat controversial, CNTF delivered via a CNTF-transfected

BDNFEFFECTSONMOTORNEURONS 55

tumor cell line (to overcome the peptide’s short half-life in vivo) was reported to ameliorate the dying-back motor neuronopathy that occurs in the pmn mutant (Schmal- bruch et al., 1991; Sendtner et al., 1992a). Clearly, our findings as well as those of others (Snider et al., 1992; Unsicker et al., 1992) are not consistent with the effects of CNTF seen by Sendtner et al. (1992a) in degenerating facial motor neurons. An apparent reason may be the difference in the source of the cytokine [in our case, as well as in the case of other investigations, recombinant CNTF was used, whereas Sendtner et al. (1990) utilized CNTF purified from sciatic nerve]. For instance, although the in vivo stability of purified CNTF is not known, recombinant CNTF is not stable in vivo; we were unable to recover bioactive CNTF from CNTF-impregnated gelfoams after 1 day of implanta- tion. The differential retrograde transport of the native vs. recombinant CNTF may also play a role, but several published studies have failed to demonstrate the retrograde transport of either form of this neural cytokine (Smet et al., 1991; Gupta et al., 1992). It is interesting to note that recent findings suggest that the presence of CNTFRa, the binding peptide for CNTF, may augment the efficiency of the retrograde transport of this factor (Curtis et al., 1992; DiStefano et al., 1992b). In any event, an explanation for the discrepancy between our study and that of Sendtner et al. (1990) requires further experimentation.

The significant effects of BDNF on motor neurons have encouraged the consideration of this neurotrophin for treating human degenerative diseases that involve motor neurons, including motor neuropathies and amyotrophic lateral sclerosis. A first step would be to test the effects of BDNF as a therapeutic agent in animal diseases involving motor neurons, such as hereditary canine spinal muscular atrophy and certain mouse mutants (e.g., the pmnlpmn mouse; Cork et al., 1979; Schmalbruch et al., 1991). However, as was shown in the present study, some of the axotomy-induced changes in motor neurons may not be influenced by BDNF. Therefore, basic research aimed at the elucidation of the molecular mechanisms of the axon reaction or additional peptides that would be more efficient in regulating the transmitter phenotype of motor neurons should be encouraged.

ACKNOWLEDGMENTS Dr. Peter Richardson (Department of Neurosurgery,

Montreal General Hospital, Montreal, Quebec, Canada) assayed the CNTF-impregnated gelfoams. Dr. Gene Burton (Genentech, Inc., South San Francisco, CA) provided the human recombinant BDNF, and Dr. Barbara Cordell (Scios- Nova Pharmaceuticals, Baltimore, MD) provided recombinant human CNTF. Ms. Gloria Cristostomo, Ms. Venera- cion Nehus, Ms. Marilyn Peper, Ms. Vicky Gonzales, and Ms. Eleanor Brown assisted in the histological preparations of the facial axotomy experiment. Ms. Catherine Fleisch- man performed the quantitative analysis, and Mr. Frank Barksdale assisted with the electron microscopy and photog- raphy. This work was supported by grants from the U.S. Public Health Service (NS 20471, AG 05146, NS 07179) as well as the American Health Assistance Foundation and the Metropolitan Life Foundation. V.E.K. and D.L.P. are the recipients of a Leadership and Excellence in Alzheimer’s Disease (LEAD) award (AG 07914). D.L.P. is the recipient of a Javits Neuroscience Investigator Award (NS 10580).

LITERATURE CITED Abercrombie, M. (1946) Estimation of nuclear population from microtome

sections. Anat. Rec. Y4t239-247. Appel, S.H., J.L. McManaman, R. Oppenheim, L. Haverkamp, and K. Vaca

(1989) Muscle-derived trophic factors influencing cholinergic neurons in vitro and in vivo. Progr. Brain Res. 79251-256.

Armstrong, D.M., R. Brady, L.B. Hersh, R.C. Hayes, and R.G. Wiley (1991) Expression of choline acetyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury. J. Comp. Neu- rol. 304:596-607.

Barde, Y.-A., D. Edgar, and H. Thoenen (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J. 1549-553.

Barron, K.D., P.F. Doolin, and J.B. Oldershaw (1967) Ultrastructural observations on retrograde atrophy of lateral geniculate body. 1. Neuro- nal alterations. J. Neuropathol. Exp. Neurol. 26:300-326.

Berkemeier, L.R., J.W. Winslow, D.R. Kaplan, K. Nikolics, D.V. Goeddel, and A. Rosenthal (1991) Neurotrophin-5: A novel neurotrophic factor that activates trk and trkB. Neuron 7:857-866.

Cavicchioli, L., T.P. Flanigan, G. Vantini, M. Fusco, P. Polato, G, Toffano, F.S. Walsh, and A. Leon (1989) NGF amplifies expression of NGF receptor messenger RNA in forebrain cholinergic neurons of rats. Eur. J. Neurosci. 1:258-262.

Clatterbuck, R.E., D.L. Price, and V.E. Koliatsos (1993) Ciliary neurotrophic factor prevents retrograde neuronal death in the adult central nervous system. Proc. Natl. Acad. Sci. USA 90:2222-2226.

Cork, L.C., J.W. Griffin, J.F. Munnell, M.D. Lorenz, R.J. Adams, and D.L. Price (1979) Hereditary canine spinal muscular atrophy. 3. Neuropathol. Exp. Neurol. 38209-221.

Curtis, R., Y. Zhu, R.M. Lindsay, and P.S. DiStefano (1992) Axonal retrograde transport of CNTF: Modulation by prior nerve injury and soluble CNTF receptor. SOC. Neurosci. Abstr. 183517.

DiStefano, P.S., B. Friedman, C. Radziejewski, C. Alexander, P. Boland, C.M. Schick, R.M. Lindsay, and S.J. Wiegand (1992a) The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron 8:983-993.

DiStefano, P.S., R.M. Lindsay, and R. Curtis (199213) Axonal retrograde transport of CNTF and leukemia inhibitory factor (LIF) share a common receptor mechanism. Soc. Neurosci. Abstr. 18:617.

Dohrmann, U., D. Edgar, M. Sendtner, and H. Thoenen (1986) Muscle- derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture. Dev. Biol. 118r209- 221.

Dohrmann, U., D. Edgar, and H. Thoenen (1987) Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons. Dev. Biol. 124t145-152.

Ernfors, P., A. Henschen, L. Olson, and H. Persson (1989) Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons. Neuron 2:1605- 1613.

Ernfors, P., C.F. Ibez, T. Ebendal, L. Olson, and H. Persson (1990) Molecular cloning and neurotrophic activities of a protein with struc- tural similarities to nerve growth factor: Developmental and topographi- cal expression in the brain. Proc. Natl. Acad. Sci. USA 87:5454-5458.

Gasser, U.E., G. Weskamp, U. Otten, and A.R. Dravid (1986) Time course of the elevation of nerve growth factor (NGF) content in the hippocampus and septum following lesions of the septohippocampal pathway in rats. Brain Res. 376351-356.

Gupta, S.K., M. Altares, R. Benoit, R.J. Riopelle, R.J. Dunn, and P.M. Richardson (1992) Preparation and biological properties of native and recombinant ciliary neurotrophic factor. J. Neurobiol. 23.81-490.

Hallbook, F., C.F. Ibafiez, and H. Persson (1991) Evolutionary studies ofthe nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6:845-858. Hefti, F. (1986) Nerve growth factor promotes survival of septa1 cholinergic neurons after fimbrial transections. J. Neurosci. 6:2 155-2162.

Hempstead, B.L., D. Martin-Zanca, D.R. Kaplan, L.F. Parada, and M.V. Chao (1991) High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor. Nature 350578-683.

Hilton, D.J. (1992) LIF: lots of interesting functions. Trends Biochem. Sci. 17:72-76.

Hohn, A,, J. Liebrock, K. Bailey, and Y.-A. Barde (1990) Identification and characterization of a novel member of the nerve growth factoribrain- derived neurotrophic factor family. Nature 344~339-34 1.

56

Ip, N.Y., C.F. Ibanez, S.H. Nye, J. McClain, P.F. Jones, D.R. Gies, L. Belluscio, M.M. Le Beau, R. Espinosa 111, S.P. Squinto, H. Persson, and G.D. Yancopoulos (1992) Mammalian neurotrophin-4: Structure, chro- mosomal localization, tissue distribution, and receptor specificity. Proc. Natl. Acad. Sci. USA 89:3060-3064.

Iwata, M., and A. Hirano (1978) Sparing of the Onufrowicz nucleus in sacral anterior horn lesions. Ann. Neurol. 4:245-249.

Johnson, I.P., and T.A. Sears (1989) Ultrastructure of axotomized alpha and gamma motoneurons in the cat thoracic spinal cord. Neuropathol. Appl. Neurobiol. 15:149-163.

Klein, R., V. Nanduri, S. Jing, F. Lamballe, P. Tapley, S. Bryant, C. Cordon-Cardo, K.R. Jones, L.F. Reichardt, and M. Barbacid (1991) The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 66:395-403.

Koliatsos, V.E., and D.L. Price (1993) Retrograde axonal transport: Applica- tions in trophic factor research. 1nA.A. Boulton, G.B. Baker, and F. Hefti (eds): Neurotrophic Factors, Neuromethods, Vol. 27. Clifton, NJ: Hu- mana Press, pp. 247-290.

Koliatsos, V.E., T.O. Crawford and D.L. Price (1991a) Axotomy induces nerve growth factor receptor immunoreactivity in spinal motor neurons. Brain Res. 549t297-304.

Koliatsos, V.E., D.L. Shelton, W.C. Mobley, and D.L. Price (1991b) A novel group of nerve growth factor receptor-immunoreactive neurons in the ventral horn of the lumbar spinal cord. Brain Res. 541,121-128.

Koliatsos, V.E., R.E. Clatterbuck, J.W. Winslow, M.H. Cayouette, and D.L. Price (1993) Evidence that brain-derived neurotrophic factor is a trophic factor for motor neurons cn uiuo. Neuron 10:359-367.

Koliatsos, V.E., W.L. Price, C.A. Pardo, and D.L. Price (1994) Ventral root avulsion: An experimental model of death of adult motor neurons. J. Comp. Neurol. 342:3544.

Kreutzberg, G.W. (1986) Neurobiology of regeneration and degeneration. In M. May (ed): The Facial Nerve. New York: Thieme, pp. 75-83.

Leibrock, J., F. Lottspeich, A. Hohn, M. Hofer, B. Hengerer, P. Masiakowski, H. Thoenen, and Y.-A. Barde (1989) Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341:149-152.

Levi-Montalcini, R. (1987) The nerve growth factor 35 years later. Science 237:1154-1162.

Lieberman, A.R. (1971) The axon reaction: A review of the principal features ofperikaryal responses to axon injury. In C.C. Pfeiffer and J.R. Smythies (eds): International Review of Neurobiology, Vol. 14. New York: Aca- demic Press, pp. 49-124.

Lo, D.C. (1992) Signal transduction and regulation of neurotrophins. Curr. Opin. Neurobiol. 2336-340.

Lowrie, M.B., and G. Vrbova (1992) Dependence of postnatal motoneurones on their targets: review and hypothesis. Trends Neurosci. 15:80-84.

Maisonpierre, P.C., L. Belluscio, Squinto, S., N.Y. Ip, M.E. Furth, R.M. Lindsay, and G.D. Yancopoulos (1990) Neurotrophin-3: A neurotrophic factor related to NGF and BDNF. Science247:1446-1451.

Mannen, T., M. Iwata, Y. Toyokura, and K. Nagashima (1977) Preservation of a certain motoneurone group of the sacral cord in amyotrophic lateral sclerosis: its clinical significance. J. Neurol. Neurosurg. Psychiatr. 40:464-469.

Mannen, T., M. Iwata, Y. Toyokura, and K. Nagashima (1982) The Onufs nucleus and the external anal sphincter muscles in amyotrophic lateral sclerosis and Shy-Drager syndrome. Acta Neuropathol. 58:255-260.

Meakin, S.O., and E.M. Shooter (1992) The nerve growth factor family of receptors. Trends Neurosci. 15323-331.

Mesulam, M.-M. (1982) Principles of horseradish peroxidase neurohistochem- istry and their applications for tracing neural pathways-Axonal transport, enzyme histochemistry and light microscopic analysis. In M.-M. Mesulam (ed): Tracing Neural Connections with Horseradish Peroxi- dase. IBRO Handbook Series: Methods in the Neurosciences. Chichester: John Wiley & Sons, pp. 1-151.

Miyata, Y., Y. Kashihara, S. Homma, and M. Kuno (1986) Effects of nerve growth factor on the survival and synaptic function of Ia sensory neurons axotomized in neonatal rats. J. Neurosci. 6:2012-2018.

Nurcombe, V., M.A. Hill, K.L. Eagleson, and M.R. Bennett (1984) Motor neuron survival and neuritic extension from spinal cord explants induced by factors released from denervated muscle. Brain Res. 291:19-28.

Onuf (Onufrowicz), B. (1899) Notes on the arrangement and function of the cell groups in the sacral region of the spinal cord. J. Nerv. Ment. Dis. 26:498-504.

Oppenheim, R.W., J.L. Maderdrut, and D.J. Wells (1982) Cell death of motoneurons in the chick embryo spinal cord. VI. Reduction of naturally occurring cell death in the thoracolumbar column of Terni by nerve growth factor. J. Comp. Neurol. 210:174-189.

R.E. CLATTERBUCK ET AL.

Oppenheim, R.W., D. Prevette, Y. Qin-Wei, F. Collins, and J. MacDonald (1991) Control of embryonic motoneuron survival in viva by ciliary neurotrophic factor. Science 251: 1616-1618.

Oppenheim, R.W., Y. $in-Wei, D. Prevette, and Q. Yan (1992) Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death. Nature 360:755-757.

Price, D.L., and K.R. Porter (1972) The response of ventral horn neurons to axonal transection. J. Cell Biol. 53:24-37.

Rosenthal, A., D.V. Goeddel, T. Nguyen, M. Lewis, A. Shih, G.R. Laramee, K. Nikolics, and J.W. Winslow (1990) Primary structure and biological activity of a novel human neurotrophic factor. Neuron 4:767-773.

Rosenthal, A,, D.V. Goeddel, T. Nguyen, E. Martin, L.E. Burton, A. Shih, G.R. Laramee, F. Wurm, A.Mason, K. Nikolics, and J.W. Winslow (1991) Primary structure and biological activity of human brain-derived neurotrophic factor. Endocrinology 129: 1289-1294.

Schmalbruch, H. (1984) Motoneuron death after sciatic nerve section in newborn rats. J. Comp. Neurol. 224:252-258.

Schmalbruch, H., H.-J.S. Jensen, M. Bjaerg, 2. Kamieniecka, and L. Kurland (1991) A new mouse mutant with progressive motor neuronopathy. J. Neuropathol. Exp. Neurol. 50:192-204.

Schwab, M.E., U. Otten, Y. Agid, and H. Thoenen (1979) Nerve growth factor (NGF) in the rat CNS: Absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra. Brain Res. 168:473-483.

Sendtner, M., G.W. Kreutzberg, and H. Thoenen (1990) Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy. Nature 345440-441.

Sendtner, M., H. Schmalbruch, K.A. Stockli, P. Carroll, G.W. Kreutzberg, and H. Thoenen 11992a) Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy. Nature 358:502-504.

Sendtner, M., B. Holtmann, R. Kolbeck, H. Thoenen, and Y.-A. Barde (1992b) Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature 360~757-759.

Smet, P.J., I.K. Abrahamson, R.E. Ressom, and R.A. Rush (1991) A ciliaIy neuronotrophic factor from peripheral nerve and smooth muscle which is not retrogradely transported. Neurochem. Res. 16:613420.

Smith, R.G., K. Vaca, J. McManaman, and S.H. Appel(1986) Selective effects of skeletal muscle extract fractions on motoneuron development in vitro. J. Neurosci. 6~439447.

Snider, W.D., and S. Thanedar (1989) Target dependence of hypoglossal motor neurons during development and in maturity. J. Comp. Neurol. 279:489498.

Snider, W.D., J.L. Elliott, and Q. Yan (1992) Axotomy-induced neuronal death during development. J. Neurobiol. 23:1231-1246.

Soppet, D., E. Escandon, J. Maragos, D.W. Middlemas, S.W. Reid, J. Blair, L.E. Burton, B.R. Stanton, D.R. Kaplan, T. Hunter, K. Nikolics, and L.F. Parada (1991) The neurotrophic factors brain-derived neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. Cell 65895-903.

Squinto, S.P., T.N. Stitt, T.H. Aldrich, S. Davis, S.M. Bianco, C. Radziejew- ski, D.J. Glass, P. Masiakowski, M.E. Furth, D.M. Valenzuela, P.S. DiStefano, and G.D. Yancopoulos (1991) trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor. Cell 6'5:885-893.

Unsicker, K., C. Grothe, R. Westermann, and K. Wewetzer (1992) Cytokines in neural regeneration. Curr. Opin. Neurobiol. 2671-678.

Wayne, D.B., and M.B. Heaton (1988) Retrograde transport of NGF by early chick embryo spinal cord motoneurons. Dev. Biol. 127:220-223.

Wood, S.J., J. Pritchard, and M.V. Sofroniew (1990) Re-expression of nerve growth factor receptor after axonal injury recapitulates a developmental event in motor neurons: Differential regulation when regeneration is allowed or prevented. Eur. J. Neurosci. 2650-657.

Yan, Q., and E.M. Johnson Jr. (1988) An immunohistochemical study of the nerve growth factor receptor in developing rats. J. Neurosci. 8:3481- 3498.

Yan, Q., W.D. Snider, J.J. Pinzone, and E.M. Johnson, Jr. (1988) Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats. Assessment of potential neurotrophic effects. Neuron I:335-343.

Yan, H., J. Schlessinger, and M.V. Chao (1991) Chimeric NGF-EGF receptors define domains responsible for neuronal differentiation. Sci- ence 252:561-563.

Yan, Q., J. Elliott, and W.D. Snider (1992) Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 360:753-755.

Download - Further characterization of the effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on axotomized neonatal and adult mammalian motor neurons

Top Related