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Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system
Molecular Brain Research, 17 (1993) 1-8 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
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BRESM 70534
Research Reports
Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system N. R o c a m o r a
b,,
F.J. G a r c l a - L a d o n a
b
J . M . P a l a c i o s a,c a n d G. M e n g o d
a,c
a Preclinical Research Sandoz Pharma Ltd., Basel (Switzerland), h Institute of Pathology, University of Basel, Basel, (Switzerland) and c Department of Neurochemistry, CID-CSIC, Barcelona (Spain) (Accepted 11 August 1992)
Key words. Granule cell; Low-affinity nerve growth factor receptor; Brain-derived neurotrophic factor; Neurotrophin-3; Sequential expression; In situ hybridization; Local action
The spatio-temporal pattern of expression of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF) and low-affinity nerve growth factor receptor (LNGFR) genes was analyzed in the postnatal developing cerebellar system of the rat by in situ hybridization histochemistry. Different ontogenetic patterns of expression were observed for these three genes. In agreement with previously published results (Neuron, 5 (1990) 501-509; Dec. Brain Res., 55 (1990) 288-292) we found that NT3 and LNGFR mRNA peaked early, during the first 2 postnatal weeks, whereas BDNF mRNA peaked later, around postnatal day 20, in the cerebellar cortex. High levels of NT3 mRNA were found in the internal granule cell layer as early as postnatal day 5. NT3 mRNA was also present in the external-premigratory granule cell layer at postnatal day 10. From postnatal day 5 on, LNGFR mRNA was present in the proliferative area of the external granule cell layer and in the Purkinje cells. NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation, suggesting a sequential stimulation of these two genes during this developmental process. LNGFR mRNA levels decreased along the same period. Although practically undetectable in the cerebellar granule cell layer in the first two postnatal weeks, BDNF mRNA was transiently expressed in the deep cerebellar nuclei during this time and it was very abundant in the inferior olivary system from postnatal day 5 on. LNGFR mRNA was transiently expressed in the inferior olivary system, in the first postnatal week. These data are discussed in relation to the coordinated postnatal maturation of the different cells of the cerebellar system. Our results are compatible with a local delivery and role of NT3 in the early postnatal development of the cerebellar cortex. Its presence may be involved in the process of granule cell migration a n d / o r the establishment of early synaptic contacts. BDNF, on the contrary, could play a role at a later stage, perhaps as a maintenance factor.
INTRODUCTION
Target-derived neurotrophic factors have been shown to play an important role in the development and maintenance of neural cells and their networks, for a review see Barde 4. Although nerve growth factor (NGF), the best characterized and prototypic neurotrophic factor, has been, for a long time, the only isolated molecule with neurotrophic activities (for a review see Levi-Montalcini34), two new molecules, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), have been recently c l o n e d 14'22'33'39. The close structural and functional relation between
these three genes suggested that they are members of a gene family, usually referred to as neurotrophin family (NTF). NTFs share very strictly conserved domains, such as six cysteines involved in disulfide bridges formation, a requisite for their biological activity, but they also contain some variable regions which might be related to their neuronal s p e c i f i c i t y 22,39. Moreover, it has been recently demonstrated that BDNF and NGF neuronal specificities can be acquired, in vitro, by different combinations of a set of sequences that differ between the two molecules 24. NGF, BDNF and NT3 have been shown to support the survival and function of distinct but overlapping
Correspondence." G. Mengod ( c / o F. Artigas), Department of Neurochemistry, CID-CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain. Fax: 34-3-2045904. * Present address: Cellular Biology Unit, Faculty of Biology, University of Barcelona, 08071-Barcelona, Spain.
n e u r o n a l s u b p o p u l a t i o n s of the p e r i p h e r a l a n d the central nervous systems. W h i l e b o t h B D N F a n d N G F have b e e n f o u n d to e n h a n c e the survival or d i f f e r e n t i a tion, or both, of c e r t a i n p o p u l a t i o n s of n e u r a l c r e s t - d e rived sensory neurons, only N G F has similar effects in s y m p a t h e t i c n e u r o n s 5'19'3s. B D N F a n d NT3, but not N G F , have an additive effect on the s u p p o r t of n e u r a l p l a c o d e - d e r i v e d n o d o s e ganglion n e u r o n s ~3'22"36. Both B D N F a n d N G F , but not NT3, have b e e n shown to e n h a n c e the survival and d i f f e r e n t i a t i o n of c u l t u r e s of e m b r y o n i c basal f o r e b r a i n cholinergic cells ~'2'4'2s. In addition, B D N F has b e e n d e m o n s t r a t e d to have t r o p h i c effects not only on cholinergic, b u t also on m e s e n c e p h a l i c G A B A e r g i c a n d d o p a m i n e r g i c cells 23'28. T a k e n t o g e t h e r , these d a t a suggest that specific roles for the different n e u r o t r o p h i c factors could be exp e c t e d in the d e v e l o p i n g nervous system. T r o p h i c req u i r e m e n t s of d i f f e r e n t n e u r o n a l p o p u l a t i o n s may change during development. The different NTFs may act e i t h e r on different n e u r o n s at the s a m e time o r on the s a m e n e u r o n , sequentially, at different d e v e l o p mental stages. A n a l y s e s of the d i s t r i b u t i o n of neur o t r o p h i n s a n d t h e i r r e c e p t o r s d u r i n g d e v e l o p m e n t may help to d e t e r m i n e which n e u r o n a l p o p u l a t i o n may be d e p e n d e n t on each p a r t i c u l a r n e u r o t r o p h i c factor at each specific d e v e l o p m e n t a l stage. T h e trk family of tyrosine kinase r e c e p t o r s w e r e r e c e n t l y identified as m e d i a t o r s in signal t r a n s d u c t i o n by n e u r o t r o p h i n s ~7'26'27'3~. L N G F R , on the o t h e r hand, was shown to b i n d with similar (low) affinity to N G F , B D N F a n d NT314'45. H i g h affinity of N G F s e e m s to be d e p e n d e n t on the p r e s e n c e of b o t h L N G F R a n d t r k A 2°. However, t r k A a l o n e has b e e n d e m o n s t r a t e d as a high-affinity r e c e p t o r for N G F 26. A l t h o u g h L N G F R alone is a p p a r e n t l y u n a b l e to initiate signal t r a n s d u c tion, m o d u l a t o r y roles in the t r k - m e d i a t e d responses, as well as its involvement in o t h e r p a t h w a y s not r e l a t e d to trks have b e e n p r o p o s e d for this low-affinity r e c e p tor ~7. T h e c e r e b e l l u m is an i n t e r e s t i n g n e u r a l tissue to study d e v e l o p m e n t a l l y r e g u l a t e d processes, d u e to its p o s t n a t a l m a t u r a t i o n a n d its relatively simple, t h r e e l a y e r e d structure. A f t e r birth, stem cells in t h e e x t e r n a l g r a n u l e cell layer ( E G L ) m i g r a t e t o w a r d s their final position in the internal g r a n u l e cell layer ( I G L ) . Simultaneously, t h e s e cells project axons ( p a r a l l e l fibres) in the m o l e c u l a r layer. D u r i n g the s a m e p e r i o d Purkinje cells a t t a i n t h e i r final a r r a n g e m e n t as a m o n o c e l l u l a r layer, b e t w e e n the m o l e c u l a r layer a n d the I G L , a n d grow their d e n d r i t i c t r e e 3. T h e inferior olivary cells project t h e i r axons (climbing fibres) t o w a r d s the molecular layer establishing a p o l y i n n e r v a t i o n of Purkinje cells d e n d r i t i c tree, which in the a d u l t regresses, lead-
ing to a ratio of one climbing fibre c o n t a i n i n g one Purkinje cell 12. W h i l e this p e r i o d has b e e n extensively a n a l y z e d at the m o r p h o l o g i c a l and b i o c h e m i c a l levels ';, the m o l e c u l a r m e c h a n i s m s r e s p o n s i b l e for these develo p m e n t a l p r o c e s s e s are still poorly u n d e r s t o o d . In situ h y b r i d i z a t i o n h i s t o c h e m i s t r y a n d i m m u n o h i s t o c h e m i c a l studies have d e m o n s t r a t e d the p r e s e n c e of both NGF and LNGFR mRNAs and their corresponding p o l y p e p t i d e s in the d e v e l o p i n g c e r e b e l l a r cortex "~'3°'4°. However, t h e r e is no i n f o r m a t i o n available on the a n a t o m i c a l localization of B D N F and NT3 p o l y p e p t i d e a n d their c o r r e s p o n d i n g m R N A s in cereb e l l u m d u r i n g this d e v e l o p m e n t a l period. T h e aim of the p r e s e n t study was to analyze the s p a t i o - t e m p o r a l p a t t e r n of expression of B D N F , NT3 and L N G F R during the p o s t n a t a l d e v e l o p m e n t of the c e r e b e l l a r system. T h e p r e s e n t results a r e discussed in r e l a t i o n to m o r p h o g e n e t i c events such as cell m i g r a t i o n a n d synaptic r e o r g a n i z a t i o n , which t a k e place d u r i n g the p o s t n a t a l m a t u r a t i o n of the c e r e b e l l a r circuitry. MATERIALS AND METHODS Animals
Wistar rats of different postnatal ages (postnatal days: PI, P5, P10, P15 P20 and P40) were sacrificed by decapitation, their brains immediately frozen in dry ice and stored at -70°C until sectioned. Sections (20 p,m thick) were obtained in a cryostat (Leitz 1720), thaw-mounted on gelatinized slides and kept at -20°C until used. In situ hybridization histochemisto'
In situ hybridization histochemistry was essentially performed as previously described44. Briefly, tissue sections were air dried, fixed by immersion in 4% formaldehyde, 1× PBS (PBS: 2.6 mM KC1, 1.4 mM KH2PO4, 136 mM NaC1, 8 mM Na2HPO4), and incubated with predigested pronase (24 U/ml in 50 mM Tris-HCI pH 7.5, 5 mM EDTA). Finally, sections were dehydrated in a graded series of ethanol (60, 80, 95 and 100%, 2 min each) and air-dried. Two oligonucleotide probes complementary to each BDNF, NT3 and LNGFR mRNA published sequences were used. The probes used were the following: bdnf/l complementary to bases 219-266, bdnf/2 to bases 777-824 of the mouse BDNF mRNA sequence 21. nt3/l complementary to bases 91-138 and nt3/2 to bases 235-288 of the rat NT3 genomic sequence 39, Ingfr/1 complementary to bases 181-220 and Ingfr/2 to bases 1314-1361 of the rat LNGFR cDNA sequence 43. Oligonucleotides were 3' end-labeled using [a-32p]dATP (3000 Ci/mmol, New England Nuclear) and terminal deoxynucleotidyltransferase (TdT, Boehringer Mannheim) to specific activities ranging between (I.9 and 2.0× 10 4 Ci/mmol, Labeled probes were diluted to a final concentration of 2-3 x 107 cpm/ml in 0.6 M NaC1, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 × Denhardt's (0.02% ficoll, 0.02% polyvinylpyrolidine, 0.02% bovine serum albumin), 500 /~g/ml yeast tRNA, 50% formamide, 10% dextran sulphate. Tissue was covered with 65 p~l of hybridization solution, overlaid with nescofilm coverslips and incubated overnight in a humid chamber at 42°C. Sections were then washed at 60°C in 0.6 M NaCI, 20 mM Tric-HCI pH 7.5, 1 mM EDTA, for 3 h, with 4 changes of buffer. Tissue was dehydrated in 70% and 95% ethanol, both containing 0.3 M ammonium acetate pH 7.0. Hybridized tissue sections were finally exposed to /3-MAX autoradiographic films (Amersham, UK) at 70°C with intensifying screens, or dipped in autoradiographic emulsion NTB-3 (Kodak). After 3-4 weeks, dippings were developed with Kodak D-19 and counterstained with Cresyl violet.
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Fig. 1. Developmental pattern of LNGFR (A, D, G, J), NT3 (B, E, H, K) and BDNF (C, F, I, L) mRNAs in the rat cerebellum at postnatal ages P5 (A, B, C), P10 (D, E, F) P15 (G, H, I) and P20 (J, K, L). Pictures are photomicrographs from emulsion autoradiograms, obtained with dark-field ilumination. Bright areas are rich in autoradiographic grains. Different intensities in hybridization signal along different postnatal ages, for each individual oligonucleotide probe, are indicative of different mRNA levels, since pictures were taken from autoradiograms of a single experiment exposed for the same amount of time. Note in A a high density of hybridization signal for LNGFR in EGL and in Purkinje cell layer (PCL). LNGFR hybridization signal is present in EGL but not in IGL (A, D, G, J). There is a high density of NT3 hybridization signal in IGL at early postnatal ages (B) decreasing afterwards (E, H, K). BDNF hybridization signal is practically undetectable until the second postnatal week (C, F, I, L). EGL, external granule cell layer; ML, molecular layer, PCL, Purkinge cell layer; IGL, internal granule cell layer. Bar = 1 mm.
4 RESULTS In situ hybridization histochemistry with oligonucleotide probes complementary to BDNF, NT3 and L N G F R m R N A sequences was performed on 20 /xm cryosections from postnatal developing rat cerebellum. The specificity of the hybridization signal at the different postnatal ages was confirmed using several criteria, as described in our previous work 44. Briefly: (a) for each m R N A two different oligonucleotides were used, as independent hybridization probes in consecutive sections, showing an identical pattern of hybridization, (b) for each labeled probe the addition of an excess of the same unlabeled oligonucleotide, resulted in the complete elimination of the specific hybridization signal, showing background levels, and finally (c) hybridization patterns obtained with oligonucleotide probes, similar in length and base composition, but specific for each BDNF, NT3 and L N G F R m R N A s were clearly distinct among them. Three different postnatal development series were studied. In order to compare the hybridization level for each oligonucleotide probe among the different postnatal ages, sections from the postnatal ages (P1, P5, P10, P15, P20, adult) were used in each individual experiment, with the same labeling, hybridization, washing and exposure conditions.
The specific hybridization signal obtained with probe b d n f / 2 was undistinguishable from the unspecific background (not shown) in the cerebellar cortex during the first two postnatal weeks, but it increased later in the I G L reaching a peak around P20 (Fig. 1C,F,I,L). In contrast, B D N F m R N A was early and transiently present in the deep cerebellar nuclei (from P5 to 15), being afterwards undetectable in adult animals (Fig. 3). High levels of B D N F m R N A were observed in the inferior olivary complex from the first postnatal ages, with a maximum at P10 and decreasing afterwards, being still present in the adult inferior olivary complex, but at lower levels than in younger animals (Fig. 4). Hybridization with n t 3 / 2 oligonucleotide probe revealed the presence of high levels of NT3 m R N A as early as P5 in the I G L (Fig. 1). Moderate levels of NT3 m R N A were also present in the most internal part of the premigratory area of the E G L at P10 (Figs. 1 and 2). Concommitant with the granule cell migration and maturation processes, NT3 m R N A levels decreased. Low levels of NT3 m R N A could be observed in the I G L in adult animals. In contrast to BDNF, NT3 m R N A was not detected neither in the deep cerebellar nuclei, nor in the inferior olivary complex. High levels of L N G F R m R N A were found, from P5 on, in the proliferative area of the E G L and in the Purkinje cell layer. However, L N G F R m R N A was not
Fig. 2. LNGFR and the NT3 mRNA expression in the cerebellar cortex layers of P10 rat. Pictures are high-magnificationphotomicrographs from emulsion autoradiograms, obtained with dark-field ilumination, LNGFR mRNA (A) and NT3 mRNA (B); and a bright-field photomicrograph corresponding to tissue in B, counterstained with Cresyl violet (C). Note in A that LNGFR hybridization signal is present in the proliferative area (PR) of the EGL (the most external) and in the Purkinje cells layer (PCL). Note in B the presence of NT3 hybridization signal in the IGL and in the most internal part (PM) of the EGL. EGL, external granule cell layer, PR, proliferative area of the EGL; PM, premigratory area of the EGL: ML, molecular layer; IGL, internal granule cell layer; PCL, Purkinje cell layer. Bar = 400 p~m.
Fig. 3. Localization of BDNF mRNA in deep cerebellar nuclei during postnatal rat development. Pictures are photomicrographs from emulsion autoradiograms, obtained with dark-field ilumination. Note that BDNF mRNA was present at P5 (A) while it was undetectable at P20 (B). Light in choroid plexus is an artifact of the dark-field illumination and does not correspond to hybridization signal, ch. pl., choroid plexus; DCN, deep cerebellar nuclei. Bar = 1 mm.
observed neither in the premigratory area of the E G L nor in the IGL (Figs. 1 and 2). At P20 L N G F R m R N A was practically only detectable in the Purkinje cell layer. Moreover, it was heterogeneously distributed, being mainly restricted to the posterior cerebellar lobules (Figs. 1 and 5). The inferior olivary system displayed high levels of hybridization with L N G F R oligoprobes at P5 decreasing thereafter and been practically undetectable in adult animals (Fig. 4). DISCUSSION The main finding of this study is the demonstration of a sequential expression of NT3 and BDNF during the postnatal migration and maturation of the cerebellar granule cells. NT3 was found to be expressed in the IGL as early as P5 and in the most internal part of the premigratory area of the E G L at P10. BDNF mRNA, in contrast, was undetectable in E G L and it was detected in the IGL only from the second postnatal week onwards. These results agree with previously published Northern analyses data on the levels of these two neurotrophic factors 38, and provide new information
about the anatomical localization of BDNF and NT3 transcripts in the developing cerebellum. The presence of NT3 mRNA, both, in the most internal part of the E G L and in the IGL of immature cerebellum suggest that granule cells express NT3 concommitantly with the beginning of their migration. NT3 could play a role in the granule cell migration a n d / o r in the maturation of its synaptic contacts. Such effects could be mediated through L N G F R a n d / o r trkB, present in the Purkinje cell 25. Moreover, NT3 secreted by migratory granule cells may also act as a trophic factor taken up by climbing fibres from the olivary neurons. These cells express L N G F R around P5 and reach their synaptic maturity at a later stage 7. In contrast, BDNF expression by granule cells begins after their arrival into the IGL, which could indicate a trophic role for BDNF over cells sending afferents (mossy fibres) to cerebellar granule cells 18. In summary, these data support an early role for NT3 in the postnatally maturing cerebellar granule cells, may be involved in their migration a n d / o r the establishment of their synaptic contacts, while BDNF might play a late role, possibly as a maintenance factor, as it has been previously suggested 38. Similar results obtained in the postnatally maturing granule cells of the dentate gyrus of the hippocampus, where NT3 was expressed earlier (from P1 on) than B D N F (from P5 on) (data not shown, Friedman et al.~5), support a more general role for NT3, not only in the cerebellar but also in the hippocampal maturing granule cells. Moreover, the presence of B D N F m R N A mainly in the external part of the granule cell layer of the dentate gyrus, observed by us (data not shown) and other authors 16 also supports a later role of BDNF in granule cells, given the external-internal gradient of maturity in this cell layer 6. From P1 to P10, and in parallel with granule cell migration and maturation, Purkinje cells develop their dendritic tree and mature their synaptic contacts. High levels and widespread distribution of L N G F R mRNA in Purkinje cells during this period, observed by us (Fig. 1) and other authors 29'47, suggest a role of N G F a n d / o r the other neurotrophins (BDNF and NT3) in the maturation of these cells. In addition, the persistence of L N G F R in Purkinje cells of adult rats 42 suggests a role of these neurotrophins also in the mature cerebellum. Our results show that at late postnatal stages (from P20), as well as in adult rats, L N G F R m R N A is mainly located in the Purkinje cells of the posterior cerebellar lobules, which extends the previously published results from Mufson et al. 4°, showing L N G F R immunoreactivity in the lobules belonging to archi- and neo-cerebellum. These results demonstrate
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Fig. 4. Localization of LNGFR (A, C, D) and BDNF (B, E, F) mRNAs in the rat inferior olivary system at different postnatal rat development ages (P,5: A, B; P15, C, D; P20: E, F). Pictures are photomicrographs from autoradiographic films• Hybridized sections for P5 and PI5 correspond to plates 72-73 (A, B, C, D) and for P20 to plates 70-71 (E, F) of the Paxinos and Watson stereotaxic atlas41. There is a clear decrease in the levels of LNGFR mRNA from P5 to P20 (A, C, E). Although high levels of BDNF hybridization signal are present at P5 and PI5, this mRNA is still abundant at P20 (B, D, F) and it remains present in adult ages (not shown). HN, hypoglossal nucleus: IO, inferior olivary system; IOA, medial nucleus, subnucleus A; lOB, medial nucleus, subnucleus B; IOC, medial nucleus, subnucleus C; IOK, medial nucleus, cap kooy; IOBe,/3 subnucleus, IOD, dorsal nucleus. Bar - 1 ram.
a h e t e r o g e n o u s r e g u l a t i o n of L N G F R expression in the different cerebellar regions. Moreover, it is well known that the cerebellar biochemical cytoarchitecture is not h o m o g e n e o u s as d e m o n s t r a t e d by p a t t e r n s of enzymes a n d n e u r o t r a n s m i t t e r receptors 8"'. With regard to this point, it will be of interest to analyze if the differential d i s t r i b u t i o n of L N G F R is an i n d e p e n d e n t evolutionary fact or if it is also related to the p r e s e n c e of specific n e u r o t r a n s m i t t e r circuits. Interestingly, Purkinje cells
have b e e n f o u n d to r e s p o n d in an additive m a n n e r to N G F a n d excitatory n e u r o t r a n s m i t t e r s t|. N e u r o t r o p h i c factors may also play a role d u r i n g the m a t u r a t i o n of the olive-Purkinje cells-deep cerebellar nuclei pathway, as suggested by the p r e s e n c e of B D N F m R N A in the deep cerebellar nuclei. T h e inferior olivary nuclei express high levels of B D N F m R N A d u r i n g the first two weeks after birth, which may act as a trophic factor for the afferent n e u r o n s projecting to
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developing rat cerebellum. Alternative trophic functions for different neurotrophins have been suggested. Moreover, our results suggest the possibility of a coordinated up- and down-regulation of BDNF and NT3 genes, respectively, as well as the possibility of a local delivery and action of neurotrophins. Further studies on the expression of trk family of high affinity neurotrophin receptors together with the use of animal models of abnormal cerebellar development (i.e. cerebellar mutant or neurotoxin-treated animals) and in vitro analyses of cerebellar systems may help to understand the physiological role of these trophic molecules in the developing nervous system. Acknowledgements. N.R. was supported by a postdoctoral fellowship from the Comissi6 Interdepartamental de Recerca i Innovaci6 Tecnolbgica (C.I.R.I.T.) of the Generalitat de Catalunya.
ABBREVIATIONS
Fig. 5. Differential localization of LNGFR mRNA in the cerebellar cortex at different postnatal ages. Pictures are photomicrographs from autoradiographic films. Note in A (P10) the presence of LNGFR mRNA in all the PCL (Purkinje cell layer), whereas in B (P20) LNGFR mRNA was mainly restricted to the PCL of posterior cerebellar lobules. Bar = 2 mm.
the olivary system. Moreover, considering the possibility of an anterograde transport, BDNF expressed in the inferior olivary nuclei, could also act on maturing Purkinje cells, given the widespread presence of LNGFR in these cells together with their polyinnervation by the olivary climbing fibres, until the characteristic monoinnervation of maturity is reached ~2. Although it is generally accepted that neurotrophic factors function in the target synthesis/retrograde transport w a y 46, the presence of both NGF and its receptor in the same brain areas 32'37 led to the proposal of local, in addition to distant effect for NGF. In this sense, it is noteworthy to stress the transient expression of LNGFR by EGL stem cells, since these cells are neither targets nor send efferents to any known system. Furthermore, NT3 expression in the EGL, along with the presence of LNGFR and trkB in the Purkinje cell dendritic tree, also suggest a local mode of action of this neurotrophic factor. In conclusion, the results in the present work show a specific and developmentally regulated expression of NT3, BDNF and LNGFR in different cell types of the
BDNF ch. pl. DCN EGL HN IGL IO IOA IOB IOBe IOC IOD IOK ML NGF LNGFR NT3 NTF P PCL PM PR trkA, trkB
brain-derived neurotrophic factor choroid plexus deep cerebellar nuclei external granule cell layer hypoglossal nucleus internal granule cell layer inferior olivary system inferior olive, subnucleus A of medial nucleus inferior olive, subnucleus B of medial nucleus inferior olive, beta subnucleus inferior olive, subnucleus C of medial nucleus inferior olive, dorsal nucleus inferior olive, cap kooy, medial nucleus molecular layer nerve growth factor low affinity nerve growth factor receptor neurotrophin-3 neurotrophins postnatal day Purkinje cell layer EGL, premigratory area EGL, proliferative area components of the trk family of tyrosine kinase receptors
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BRESM 70534
Research Reports
Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system N. R o c a m o r a
b,,
F.J. G a r c l a - L a d o n a
b
J . M . P a l a c i o s a,c a n d G. M e n g o d
a,c
a Preclinical Research Sandoz Pharma Ltd., Basel (Switzerland), h Institute of Pathology, University of Basel, Basel, (Switzerland) and c Department of Neurochemistry, CID-CSIC, Barcelona (Spain) (Accepted 11 August 1992)
Key words. Granule cell; Low-affinity nerve growth factor receptor; Brain-derived neurotrophic factor; Neurotrophin-3; Sequential expression; In situ hybridization; Local action
The spatio-temporal pattern of expression of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF) and low-affinity nerve growth factor receptor (LNGFR) genes was analyzed in the postnatal developing cerebellar system of the rat by in situ hybridization histochemistry. Different ontogenetic patterns of expression were observed for these three genes. In agreement with previously published results (Neuron, 5 (1990) 501-509; Dec. Brain Res., 55 (1990) 288-292) we found that NT3 and LNGFR mRNA peaked early, during the first 2 postnatal weeks, whereas BDNF mRNA peaked later, around postnatal day 20, in the cerebellar cortex. High levels of NT3 mRNA were found in the internal granule cell layer as early as postnatal day 5. NT3 mRNA was also present in the external-premigratory granule cell layer at postnatal day 10. From postnatal day 5 on, LNGFR mRNA was present in the proliferative area of the external granule cell layer and in the Purkinje cells. NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation, suggesting a sequential stimulation of these two genes during this developmental process. LNGFR mRNA levels decreased along the same period. Although practically undetectable in the cerebellar granule cell layer in the first two postnatal weeks, BDNF mRNA was transiently expressed in the deep cerebellar nuclei during this time and it was very abundant in the inferior olivary system from postnatal day 5 on. LNGFR mRNA was transiently expressed in the inferior olivary system, in the first postnatal week. These data are discussed in relation to the coordinated postnatal maturation of the different cells of the cerebellar system. Our results are compatible with a local delivery and role of NT3 in the early postnatal development of the cerebellar cortex. Its presence may be involved in the process of granule cell migration a n d / o r the establishment of early synaptic contacts. BDNF, on the contrary, could play a role at a later stage, perhaps as a maintenance factor.
INTRODUCTION
Target-derived neurotrophic factors have been shown to play an important role in the development and maintenance of neural cells and their networks, for a review see Barde 4. Although nerve growth factor (NGF), the best characterized and prototypic neurotrophic factor, has been, for a long time, the only isolated molecule with neurotrophic activities (for a review see Levi-Montalcini34), two new molecules, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), have been recently c l o n e d 14'22'33'39. The close structural and functional relation between
these three genes suggested that they are members of a gene family, usually referred to as neurotrophin family (NTF). NTFs share very strictly conserved domains, such as six cysteines involved in disulfide bridges formation, a requisite for their biological activity, but they also contain some variable regions which might be related to their neuronal s p e c i f i c i t y 22,39. Moreover, it has been recently demonstrated that BDNF and NGF neuronal specificities can be acquired, in vitro, by different combinations of a set of sequences that differ between the two molecules 24. NGF, BDNF and NT3 have been shown to support the survival and function of distinct but overlapping
Correspondence." G. Mengod ( c / o F. Artigas), Department of Neurochemistry, CID-CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain. Fax: 34-3-2045904. * Present address: Cellular Biology Unit, Faculty of Biology, University of Barcelona, 08071-Barcelona, Spain.
n e u r o n a l s u b p o p u l a t i o n s of the p e r i p h e r a l a n d the central nervous systems. W h i l e b o t h B D N F a n d N G F have b e e n f o u n d to e n h a n c e the survival or d i f f e r e n t i a tion, or both, of c e r t a i n p o p u l a t i o n s of n e u r a l c r e s t - d e rived sensory neurons, only N G F has similar effects in s y m p a t h e t i c n e u r o n s 5'19'3s. B D N F a n d NT3, but not N G F , have an additive effect on the s u p p o r t of n e u r a l p l a c o d e - d e r i v e d n o d o s e ganglion n e u r o n s ~3'22"36. Both B D N F a n d N G F , but not NT3, have b e e n shown to e n h a n c e the survival and d i f f e r e n t i a t i o n of c u l t u r e s of e m b r y o n i c basal f o r e b r a i n cholinergic cells ~'2'4'2s. In addition, B D N F has b e e n d e m o n s t r a t e d to have t r o p h i c effects not only on cholinergic, b u t also on m e s e n c e p h a l i c G A B A e r g i c a n d d o p a m i n e r g i c cells 23'28. T a k e n t o g e t h e r , these d a t a suggest that specific roles for the different n e u r o t r o p h i c factors could be exp e c t e d in the d e v e l o p i n g nervous system. T r o p h i c req u i r e m e n t s of d i f f e r e n t n e u r o n a l p o p u l a t i o n s may change during development. The different NTFs may act e i t h e r on different n e u r o n s at the s a m e time o r on the s a m e n e u r o n , sequentially, at different d e v e l o p mental stages. A n a l y s e s of the d i s t r i b u t i o n of neur o t r o p h i n s a n d t h e i r r e c e p t o r s d u r i n g d e v e l o p m e n t may help to d e t e r m i n e which n e u r o n a l p o p u l a t i o n may be d e p e n d e n t on each p a r t i c u l a r n e u r o t r o p h i c factor at each specific d e v e l o p m e n t a l stage. T h e trk family of tyrosine kinase r e c e p t o r s w e r e r e c e n t l y identified as m e d i a t o r s in signal t r a n s d u c t i o n by n e u r o t r o p h i n s ~7'26'27'3~. L N G F R , on the o t h e r hand, was shown to b i n d with similar (low) affinity to N G F , B D N F a n d NT314'45. H i g h affinity of N G F s e e m s to be d e p e n d e n t on the p r e s e n c e of b o t h L N G F R a n d t r k A 2°. However, t r k A a l o n e has b e e n d e m o n s t r a t e d as a high-affinity r e c e p t o r for N G F 26. A l t h o u g h L N G F R alone is a p p a r e n t l y u n a b l e to initiate signal t r a n s d u c tion, m o d u l a t o r y roles in the t r k - m e d i a t e d responses, as well as its involvement in o t h e r p a t h w a y s not r e l a t e d to trks have b e e n p r o p o s e d for this low-affinity r e c e p tor ~7. T h e c e r e b e l l u m is an i n t e r e s t i n g n e u r a l tissue to study d e v e l o p m e n t a l l y r e g u l a t e d processes, d u e to its p o s t n a t a l m a t u r a t i o n a n d its relatively simple, t h r e e l a y e r e d structure. A f t e r birth, stem cells in t h e e x t e r n a l g r a n u l e cell layer ( E G L ) m i g r a t e t o w a r d s their final position in the internal g r a n u l e cell layer ( I G L ) . Simultaneously, t h e s e cells project axons ( p a r a l l e l fibres) in the m o l e c u l a r layer. D u r i n g the s a m e p e r i o d Purkinje cells a t t a i n t h e i r final a r r a n g e m e n t as a m o n o c e l l u l a r layer, b e t w e e n the m o l e c u l a r layer a n d the I G L , a n d grow their d e n d r i t i c t r e e 3. T h e inferior olivary cells project t h e i r axons (climbing fibres) t o w a r d s the molecular layer establishing a p o l y i n n e r v a t i o n of Purkinje cells d e n d r i t i c tree, which in the a d u l t regresses, lead-
ing to a ratio of one climbing fibre c o n t a i n i n g one Purkinje cell 12. W h i l e this p e r i o d has b e e n extensively a n a l y z e d at the m o r p h o l o g i c a l and b i o c h e m i c a l levels ';, the m o l e c u l a r m e c h a n i s m s r e s p o n s i b l e for these develo p m e n t a l p r o c e s s e s are still poorly u n d e r s t o o d . In situ h y b r i d i z a t i o n h i s t o c h e m i s t r y a n d i m m u n o h i s t o c h e m i c a l studies have d e m o n s t r a t e d the p r e s e n c e of both NGF and LNGFR mRNAs and their corresponding p o l y p e p t i d e s in the d e v e l o p i n g c e r e b e l l a r cortex "~'3°'4°. However, t h e r e is no i n f o r m a t i o n available on the a n a t o m i c a l localization of B D N F and NT3 p o l y p e p t i d e a n d their c o r r e s p o n d i n g m R N A s in cereb e l l u m d u r i n g this d e v e l o p m e n t a l period. T h e aim of the p r e s e n t study was to analyze the s p a t i o - t e m p o r a l p a t t e r n of expression of B D N F , NT3 and L N G F R during the p o s t n a t a l d e v e l o p m e n t of the c e r e b e l l a r system. T h e p r e s e n t results a r e discussed in r e l a t i o n to m o r p h o g e n e t i c events such as cell m i g r a t i o n a n d synaptic r e o r g a n i z a t i o n , which t a k e place d u r i n g the p o s t n a t a l m a t u r a t i o n of the c e r e b e l l a r circuitry. MATERIALS AND METHODS Animals
Wistar rats of different postnatal ages (postnatal days: PI, P5, P10, P15 P20 and P40) were sacrificed by decapitation, their brains immediately frozen in dry ice and stored at -70°C until sectioned. Sections (20 p,m thick) were obtained in a cryostat (Leitz 1720), thaw-mounted on gelatinized slides and kept at -20°C until used. In situ hybridization histochemisto'
In situ hybridization histochemistry was essentially performed as previously described44. Briefly, tissue sections were air dried, fixed by immersion in 4% formaldehyde, 1× PBS (PBS: 2.6 mM KC1, 1.4 mM KH2PO4, 136 mM NaC1, 8 mM Na2HPO4), and incubated with predigested pronase (24 U/ml in 50 mM Tris-HCI pH 7.5, 5 mM EDTA). Finally, sections were dehydrated in a graded series of ethanol (60, 80, 95 and 100%, 2 min each) and air-dried. Two oligonucleotide probes complementary to each BDNF, NT3 and LNGFR mRNA published sequences were used. The probes used were the following: bdnf/l complementary to bases 219-266, bdnf/2 to bases 777-824 of the mouse BDNF mRNA sequence 21. nt3/l complementary to bases 91-138 and nt3/2 to bases 235-288 of the rat NT3 genomic sequence 39, Ingfr/1 complementary to bases 181-220 and Ingfr/2 to bases 1314-1361 of the rat LNGFR cDNA sequence 43. Oligonucleotides were 3' end-labeled using [a-32p]dATP (3000 Ci/mmol, New England Nuclear) and terminal deoxynucleotidyltransferase (TdT, Boehringer Mannheim) to specific activities ranging between (I.9 and 2.0× 10 4 Ci/mmol, Labeled probes were diluted to a final concentration of 2-3 x 107 cpm/ml in 0.6 M NaC1, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 × Denhardt's (0.02% ficoll, 0.02% polyvinylpyrolidine, 0.02% bovine serum albumin), 500 /~g/ml yeast tRNA, 50% formamide, 10% dextran sulphate. Tissue was covered with 65 p~l of hybridization solution, overlaid with nescofilm coverslips and incubated overnight in a humid chamber at 42°C. Sections were then washed at 60°C in 0.6 M NaCI, 20 mM Tric-HCI pH 7.5, 1 mM EDTA, for 3 h, with 4 changes of buffer. Tissue was dehydrated in 70% and 95% ethanol, both containing 0.3 M ammonium acetate pH 7.0. Hybridized tissue sections were finally exposed to /3-MAX autoradiographic films (Amersham, UK) at 70°C with intensifying screens, or dipped in autoradiographic emulsion NTB-3 (Kodak). After 3-4 weeks, dippings were developed with Kodak D-19 and counterstained with Cresyl violet.
i '
iii
Fig. 1. Developmental pattern of LNGFR (A, D, G, J), NT3 (B, E, H, K) and BDNF (C, F, I, L) mRNAs in the rat cerebellum at postnatal ages P5 (A, B, C), P10 (D, E, F) P15 (G, H, I) and P20 (J, K, L). Pictures are photomicrographs from emulsion autoradiograms, obtained with dark-field ilumination. Bright areas are rich in autoradiographic grains. Different intensities in hybridization signal along different postnatal ages, for each individual oligonucleotide probe, are indicative of different mRNA levels, since pictures were taken from autoradiograms of a single experiment exposed for the same amount of time. Note in A a high density of hybridization signal for LNGFR in EGL and in Purkinje cell layer (PCL). LNGFR hybridization signal is present in EGL but not in IGL (A, D, G, J). There is a high density of NT3 hybridization signal in IGL at early postnatal ages (B) decreasing afterwards (E, H, K). BDNF hybridization signal is practically undetectable until the second postnatal week (C, F, I, L). EGL, external granule cell layer; ML, molecular layer, PCL, Purkinge cell layer; IGL, internal granule cell layer. Bar = 1 mm.
4 RESULTS In situ hybridization histochemistry with oligonucleotide probes complementary to BDNF, NT3 and L N G F R m R N A sequences was performed on 20 /xm cryosections from postnatal developing rat cerebellum. The specificity of the hybridization signal at the different postnatal ages was confirmed using several criteria, as described in our previous work 44. Briefly: (a) for each m R N A two different oligonucleotides were used, as independent hybridization probes in consecutive sections, showing an identical pattern of hybridization, (b) for each labeled probe the addition of an excess of the same unlabeled oligonucleotide, resulted in the complete elimination of the specific hybridization signal, showing background levels, and finally (c) hybridization patterns obtained with oligonucleotide probes, similar in length and base composition, but specific for each BDNF, NT3 and L N G F R m R N A s were clearly distinct among them. Three different postnatal development series were studied. In order to compare the hybridization level for each oligonucleotide probe among the different postnatal ages, sections from the postnatal ages (P1, P5, P10, P15, P20, adult) were used in each individual experiment, with the same labeling, hybridization, washing and exposure conditions.
The specific hybridization signal obtained with probe b d n f / 2 was undistinguishable from the unspecific background (not shown) in the cerebellar cortex during the first two postnatal weeks, but it increased later in the I G L reaching a peak around P20 (Fig. 1C,F,I,L). In contrast, B D N F m R N A was early and transiently present in the deep cerebellar nuclei (from P5 to 15), being afterwards undetectable in adult animals (Fig. 3). High levels of B D N F m R N A were observed in the inferior olivary complex from the first postnatal ages, with a maximum at P10 and decreasing afterwards, being still present in the adult inferior olivary complex, but at lower levels than in younger animals (Fig. 4). Hybridization with n t 3 / 2 oligonucleotide probe revealed the presence of high levels of NT3 m R N A as early as P5 in the I G L (Fig. 1). Moderate levels of NT3 m R N A were also present in the most internal part of the premigratory area of the E G L at P10 (Figs. 1 and 2). Concommitant with the granule cell migration and maturation processes, NT3 m R N A levels decreased. Low levels of NT3 m R N A could be observed in the I G L in adult animals. In contrast to BDNF, NT3 m R N A was not detected neither in the deep cerebellar nuclei, nor in the inferior olivary complex. High levels of L N G F R m R N A were found, from P5 on, in the proliferative area of the E G L and in the Purkinje cell layer. However, L N G F R m R N A was not
Fig. 2. LNGFR and the NT3 mRNA expression in the cerebellar cortex layers of P10 rat. Pictures are high-magnificationphotomicrographs from emulsion autoradiograms, obtained with dark-field ilumination, LNGFR mRNA (A) and NT3 mRNA (B); and a bright-field photomicrograph corresponding to tissue in B, counterstained with Cresyl violet (C). Note in A that LNGFR hybridization signal is present in the proliferative area (PR) of the EGL (the most external) and in the Purkinje cells layer (PCL). Note in B the presence of NT3 hybridization signal in the IGL and in the most internal part (PM) of the EGL. EGL, external granule cell layer, PR, proliferative area of the EGL; PM, premigratory area of the EGL: ML, molecular layer; IGL, internal granule cell layer; PCL, Purkinje cell layer. Bar = 400 p~m.
Fig. 3. Localization of BDNF mRNA in deep cerebellar nuclei during postnatal rat development. Pictures are photomicrographs from emulsion autoradiograms, obtained with dark-field ilumination. Note that BDNF mRNA was present at P5 (A) while it was undetectable at P20 (B). Light in choroid plexus is an artifact of the dark-field illumination and does not correspond to hybridization signal, ch. pl., choroid plexus; DCN, deep cerebellar nuclei. Bar = 1 mm.
observed neither in the premigratory area of the E G L nor in the IGL (Figs. 1 and 2). At P20 L N G F R m R N A was practically only detectable in the Purkinje cell layer. Moreover, it was heterogeneously distributed, being mainly restricted to the posterior cerebellar lobules (Figs. 1 and 5). The inferior olivary system displayed high levels of hybridization with L N G F R oligoprobes at P5 decreasing thereafter and been practically undetectable in adult animals (Fig. 4). DISCUSSION The main finding of this study is the demonstration of a sequential expression of NT3 and BDNF during the postnatal migration and maturation of the cerebellar granule cells. NT3 was found to be expressed in the IGL as early as P5 and in the most internal part of the premigratory area of the E G L at P10. BDNF mRNA, in contrast, was undetectable in E G L and it was detected in the IGL only from the second postnatal week onwards. These results agree with previously published Northern analyses data on the levels of these two neurotrophic factors 38, and provide new information
about the anatomical localization of BDNF and NT3 transcripts in the developing cerebellum. The presence of NT3 mRNA, both, in the most internal part of the E G L and in the IGL of immature cerebellum suggest that granule cells express NT3 concommitantly with the beginning of their migration. NT3 could play a role in the granule cell migration a n d / o r in the maturation of its synaptic contacts. Such effects could be mediated through L N G F R a n d / o r trkB, present in the Purkinje cell 25. Moreover, NT3 secreted by migratory granule cells may also act as a trophic factor taken up by climbing fibres from the olivary neurons. These cells express L N G F R around P5 and reach their synaptic maturity at a later stage 7. In contrast, BDNF expression by granule cells begins after their arrival into the IGL, which could indicate a trophic role for BDNF over cells sending afferents (mossy fibres) to cerebellar granule cells 18. In summary, these data support an early role for NT3 in the postnatally maturing cerebellar granule cells, may be involved in their migration a n d / o r the establishment of their synaptic contacts, while BDNF might play a late role, possibly as a maintenance factor, as it has been previously suggested 38. Similar results obtained in the postnatally maturing granule cells of the dentate gyrus of the hippocampus, where NT3 was expressed earlier (from P1 on) than B D N F (from P5 on) (data not shown, Friedman et al.~5), support a more general role for NT3, not only in the cerebellar but also in the hippocampal maturing granule cells. Moreover, the presence of B D N F m R N A mainly in the external part of the granule cell layer of the dentate gyrus, observed by us (data not shown) and other authors 16 also supports a later role of BDNF in granule cells, given the external-internal gradient of maturity in this cell layer 6. From P1 to P10, and in parallel with granule cell migration and maturation, Purkinje cells develop their dendritic tree and mature their synaptic contacts. High levels and widespread distribution of L N G F R mRNA in Purkinje cells during this period, observed by us (Fig. 1) and other authors 29'47, suggest a role of N G F a n d / o r the other neurotrophins (BDNF and NT3) in the maturation of these cells. In addition, the persistence of L N G F R in Purkinje cells of adult rats 42 suggests a role of these neurotrophins also in the mature cerebellum. Our results show that at late postnatal stages (from P20), as well as in adult rats, L N G F R m R N A is mainly located in the Purkinje cells of the posterior cerebellar lobules, which extends the previously published results from Mufson et al. 4°, showing L N G F R immunoreactivity in the lobules belonging to archi- and neo-cerebellum. These results demonstrate
2 5
.
:..
F
Fig. 4. Localization of LNGFR (A, C, D) and BDNF (B, E, F) mRNAs in the rat inferior olivary system at different postnatal rat development ages (P,5: A, B; P15, C, D; P20: E, F). Pictures are photomicrographs from autoradiographic films• Hybridized sections for P5 and PI5 correspond to plates 72-73 (A, B, C, D) and for P20 to plates 70-71 (E, F) of the Paxinos and Watson stereotaxic atlas41. There is a clear decrease in the levels of LNGFR mRNA from P5 to P20 (A, C, E). Although high levels of BDNF hybridization signal are present at P5 and PI5, this mRNA is still abundant at P20 (B, D, F) and it remains present in adult ages (not shown). HN, hypoglossal nucleus: IO, inferior olivary system; IOA, medial nucleus, subnucleus A; lOB, medial nucleus, subnucleus B; IOC, medial nucleus, subnucleus C; IOK, medial nucleus, cap kooy; IOBe,/3 subnucleus, IOD, dorsal nucleus. Bar - 1 ram.
a h e t e r o g e n o u s r e g u l a t i o n of L N G F R expression in the different cerebellar regions. Moreover, it is well known that the cerebellar biochemical cytoarchitecture is not h o m o g e n e o u s as d e m o n s t r a t e d by p a t t e r n s of enzymes a n d n e u r o t r a n s m i t t e r receptors 8"'. With regard to this point, it will be of interest to analyze if the differential d i s t r i b u t i o n of L N G F R is an i n d e p e n d e n t evolutionary fact or if it is also related to the p r e s e n c e of specific n e u r o t r a n s m i t t e r circuits. Interestingly, Purkinje cells
have b e e n f o u n d to r e s p o n d in an additive m a n n e r to N G F a n d excitatory n e u r o t r a n s m i t t e r s t|. N e u r o t r o p h i c factors may also play a role d u r i n g the m a t u r a t i o n of the olive-Purkinje cells-deep cerebellar nuclei pathway, as suggested by the p r e s e n c e of B D N F m R N A in the deep cerebellar nuclei. T h e inferior olivary nuclei express high levels of B D N F m R N A d u r i n g the first two weeks after birth, which may act as a trophic factor for the afferent n e u r o n s projecting to
7
•
developing rat cerebellum. Alternative trophic functions for different neurotrophins have been suggested. Moreover, our results suggest the possibility of a coordinated up- and down-regulation of BDNF and NT3 genes, respectively, as well as the possibility of a local delivery and action of neurotrophins. Further studies on the expression of trk family of high affinity neurotrophin receptors together with the use of animal models of abnormal cerebellar development (i.e. cerebellar mutant or neurotoxin-treated animals) and in vitro analyses of cerebellar systems may help to understand the physiological role of these trophic molecules in the developing nervous system. Acknowledgements. N.R. was supported by a postdoctoral fellowship from the Comissi6 Interdepartamental de Recerca i Innovaci6 Tecnolbgica (C.I.R.I.T.) of the Generalitat de Catalunya.
ABBREVIATIONS
Fig. 5. Differential localization of LNGFR mRNA in the cerebellar cortex at different postnatal ages. Pictures are photomicrographs from autoradiographic films. Note in A (P10) the presence of LNGFR mRNA in all the PCL (Purkinje cell layer), whereas in B (P20) LNGFR mRNA was mainly restricted to the PCL of posterior cerebellar lobules. Bar = 2 mm.
the olivary system. Moreover, considering the possibility of an anterograde transport, BDNF expressed in the inferior olivary nuclei, could also act on maturing Purkinje cells, given the widespread presence of LNGFR in these cells together with their polyinnervation by the olivary climbing fibres, until the characteristic monoinnervation of maturity is reached ~2. Although it is generally accepted that neurotrophic factors function in the target synthesis/retrograde transport w a y 46, the presence of both NGF and its receptor in the same brain areas 32'37 led to the proposal of local, in addition to distant effect for NGF. In this sense, it is noteworthy to stress the transient expression of LNGFR by EGL stem cells, since these cells are neither targets nor send efferents to any known system. Furthermore, NT3 expression in the EGL, along with the presence of LNGFR and trkB in the Purkinje cell dendritic tree, also suggest a local mode of action of this neurotrophic factor. In conclusion, the results in the present work show a specific and developmentally regulated expression of NT3, BDNF and LNGFR in different cell types of the
BDNF ch. pl. DCN EGL HN IGL IO IOA IOB IOBe IOC IOD IOK ML NGF LNGFR NT3 NTF P PCL PM PR trkA, trkB
brain-derived neurotrophic factor choroid plexus deep cerebellar nuclei external granule cell layer hypoglossal nucleus internal granule cell layer inferior olivary system inferior olive, subnucleus A of medial nucleus inferior olive, subnucleus B of medial nucleus inferior olive, beta subnucleus inferior olive, subnucleus C of medial nucleus inferior olive, dorsal nucleus inferior olive, cap kooy, medial nucleus molecular layer nerve growth factor low affinity nerve growth factor receptor neurotrophin-3 neurotrophins postnatal day Purkinje cell layer EGL, premigratory area EGL, proliferative area components of the trk family of tyrosine kinase receptors
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