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Localization of low affinity nerve growth factor receptor in the rat inferior olivary complex during development and plasticity of climbing fibres 1 1 Published on the World Wide Web on 31 January 2001

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Localization of low affinity nerve growth factor receptor in the rat inferior olivary complex during development and plasticity of climbing fibres 1 1 Published on the World Wide Web on 31 January 2001
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  Developmental Brain Research 126 (2001) 229–239www.elsevier.com/locate/bres Interactive report Localization of low affinity nerve growth factor receptor in the ratinferior olivary complex during development and plasticity of climbing 1 fibres b a a, *Megan Nitz , Adrian J. Bower , Rachel M. Sherrard a  Developmental Neuroplasticity Laboratory ,  Department of Anatomy and Pathology ,  School of Medicine ,  James Cook University ,  Townsville , Queensland   4811,  Australia b School of Life Sciences ,  Queensland University of Technology ,  Brisbane ,  Queensland  ,  Australia Accepted 18 January 2001 Abstract The rat olivocerebellar pathway has a precise topography from an inferior olive (IOC) to Purkinje cells in the contralateralhemicerebellum. While its development and plasticity have been documented, the molecular mechanisms underlying these events are notfully elucidated. Neurotrophins are a family of growth factors with diverse roles in development and neuronal plasticity, acting through atwo-receptor system, including a low affinity receptor (LNGFR) which binds all neurotrophins with similar affinity. Since neurotrophinsare present in the cerebellum during early postnatal development when LNGFR is synthesized in the IOC, they may act as target-derivedtrophic agents for climbing fibres during development and plasticity. To assess this, standard immunohistochemistry was used todocument the distribution of LNGFR in the rat IOC during climbing fibre development and until cerebellar development was complete atpostnatal day 28 (P28). LNGFR immunoreactivity (LNGFR-IR) was detected in the IOC from P0 until P15, however after P7 itdiminished in intensity and distribution, a change which indicates a relationship between cerebellar neurotrophins and climbing fibredevelopment. After denervation of the left hemicerebellum, there was an apparent increase in inferior olivary LNGFR-IR that wasconcurrent with climbing fibre re-innervation. Thus the results of this study support the hypothesis that neurotrophins are involved inclimbing fibre development and suggest a possible contribution to the plasticity of the olivocerebellar pathway.  ©  2001 Elsevier ScienceB.V. All rights reserved. Theme :   Development and regeneration Topic :   Neurotrophic factors: biological effects Keywords :   LNGFR; Inferior olivary complex; Climbing fibres; Plasticity; Development; Neurotrophins 1. Introduction  cerebellum via the inferior cerebellar peduncle and synaps-ing in parasagittal zones on Purkinje cells of the contrala-Motor co-ordination and motor learning are important teral hemicerebellum [8,12]. Although key developmentalfunctions of the cerebellum [27]. Normal cerebellar func- stages have been recognized for IOC neurons and climbingtion requires climbing fibre input from the inferior olivary fibre–Purkinje cell synaptogenesis, the molecular mecha-complex (IOC) [25]. The olivocerebellar projection has a nisms underlying climbing fibre development remain poor-well defined topography with climbing fibre axons crossing ly defined.the midline in the medulla oblongata, ascending to the Since target-derived trophic agents are known to facili-tate axonal growth [35,55], it is possible they are involvedin climbing fibre development. One important group of  1 Published on the World Wide Web on 31 January 2001. such factors is the neurotrophin family [22,51]. Four of  *Corresponding author. Tel.:  1 61-7-4781-5022; fax:  1 61-7-4781- these, nerve growth factor (NGF) [29], brain-derived 6986.  E  - mail address :   rachel.sherrard@jcu.edu.au (R.M. Sherrard).  neurotrophic factor (BDNF), neurotrophin-3 (NT-3) [31] 0165-3806/01/$ – see front matter  ©  2001 Elsevier Science B.V. All rights reserved.PII: S0165-3806(01)00100-6  230  M  .  Nitz et al .  /   Developmental Brain Research  126 (2001) 229  – 239  and neurotrophin-4/5 (NT-4/5) [37], are synthesized in fibre–Purkinje cell synaptogenesis [20]. In the presentthe developing cerebellum at the same ages that both the study using immunohistochemistry, the distribution of low affinity (LNGFR) [28,42] and high affinity  Trk   [40,41] LNGFR was mapped in the inferior olive throughoutneurotrophin receptors are synthesized in the inferior olive. postnatal development in normal and pedunculotomizedThis may indicate a possible interaction between the rats.cerebellar neurotrophins and climbing fibre development.Since neurotrophins can be delivered either anterogradelyby afferent axons [46,53] or retrogradely from the target  2. Materials and methods tissue [47], the presence of neurotrophin receptors in theinferior olive does not automatically indicate responsive-  2.1.  Animals ness to cerebellar (vs. afferent delivered) neurotrophins. Toconfirm any role of cerebellar neurotrophins in climbing All experiments were carried out under license of thefibre development, olivary expression of neurotrophin Queensland University of Technology University Researchreceptors must be studied not only during normal climbing Ethics Committee and were in accordance with the Euro-fibre development but also in animals in which the normal pean Communities Council directive for animal proce-development has been perturbed, e.g. when a new climbing dures. Pregnant Wistar rats were obtained from the centralfibre pathway has been induced to grow. animal breeding house at the University of Queensland andIn the rat, climbing fibre axons reach the cerebellar housed under standard conditions of 12 h light/12 h dark anlage by embryonic day 17 (E17) [15] and there are a few cycles. The day of birth corresponds to P0. Groups of fourimmature synapses on Purkinje cells by birth (P0) [16]. By to seven pups aged 0, 3, 7, 10, 15, 21 or 28 days were usedpostnatal day 3 (P3), Purkinje cell responses to climbing to document the spatio-temporal distribution of LNGFR infibre stimulation have begun [20,32] through temporary the IOC during normal development.synapses on perisomatic processes [1,34]. It is between P7and P10 that climbing fibres ascend to their permanent  2.2.  Transection of climbing fibres sites on the Purkinje cell primary dendrites [1,34]. FromP10 to P15, climbing fibre terminals regress to the adult In order to induce the growth of a new climbing fibreconfiguration when there is only one climbing fibre per pathway, neonatal rats of either sex were anaesthetizedPurkinje cell [20,32]. In addition to their normal develop- with diethyl ether (BDH, UK) and underwent unilateralment, the rat climbing fibre projection is also capable of transection of the inferior cerebellar peduncle [10]. Sixdevelopmental plasticity. Following unilateral destruction litters of 10–12 pups were used, from which five pupsof climbing fibres by inferior cerebellar pedunculotomy underwent pedunculotomy and the remaining five were[10] early in development, the contralateral IOC degener- used as sham-operated controls. There were two ex-ates and new climbing fibre projections arise from the perimental groups: one lesioned aged 3 days, which isremaining IOC to re-innervate the denervated hemicerebel- followed by climbing fibre plasticity, and a secondlum [3,4,45,56]. However, plasticity is limited to the first lesioned at P10, an age when no compensatory projection10 postnatal days [45]. The compensatory projection arises is produced [45]. This allowed documentation of anymainly, but not exclusively, from neurons of the medial changes in LNGFR distribution during plasticity and whenaccessory olive (MAO) [4,45] and re-establishes normal plasticity was lost due to advanced maturation. After fullolivocerebellar topography within a few days of the lesion recovery from the anaesthetic in a warm box, the animals[56]. were returned to the dam. Animals survived for 24 h, 48 hTherefore, in order to assess the involvement of cerebel- or 4 days (5 days for the 10-day animals) post-operation.lar neurotrophins in climbing fibre development, thespatio-temporal distribution of neurotrophin receptors in  2.3.  Tissue preparation the IOC at ages critical to climbing fibre development andplasticity needs to be documented. Although olivary At the endpoint for both the normal and post-operativesynthesis of LNGFR [28,42] and the  Trk   receptors [40,41] experimental animals, they were euthanazed with anare high in the first two postnatal weeks, there is in- overdose of diethyl ether and perfused transcardially withsufficient data on the spatio-temporal distribution of these an age appropriate volume, 10–20 ml, of heparinizedreceptors in the inferior olive to confirm any specific role saline (5 IU/ml) and 50–200 ml of Bouin’s fixative (2%of cerebellar neurotrophins in normal climbing fibre de- picric acid, 5% glacial acetic acid and 10% formaldehyde).velopment. As LNGFR is capable of binding all neuro- The cerebellum and brainstem were removed, dehydratedtrophins with similar affinity [38,43] the presence of and processed to wax. Two parallel sets of coronalLNGFR is indicative of neurons responsive to any of the sections, 10  m m thick, were taken every 100  m m through-neurotrophin family. Previous studies on olivary LNGFR out the rostrocaudal extent of the IOC and cerebellum andhave been limited to very few developmental ages [54], mounted on Vectabond (Vector Laboratories, CA, USA)which do not correspond to those important for climbing coated slides.   M  .  Nitz et al .  /   Developmental Brain Research  126 (2001) 229  – 239   231 2.4.  Immunohistochemical analysis  and each area contained both labelled and unlabelled cells.Instead, each subnucleus containing neurons with LNGFR-LNGFR was detected immunohistochemically on one IR was noted, with frequency of labelling being defined asset of sections using either of two different antibodies: (1) scattered or numerous and intensity of immunoreactivitymouse monoclonal antibody 192-IgG (Alomone Labs, defined as weak, moderate or strong. This allowed theIsrael), which has been documented as specific for the rat spatio-temporal distribution of the LNGFR-IR location inLNGFR [14], and (2) rabbit polyclonal anti-human p75 the IOC to be determined both during normal develop-(Promega, USA). Immunohistochemistry was performed so ment, during generation of new climbing fibre efferentsthat both antibodies were used on tissue from each age/ after pedunculotomy and when new projections do notexperimental group and that tissue from more than one arise following climbing fibre transection, due to advancedage/experimental group was processed at any one time. maturation. To ensure consistency of interpretation, allThe sections were de-waxed and rehydrated before non- analyses were performed by two authors (MN and RS),specific binding was blocked by 30 min incubation with who did not know the group to which the animal belonged.20% normal swine serum in 0.1 M phosphate buffered In addition, as neurotrophin receptors are transportedsaline (PBS, pH 7.4) with 1% bovine serum albumin from neuronal somata to axon terminals [51], representa-(BSA; Sigma, USA) (PBS 1 BSA). Sections were rinsed in tive sections from the cerebellum of P0, P3, P7 and P10PBS and then incubated overnight at 4 8 C with primary animals were also examined to determine whetherantibody diluted 1:200 for 192-IgG or 1:250 for p75 in LNGFR-IR in the cerebellum was anatomically consistentPBS 1 BSA. The following day the tissue was rinsed in with climbing fibre terminals. The cerebellar distributionPBS three times, followed by 20 min incubation at room of LNGFR-IR was also investigated in animals whichtemperature with biotinylated goat anti-mouse antibody underwent left unilateral pedunculotomy at P3, to see if (Dako, Carpenteria CA, USA) diluted 1:600 in PBS 1 BSA any visible climbing fibre terminals were removed fromfor 192-IgG, or 30 min incubation with biotinylated goat the left hemicerebellum.anti-rabbit antibody (Dako) diluted 1:400 in PBS 1 BSA for Sections were photographed using a Nikon microscopep75. Sections were rinsed in PBS and endogenous per- fitted with a Nikon HC300 digital camera. Images wereoxidase activity was quenched by incubation with 0.3% imported into Adobe Photoshop 5.2 and converted tohydrogen peroxide in methanol for 30 min. The tissue was greyscale for printing.rinsed and treated with a preparation of avidin–biotin–peroxidase complex (Vectastain ABC elite kit, VectorLaboratories) for 30 min and rinsed in PBS. The antibody–  3. Results peroxidase conjugate was visualized using 0.06% 3,3-diaminobenzidine in PBS with 0.01% hydrogen peroxide  3.1.  Specificity of the antibody reaction for 4 min. Following dehydration, slides were mounted inDePeX (BDH) and coverslipped. Controls for antibody LNGFR-IR was demonstrated in the IOC until P15.specificity were the omission of primary antibody and lack Immunoreactivity was consistent for both antibodies usedof immunoreactivity with a tissue known to be LNGFR (192-IgG and p75) and reproducible for animals withinnegative (P0 spleen) [30]. each age group. Antibody specificity was indicated byabolition of LNGFR-IR when the primary antibody was 2.5.  Histological analysis  omitted and lack of detectable LNGFR-IR in tissue whichis known to be negative for LNGFR; P0 rodent spleenFor histological analysis, the second set of sections were [30].rehydrated and stained with Harris’s haematoxylin,mounted in DePeX (BDH) and coverslipped. Specific  3.2.  Anatomical distribution of LNGFR -  IR in the inferior  criteria were used to assess the success of the surgery for  olive during normal development  pedunculotomy: (1) histological examination of the lesionsite and (2) evidence of neuronal degeneration within the In the IOC, neurons possessed LNGFR-IR between P0axotomized right IOC [7]. Only those animals which and P15. The staining pattern was identical in both olives,fulfilled both of these criteria were used for subsequent being symmetrical about the midline. In every develop-analysis. mental age group the IOC contained a mixture of neuronsA semi-quantitative assessment of the distribution of displaying LNGFR-IR and unlabelled neurons (Fig. 1c).LNGFR-like immunoreactivity (LNGFR-IR) was per- Although from P0 to P15 all areas of the IOC containedformed by examination of the sections using light micro- neurons displaying LNGFR-IR, the distribution of neuronsscopy ( 3 10 or  3 25 objective). At all experimental ages, and intensity of labelling was not uniform.the distribution of LNGFR-IR from rostral to caudal IOC The distribution and intensity of LNGFR-IR was identi-was mapped onto outlines of the IOC. Individual neurons cal in all animals in the P0 ( n 5 5), P3 ( n 5 6) and P7were not mapped as there were too many neurons to count ( n 5 7) groups. All subnuclei of the IOC [medial accessory  232  M  .  Nitz et al .  /   Developmental Brain Research  126 (2001) 229  – 239  Fig. 1. LNGFR immunoreactivity in the inferior olive between P0 and P7 is present in both inferior olives, being symmetrical about the midline. (a) At day0 in the caudal MAO, LNGFR-IR is differentially distributed, being intense in subnuclei ‘a’ and ‘c’, weak in subnucleus ‘b’, but was not detected in thedorsal cap (dc). Bar 150  m m. (b) In the midrostral olive at birth, LNGFR is intense in the MAO, including its ventrolateral outgrowth (vlo), but only weak in the PO and DAO. Also in the MAO the neuropil is immunoreactive for LNGFR. Bar 130  m m. (c) At higher magnification it can be seen that not allinferior olive neurons are immunoreactive for LNGFR. In the DAO and PO (below) from an animal at P7, there is a mix of both positive (black arrows)and unlabelled (white arrows) neurons. Bar 20  m m.   M  .  Nitz et al .  /   Developmental Brain Research  126 (2001) 229  – 239   233 olive (MAO), principal olive (PO) and dorsal accessoryolive (DAO)] contained neurons displaying LNGFR-IR,but within the MAO there was differential labellingbetween its subdivisions. In the caudal MAO, almost allneurons displayed LNGFR-IR. Subnuclei ‘a’ and ‘b’ of thehorizontal lamella and ‘c’ and nucleus beta (n b ) of thevertical lamella were positive, although the dorsal cap (dc)was not labelled (Fig. 1a). Differential labelling intensitywas noted between the neurons of subnuclei ‘a’, ‘c’ andn b , which were strongly labelled, and subnucleus ‘b’,which displayed weak LNGFR-IR (Fig. 1a). Also, in themid-rostral MAO the neurons of the ventrolateral out-growth (vlo) were strongly labelled, while the dorsomedialcell column (dmcc) was negative. The majority of neuronsof the rostral lamella of the MAO were strongly positive.Furthermore, in the subdivisions displaying positive neu-rons, the MAO demonstrated strong neuropil LNGFR-IR(Fig. 1b). Neurons of the PO and DAO displayed weak LNGFR-IR, with scattered positive neurons being random-ly distributed throughout these subnuclei (Fig. 1b).From P10 ( n 5 5) onwards, only a few cells werepositive in the IOC with apparent decreasing numbers of immunoreactive neurons with increasing age and loss of neuropil staining from the entire rostrocaudal length of theIOC. By P15 ( n 5 5), the IOC displayed only very weaklypositive neurons distributed throughout the entire ros-trocaudal extent of the IOC (Fig. 2a) and at P21 ( n 5 4)and P28 ( n 5 4) all neurons of the IOC were negative. Thedistribution of LNGFR-IR within the IOC is summarizedin Fig. 2b. 3.3.  Inferior olivary complex LNGFR -  IR after left inferior cerebellar pedunculotomy at P 3  These results describe only the left inferior olive as theaxotomized right inferior olive degenerates post-pedun-culotomy [7].Twenty-four hours post-pedunculotomy on day 3 thepattern of LNGFR-IR was identical in both pedunculotom-ized ( n 5 5) and sham-operated controls ( n 5 4). Further-more, the distribution of immunoreactivity was the same asdescribed above for P0–P7 animals. Forty-eight hourspost-pedunculotomy, the pattern of LNGFR-IR in operated( n 5 5) animals was different from controls ( n 5 5): thereappeared to be a greater number of LNGFR-IR neurons in Fig. 2. The distribution of LNGFR-IR in the inferior olive duringdevelopment. (a) The right inferior olive at P15. Weakly LNGFR  the left IOC (compare Fig. 3a and b). This change was immunoreactive neurons (arrows) are scattered in all divisions of the most evident in the mid-rostral PO and DAO. The majority inferior olive. The midline is indicated by the vertical line. Bar 50  m m. of neurons of the right IOC were degenerating. In addition, (b) A diagram showing a series of coronal sections through the inferior in this experimental group, in contrast to the 24 h survival olive (adapted from Buisseret-Delmas and Angaut [11]) showing the group and control animals, no differential labelling was distribution of LNGFR-IR in the inferior olive. Between P0 and P7 (leftcolumn) there is LNGFR-IR in all divisions with the MAO being strongly  noted between subnuclei ‘a’, ‘b’ or ‘c’ or n b  of the MAO labelled (black shading) and the PO and DAO weakly–moderately as the majority of neurons and neuropil were strongly labelled (medium shading). From P10 (right column illustrating P15), positive in all subnuclei. However, the neurons of the dc LNGFR immunoreactivity is weak but still distributed throughout the and dmcc remained negative. olivary divisions. a, subnucleus ‘a’; b, subnucleus ‘b’; c, subnucleus ‘c’; As previously described [7], 4 days after pedunculotomy DAO, dorsal accessory olive; MAO, medial accessory olive; n b , nucleusbeta; PO, principal olive; vlo, ventrolateral outgrowth.  the right IOC had completely degenerated. In the remain-
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