Expression of Brain-Derived Neurotrophic Factor and TrkB NeurotrophinReceptors after Striatal Injury in the Mouse

J. Y. F. Wong, G. T. Liberatore, G. A. Donnan, and D. W. HowellsDepartments of Neurology and Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg,

Victoria 3084, Australia

Received April 14, 1997; accepted August 12, 1997

Brain-derived neurotrophic factor (BDNF) promotesthe survival and differentiation of nigral dopaminer-gic neurons and supports the activity of dopaminergiccells grafted into the striatum. However, little atten-tion has been given to the physiological role of endog-enous BDNF and its receptor TrkB within the nigro-striatal dopamine system. We know that striatal injuryis followed by long-term stimulation of dopaminergicactivity in the striatum, could BDNF play a role in thisphenomenon? One week after physical injury to thestriatum of C57/Black mice, just before dopaminergicactivation becomes obvious, in situ hybridization oncoronal sections through mouse striatum reveals thatBDNF mRNA expression increases significantly beforereturning to basal levels within 1 month. Expression ofmRNA for TrkB follows a very different pattern. Nochange of expression of the full-length and catalyti-cally competent TrkBTK1 receptor is seen. However,expression of the truncated form of the receptor TrkTK2,which lacks the catalytic tyrosine kinase domain, doesincrease and stays elevated for at least 2 months afterinjury. When combined with observations of dopamin-ergic activation after striatal injury and the neuropro-tective effects of BDNF introduced into the striatum,our findings suggest that BDNF and TrkBTK2 do indeedplay a role in dopaminergic regeneration andrepair. r 1997 Academic Press

Key Words: brain-derived neurotrophic factor; TrkB;striatum; injury; dopamine

INTRODUCTION

Until recently the central nervous system was thoughtto have little capacity for regeneration and repair.However, there is now abundant evidence to the con-trary and many of the neurotrophic and growth factorswhich regulate brain development also play an impor-tant role in neural regeneration. Brain-derived neuro-trophic factor (BDNF), a member of the nerve growthfactor gene family (18), and its receptor TrkB maycontribute to this role in a number of key neuronal

pathways, of which the nigrostriatal dopaminergicsystem is of particular interest.

Degeneration of nigrostriatal dopaminergic neuronsand loss of dopamine terminals in the striatum plays akey role in the motor disturbances of idiopathic Parkin-son’s disease (14). However, it appears that more than50% of these neurons must be lost before symptomsbecome apparent (1). Either we are blessed with anoversupply of neurons in this pathway or the survivingdopamine neurons compensate for the loss of theirneighbors. After physical injury one compensatorymechanism available to these neurons may be sprout-ing of new processes within the striatum (34) with aconcomitant increase in tyrosine hydroxylase activity(22) and immunoreactivity (15), increased concentra-tions of dopamine and its metabolites, dihydroxyphen-ylacetic acid and homovanillic acid (22), and an in-creased density of dopamine uptake sites (21, 48).Based on data from patients with Parkinson’s disease itseems likely that these compensatory mechanisms mayalso occur in the human brain. For example, humanautopsy studies suggest that adrenal medullary im-plants in patients with Parkinson’s disease can inducesprouting of host tyrosine hydroxylase-immunoreactivefibers, even when there is poor survival of the graft (19,31).

A wealth of information suggests that BDNF has theability to play an important role in this regenerativeprocess. We know that messenger RNAs (mRNAs) forBDNF and TrkB are present in both the substantianigra and the striatum (8, 20, 35, 36, 50, 56) and thatnigral dopaminergic neurons have the ability to bothproduce (56) and transport (4) BDNF. Furthermore,application of exogenous BDNF stimulates dopaminer-gic activity in models of Parkinson’s disease both invitro (10, 54) and in vivo (2, 3) and protects dopaminer-gic neurons against 6-OHDA and MPTP toxicity (24,33, 53). Importantly, in retinal ganglion cells there isclear evidence that application of exogenous BDNFstimulates axonal sprouting and regeneration (39, 52).However, we know very little about the role of endog-enous BDNF within the nigrostriatal dopamine sys-

EXPERIMENTAL NEUROLOGY 148, 83–91 (1997)ARTICLE NO. EN976670

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tem. Does locally produced BDNF mediate the neuro-nal sprouting and dopaminergic activation seen afterstriatal injury? It would certainly explain why exog-enous BDNF is able to stimulate the dopaminergicsystem. To examine this possibility we have used in situhybridization to determine whether the dopaminergicactivation associated with striatal injury is also accom-panied by increased expression of BDNF and TrkBmRNAs.

METHODS

Experimental Animals

Two groups of 5- to 6-week old (17–25 g) maleC57/Black/6J mice (28 control and 60 surgery) werekept on a 12-h light/dark cycle with free access to foodand water. The surgical group was anesthetised withintraperitoneal Nembutal (70 mg/kg, Boehringer Ingel-heim, Artarmon, NSW, Australia), followed by theadministration of atropine sulfate (0.5 mg/kg, ip; DeltaWest, Bently, Western Australia), and placed in a ratstereotaxic frame (Model 900, David Kopf Instruments,Tujunga, CA). A Scouten wire knife (Model 120, Blade121-A, David Kopf Instruments) was inserted into thestriatum via a 1-mm-diameter burr hole created overthe left hemisphere (1 mm anterior to Bregma, 2 mmfrom the midline, and 3 mm from the dura surface) andused to reproducably injure the striatum. After surgery,the skin was closed using silk thread and an antibioticcream containing bacitracin and neomycin applied tothe operation site. The age-matched control groupreceived no treatment.

Tissue Preparation

Mice from both groups were killed at 0 h, 6 h, 1 day, 3days, 1 week, 1 month, and 2 months postsurgery. Thebrains were removed and snap frozen in isopentanecooled in dry ice and then stored at 280°C before use. Aseries of 20-µm frozen coronal sections was cut using acryostat (Bright Instruments, Huntingdon, UK, Model5030) and then thaw mounted onto 3-aminopropyltrieth-oxysilane (Sigma Chemical Co., St. Louis, MO) coatedslides.

Oligonucleotide Probes

These were synthesized using a Bio-Rad PCR Matesynthesiser (Murdoch Institute, Melbourne, Australia)and detritylated overnight at 55°C in 25% NH3 beforedrying and dissolving in dH2O to give a stock concentra-tion of approximately 3 µg/ml. The antisense mouseBDNF sequence comprised the 50 bases located betweennucleotides 650 and 699 (58-AGTTCCAGTGCCTTTTGTC-TATGCCCCTGCAGCCTTCCTTGGTGTAAC-CC-38) fromthe sequence reported by Hofer and colleagues (20). For theTrkB detection, 45-base probes were based on the sequence

published by Klein and colleagues (29, 30). The oligonucleo-tide to detect only truncated TrkB (TrkBTK2) correspondedto nucleotides 1630–1674 (58-GTGAATCTAAG-TGTGT-TCTTCTGCTGCTTCTCAGCTGCCTGACCC-38), whilethe oligonucleotide used to detect only the catalytic,full-length TrkB (TrkBTK1) corresponded to nucleotides2781–2829 (58-GAAGGACTCTTCCCTGGGTGATG-CACTCTATCACCTCATTGTTCG-38). For each of theseoligonucleotides a complimentary ‘‘sense’’ oligonucleo-tide was synthesized for use as a control.

The oligonucleotide probes were end labeled using astandard kinase protocol (51) with [g-33P]ATP (Amer-sham International,Amersham, UK) and T4 polynucleo-tide kinase (New England Biolabs, Beverly, MA). Thelabeled oligonucleotides were separated from unincor-porated radiolabel by chromatography using S200 Mi-crospin columns (Pharmacia Biotechnology, Milwau-kee, WI) according to the manufacturer’s instructions.

In Situ Hybridization

Hybridization of 33P-labeled oligonucleotide probeswas performed in a drop of 20 mM sodium phosphatebuffer, pH 7.0 (containing 600 mM NaCl, 60 mMsodium citrate, 0.02% Ficoll, 0.02% bovine serum albu-min, 0.02% poly(vinylpyrrolidone), 10% dextran sul-fate, 0.1% degraded salmon sperm DNA, 1 mM dithio-threitol, and 50% deionized formamide), placed on thesection and incubated at 42°C for 18 h in a humidifiedcontainer. Sections hybridized for the different forms ofTrkB were then washed for 4 3 15 min in 13 SSC at55°C before dehydration in ethanol and drying at roomtemperature. For BDNF, the sections were washed for4 3 60 min.

Specificity of Binding

To determine whether the autoradiographic signalsseen using the antisense probes for BDNF, TrkBTK1,and TrkBTK2 represented specific binding to the targetsequences, the autoradiographs were compared withthose generated using the corresponding sense probes.As a further check, a 100-fold excess of unlabeledantisense oligonucleotide was added to the in situhybridization reactions to competitively inhibit probebinding. Pretreatment of sections with 20 µg/ml RNaseA (Boehringer Mannheim, Mannheim, Germany) wasused to demonstrate that labeled probes were bindingspecifically to RNAs.

Autoradiography

Autoradiographic detection was carried out by expos-ing the slides (together with laboratory-prepared 33Pstandards) to Hyperfilm (Amersham International) for1 week (BDNF) and 4 weeks (TrkB), respectively.

84 WONG ET AL.

Image Analysis

Quantitation was performed using a microcomputerimaging device (Imaging Research Inc., Brock Univer-sity, St. Catherine, Canada). In each section the dam-age site in the ipsilateral striatum was located bysuperimposing the corresponding thionin-stained sec-tion over the autoradiograph and densitometry wasperformed in a 0.2-mm wide band of tissue around thewound. Standardization was achieved by comparingbinding densities with a standard curve created fromthe 33P standard exposed with each film.

Statistical Analysis

The statistical significance of differences betweenand within groups was assessed using Student’s t testand a paired t test, respectively.

RESULTS

Distribution of BDNF, TrkBTK1, and TrkBTK2 mRNAsin Control Mice

The basal distribution of BDNF, TrkBTK1, and Trk-BTK2 mRNAs in coronal sections through brains ofnormal mice at the level of hippocampus and striatumare in agreement with other studies. We found thehighest BDNF expression in the hippocampus anddentate gyrus, followed by cerebral cortex and substan-tia nigra. In striatal sections BDNF expression wasgreatest in the cortex particularly the piriform cortex,ependymal linings of the ventricles, septohippocampalnucleus, lateral septum, medial preoptic area, andsuprachiasmatic nucleus, with relatively little expres-sion in the striatum under basal conditions (20, 45, 57).

The pattern of expression of TrkBTK1 was similar toBDNF with high levels in the hippocampus, dentategyrus, hypothalamic nuclei, cortex, and, particularly,piriform cortex (8, 17, 29, 30, 40). Very little expressionwas detected in the striatum under basal conditions.Expression of TrkBTK2 was similar to TrkBTK1 but withadditional expression in the choroid plexus and ependy-mal linings of the ventricles (29). When taken togetherwith the observation that pretreatment with RNase Aand the appropriate control (sense) probes includedwith every experiment did not give a signal in theseregions and that the antisense signal could be displacedby unlabeled antisense oligonucleotide, we believe thisprovides good evidence that the signals detected repre-sent specific signals for the BDNF, TrkBTK1, and Trk-

BTK2 mRNAs. Examples of the control experiments forBDNF are shown in Fig. 1; the results for the TrkBprobes are essentially the same but are not shown.

Expression of BDNF mRNA after Striatal Injury

In the striatum, BDNF mRNA expression is approxi-mately 70% of that seen in the overlying cortex withlittle variation along either the dorsoventral or rostro-caudal axis. Immediately after striatal surgery BDNFexpression in the injured hemisphere is unaltered andshows no variation either from the contralateral hemi-sphere (used as an internal control) or from the homo-typic region in untreated control animals. BDNF expres-sion remains unaltered for 3 days but after 1 week thedensity of probe binding increases from 120 to 180cpm/mm2 (P , 0.005) in the region of striatum sur-rounding the site of injury. After 1 month, BDNFexpression had returned to near control levels (Figs. 2and 5A).

Expression of TrkBTK1 mRNA after Striatal Injury

Using the oligonucleotide probe which detects onlyTrkBTK1, we see no change in expression followingsurgery (Figs. 3 and 5B).

Expression of TrkBTK2 mRNA after Striatal Injury

Expression of TrkBTK2 increases after striatal injury,peaks 1 week after surgery, and remains high for 2months (Figs. 4 and 5C). This increased expression issignificant when compared with the same region of theunoperated striatum in the contralateral hemisphere ofthe same animal and the homotypic region of thestriatum in unoperated controls (P , 0.005). The spa-tial distribution, however, is similar to BDNF, involvingmost of the striatum at 1 week but only a muchnarrower band around the wound after 2 months.Expression of TrkBTK2 also appears to increase in anumber of midline structures between 6 h and 1 weekafter striatal injury but this is not discussed furtherhere.

DISCUSSION

In culture, application of BDNF specifically supportsthe survival of tyrosine hydroxylase-immunoreactivemesencephalic cells (25) and doubles dopamine uptake(32). In vivo, application of BDNF to the striatumreduces apomorphine-induced contraversive rotations

FIG. 1. Autoradiographs showing expression of mRNA for BDNF at the level of the hippocampus using antisense oligonucleotide, senseoligonucleotide, and antisense oligonucleotide in the presence of 100-fold excess of unlabeled antisense oligonucleotide and after RNasepretreatment.

FIG. 2. Autoradiographs showing expression of mRNA for BDNF in uninjured controls and 6 h, 1 week, and 1 month after striatal injury.

85EXPRESSION OF BDNF AND TrkB AFTER STRIATAL INJURY

86 WONG ET AL.

87EXPRESSION OF BDNF AND TrkB AFTER STRIATAL INJURY

and loss of striatal dopaminergic terminals after 6-hydroxydopamine lesions to the nigrostriatal system(33, 53). Similarly, striatal injury stimulates a sprout-ing response that leads to increased dopaminergicfunction in normal, 1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine- and 6-hydroxydopamine-lesioned animals(7, 15, 21, 27, 34, 47, 48). Together these observationssuggest that the effects of exogenous BDNF may reflectstimulation of endogenous processes concerned primar-ily with the regenerative capacity of nigrostriatal dopa-mine neurones. If this is the case, induction of endog-enous BDNF expression would be expected after striatalinjury.

Using in situ hybridization we have found thatstriatal injury does indeed induce a marked increase ofBDNF mRNA expression in the striatum, suggestingthat local production of BDNF rather than transportedBDNF is responsible for mediating neuronal sproutingand dopaminergic activation. This induction, whichinitially involves a large proportion of the striatum,peaks 1 week after injury before contracting to involveonly a thin band of tissue immediately adjacent to thewound after 1 month with the overall density of probebinding returning nearly to normal (Figs. 2 and 5A).This is consistent with the recent Northern blottingexperiments by Plunkett and colleagues showing upreg-ulation of BDNF and CNTF mRNA expression aftertrauma to rat striatum (46). The finding that gel foamimplants removed from rat striatum provide trophicsupport for cultured dorsal root ganglia explants whichis inhibited by an anti-BDNF antibody (5) suggests thatthe increased BDNF mRNA expression detected in ourstudy is accompanied by increased production of BDNFprotein. Importantly, maximum trophic support is pro-vided by gel foam implants removed from the striatum1 week after trauma (5); at the same time we see themost extensive induction of BDNF mRNA expression.

The low basal levels of striatal BDNF mRNA expres-sion and the transient nature of the increase in BDNFmRNA coupled with ‘‘BDNF-like’’ trophic activity (5) inthe presence of a slow and long-term stimulation ofdopaminergic activity in response to injury (21, 22) arenot consistent with BDNF acting as a ‘‘classical’’ target-derived neurotrophin for dopaminergic neurons afterinjury. Instead, a triggering role would appear morelikely. Similar transient increases of BDNF expressionassociated with neurochemical and morphological plas-ticity (38) elsewhere in the CNS (6, 13, 23, 26, 37, 55)support this suggestion.

The TrkB protooncogene exhibits a complex transcrip-

tion pattern encoding a full-length catalytically activereceptor, TrkBTK1 (gp145TrkB) (28, 43), and a truncatedreceptor, TrkBTK2, which has the same extracellularand transmembrane domains as TrkBTK1 but has avery short cytoplasmic domain lacking the tyrosinekinase catalytic region (29, 43). These two transcriptsare differentially regulated after striatal injury. Usingtranscript-specific probes we saw no change in expres-sion of the catalytically competent TrkBTK1 after stria-tal injury (Figs. 3 and 5b). The noncatalytic TrkBTK2

shows a pattern initially very similar to BDNF, increas-ing rapidly after striatal injury and reaching its peak 1week after surgery in keeping with previous reports ofincreased total-TrkB (using probes which do not dis-criminate between TrkBTK1 and TrkBTK2) expressionafter central nervous system injury (16, 41, 44). How-ever unlike BDNF, the overall density of expressionthen remains high for at least 2 months although it alsobecomes concentrated close to the wound site (P , 0.005,Figs. 4 and 5C). Since TrkBTK1 transcripts are reportedto be preferentially located in neurons while TrkBTK2

transcripts are found in astrocytes and oligodendro-cytes (17), astrocytic activation could be responsible forincreased expression of TrkBTK2 after injury.

Similar increases of TrkBTK2 mRNA in associationwith axonal sprouting in the hippocampus (11) andspinal cord (16) have lead to conflicting suggestionsabout the role of TrkBTK2. One suggestion is thatTrkBTK2 plays a role in BDNF recruitment/presenta-tion during axon growth and/or regeneration (11). SinceBDNF occurs as a homodimer (49) it has been sug-gested that one subunit binds with a TrkBTK2 on glialcells and the other with TrkBTK1 on a growing axon/dendrite, prolonging the availability of BDNF to respon-sive axons/dendrites by acting as a local storage depot(11). Intuitively this seems a plausible explanation forour observations of BDNF and TrkBTK2 mRNA induc-tion in the presence of injury induced gliosis (notshown). An alternate suggestion is that TrkBTK2 isinvolved in ligand clearance (29), sequestering BDNFand inhibiting neurotrophin responsiveness. The domi-nant inhibitory effect of TrkB.T1 and TrkB.T2 (twoisoforms of TrkBTK2) on BDNF-dependent signaling inXenopus oocytes supports this suggestion (12). Clearlyit is important to determine precisely which striatalcells are expressing BDNF and TrkBTK2. Neither hy-pothesis makes sense if the same cell type is producingboth proteins. The recent observation that binding ofBDNF to truncated isoforms of TrkB expressed in Ltk2

cells has profound effects upon release of acidic metabo-

FIG. 3. Autoradiographs showing expression of mRNA for TrkBTK1 in uninjured controls and 6 h, 1 week, and 2 months after striatalinjury.

FIG. 4. Autoradiographs showing expression of mRNA for TrkBTK2 in uninjured controls and 6 h, 1 week, and 2 months after striatalinjury.

88 WONG ET AL.

lites strongly suggests that these truncated TrkB recep-tors mediate the actions of an as yet undeterminedsignal transduction pathway (9).

The transient induction of BDNF expression does notsupport a classical neurotrophic role for BDNF afterinjury but suggests instead that BDNF acts as atriggering agent. Our observation of increased expres-sion of BDNF and TrkBTK2 mRNAs after injury, com-bined with reports that both injury and application ofexogenous BDNF enhance dopaminergic function sug-gest that BDNF and TrkBTK2 may play an importantrole in the regenerative capacity of nigrostriatal dopa-mine neurons. Furthermore, improved motor functionafter resection of large parts of the caudate nucleus (42)and proliferation and sprouting of host tyrosine hydroxy-lase-immunoreactive fibers in the striatum after adre-nal medullary implants in patients with Parkinson’sdisease (19) suggests that the human nigrostriatal alsohas this capacity for regeneration and repair.

ACKNOWLEDGEMENTS

This study was supported by the Australian National Health andMedical Research Council and the Austin Hospital Medical ResearchFoundation. J.Y.F.W. was supported by a Leslie Eric Paddle Ph.D.Scholarship from the University of Melbourne. We gratefully acknowl-edge the assistance of Drs. Andrew Churchyard and Siew Yeen Chai.

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FIG. 5. Striatal mRNA expression for (A) BDNF, (B) TrkBTK1,and (C) TrkBTK2 0 h, 6 h, 1 day, 3 days, 1 week, 1 month, and 2 months(where stated) postinjury and in age-matched control mice. Forstriatal BDNF mRNA expression: n 5 3, 8, 7, 6, 5, and 8 at each timepoint postinjury, respectively, and n 5 3, 3, 3, 3, 4, and 4 at each timepoint, respectively, in age-matched controls. For striatal TrkBTK1

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91EXPRESSION OF BDNF AND TrkB AFTER STRIATAL INJURY