Experimental Neurology 176, 289–307 (2002)doi:10.1006/exnr.2002.7943

Long-Term Locomotor Training Up-Regulates TrkB Receptor-likeProteins, Brain-Derived Neurotrophic Factor, and Neurotrophin 4

with Different Topographies of Expression in Oligodendrogliaand Neurons in the Spinal Cord

Malgorzata Skup,1 Anna Dwornik, Matylda Macias, Dorota Sulejczak,Maciej Wiater, and Julita Czarkowska-Bauch

Department of Neurophysiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences,3 Pasteur St., 02-093 Warsaw, Poland

Received July 2, 2001; accepted March 21, 2002

Neurotrophins are potent regulators of neuronalsurvival, maintenance, and synaptic strength. Inparticular, brain-derived neurotrophic factor(BDNF), acting through full-length TrkB receptor(TrkBFL), is implicated in the stimulation of neuro-transmission. Physical activity has been reported toincrease BDNF expression in the brain and spinalcord. In this study we have evaluated the hypothesisthat activation of a spinal neuronal network, due toexercise, affects the entire spinal neurotrophin sys-tem acting via TrkB receptors by modulation ofBDNF, neurotrophin 4 (NT-4), and their TrkB recep-tor proteins. We investigated the effect of treadmillwalking (4 weeks, 1 km daily) on distribution pat-terns and response intensity of these proteins in thelumbar spinal cord of adult rats. Training enhancedimmunoreactivity (IR) of both neurotrophins. BDNFIR increased in cell processes of spinal gray matter,mainly in dendrites. NT-4 IR was augmented in thewhite matter fibers, which were, in part, of astro-cytic identity. Training strongly increased bothstaining intensity and number of TrkBFL-like IRsmall cells of the spinal gray matter. The majority ofthese small cells were oligodendrocytes, represent-ing both their precursor and their mature forms. Incontrast, training did not exert an effect on expres-sion of the truncated form of TrkB receptor in thespinal cord. These results show that both neuronaland nonneuronal cells may be actively recruited toBDNF/NT-4/TrkBFL neurotrophin signaling whichcan be up-regulated by training. Oligodendrocytesof the spinal gray matter were particularly respon-sive to exercise, pointing to their involvement inactivity-driven cross talk between neurons and glia.© 2002 Elsevier Science (USA)

1 To whom correspondence should be addressed. Fax: (4822) 822 5342. E-mail: mskup@nencki.gov.pl.

Key Words: locomotor training; TrkB; BDNF; NT-4;oligodendroglia; spinal cord; immunohistochemistry;adult rat.

INTRODUCTION

Locomotor training leads to improvement of steppingability in animals after complete spinal cord transec-tion (11, 28, 53). The mechanism of such improvementremains unclear, although recent data point to theinvolvement of neurotrophins as a possible factor. Sev-eral recent observations support such a possibility. Anumber of spinal neurons, including motoneurons, ac-cumulate neurotrophins (22, 96) and synthesize brain-derived neurotrophic factor (BDNF), neurotrophin 3(NT-3), and neurotrophin 4 (NT-4) (15, 24, 32). Sinceneurotrophins can be transported both anterogradely(4, 79, 87) and retrogradely (29, 45, 59, 72, 80),motoneurons might be a potent source of neurotro-phins both for the periphery and for the spinal inter-neuronal network terminating upon them. Neurotro-phins present in peripheral tissues and synthesized bysensory neurons of the dorsal root ganglia are otherimportant sources of these proteins for the spinal cord(68, 98). Moreover, glial cells, and reactive astrocytesin particular, can express and release neurotrophins(9, 16, 21, 30, 44, 68). Thus, adequate pharmacologicalor physiological stimulation, affecting the activity ofneuronal circuitries, may induce multiple cellulartypes to produce, transport, and release neurotrophins,supporting and modulating the activity of the spinalnetworks.

There is an increasing amount of evidence that neu-rotrophins are involved in regulation of neuronal ac-tivity (8, 10, 48–50, 65, 82). However, only BDNF hasbeen shown to exert complex action at multiple synap-tic levels, causing both translational and posttransla-

FL

289 0014-4886/02 $35.00© 2002 Elsevier Science (USA)

All rights reserved.

tional changes in presynaptic proteins associated withexocytosis and in multiple postsynaptic receptors (49,82). Evidence of a central role for BDNF/TrkB in thedevelopment of hippocampal kindling has been pre-sented (48). The mechanisms for the involvement ofBDNF/TrkB in long-term potentiation have been re-cently explained in the elegant work of Patterson et al.(63). BDNF may enhance the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) and N-methyl-D-aspartate (NMDA) receptorsdue to the increased release of glutamate (14, 17, 83,84). A recent study by Sherwood and Lo (73) indicatesthat chronic BDNF treatment leads to a long-lastingup-regulation of non-NMDA receptor-mediated gluta-matergic transmission.

The role of glutamate-related amino acids in thecontrol of locomotion is well documented through bothin vitro and in vivo studies (7, 26, 31, 89). The NMDAreceptor agonists (glutamate, NMDA, or aspartate) canactivate locomotion in an in vitro brain stem/spinalcord preparation (7). It is thus possible that the neu-rotrophin-driven mechanism of increasing excitabilityof the spinal neuronal network might be partly respon-sible for the improvement observed following locomotortraining in spinal animals (11). Finally, BDNF synthe-sis is known to be modulated in an activity-dependentmanner (60). Physical exercise increases BDNF mRNAexpression in the rat hippocampus and cerebral cortex(60, 61, 69). It has been recently shown that short-lastinglocomotor training also leads to up-regulation of BDNFsynthesis in the spinal neurons and muscles (41).

To exert their biological effect, neurotrophins actthrough their high-affinity full-length Trk receptors(TrkFL). BDNF and NT-4 transmit neurotrophic mes-sages acting specifically through TrkB catalytic recep-tors (TrkBFL) while other forms of TrkB (truncated;TrkBTK�) serve predominantly as regulators of the ex-tracellular contents of these neurotrophins (1, 42, 43,51, 92–94). In the spinal cord both forms of this recep-tor are expressed (6, 33, 37).

The assumption of our study was that, to exert ben-eficial effects on the spinal network, physical exercisewould affect the neurotrophin system of the spinalcord, modulating both the receptors and their ligands.It was expected that the clearest effect of trainingwould be detected in the spinal interneuronal networkinvolved in the control of locomotion. Here we demon-strate that moderate, long-term locomotor trainingclearly enhances both the number and the intensity oflabeling of TrkBFL-positive cells in the spinal gray mat-ter at lumbar segments of the spinal cord in intact rats.However, the effect was the strongest in the populationof small cells of oligodendroglial origin. The trainingalso affected TrkB ligands, changing the patterns andintensity of BDNF and NT-4 expression, predomi-nantly in fibers of the gray and white matter. Prelim-inary reports have previously appeared (27, 76–78).

MATERIAL AND METHODS

Animals. Twelve adult male Wistar rats, weighing360–540 g at the end of the experiment, were used in thework described here. The animals were bred in the ani-mal house of the Nencki Institute, Warsaw, Poland. Theywere given free access to water and pellet food and werehoused under standard humidity and temperature and12-h light/dark cycle. Procedures involving animals andtheir care were conducted in conformity with the institu-tional guidelines of the Ethical Council of the NenckiInstitute of the Polish Academy of Sciences, which are incompliance with the national rules established by the Po-lish Council on Animal Care on use of laboratory animals.

Materials. The primary antibodies used in thisstudy were the anti-TrkB full-length (794; sc-12), anti-TrkBTK� (C-13; sc-119), anti-BDNF (N-20; sc-546), andanti-NT-4 (N-20; sc-545) rabbit polyclonal and respec-tive control peptides (sc-12P, sc-119P, N20P 546, andN20P 545), purchased from Santa Cruz Biotechnology,Inc. (Santa Cruz, CA, USA); anti-NeuN mouse mono-clonal Chemicon, USA); anti-GFAP mouse monoclonalobtained from Boehringer (Mannheim, Germany); anti-GFAP rabbit polyclonal purchased from DAKO (Den-mark); anti-RIP mouse monoclonal purchased from De-velopmental Studies Hybridoma Bank (Iowa, USA);anti-NG2 mouse monoclonal (a gift of Dr. WilliamStallcup, La Jolla, CA); anti-GalC and O4 mouse mono-clonal; and anti-MAP2 (clone HM-2) mouse monoclonal(Sigma Chemicals, St. Louis, MO). Microglial cellswere stained specifically using peroxidase-labeledisolectin B4 isolated from Bandeiraea simplicifolia(Sigma). Fluorescein isothiocyanate and tetramethyl-rhodamine isothiocyanate fluorescent conjugates withsecondary antibodies raised against rabbit whole-mol-ecule immunoglobulins and mouse Fab fragments, re-spectively, were from Sigma, whereas Texas red andfluorescein conjugates with streptavidin used for theamplification of fluorescent signal were from VectorLaboratories (Burlingame, CA). Vectastain ABC detec-tion kits, standard and Elite, were purchased fromVector Laboratories. Hoechst 33458 (bis-benzimide)dye used for the detection of cell nuclei and cresyl violetused for histological staining were from Sigma. Allother chemicals and reagents were from Sigma, exceptfor PFA (Merck, Germany), DPX (Park, UK), and alco-hols and xylene (POCh, Poland).

Behavioral training. Six adult male Wistar ratswalked on a treadmill about 1000 m daily at a speedbetween 20 and 25 cm/s. The locomotor training wascarried out for 4 weeks, 5 days a week. Both the totaldaily walking distance and the speed of locomotionwere gradually increased after the animals becameaccustomed to the treadmill. The daily training con-sisted of three to four 20-min walking sessions sepa-rated by about 1 h rest in the animal cages. The ani-

290 SKUP ET AL.

mals were rewarded with their preferred food aftereach session. The control group consisted of six ani-mals that were never trained, but were handled andrewarded in the same way as the trained group. Tocontrol the effect of training on body and muscleweight, the animals were weighed before and at theend of the training. After perfusion the following hind-limb muscles were isolated and weighed: soleus, gas-trocnemius (lateral and medial heads), plantaris, tibi-alis anterior, and extensor digitorum longus. The ratioof muscle to body weight was then calculated. After 4experimental weeks the trained animals increasedtheir initial body weight by about 4%, whereas thenontrained animals gained about 11%. This type oftraining did not influence the muscle to body weightratios significantly (Mann–Whitney U test; (74)).

Immunocytochemistry and isolectin binding. After4 weeks of training the rats were anesthetized withlethal doses of sodium pentobarbital (80 mg/kg, i.p.)and perfused via the ascending aorta with 200 ml 0.01M phosphate-buffered saline (PBS), pH 7.4, for 2–3min and, subsequently, with 400–500 ml of ice-coldfixative (2% paraformaldehyde plus 0.2% parabenzo-quinone in 0.1 M PB) for the next 20 min. Spinal cordswere removed from the vertebral columns and werepostfixed in the fixative for 1.5 h at room temperature.The tissue was then cryoprotected overnight in 10%sucrose in 0.1 M PB at 4°C followed by 30% sucroseuntil the tissue sank. The spinal cord L1–S1 segmentswere frozen with precooled heptane (temperaturearound �30°C), placed on tissue holders, surroundedby Jung tissue-freezing medium (Leica), and cut on acryostat. Forty-micrometer transverse sections werecollected free-floating in PBS, pH 7.4, to perform sin-gle-immunolabeling and complementary cresyl violetstaining. Consecutive sections were collected to neigh-boring wells to assure that patterns of TrkB receptorand BDNF/NT-4 neurotrophin labeling were analyzedon adjacent tissue areas. For a given marker eachsection was spaced from another one by 640 �m. Thus,about five 40-�m sections represented each L3 and L4segment. For double-labeling studies, 14-�m glass-mounted transverse sections were collected and frozenat �20°C until used. The sections were washed in PBSwith 0.2% Triton X-100, pH 7.4 (PBS � T), incubated ina solution of 0.3% H2O2 in water for 20 min to quenchendogenous peroxidase activity, washed extensively inPBS � T, and finally incubated with 3% normal serumin PBS � T for 60 min to reduce nonspecific staining.Higher concentrations of the normal serum were usedin anti-MAP-2 (5%) and in anti-RIP and anti-NeuN(10%) labeling protocols, whereas no blocking serumwas used with anti-GFAP monoclonal antibody.Quenching of endogenous peroxidases was omitted inthe anti-RIP labeling protocol, as it significantly dete-riorated the staining. In addition, the anti-RIP labeling

protocol required exclusion of Triton X-100 from thebuffers, as the detergent enhanced myelin staining,thus masking RIP detection in oligodendroglial cellbodies. The sections were subsequently incubated over-night at 4°C with the respective primary antibodies.Anti-BDNF, anti-NT-4, and anti-TrkB antibodies wereused at a 1:1000 dilution when developed with 3,3�-diaminobenzidine tetrahydrochloride (DAB); 1:400was used for fluorescence (IF). For single-labeling ofastrocytes, anti-GFAP polyclonal antibody was used ata 1:5000 dilution. Anti-NeuN (1:10,000 when devel-oped with DAB; 1:1000 was used for IF) and anti-MAP-2 (1:25,000) were used to identify neuronal phe-notype of TrkB immunoreactive (IR) cells and to definecellular compartments colocalizing BDNF or NT-4. Inthe search for the phenotype of TrkB IR nonneuronalcells we used four antibodies recognizing both adult(anti-RIP supernatant, 1:100; anti-GalC, 1:25) and im-mature (anti-O4; 1:10) and precursor (anti-NG2,1:1000 if developed with DAB; 1:200 if developed withIF) forms of oligodendroglia. In addition, anti-GFAP(Boehringer, 1:10) antibody and B. simplicifolia isolec-tin (1:50) were applied to recognize astrocytic or micro-glial cells, respectively. The sections were then rinsedin PBS � T prior to a 1-h incubation at room temper-ature with the respective secondary biotinylated anti-bodies from the ABC kit. Subsequently, after extensivewashings with PBS � T, sections were incubated for1 h with AB complex containing avidin–HRP conju-gate. The sections were then washed with PBS � T andthe antigenic sites were revealed by treating with0.05% DAB and 0.01% H2O2. When double-labelingwas performed, the first antigen was detected withnickel intensification of DAB reaction resulting ingrayish-blue product, and the second antigen was vi-sualized with DAB only, resulting in brown precipitate.The reaction was terminated by addition of extensivePBS � T and by subsequent PBS washings. The sec-tions were mounted on gelatin-subbed slides, dehy-drated in ascending alcohol concentrations, clearedthrough xylene, and covered with DPX resin. The pro-cedure used for fluorescent detection of antigens wasbasically the same, except that peroxidase quenchingwas omitted and following incubation with secondaryantibodies carried out in the dark, sections were driedand kept at 4°C.

Within each experiment, immunohistochemical pro-cessing of tissue sections from trained and nontrainedgroups was carried out simultaneously. The conditionsof the procedure (dilutions of reagents and antibodies,washings, incubation time and temperature, blockingof nonspecific staining, reaction development regimen),kept rigorously throughout the assays, were identicalfor the sections from both treatment groups.

Control of immunolabeling specificity. (1) Controlof immunolabeling specificity was routinely performed

291TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

by omitting a primary antibody in the incubation mix-ture. Under these conditions, no immunolabeling wasever detected. (2) In the initial series of experimentscontrol immunostainings with Santa Cruz antibodiespreincubated with blocking peptides were performed.The blocking peptides were supplied by the manufac-turers of immunogens against which the respectiveantibodies were raised. No nonspecific staining in sec-tions was registered for any of the antibodies used inthe tests except for a very weak background labelingdetected in the outermost part of the spinal funiculi. (3)Additionally, for anti-TrkBFL antibody dot blots andpreabsorption tests with different preincubation/incu-bation conditions have been carried out. They showedthe stability of antigen–antibody complexes and con-firmed immunological specificity of the anti-TrkBFL an-tibody. (4) The biochemical specificity of TrkBFL anti-body has been characterized with Western blottinganalysis following 7% SDS–polyacrylamide gel electro-phoresis of the lumbar spinal cord tissue (see Fig. 1).Tissue homogenized or sonicated with or without de-tergents (1% Triton, 1% tergitol Nonidet-P40 (NP-40),or 1% SDS) was tested. Various assays were carriedout with the following detection systems: (i) HRP-con-jugated secondary antibody developed with DAB, (ii)ABC kit developed with DAB, (iii) HRP-conjugatedsecondary antibody developed with ECL chemilumi-nescence kit, and (iv) ABC kit developed with ECLchemiluminescence kit. As shown in Fig. 1, lane 2, theantibody recognized, with different intensity (depend-ing on the detection system used), three protein bandswith estimated molecular masses of 145, 125, and80 kDa (Fig. 1). A different antibody raised againstTrkBFL receptor (Oncogene Ab-1, No. PC-86, epitope:807–822 amino acids of the carboxy terminus) usedwith the same tissue preparations recognized the same

protein bands (Fig. 1, lane 3). We assume that thebands recognized by both antibodies corresponded tofully glycosylated, partly glycosylated, and unglycosy-lated forms, respectively. Another possibility is that adensely stained band at 79–80 kDa, that is alsostained albeit with less intensity by Oncogene Ab, is anunknown protein structurally related to TrkB. BecauseTrkB immunoreactivity obtained with the anti-TrkBfull-length (794; sc-12) antibody used may be not ex-clusive to full-length TrkB protein, in this study suchstain will be referred to as TrkBFL IR (TrkBFL-likeimmunoreactivity or TrkBFL-like immunoreactive)hereafter. (5) In fluorescent-labeling assays the speci-ficity of staining was verified in two ways, first byomission of the primary antibodies. These tests re-sulted in total disappearance of fluorescent staining.Second, labeling was controlled for each antibody sep-arately, by omission of the second antibody, with allother steps included. These assays proved that none ofthe staining described by us was due to nonspecificfluorescence or filter bleed-through.

Quantitative, morphometrical, and densitometricanalyses of the images. The sections were examinedusing a Nikon Optiphot-2 light microscope and anEclipse 400 fluorescent microscope and were photo-graphed with a Nikon FDX-35 camera. Data were an-alyzed under the microscope by two observers. Morpho-metric and densitometric analyses were performedwith the aid of a computer-assisted image analysissystem consisting of a Nikon Optiphot-2 microscope(20 � 10 magnification) equipped with an x/y move-ment-sensitive stage and a Sony 3CCD color videocamera. Image Pro Plus 4.0 (UK) digitizer and softwareand Scion Corp. (NIH, Frederick, MD) software wereused. During image capturing shading correction wasapplied, the light source was stabilized, and to main-tain the same illumination level at each imaging ses-sion the settings of the camera and lamp were constant(see Mize et al. (58)). In the case of BDNF and NT-4densitometry, all images were captured during onesession, to ensure the same illumination level. Thesections used for quantification and morphometry werecoded to ensure that the observer was blind to allsubjects. The codes were broken only immediatelyprior to statistical analyses.

Three of five sections representing each of L3 and L4segments were chosen from each rat for TrkBFL IRquantification, morphometry, and densitometry. Thesections best matching the L3 or L4 segment diagramsin the stereotaxic atlas were selected (64). Cell size wasdefined from the cell body profiles outlined manually.Only cell bodies with sharp, well-defined edges weretaken into account. Counting included all the labeledcells located within the gray matter of half of eachsection. The same population of labeled cells was thensubjected to densitometric analysis.

FIG. 1. Western blot of TrkBFL immunoreactivity in the lumbarspinal cord tissue derived from naive rat. Tissue was homogenized in0.05 M Tris–HCl buffer with 1% Nonidet-P40, 0.04% mercaptoetha-nol, and protease inhibitors. The homogenates were centrifuged at6000g, 10 min, and the protein (30 �g per lane) from the superna-tants was subjected to 7% SDS–polyacrylamide gel electrophoresis,transferred to nitrocellulose membrane, and probed with TrkBFL

antiserum. Antigen was detected with secondary Ab conjugated toHRP and the reaction was developed with chemiluminescent system(ECL, Amersham). Lanes 2, TrkB (794) Santa Cruz No. sc-12 Ab,1:1500; 3, TrkB Oncogene No. PC-86 Ab, 1:400; 4, TrkA Ab providedby L. F. Reichardt, 1:1000; 5, blot developed without primary anti-body.

292 SKUP ET AL.

Six to 10 consecutive sections of L3 and L4 segments,chosen from each rat, were used for densitometric anal-yses of BDNF and NT-4 immunostaining. LabeledNT-4 fibers were examined using a segmented fiberanalysis program, involving skeletonization, as previ-ously described by Mize and co-workers (58). Briefly,with this method, detected fibers are extracted fromthe background and reduced to a single pixel in width.The boundaries of the spinal laminae were verifiedmicroscopically at Nissl stained cross sections. Figureswere assembled using the Adobe Photoshop software.

Statistical analysis. Quantitative analysis of TrkBFL

IR cell profile number was performed with the Mann–Whitney U test for two independent samples (74). Twogroups of cell profiles were arbitrarily distinguished forthe analysis: (i) up to 160 �m2 and (ii) above 160 �m2. Fordensitometric analysis additional subclasses within thesegroups were distinguished.

Since the results of two independent experimentswere combined, data were normalized prior to statisti-cal analysis because the number of TrkBFL-labeled cellsin the second experiment outnumbered those in thefirst one. Data were normalized as follows: the ratiobetween mean numbers of immunoreactive cells de-rived from both control, nontrained groups was calcu-lated. Then, the number of labeled cells obtained foreach animal in the control and trained groups of thesecond experiment was divided by the obtained factor.

RESULTS

TrkB Expression in Lumbar Spinal Cord

In nontrained rats the most intense TrkBFL IR wasdetected in the perikarya and processes of small cellsscattered throughout the spinal gray matter (Figs. 2Aand 2C). The average soma size of these cells rangedbetween 20 and 80 �m2 (Fig. 3A). In the ventral horn,small TrkBFL IR cells were frequently seen in closeapposition to neighboring large cells, forming clustersaround them (see Fig. 2C). TrkBFL was also expressedby other subpopulations of spinal cells, including largecells of lamina IX, which generally showed weakerlabeling.

Intracellularly, TrkBFL immunodeposits were dis-tributed throughout the cytoplasm of the cell bodieswith condensation of the staining within processes.Cell nuclei were devoid of TrkBFL IR, but in some cellsTrkBFL immunodeposits were detected around nuclei(Figs. 2A and 2C).

TrkBFL IR cells were also scattered throughout thespinal funiculi as illustrated in Fig. 5J. Their densitywas similar both in ventral and in dorsal funiculi innontrained rats (not shown). The average soma size ofthese TrkB IR cells was similar to that of small cells ofthe spinal gray matter.

TrkBTK� IR was detected in some medium-size neu-rons of the spinal gray matter. These neurons werelocated mostly in laminae VIII and IX. In contrast toTrkBFL, only single TrkBTK� IR small cells were de-tected in the spinal gray matter. The large neurons oflamina IX were TrkBTK�-negative. Intracellularly,TrkBTK� immunodeposits were distributed throughoutthe cytoplasm of the cell bodies and not clearly distin-guished in cell processes. TrkBTK� IR neuropil of thespinal gray matter formed a dense mesh particularly inthe superficial laminae of the dorsal horn, in the ven-tral horn, and in lamina X. In the white matterTrkBTK� was localized in cells resembling astrocytes.

Effect of Training on TrkB Expression

The locomotor training enhanced both TrkBFL IRintensity and number of immunopositive cells (Figs. 2Band 2D). As shown in Fig. 3B, the training enhancedTrkBFL IR intensity in the cells of the gray matter byabout 25% (P � 0.01). When the effect was analyzedwith respect to various cell categories (based on cellbody size), the most prominent influence of trainingwas found in small cells, between 40 and 80 �m2 in size(P � 0.008). It was particularly pronounced in theventral horn and intermediate zone (Fig. 2D). The en-hancement of TrkBFL expression was clearly visible insome large neurons of lamina IX (Fig. 2D). However,densitometry of TrkBFL IR carried out in a populationof cells of lamina IX larger than 800 �m2 (ca. 100 cellsin each group) revealed that the effect of training didnot reach statistical significance.

Training caused some changes of intracellular local-ization of TrkBFL immunodeposits. In a number oflarge cells of lamina IX we observed intensification ofTrkBFL IR concentration around the nuclei (Figs. 2D,4A, and 4B).

The number of TrkBFL IR small cells (with profileareas less than 160 �m2) was significantly higher intrained than in nontrained animals (P � 0.001). Fig-ure 3A shows that twice as many TrkBFL IR small cellswere found in trained animals. The number of largercells expressing TrkBFL did not change due to the train-ing (Fig. 3A). The training did not cause a significanteffect on TrkBFL immunoreactivity in small cells of thewhite matter.

We did not observe any changes either in the numberof TrkBTK� IR cells or in the immunostaining intensityin the gray and white matter due to the training. Inparticular, no TrkBTK� IR induction was detected insmall cells of the spinal gray matter, indicating thattraining differentially influenced TrkBTK� and TrkBFL

signaling pathways.

Identity of TrkBFL Immunopositive Cells

It appeared that in the spinal gray matter a numberof TrkBFL immunopositive cells were of neural origin.

293TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

294 SKUP ET AL.

Double-immunostaining of TrkBFL and NeuN, a neu-ron-specific marker, demonstrated that numerousTrkBFL-positive cells of various sizes coexpressedNeuN. Figure 4 shows an example of the expression ofTrkBFL in large neurons of lamina IX. It also indicatesthat a number of nonneuronal small cells surroundingthese neurons were strongly TrkBFL immunopositive.

To reveal the phenotype of TrkBFL IR nonneuronalcells, double-immunostaining with specific antibodiesagainst TrkBFL and various types of glia was per-formed. Figures 5A–5C show that astrocytic marker(GFAP) labeling in the lumbar spinal gray matter doesnot colocalize with TrkB. The number and morphologyof astrocytes was similar in both nontrained and

FIG. 3. The effect of training on the number (A) and staining intensity (B) of TrkB IR cells in the gray matter of lumbar spinal cord. Thenumber of small TrkB IR cells, with soma profile ranging from 20 to 160 �m2, doubled due to locomotor training (P � 0.001; Mann–WhitneyU test). Staining intensity was higher in trained than in control, nontrained animals (P � 0.01). Half of each lumbar cross section underwentanalysis, with three sections per animal. Bars represent means � SEM of the six animals per group.

FIG. 2. Brightfield photomicrographs showing transverse sections of the L3 lumbar spinal cord after immunocytochemical labeling ofTrkB receptor in nontrained (A, C) and trained (B, D) rats. In the dorsal horn (A, B), small polygonal and oval TrkB IR cells indicated byarrowheads are randomly scattered in a densely labeled neuropil. In the ventral horn (C, D), TrkB IR is detected in large cells (arrows), inmuch more intensely stained small cells (thin arrowheads), and in fibers (thin arrows). The small cells formed clusters (thick arrowheads)and were often detected in a close apposition to large cells of lamina IX. Note that in the trained animal an enhancement of TrkB IR occursboth in large and in small cells (B and D). Scale bar, 20 �m.

295TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

FIG. 4. Photomicrographs showing ventral horns out of transverse sections of L3 segment of the spinal cord after immunocytochemicalfluorescent double-labeling of TrkB receptor (A) overlaid with neuron-specific antigen NeuN (B) and combined with Hoechst staining tovisualize cell nuclei (C). Note the coexpression of TrkB and NeuN in the large neurons of lamina IX (solid arrowheads) and the lack of NeuNexpression in strongly TrkB IR small cells (hollow arrowheads), indicating their nonneuronal phenotype. Scale bar, 10 �m.

FIG. 7. Colocalization of BDNF and MAP-2 at the border of the spinal gray and white matter of the ventral horn (D). Double-labelingrevealed neuronal origin of BDNF IR fibers. Arrows point to an example of double-labeled dendrite. (A) BDNF IR; (B) MAP-2 IR; (C) Hoechststaining to visualize cell nuclei. Asterisk indicates a row of cell bodies in apposition to another double-labeled dendrite. Scale bar, 10 �m.

296 SKUP ET AL.

FIG. 5. Photomicrographs showing spinal gray (A–I) and white (J–L) matter at transverse sections of L3 lumbar spinal cord afterimmunocytochemical fluorescent double-labeling of TrkB receptor (A, D, G, J) with astrocyte marker, GFAP, (B), oligodendroglia markers,RIP (E), O4 (H), and NG2 (K), combined with Hoechst staining to visualize cell nuclei (C, F, I, L). Note that TrkB IR is not present inastrocytes (A, B) but is present in oligodendrocytes both in their mature (D, E) and in their immature (G, H) forms. In the white matterabundant oligodendrocyte precursor cells are TrkB IR (J, K). Hollow arrowheads indicate single-labeled cells; solid arrowheads point todouble-labeled cells. Arrows indicate astrocytic fibers. In D–F, dotted lines outline free of myelin lamina II. Scale bar, 10 �m.

FIG. 10. Photomicrographs showing transverse sections of L3 lumbar spinal cord after immunocytochemical fluorescent double-labelingof NT-4 with astrocytic marker, GFAP (top), and NT-4 with dendritic marker, MAP-2 (bottom), proteins. Immunolabeling was combined withHoechst staining to visualize cell nuclei (C, G). NT-4 IR (A, E); GFAP IR (B); MAP-2 IR (F) Note that overlaid images of NT-4 IR and GFAP(D) reveal NT-4 expression in some astrocytes (solid arrowheads) and their processes (solid arrows). Overlaid images of NT-4 and MAP-2 (H)prove a dendritic localization of NT-4 (solid arrows). Hollow arrowheads, single-labeled cells (A–H); hollow arrow, single GFAP-labeled fiber.Scale bar, 10 �m.

297TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

trained animals. No signs of reactive hypertrophy wereobserved due to the training. All of these observationsindicate that TrkBFL IR small cells of the spinal graymatter are unlikely to belong to an astroglial subpopu-lation. However, a small fraction of TrkBFL IR cells inthe white matter was found to be of astroglial origin.

Double-labeling with anti-TrkBFL antibody and a mi-croglial marker (B. simplicifolia isolectin) showed thatthey did not colocalize, thus excluding microglial originof the TrkBFL IR cells (not shown). On the other hand,the morphology, soma size, and distribution of TrkBFL

IR small cells resembled those of oligodendroglia. Dou-ble-immunostaining demonstrated that small cells co-localized TrkBFL and RIP or TrkBFL and O4, indicatingthe oligodendroglial phenotype of the majority ofTrkBFL IR small cells (Figs. 5D, 5E, 5G, and 5H). Ad-ditionally, colocalization of TrkBFL and GalC in multi-ple small cells confirmed their oligodendroglial identity(not shown). Moreover, some TrkBFL IR small cellswere demonstrated to be oligodendroglial precursorcells, as indicated by double-labeling with TrkBFL andNG2 antibodies (Figs. 5J and 5K). This raises the ques-tion of whether the effect of physical exercise on TrkBFL

expression in small cells is due to increased receptorexpression or to a generation of newborn oligodendro-cytes.

BDNF Expression in the Lumbar Spinal Cord

BDNF immunoreactivity was found mostly in theneuropil of the spinal gray matter, with numerousfibers forming a dense mesh. Figure 6 shows an exam-ple of BDNF IR distribution in neuropil of the dorsal(Fig. 6A) and ventral (Figs. 6C and 6E) horns in anontrained animal. The most extensive accumulationof the reaction product appeared in the lateral aspect oflaminae I–III and in lamina IX. In contrast to strongBDNF expression in the spinal neuropil, perikaryonallabeling was weak (Figs. 6A, 6C, and 6E).

Effect of Training on BDNF Expression

Locomotor training enhanced expression of BDNF inthe spinal gray matter (Figs. 6B, 6D, and 6F). A cleareffect was observed in large neurons of lamina IX (Figs.6D and 6F). Particularly strong enhancement of BDNFIR was found in fibers surrounding large neurons. Inaddition, numerous granular, strongly BDNF IR de-posits resembling terminal swellings were detected inapposition to neuronal perikarya (Figs. 6D and 6F). Anumber of BDNF IR fibers in lamina IX were alsoMAP-2 immunopositive, indicating that many of themwere dendrites (Figs. 7A–7D). The training had aneffect also on BDNF IR laminar distribution in thedorsal horn (Fig. 6B). Densitometric analysis revealeda reduction of BDNF IR in lamina II, particularly in itslateral part, after the training (Fig. 6B). In two of thesix trained animals, a decrease of labeling density in

lamina II was accompanied by an increase of immuno-reactivity in lamina III. In deeper laminae, clear en-hancement of BDNF IR was found in many fibers(Fig. 6B).

NT-4 Expression in the Lumbar Spinal Cord

NT-4 IR was detected predominantly in fibers of thewhite matter with the most intense labeling at itsperiphery (Figs. 8 and 9). Double-staining with MAP-2revealed that some NT-4 IR fibers were dendrites, par-ticularly those located at the border of the white andgray matter (Fig. 10). Dendritic NT-4 expression wasweak. In contrast, the vast majority of strongly NT-4IR fibers at the periphery were not dendrites. Some ofthem coexpressed GFAP, indicating their astrocyticidentity.

Perikaryonal NT-4 IR was detected in the white mat-ter (Fig. 10E). Some NT-4 IR cells were of astrocyticorigin, as indicated by double-labeling with GFAP(Figs. 10A–10D). NT-4 IR cells were often located alongdendrites in close proximity to the spinal gray matter(see Figs. 10E–10H).

Effect of Training on NT-4 Expression

The number of NT-4 IR fibers and their stainingintensity increased at the periphery of the white mat-ter after locomotor training (Figs. 8A and 8B). Thiseffect was slightly stronger in ventral than in dorsalfuniculi, as revealed by densitometric analysis and byskeletonization of the immunolabeled fibers (Fig. 9).No detectable change in NT-4 IR was found inperikarya. Double-labeling with GFAP revealed thatan enhancement could have occurred in both astroglialand nonastroglial fibers.

Taken together, our immunocytochemical resultsshow that long-lasting, moderate locomotor trainingenhanced BDNF and NT-4 expression and increasedthe potential for their signaling due to up-regulation oftheir high-affinity TrkBFL receptors in neurons and innumerous oligodendroglial cells in the spinal cord.

DISCUSSION

The most striking findings of our study are thatTrkBFL neurotrophin receptor is present in the oligo-dendroglial cells of the spinal gray matter and that thisoligodendroglial subpopulation responds to long-term,moderate training with strong up-regulation of TrkBFL

receptor. Our results show for the first time that train-ing up-regulates TrkBFL receptor and its BDNF andNT-4 ligands, facilitating signaling through this neu-rotrophin system both in neurons and in oligodendro-glia. This study confirms our assumption that trainingnot only causes up-regulation of BDNF and NT-4 ex-pression, as indicated earlier for BDNF by Gomez-Pinilla and co-workers (41) after short training, but

298 SKUP ET AL.

also enhances their TrkBFL receptor expression. Wehave also demonstrated that this type of training ef-fectively stimulates neurotrophin proteins and not onlytheir mRNAs, as shown by others (41, 61).

The effect of training on TrkBFL IR was particularlystrong in oligodendrocytes of the spinal gray matter,affecting the staining intensity and doubling the num-ber of TrkBFL immunoreactive cells. In the gray mattersome oligodendrocytes are associated with groups ofmyelinated nerve fibers, but they also appear as satel-lite cells in apposition to neuronal perikarya. Thesesatellite cells are believed to be involved in the nutri-tion and maintenance of neurons and cell homeostasis(66). Although the concept was elaborated by Hyden’sgroup years ago (57), data showing that oligodendro-glia contain neurotransmitter receptors, thus render-ing possible a cross talk between neurons and glialcells, started to appear only recently (12, 71, 99). As-suming that oligodendroglial satellite cells are rich inTrkBFL receptors, which up-regulate due to activationof the spinal network, we suggest that these cells mayparticipate in neuron–glia interaction through theneurotrophin signaling.

TrkBFL Expression on Oligodendrocytes

The oligodendroglial phenotype of small cells thatboth showed enhanced TrkBFL IR and increased innumber following training is rather surprising. To ourknowledge, full-length TrkB receptors have not beenreported to be present in oligodendroglial cells, in con-trast to the high-affinity TrkC and TrkA receptors (13,20, 52, 90; see also 54). On the other hand, a truncatedform of this receptor was found both in neurons and inglial cells (5, 19, 36). This raises the question of thespecificity of anti- TrkBFL antibody used in our study.

The antibody used by us was raised against a peptide15 amino acids long, mapping adjacent to the carboxyterminus of the precursor form of TrkB gp145, thusrecognizing the full-length TrkB isoform. Western blotanalysis carried out to identify the proteins recognizedby this antibody revealed three protein bands withestimated molecular masses of 145, 125, and 80 kDa.Our interpretation is that the antibody specifically rec-ognizes the fully glycosylated form of TrkBFL (ca. 145kDa), the partly glycosylated form of ca. 125 kDa, andthe unglycosylated form of ca. 80 kDa (25), whereas itdoes not recognize TrkBTK� (95-kDa protein). It has tobe noted here that, based on our assays, without addi-tional experiments we cannot exclude that the anti-body also detects cross-reacting TrkB-like protein. Al-though this possibility seems minor, as data banks donot report any other proteins with the sequence recog-nized by anti-TrkBFL antibody, further studies to doc-ument the specificity and significance of the 80-kDaprotein detected with Western blotting in the spinaltissue are needed. Therefore, although other histo-

chemical data indicate that this antibody has a patternof cellular labeling comparable to that of other anti-TrkB antibodies (38, 95), its specificity toward TrkBFL

exclusively has to be confirmed.It has to be stressed that anti-TrkBTK� antibody rec-

ognizing the truncated form of the TrkB receptor pro-duced a different pattern of labeling than anti-TrkBFL

antibody in the spinal cord of nontrained animals.Moreover, in contrast to TrkBFL, we did not find anyeffect of training on TrkBTK� labeling. We also foundthat there is no cross-reactivity of TrkB with TrkA orTrkC receptors, as confirmed by different patterns ofTrkA and TrkC staining in the spinal cord, cerebralcortex, and hippocampus (56, 75). As we did not ob-serve either truncated TrkB or TrkC and TrkA up-regulation due to the exercise, our results stronglyindicate that locomotor training selectively up-regu-lated the full-length form of TrkB.

The increased number of TrkBFL IR cells in trainedrats may have been caused by an enhancement ofTrkBFL synthesis, rendering detectable those oligoden-drocytes that, in nontrained rats, synthesized TrkBFL

at levels below the sensitivity threshold of immunocy-tochemical detection. An alternative hypothesis, how-ever, is that exercise causes the recruitment of newoligodendrocytes. Proliferative oligodendrocyte progen-itors are known to be present in the adult centralnervous system (86, this paper). Moreover, it was dem-onstrated that cellular proliferation leads to the forma-tion of new oligodendrocytes in normal adult spinalcord (47). In addition, neurotrophins have been shownto induce oligodendrogliagenesis in vitro and in vivo(55, 70). It has been recently demonstrated that short-lasting locomotor training is able to induce BDNF andNT-3 message in the spinal cord (41). Our results showthat 1 month of training not only increases neurotro-phic spinal protein expression but also substantiallyenhances their receptor expression, resulting in pow-erful reinforcement of neurotrophic signaling throughTrkBFL receptor. This period of exercise is long enoughfor newly generated cells and oligodendrocyte precur-sors to differentiate. Further studies will examinewhether an increase in the number of oligodendrocytesexpressing TrkBFL receptors was caused by prolifera-tion of endogenous oligodendrocyte progenitors.

TrkB Receptors on Neurons

The presence of TrkB receptors in motoneurons waselegantly documented by Copray and Kernell (24).They found that practically all identified motoneuronsexpress TrkB, but there was considerable variation inthe intensity of mRNA expression for TrkB in mo-toneurons, which did not match electrical properties ofthe corresponding muscles. Their results demonstratethat there is no correlation between functional diver-sity of motoneurons (“slow” versus “fast”) and TrkB

299TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

300 SKUP ET AL.

expression. Our results show, for the first time, thatlocomotor exercise enhanced TrkBFL expression insome large neurons of lamina IX, presumably �-mo-toneurons. Interestingly, the TrkBFL immunoreactivedeposits were concentrated around their nuclei. How-ever, not all of the largest cells of lamina IX showedenhancement of TrkBFL immunoreactivity, in agree-ment with the observation of Copray and Kernell (24)on a variation of TrkB expression in motoneurons.

Influence of Training on BDNF and NT-4 ExpressionPatterns

The effect of training on both tested neurotrophinswas expressed differentially, being most prominent infibers. Locomotor training evidently affected the accu-mulation of BDNF in the spinal gray matter fibers and,to a lesser extent, caused an increase of that proteinwithin neuronal perikarya. In contrast, it enhancedNT-4 IR primarily in the white matter fibers, having no

detectable effect on the number of white matter astro-glial and other glial cells.

In our hands BDNF immunoreactivity was easilydetected in nerve fibers and terminals. Its detection incell bodies of the spinal gray matter was more difficultto achieve, due to a weak perikaryonal labeling, inagreement with earlier observations of Dreyfus et al.(32). Our complementary in situ hybridization studydetected mRNA BDNF both in large and in small cellsof the spinal gray matter (unpublished data), extend-ing the results of Buck et al. (15) and Gomez-Pinilla etal. (41). Therefore we conclude that spinal cells pro-duce BDNF peptide but it does not occur in high con-centration in cell bodies.

The anti-BDNF antibody used by us was of the sametype as the one applied by Fawcett and co-workers (34).A specificity test carried out on BDNF knock-outs in-dicates that the antibody reacts with BDNF protein perse but also recognizes an unknown protein of molecular

FIG. 8. NT-4 immunoreactivity in the ventromedial funiculus at lumbar spinal cord of nontrained (A) and trained (B) rats. Arrowheadsindicate NT-4-positive fibers. Scale bar, 40 �m.

FIG. 6. Brightfield photomicrographs showing transverse sections of the L3 segment of the spinal cord after immunocytochemicallabeling of BDNF in nontrained (A, C, E), and in trained (B, D, F) rats. Note the differences of distribution of BDNF IR in the dorsal hornin nontrained (A) and trained (B) animals (asterisks indicate lamina II). Training led to the enhancement of BDNF IR in dense plexus offibers and terminal swellings surrounding large neurons of lamina IX in its lateral (C, D) and medial (E, F) aspects. Thin arrowheads pointto BDNF immunopositive fibers; arrows indicate large neurons of lamina IX; thick arrowheads show terminal swellings around largeneurons. Scale bar, 20 �m.

301TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

mass 28 kDa. Thus we cannot exclude the possibilitythat a fraction of the protein undergoing training-in-duced up-regulation may not be BDNF. However, sincetissue levels of that unknown peptide are not sensitiveto even strong seizure-evoked stimuli, in the interpre-tation of our data we ascribe an increase of BDNFimmunoreactivity to BDNF protein itself (3).

The spatially different patterns of BDNF and NT-4expression described herein support a previously for-mulated hypothesis that these neurotrophins playcomplementary roles in the normal spinal cord (71). Incontrast to strong NT-4 expression in white mattercells and fibers, the expression of this protein in thespinal gray matter was restricted to fibers. Whereas afinding that white matter glial cells express NT-4 is inaccordance with previous studies by Scarisbrick et al.(71), barely detectable NT-4 IR in neuronal cells indi-cates that protein expression in cell perikarya is notmatched by strong mRNA expression documented byothers (15, 71).

A training-induced increase of BDNF neurotrophinwithin dendrites (as revealed by coexpression of BDNFand MAP-2) may suggest that exercise influences pro-

duction/translocation of BDNF within this compart-ment. This points to possible activation of an intrinsicspinal neurotrophin system by the exercise. However,a topography of fibers showing enhanced expression ofBDNF and NT-4 IR due to the training indicates thatexercise not only influences the neurotrophin system inthe spinal cord, but also involves its peripheral andsupraspinal components. Further studies are needed todefine the neurotransmitter systems that colocalizeBDNF and NT-4 proteins.

Functional Implications of Activity-DependentRegulation of the Neurotrophin System

The expression of TrkB receptor and its ligands areregulated in an activity-dependent manner. Theamount of NT-4 mRNA in the rat soleus muscle isup-regulated after tetanic stimulation of the sciaticnerve and down-regulated after local blocking of neu-romuscular transmission with �-bungarotoxin (39).This points to the possibility of peripheral supply ofneurotrophin to the target motoneurons. However, theeffect of physical exercise on the neurotrophin system

FIG. 9. Distribution of NT-4 IR density in the ventral (A) and dorsolateral (B) funiculi in nontrained (gray line) and trained (black line)animals. Optical density is expressed in arbitrary units. (C and D) Skeletonization of the NT-4 immunolabeled fibers. The areas from whichthe data were collected are shown in the insets of halves of spinal cord. Note the higher number of NT-4 IR fibers in trained than in controlanimals, particularly in the ventral funiculi (C).

302 SKUP ET AL.

is not limited to the peripheral nervous system and thespinal cord. As little as 1 week of physical activity ledto up-regulation of BDNF mRNA in the hippocampusand neocortex in rats running 1–2 km per night andthis effect was positively correlated with the distancethat the animals ran (60, 61). Long-distance runningwas also shown to enhance not only BDNF but alsoTrkB mRNA expression in the hippocampus of hyper-tensive rats (91). Interestingly, activity-dependent tar-geting of BDNF and TrkB mRNAs was reported indendrites of hippocampal neurons (85). This, togetherwith our observation that training causes BDNF accu-mulation in spinal dendrites, may suggest that similarmechanisms function in the hippocampus and spinalnetwork.

It is quite likely that the hypothesized easier accessof spinal neurons to neurotrophins following exercisemight lead to potentiation of excitatory drive to mo-toneurons through neurotrophin interaction with glu-tamatergic receptors (62, 83, 84). The results obtainedhere gain an additional value in light of very recentdata by Gonzales et al. (42) who showed that sustain-ing of TrkB-mediated signals is crucial for neurotrans-mission, as they maintain the stability and integrity ofneurotransmitter receptors in the neuromuscular junc-tion. This may be of particular importance in spinal-ized animals, when neurotransmitter signaling is im-paired. It would, to some extent, compensate for thedeficit of descending influences in spinalized animals.It is worth noting that locomotor training of compara-ble duration and slightly higher intensity was recentlyfound to result in an increase in somal and axonalcontent of CGRP in the spinal cord and in sciatic nerveterminals (40). Taken together, it may be concludedthat increased neuromuscular activity might be instru-mental not only in the modulation of spinal neuronalcircuitry, but also in the remodeling and stabilizing ofneuromuscular junctions. This study has establishedthat an increase of neurotrophin expression is accom-panied by TrkBFL IR enhancement, thus indicatingthat moderate, long-lasting training is a powerful toolfor increasing potential efficacy of the entire neurotro-phin system. Although our study does not establishunambiguously that extracellular spinal neurotrophinpools are increased, since a diffusible fraction of bothpeptides might be too small to be detected immunocy-tochemically (as previously shown for NGF; 23, 46, 54),a strong enhancement of BDNF and NT-4 IR found inthe fiber compartments indicates such a possibility. Ifthat were the case, prolonged increase in the supply ofBDNF and NT-4 due to long-lasting training wouldcause effects on TrkB receptor opposite to those de-scribed by Sommerfeld et al. (81). Those in vitro studieshave shown ligand-induced down-regulation of TrkBreceptors. A likely explanation of such a discrepancymay be provided by different regulatory mechanismsoperating in our in vivo experimental situation, as also

shown for other Trk receptors (35, 67). Another expla-nation of this phenomenon might be that stimulation ofneuronal circuits might, by itself, cause direct increaseof TrkB expression. Such an effect was recently de-scribed for TrkB expression in stimulated regeneratingmotoneurons (2).

Finally, the issue of the physiological importance ofTrkB up-regulation in oligodendroglial cells remains tobe solved. Intense and long-lasting activity of neuronalnetworks requires increased activity of satellite cells,which are responsible for neuronal maintenance andionic balance. Our result points to oligodendroglia asan important player in that regulation. It challengesthe generally accepted fact of the main role of astrogliain these processes.

The other indication of the role of TrkBFL in oligo-dendroglial cells may be derived from the recent re-ports showing that BDNF stimulates oligodendrocyteproliferation and myelinization following spinal cordcontusion (55) and is required for myelination and re-generation of injured peripheral neurons (97). If weassume that training-induced increase of BDNF andNT-4 immunoreactivity reflects an enhancement ofBDNF/NT-4 content and release, stimulating TrkBFL

signaling in the myelinating oligodendrocytes, we mayspeculate that it affects their survival and maintainsoligodendroglial myelinating activity. There is recentevidence on particular vulnerability of oligodendroglialcells to tissue damage (18, 88). The mechanism under-lying this sensitivity is not known, but the result maybe myelin deficit due to oligodendroglial death, at leastin some forms of spinal injury. Therefore, if oligoden-drocytes are regulated by neurotrophic supply, and ifequivalent mechanisms are operative under patho-physiological conditions, training started soon post le-sion might rescue these cells from elimination, thusattenuating possible demyelinization process.

ACKNOWLEDGMENTS

This study was supported by grants of the State Committee forScientific Research (No. 1305/P05/98/14, 1030/P05/96/11) and statu-tory for the Nencki Institute. We express our gratitude to ProfessorJ. Skangiel-Kramska (Nencki Institute of Experimental Biology,Polish Academy of Sciences, Poland), Dr. Jennifer Cook (Founda-tion for Undergraduate Research, Albion College, USA), and Profes-sor Alexander Wlodawer (National Cancer Institute, Frederick,USA) for critical reading of the manuscript. We thank also ProfessorWilliam Stallcup (La Jolla Cancer Research Foundation, CA, USA)for a generous gift of anti-NG2 antibody and Professor LouisReichardt (University of California San Francisco, CA, USA) for agenerous gift of anti-TrkA antibody. We thank our colleagues forkind donations of antibody samples: Dr. L. Buzanska (Medical Re-search Center, Polish Academy of Sciences, Warsaw) for anti-GalCand anti-O4 antibodies, Professor L. Kaczmarek (Nencki Institute ofExperimental Biology, Polish Academy of Sciences, Warsaw) foranti-NeuN antibody, and Professor J. Skangiel-Kramska (NenckiInstitute of Experimental Biology, Polish Academy of Sciences, War-saw) for anti-MAP-2 antibody.

303TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

REFERENCES

1. Alderson, R. F., R. Curtis, A. L. Alterman, R. M. Lindsay, andP. S. DiStefano. 2000. Truncated TrkB mediates the endocyto-sis and release of BDNF and neurotrophin-4/5 by rat astrocytesand schwann cells in vitro. Brain Res. 871: 210–222.

2. Al-Majed, A. A., T. M. Brushart, and T. Gordon. 2000. Electricalstimulation accelerates and increases expression of BDNF andtrkB mRNA in regenerating rat femoral motoneurons. Eur.J. Neurosci. 12: 4381–4390.

3. Aloyz, R., J. P. Fawcett, D. R. Kaplan, R. A. Murphy, and F. D.Miller. 1999. Activity-dependent activation of TrkB neurotro-phin receptors in the adult CNS. Learn. Mem. 6: 216–231.

4. Altar, C. A., N. Cai, T. Bliven, M. Juhasz, J. M. Conner, A. L.Acheson, R. M. Lindsay, and S. J. Wiegand. 1997. Anterogradetransport of brain-derived neurotrophic factor and its role in thebrain. Nature 389: 856–860.

5. Altar, C. A., J. A. Siuciak, P. Wright, N. Y. Ip, R. M. Lindsay,and S. J. Wiegand. 1994. In situ hybridization of trkB and trkCreceptor mRNA in rat forebrain and association with high-affinity binding of [125I]BDNF, [125I]NT-4/5 and [125I]NT-3.Eur. J. Neurosci. 6: 1389–1405.

6. Armanini, M. P., S. B. McMahon, J. Sutherland, D. L. Shelton,and H. S. Phillips. 1995. Truncated and catalytic isoforms oftrkB are co-expressed in neurons of rat and mouse CNS. Eur.J. Neurosci. 7: 1403–1409.

7. Atsuta, Y., P. Abraham, T. Iwahara, E. Garcia-Rill, and R. D.Skinner. 1991. Control of locomotion in vitro: II. Chemical stim-ulation. Somatosens. Mot. Res. 8: 55–63.

8. Auld, D. S., F. Mennicken, and R. Quirion. 2001. Nerve growthfactor rapidly induces prolonged acetylcholine release from cul-tured basal forebrain neurons: Differentiation between neuro-modulatory and neurotrophic influences. J. Neurosci. 21: 3375–3382.

9. Baas, D., K. Prufer, M. E. Ittel, S. Kuchler-Bopp, G. Labour-dette, L. L. Sarlieve, and P. Brachet. 2000. Rat oligodendrocytesexpress the vitamin D(3) receptor and respond to 1,25-dihy-droxyvitamin D(3). Glia 31: 59–68.

10. Baldelli, P., P. E. Forni, and E. Carbone. 2000. BDNF, NT-3 andNGF induce distinct new Ca2� channel synthesis in developinghippocampal neurons. Eur. J. Neurosci. 12: 4017–4032.

11. Barbeau, H., and S. Rossignol. 1987. Recovery of locomotionafter chronic spinalization in the adult cat. Brain Res. 412:84–95.

12. Barres, B. A., W. J. Koroshetz, K. J. Swartz, L. L. Chun, andD. P. Corey. 1990. Ion channel expression by white matter glia:The O-2A glial progenitor cell. Neuron 4: 507–524.

13. Barres, B. A., M. C. Raff, F. Gaese, I., Bartke, G. Dechant, andY. A. Barde. 1994. A crucial role for neurotrophin-3 in oligoden-drocyte development. Nature 367: 371–375.

14. Bonanno, G., A. Fassio, G. Schmid, P. Severi, R. Sala, and M.Raiteri. 1997. Pharmacologically distinct GABAB receptorsthat mediate inhibition of GABA and glutamate release inhuman neocortex. Br. J. Pharmacol. 120: 60–64.

15. Buck, C. R., K. L. Seburn, and T. C. Cope. 2000. Neurotrophinexpression by spinal motoneurons in adult and developing rats.J. Comp. Neurol. 416: 309–318.

16. Byravan, S., L. M. Foster, T. Phan, A. N. Verity, and A. T.Campagnoni. 1994. Murine oligodendroglial cells express nervegrowth factor. Proc. Natl. Acad. Sci. USA 91: 8812–8816.

17. Carmignoto, G., T. Pizzorusso, S. Tia, and S. Vicini. 1997.Brain-derived neurotrophic factor and nerve growth factor po-tentiate excitatory synaptic transmission in the rat visual cor-tex. J. Physiol. (Lond.) 498: 153–164.

18. Casha, S., W. R. Yu, and M. G. Fehlings. 2001. Oligodendroglialapoptosis occurs along degenerating axons and is associatedwith FAS and p75 expression following spinal cord injury in therat. Neuroscience 103: 203–218.

19. Chalazonitis, A., T. P. Rothman, J. Chen, F. Lamballe, M.Barbacid, and M. D. Gershon. 1994. Neurotrophin-3 inducesneural crest-derived cells from fetal rat gut to develop in vitroas neurons or glia. J. Neurosci. 14: 6571–6584.

20. Cohen, R. I., R. Marmur, W. T. Norton, M. F. Mehler, and J. A.Kessler. 1996. Nerve growth factor and neurotrophin-3 differ-entially regulate the proliferation and survival of developingrat brain oligodendrocytes. J. Neurosci. 16: 6433–6442.

21. Condorelli, D. F., T. Salin, P. Dell’ Albani, G. Mudo, M. Corsaro,T. Timmusk, M. Metsis, and N. Belluardo. 1995. Neurotrophinsand their trk receptors in cultured cells of the glial lineage andin white matter of the central nervous system. J. Mol. Neurosci.6: 237–248.

22. Conner, J. M., J. C. Lauterborn, Q. Yan, C. M. Gall, and S.Varon. 1997. Distribution of brain-derived neurotrophic factor(BDNF) protein and mRNA in the normal adult rat CNS: Evi-dence for anterograde axonal transport. J. Neurosci. 17: 2295–2313.

23. Conner, J. M., and S. Varon. 1992. Distribution of nerve growthfactor-like immunoreactive neurons in the adult rat brain fol-lowing colchicine treatment. J. Comp. Neurol. 326: 347–362.

24. Copray, S., and D. Kernell. 2000. Neurotrophins and trk-recep-tors in adult rat spinal motoneurons: Differences related to cellsize but not to ‘slow/fast’ specialization. Neurosci. Lett. 289:217–220.

25. Coulier, F., R. Kumar, M. Ernst, R. Klein, D. Martin-Zanca, andM. Barbacid. 1990. Human trk oncogenes activated by pointmutation, in-frame deletion, and duplication of the tyrosinekinase domain. Mol. Cell. Biol. 10: 4202–4210.

26. Cowley, K. C., and B. J. Schmidt. 1994. A comparison of motorpatterns induced by N-methyl-D-aspartate, acetylcholine andserotonin in the in vitro neonatal rat spinal cord. Neurosci. Lett.171: 147–150.

27. Czarkowska-Bauch, J., A. Dwornik, M. Macias, D. Sulejczak,M. Wiater, and M. Skup. 2001. BDNF and NT-4 immunoreac-itvity increase in spinal cord fibers following locomotor trainingin the adult rat. In Higher Nervous Control of Posture andLocomotion: Parallel and Centralized Control Mechanisms. Na-tional Center for Physiological Sciences, Okazaki, Japan.

28. de Leon, R. D., J. A. Hodgson, R. R. Roy, and V. R. Edgerton.1998. Locomotor capacity attributable to step training versusspontaneous recovery after spinalization in adult cats. J. Neu-rophysiol. 79: 1329–1340.

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

30. Dougherty, K. D., C. F. Dreyfus, and I. B. Black. 2000. Brain-derived neurotrophic factor in astrocytes, oligodendrocytes, andmicroglia/macrophages after spinal cord injury. Neurobiol. Dis.7: 574–585.

31. Douglas, J. R., B. R. Noga, X. Dai, and L. M. Jordan. 1993. Theeffects of intrathecal administration of excitatory amino acidagonists and antagonists on the initiation of locomotion in theadult cat. J. Neurosci. 13: 990–1000.

32. Dreyfus, C. F., X. Dai, L. D. Lercher, B. R. Racey, W. J. Fried-man, and I. B. Black. 1999. Expression of neurotrophins in theadult spinal cord in vivo. J. Neurosci. Res. 56: 1–7.

33. Ernfors, P., C. M. Rosario, J. P. Merlio, G. Grant, H. Aldskogius,and H. Persson. 1993. Expression of mRNAs for neurotrophin

304 SKUP ET AL.

receptors in the dorsal root ganglion and spinal cord duringdevelopment and following peripheral or central axotomy.Brain Res. Mol. Brain Res. 17: 217–226.

34. Fawcett, J. P., R. Aloyz, J. H. McLean, S. Pareek, F. D. Miller,P. S. McPherson, and R. A. Murphy. 1997. Detection of brain-derived neurotrophic factor in a vesicular fraction of brainsynaptosomes. J. Biol. Chem. 272: 8837–8840.

35. Figueiredo, B. C., M. Skup, A. M. Bedard, W. Tetzlaff, and A. C.Cuello. 1995. Differential expression of p140trk, p75NGFR andgrowth-associated phosphoprotein-43 genes in nucleus basalismagnocellularis, thalamus and adjacent cortex following neo-cortical infarction and nerve growth factor treatment. Neuro-science 68: 29–45.

36. Frisen, J., V. M. Verge, S. Cullheim, H. Persson, K. Fried, D. S.Middlemas, T. Hunter, T. Hokfelt, and M. Risling. 1992. In-creased levels of trkB mRNA and trkB protein-like immunore-activity in the injured rat and cat spinal cord. Proc. Natl. Acad.Sci. USA 89: 11282–11286.

37. Frisen, J., V. M. Verge, K. Fried, M. Risling, H. Persson, J.Trotter, T. Hokfelt, and D. Lindholm. 1993. Characterization ofglial trkB receptors: Differential response to injury in the cen-tral and peripheral nervous systems. Proc. Natl. Acad. Sci. USA90: 4971–4975.

38. Fryer, R. H., D. R. Kaplan, S. C. Feinstein, M. J. Radeke, D. R.Grayson, and L. F. Kromer. 1996. Developmental and matureexpression of full-length and truncated TrkB receptors in therat forebrain. J. Comp. Neurol. 374: 21–40.

39. Funakoshi, H., N. Belluardo, E. Arenas, Y. Yamamoto, A. Casa-bona, H. Persson, and C. F. Ibanez. 1995. Muscle-derived neu-rotrophin-4 as an activity-dependent trophic signal for adultmotor neurons. Science 268: 1495–1499.

40. Gharakhanlou, R., S. Chadan, and P. Gardiner. 1999. Increasedactivity in the form of endurance training increases calcitoningene-related peptide content in lumbar motoneuron cell bodiesand in sciatic nerve in the rat. Neuroscience 89: 1229–1239.

41. Gomez-Pinilla, F., Z. Ying, P. Opazo, R. R. Roy, and V. R.Edgerton. 2001. Differential regulation by exercise of BDNFand NT-3 in rat spinal cord and skeletal muscle. Eur. J. Neu-rosci. 13: 1078–1084.

42. Gonzalez, M., F. P. Ruggiero, Q. Chang, Y. J. Shi, M. M. Rich,S. Kraner, and R. J. Balice-Gordon. 1999. Disruption of Trkb-mediated signaling induces disassembly of postsynaptic recep-tor clusters at neuromuscular junctions. Neuron 24: 567–583.

43. Gooney, M., and M. A. Lynch. 2001. Long-term potentiation inthe dentate gyrus of the rat hippocampus is accompanied bybrain-derived neurotrophic factor-induced activation of TrkB.J. Neurochem. 77: 1198–1207.

44. Goutan, E., E. Marti, and I. Ferrer. 1998. BDNF, and full lengthand truncated TrkB expression in the hippocampus of the ratfollowing kainic acid excitotoxic damage: Evidence of complextime-dependent and cell-specific responses. Brain Res. Mol.Brain Res. 59: 154–164.

45. Hendry, I. A., K. Stockel, H. Thoenen, and L. L. Iversen. 1974.The retrograde axonal transport of nerve growth factor. BrainRes. 68: 103–121.

46. Hoener, M. C., E. Hewitt, J. M. Conner, J. W. Costello, and S.Varon. 1996. Nerve growth factor (NGF) content in adult ratbrain tissues is several-fold higher than generally reported andis largely associated with sedimentable fractions. Brain Res.728: 47–56.

47. Horner, P. J., A. E. Power, G. Kempermann, H. G. Kuhn, T. D.Palmer, J. Winkler, L. J. Thal, and F. H. Gage. 2000. Prolifer-ation and differentiation of progenitor cells throughout theintact adult rat spinal cord. J. Neurosci. 20: 2218–2228.

48. Hughes, P. E., T. Alexi, M. Walton, C. E. Williams, M. Dra-gunow, R. G. Clark, and P. D. Gluckman. 1999. Activity andinjury-dependent expression of inducible transcription factors,growth factors and apoptosis-related genes within the centralnervous system. Prog. Neurobiol. 57: 421–450.

49. Johnston, A. N., M. P. Clements, and S. P. Rose. 1999. Role ofbrain-derived neurotrophic factor and presynaptic proteins inpassive avoidance learning in day-old domestic chicks. Neuro-science 88: 1033–1042.

50. Kafitz, K. W., C. R. Rose, H. Thoenen, and A. Konnerth. 1999.Neurotrophin-evoked rapid excitation through TrkB receptors.Nature 401: 918–921.

51. Kryl, D., T. Yacoubian, A. Haapasalo, E. Castren, D. Lo, andP. A. Barker. 1999. Subcellular localization of full-length andtruncated Trk receptor isoforms in polarized neurons and epi-thelial cells. J. Neurosci. 19: 5823–5833.

52. Kumar, S., and J. de Vellis. 1996. Neurotrophin activates signaltransduction in oligodendroglial cells: Expression of functionalTrkC receptor isoforms. J. Neurosci. Res. 44: 490–498.

53. Lovely, R. G., R. J. Gregor, R. R. Roy, and V. R. Edgerton. 1986.Effects of training on the recovery of full-weight-bearing step-ping in the adult spinal cat. Exp. Neurol. 92: 421–435.

54. McAllister, A. K., L. C. Katz, and D. C. Lo. 1999. Neurotrophinsand synaptic plasticity. Annu. Rev. Neurosci. 22: 295–318.

55. McTigue, D. M., P. J. Horner, B. T. Stokes, and F. H. Gage.1998. Neurotrophin-3 and brain-derived neurotrophic factorinduce oligodendrocyte proliferation and myelination of regen-erating axons in the contused adult rat spinal cord. J. Neurosci.18: 5354–5365.

56. Michael, G. J., E. Kaya, S. Averill, M. Rattray, D. O. Clary, andJ. V. Priestley. 1997. TrkA immunoreactive neurones in the ratspinal cord. J. Comp. Neurol. 385: 441–455.

57. Mihailovic, L. J., and H. Hyden. 1969. On antigenic differencesbetween nerve cells and glia. Brain Res. 16: 243–256.

58. Mize, R. R., R. N. Holdefer, and L. B. Nabors. 1988. Quantita-tive immunocytochemistry using an image analyzer. I. Hard-ware evaluation, image processing, and data analysis. J. Neu-rosci. Methods 26: 1–23.

59. Mufson, E. J., J. S. Kroin, T. Sobreviela, M. A. Burke, J. H.,Kordower, R. D. Penn, and J. A. Miller. 1994. Intrastriatalinfusions of brain-derived neurotrophic factor: Retrogradetransport and colocalization with dopamine containing sub-stantia nigra neurons in rat. Exp. Neurol. 129: 15–26.

60. Neeper, S. A., F. Gomez-Pinilla, J. Choi, and C. Cotman. 1995.Exercise and brain neurotrophins [letter]. Nature 373: 109.

61. Neeper, S. A., F. Gomez-Pinilla, J. Choi, and C. W. Cotman.1996. Physical activity increases mRNA for brain-derived neu-rotrophic factor and nerve growth factor in rat brain. Brain Res.726: 49–56.

62. Pascual, M., E. Climent, and C. Gueri. 2001. BDNF inducesglutamate release in cerebrocortical nerve terminals and incortical astrocytes. Neuroreport 12: 2673–2677.

63. Patterson, S. L., C. Pittenger, A. Morozov, K. C. Martin, H.Scanlin, C. Drake, and E. R. Kandel. 2001. Some forms ofcAMP-mediated long-lasting potentiation are associated withrelease of BDNF and nuclear translocation of phospho-MAPkinase. Neuron 32: 123–140.

64. Paxinos, G., and C. Watson. 1997. The Rat Brain in StereotaxicCoordinates, compact 3rd ed. Academic Press, San Diego.

65. Pesavento, E., E. Margotti, M. Righi, A. Cattaneo, and L. Do-menici. 2000. Blocking the NGF–TrkA interaction rescues thedevelopmental loss of LTP in the rat visual cortex: Role of thecholinergic system. Neuron 25: 165–175.

66. Rash, J. E., H. S. Duffy, F. E. Dudek, B. L. Bilhartz, L. R.Whalen, and T. Yasumura. 1997. Grid-mapped freeze-fracture

305TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD

analysis of gap junctions in gray and white matter of adult ratcentral nervous system, with evidence for a �panglial syncy-tium� that is not coupled to neurons. J. Comp. Neurol. 388:265–292.

67. Righi, M., E. Tongiorgi, and A. Cattaneo. 2000. Brain-derivedneurotrophic factor (BDNF) induces dendritic targeting ofBDNF and tyrosine kinase B mRNAs in hippocampal neuronsthrough a phosphatidylinositol-3 kinase-dependent pathway.J. Neurosci. 20: 3165–3174.

68. Rubio, N. 1997. Mouse astrocytes store and deliver brain-de-rived neurotrophic factor using the non-catalytic gp95trkB re-ceptor. Eur. J. Neurosci. 9: 1847–1853.

69. Russo-Neustadt, A., R. C. Beard, and C. W. Cotman. 1999.Exercise, antidepressant medications, and enhanced brain de-rived neurotrophic factor expression. Neuropsychopharmacol-ogy 21: 679–682.

70. Scarisbrick, I. A., K. Asakura, and M. Rodriguez. 2000. Neuro-trophin-4/5 promotes proliferation of oligodendrocyte-type-2 as-trocytes (O-2A). Brain Res. Dev. Brain Res. 123: 87–90.

71. Scarisbrick, I. A., P. J. Isackson, and A. J. Windebank. 1999.Differential expression of brain-derived neurotrophic factor,neurotrophin-3, and neurotrophin-4/5 in the adult rat spinalcord: Regulation by the glutamate receptor agonist kainic acid.J. Neurosci. 19: 7757–7769.

72. Schwab, M. E., U. Otten, Y. Agid, and H. Thoenen. 1979. Nervegrowth factor (NGF) in the rat CNS: Absence of specific retro-grade axonal transport and tyrosine hydroxylase induction inlocus coeruleus and substantia nigra. Brain Res. 168: 473–483.

73. Sherwood, N. T., and D. C. Lo. 1999. Long-term enhancement ofcentral synaptic transmission by chronic brain-derived neuro-trophic factor treatment. J. Neurosci. 19: 7025–7036.

74. Siegel, S. 1956. Nonparametric Statistics for the BehavioralSciences. McGraw–Hill, New York.

75. Skup, M., D. Koczyk, M. Zaremba, and B. Oderfeld-Nowak.1995. Differential changes in TrkA and TrkB neurotrophinreceptors in the rat hippocampus following trimethylin intoxi-cation. Soc. Neurosci. Abstract, p. 1550.

76. Skup, M., J. Czarkowska-Bauch, A. Dwornik, M. Macias, D.Sulejczak, and M. Wiater. 2000. Locomotion induces changes inTrk B receptors in small diameter cells of the spinal cord. ActaNeurobiol. Exp. 60: 371.

77. Skup, M., M. Macias, A. Dwornik, D. Sulejczak, M. Wiater, andJ. Czarkowska-Bauch. 2001. Identification of spinal cells re-sponding with an increased expression of TrkB receptor proteinto locomotor training in adult rats. In Higher Nervous Controlof Posture and Locomotion: Parallel and Centralized ControlMechanisms. National Center for Physiological Sciences, Oka-zaki, Japan.

78. Skup, M., M. Macias, A. Dwornik, D. Sulejczak, M. Wiater, andJ. Czarkowska-Bauch. 2001. Locomotor training induceschanges in TrkB expression in the spinal cord of adult rats:Involvement of glial cells. In Spinal Cord Trauma: NeuralRepair and Functional Recovery, p. 101. XXIII CRSN Symp.Int., Montreal, Canada.

79. Smith, M. A., L. X. Zhang, W. E. Lyons, and L. A. Mamounas.1997. Anterograde transport of endogenous brain-derived neu-rotrophic factor in hippocampal mossy fibers. Neuroreport 8:1829–1834.

80. Sobreviela, T., M. Pagcatipunan, J. S. Kroin, and E. J. Mufson.1996. Retrograde transport of brain-derived neurotrophic factor(BDNF) following infusion in neo- and limbic cortex in rat:Relationship to BDNF mRNA expressing neurons. J. Comp.Neurol. 375: 417–444.

81. Sommerfeld, M. T., R. Schweigreiter, Y. A. Barde, and E.Hoppe. 2000. Down-regulation of the neurotrophin receptor

TrkB following ligand binding: Evidence for an involvement ofthe proteasome and differential regulation of TrkA and TrkB.J. Biol. Chem. 275: 8982–8990.

82. Suen, P. C., K. Wu, E. S. Levine, H. T. Mount, J. L. Xu, S. Y.Lin, and I. B. Black. 1997. Brain-derived neurotrophic factorrapidly enhances phosphorylation of the postsynaptic N-methyl-D-aspartate receptor subunit I. Proc. Natl. Acad. Sci.USA 94: 8191–8195.

83. Takei, N., T. Numakawa, S. Kozaki, N. Sakai, Y. Endo, M.Takahashi, and H. Hatanaka. 1998. Brain-derived neurotro-phic factor induces rapid and transient release of glutamatethrough the non-exocytotic pathway from cortical neurons.J. Biol. Chem. 273: 27620–27624.

84. Takei, N., K. Sasaoka, K. Inoue, M. Takahashi, Y. Endo, and H.Hatanaka. 1997. Brain-derived neurotrophic factor increasesthe stimulation-evoked release of glutamate and the levels ofexocytosis-associated proteins in cultured cortical neurons fromembryonic rats. J. Neurochem. 68: 370–375.

85. Tongiorgi, E., M. Righi, and A. Cattaneo. 1997. Activity-depen-dent dendritic targeting of BDNF and TrkB mRNAs in hip-pocampal neurons. J. Neurosci. 17: 9492–9505.

86. Vick, R. S., J. M. Collins, and G. H. DeVries. 1992. Cell cycleand proliferation dynamics of adult rat oligodendrocytes.J. Neurosci. Res. 33: 75–81.

87. von Bartheld, C. S., M. R. Byers, R. Williams, and M. Bothwell.1996. Anterograde transport of neurotrophins and axodendritictransfer in the developing visual system. Nature 379: 830–833.

88. Wada, S., K. Yone, Y. Ishidou, T. Nagamine, S. Nakahara, T.Niiyama, and T. Sakou. 1999. Apoptosis following spinal cordinjury in rats and preventative effect of N-methyl-D-aspartatereceptor antagonist. J. Neurosurg. 91: 98–104.

89. Walwyn, W. M., J. Ta-Haung, L. Ackerson, N. T. Maidment,and V. R. Edgerton. 1999. Extracellular glutamate in the dorsalhorn of the lumbar spinal cord in the freely moving rat duringhindlimb stepping. Pharmacol. Biochem. Behav. 63: 581–588.

90. Wetmore, C., and L. Olson. 1995. Neuronal and nonneuronalexpression of neurotrophins and their receptors in sensory andsympathetic ganglia suggest new intercellular trophic interac-tions. J. Comp. Neurol. 353: 143–159.

91. Widenfalk, J., L. Olson, and P. Thoren. 1999. Deprived of ha-bitual running, rats downregulate BDNF and TrkB messagesin the brain. Neurosci. Res. 34: 125–132.

92. Wu, K., J. L. Xu, P. C. Suen, E. Levine, Y. Y. Huang, H. T.Mount, S. Y. Lin, and I. B. Black. 1996. Functional trkB neu-rotrophin receptors are intrinsic components of the adult brainpostsynaptic density. Brain Res. Mol. Brain Res. 43: 286–290.

93. Wyneken, U., K. H. Smalla, J. J. Marengo, D. Soto, A. de laCerda, W. Tischmeyer, R. Grimm, T. M. Boeckers, G. Wolf, F.Orrego, and E. D. Gundelfinger. 2001. Kainate-induced sei-zures alter protein composition and N-methyl-D-aspartate re-ceptor function of rat forebrain postsynaptic densities. Neuro-science 102: 65–74.

94. Xu, B., W. Gottschalk, A. Chow, R. I. Wilson, E. Schnell, K.Zang, D. Wang, R. A. Nicoll, B. Lu, and L. F. Reichardt. 2000.The role of brain-derived neurotrophic factor receptors in themature hippocampus: Modulation of long-term potentiationthrough a presynaptic mechanism involving TrkB. J. Neurosci.20: 6888–6897.

95. Yan, Q., M. J. Radeke, C. R. Matheson, J. Talvenheimo, A. A.Welcher, and S. C. Feinstein. 1997. Immunocytochemical local-ization of TrkB in the central nervous system of the adult rat.J. Comp. Neurol. 378: 135–157.

96. Yan, Q., R. D. Rosenfeld, C. R. Matheson, N. Hawkins, O. T.Lopez, L. Bennett, and A. A. Welcher. 1997. Expression of

306 SKUP ET AL.

brain-derived neurotrophic factor protein in the adult rat cen-tral nervous system. Neuroscience 78: 431–448.

97. Zhang, J. Y., X. G. Luo, C. J. Xian, Z. H. Liu, and X. F. Zhou.2000. Endogenous BDNF is required for myelination and re-generation of injured sciatic nerve in rodents. Eur. J. Neurosci.12: 4171–4180.

98. Zhou, X. F., and R. A. Rush. 1996. Endogenous brain-derivedneurotrophic factor is anterogradely transported in primarysensory neurons. Neuroscience 74: 945–953.

99. Ziak, D., A. Chvatal, and E. Sykova. 1998. Glutamate-, kainate-and NMDA-evoked membrane currents in identified glial cellsin rat spinal cord slice. Physiol. Res. 47: 365–375.

307TRAINING UP-REGULATES TrkB BDNF NT-4 IN SPINAL CORD