BDNF overexpression increases dendrite complexity in hippocampal dentate gyrus

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Neuroscience Vol. 114, No. 3, pp. 795^805, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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BDNF OVEREXPRESSION INCREASES DENDRITE COMPLEXITY IN HIPPOCAMPAL DENTATE GYRUS R. J. TOLWANI,a P. S. BUCKMASTER,a S. VARMA,a J. M. COSGAYA,b Y. WU,b C. SURIc and E. M. SHOOTERb a

Department of Comparative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA b

Department of Neurobiology, Stanford University School of Medicine, Fairchild Building, Room D200, Stanford, CA 94305-5125, USA c

Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA

Abstract9There is increasing evidence that brain-derived neurotrophic factor (BDNF) modulates synaptic and morphological plasticity in the developing and mature nervous system. Plasticity may be modulated partially by BDNF’s e¡ects on dendritic structure. Utilizing transgenic mice where BDNF overexpression was controlled by the L-actin promoter, we evaluated the e¡ects of long-term overexpression of BDNF on the dendritic structure of granule cells in the hippocampal dentate gyrus. BDNF transgenic mice provided the opportunity to investigate the e¡ects of modestly increased BDNF levels on dendrite structure in the complex in vivo environment. While the elevated BDNF levels were insu⁄cient to change levels of TrkB receptor isoforms or downstream TrkB signaling, they did increase dendrite complexity of dentate granule cells. These cells showed an increased number of ¢rst order dendrites, of total dendritic length and of total number of branch points. These results suggest that dendrite structure of granule cells is tightly regulated and is sensitive to modest increases in levels of BDNF. This is the ¢rst study to evaluate the e¡ects of BDNF overexpression on dendrite morphology in the intact hippocampus and extends previous in vitro observations that BDNF in£uences synaptic plasticity by increasing complexity of dendritic arbors. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: brain-derived neurotrophic factor, dendrite structure, granule cells, dentate gyrus, plasticity, TrkB receptor.

plexity of dendritic arbors (Cohen-Cory and Fraser, 1994; Cohen-Cory, 1999; Lom and Cohen-Cory, 1999; McAllister et al., 1995, 1997; Horch et al., 1999). Also in the ferret cortex, brain-derived neurotrophic factor (BDNF) overexpression induced structural instability of newly formed dendrites and increased turnover of spines (Horch et al., 1999). The mode of dendritic growth can be modulated not only by neurotrophins but also by TrkB receptors. There are three mammalian TrkB receptor isoforms produced by alternative splicing that bind BDNF, a full-length TrkB receptor isoform with the tyrosine kinase domain intact and two truncated isoforms, T1 and T2, that lack the tyrosine kinase domain (Barbacid, 1994). In vitro studies suggest that truncated TrkB isoforms may decrease the BDNF response (Bi¡o et al., 1995; Eide et al., 1996; Ninkina et al., 1996; Fryer et al., 1997) or may have their own signaling cascade (Baxter et al., 1997). A role for TrkB in the e¡ects of BDNF in dendritic complexity is suggested by the observation that deletion of TrkB receptors reduced the dendritic complexity of pyramidal cells and compressed cortical layers in vivo (Xu et al., 2000b). Moreover, full-length and T1 TrkB receptor isoforms appear to regulate dendritic structure in organotypic ferret cortical slices (Yacoubian and Lo, 2000). The in vivo and in vitro studies noted above demonstrated that short-term increases in BDNF levels in£u-

Neurotrophins consist of a family of related proteins that exert biological actions primarily on cells of the nervous system (Lewin and Barde, 1996). Besides their classical role in supporting survival of neuronal populations, neurotrophins in the CNS modulate morphological plasticity and potentiate synaptic transmission (Kang and Schuman, 1995a,b; Cabelli et al., 1995; Katz and Shatz, 1996). Neurotrophins are secreted by activity dependent mechanisms in CNS neurons (Blochl and Thoenen, 1995; Moller et al., 1998) and strengthen active synaptic connections. Structural changes in neurons, in£uenced by neurotrophins, alter the network of synaptic connections between neurons and modify neuronal circuitry (Katz and Shatz, 1996). In vivo studies in the Xenopus tadpole and in vitro studies in mammalian systems using ferret cortical slices demonstrated that neurotrophins modulate neuronal architecture by promoting changes in growth and com-

*Corresponding author. Tel. : +1-650-723-6638; fax: +1-650-7253958. E-mail address: [email protected] (E. M. Shooter). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; BDNF, brainderived neurotrophic factor; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase ; NGF, nerve growth factor; PBS, phosphate-bu¡ered saline; PC12, pheochromocytoma; PC12-TrkB, PC12 cell-derived line; PI-3K, phosphatidylinositol-3 kinase ; SDS, sodium dodecyl sulfate. 795

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ence dendritic morphology at speci¢c early development stages when dendrites are rapidly forming (Cohen-Cory, 1999; Lom and Cohen-Cory, 1999; McAllister et al., 1995, 1997; Horch et al., 1999) but have not addressed the e¡ects of long-term increases in BDNF on dendritic structure in the mature brain. Using transgenic mice overexpressing BDNF under the control of the L-actin promoter, we examined the e¡ects of long-term BDNF overexpression on dendritic structure of granule cells of the hippocampal dentate gyrus. The transgenic mice provided a means to examine the e¡ects of sustained modest increases in levels of BDNF in an in vivo system where the three-dimensional environment and complex in£uences of their network interactions are preserved.

EXPERIMENTAL PROCEDURES

Animals All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All e¡orts were made to minimize the number of animals used and their su¡ering. Transgenic mice overexpressing BDNF with a carboxy-terminus myc-epitope tag under the control of the human L-actin promoter were provided by Regeneron Pharmaceuticals (Tarrytown, NY, USA) (Croll et al., 1999). The BDNF transgenic mice express BDNFmyc in many regions of the brain and other tissues (Croll et al., 1999). The mice were produced on a CBAUC57BL/6 background and were subsequently backcrossed to C57BL/6 mice for six generations. Mice were genotyped by Southern blot analysis using BamHI-digested genomic DNA probed with 32 P-labeled 800-bp full-length BDNF probe using standard techniques. The L-actin promoter controlling BDNF overexpression is active during early development (DePrimo et al., 1996) and into adulthood and is active in neuronal, glial and other non-neuronal cells. Western analysis of TrkB receptors Hippocampi were isolated from 2-day-old (n = 5 BDNF and n = 5 control) and 8-week-old (n = 8 BDNF and n = 8 control) mice for TrkB receptor protein analysis and immediately frozen. Tissue was homogenized and lysed in RIPA bu¡er (1Uphosphate-bu¡ered saline (PBS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS)) with 10% glycerol and Complete1 Protease Inhibitor (Boehringer Mannheim), 1 mM phenylmethylsulfonyl £uoride and 1 mM sodium orthovanadate. Lysates were quanti¢ed by Bradford BCA protein assay. Protein lysates were denatured, separated in 8% SDS^PAGE and transferred overnight onto a 0.45-Wm nitrocellulose membrane (SchleicherpSchuell, Keene, NH, USA). After blocking with 5% non-fat milk in PBS with 0.1% Tween 20, the membrane was immunoblotted overnight at 4‡C with a 1:500 dilution of a mouse monoclonal anti-TrkB antibody (Transduction Laboratories, Lexington, KY, USA). Blots were incubated in anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody for 2^4 h at room temperature using standard procedures and developed by chemiluminescence (Renaissance1, NEN Lifesciences Products, Boston, MA, USA). The anti-TrkB antibody recognized the 95-kDa truncated and 145-kDa full-length TrkB bands. Truncated and full-length TrkB bands were quanti¢ed using NIH Imaging software (NIH, Bethesda, MD, USA). Western analysis of TrkB downstream signaling cascades We examined two downstream signaling transduction pathways of TrkB receptor activation. The extent of activation of

the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3 kinase (PI-3K) pathways was determined with speci¢c antibodies against phospho-ERK and phospho-Akt, respectively. In both instances, the blots were stripped and reprobed with antibodies against ERK or Akt to determine their absolute levels of expression. Also as controls, pheochromocytoma (PC12) cells (that express TrkA but not TrkB) and a TrkB stably transfected PC12 cell-derived line (PC12-TrkB) were stimulated for 10 min with medium alone, 200 ng/ml of NGF (nerve growth factor), or 200 ng/ml of BDNF. Cells were washed and lysed in ice-cold RIPA lysis bu¡er. Lysates were boiled with sample bu¡er and loaded onto the SDS^PAGE gel along with hippocampal lysates from 2-day-old (n = 3 BDNF, n = 3 control) and 8-week-old (n = 8 BDNF and n = 8 control) mice as above. After transfer, the membranes were blocked and sequentially probed with the di¡erent antibodies: 1:500 dilution of anti-phospho (Thr-183/Thr-185)-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 dilution of anti-ERK2 (Santa Cruz Biotechnology), 1:2000 dilution of anti-phospho (Ser-473)-Akt 4E3 monoclonal antibody (Cell Signaling Technology1, Beverly, MA, USA) and 1:1000 dilution of anti-Akt antibody (Cell Signaling Technology). Blots were incubated in HRP-conjugated secondary antibody for 2^4 h at room temperature using standard procedures. The bands were visualized by the chemiluminescence assay method and quanti¢ed with NIH Imaging as described above. Hippocampal slice preparation and biocytin labeling Four BDNF transgenic mice and three littermate controls at 8 weeks of age were used for biocytin labeling. Mice were anesthetized with pentobarbital (60 mg/kg, i.p.). The brain was removed, placed in ice-cold oxygenated arti¢cial cerebrospinal £uid (ACSF) for 60 s, blocked, and sliced at 400 Wm on a vibratome with a refrigeration unit (Technical Products, St. Louis, MO, USA) to yield horizontal slices. The slices were placed in an interface-type recording chamber (Fine Science Tools, Foster City, CA, USA) perfused with oxygenated ACSF at 1^2 ml/min at 34‡C. Intracellular recording electrodes (70^150 M6), prepared with a horizontal puller (P-97, Sutter Instruments, Novato, CA, USA) and ¢lled with 1 M potassium acetate and 2% biocytin (Sigma, St. Louis, MO, USA), were inserted into the granule cell layer in the dentate gyrus. The membrane potential of the dentate cells was ampli¢ed and recorded (Axoclamp-2B, CyberAmp 380, Axon Instruments, Foster City, CA, USA). Impaled cells with resting membrane potentials of at least 355 mV were used for labeling. Cells from acute slice preparations were iontophoretically injected with biocytin by passing 300-ms pulses, 50% duty cycle, of 0.4^1.0-nA hyperpolarizing current for an average of 8 min. Slices were ¢xed immediately after ¢lling by placing them between ¢lter papers in 4% paraformaldehyde in 0.1 M phosphate bu¡er pH 7.2 for 36^60 h. Cells were labeled and slices ¢xed within 6 h of slice preparation. Slices were cryoprotected in 30% sucrose and sectioned at 60 Wm with a sliding microtome. Free-£oating sections were processed using the avidin biotinylated enzyme complex method and diaminobenzidine after suppressing endogenous peroxidases as described (Buckmaster and Amaral, 2001). Sections were mounted in series on gelatincoated slides and coverslipped following ethanol dehydration. Dendritic reconstruction Dendritic arbors of granule cells were reconstructed threedimensionally with the Neurolucida image analysis system (MicroBrightField, Colchester, VT) using a 100U oil objective on an Axioskop microscope (Zeiss, Germany). The criteria used to determine if granule cells were adequately labeled for accurate dendritic reconstructions included: (1) dendrites were darkly labeled and easily visible throughout the molecular layer, (2) all dendrites within the proximal third of the molecular layer were contained within the adjacent serial histological sections, and (3) no more than two dendrites from the distal third of the molecular layer were cut at the surface of the slice. Only

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labeled granule cells meeting these criteria were used for reconstructions. Sholl analysis Three-dimensional dendritic tree reconstructions were used for Sholl analysis (Sholl, 1953). The Neurolucida software calculated the cumulative number of dendritic intersections at 25-Wm interval distance points from each granule cell. The average total number of intersections from each granule cell was plotted against the distance from the cell body. Statistical tests Student’s t-test and M2 test were used for statistical analysis. The null hypothesis was rejected at P 6 0.05.

RESULTS

BDNF transgenic mice To study the e¡ects of BDNF on dendritic morphology we examined granule cells of the hippocampus in young adult transgenic mice where the expression of BDNF, with a carboxy-terminus myc-epitope tag, is under the control of the L-actin promoter (Croll et al., 1999). It has been previously determined that the carboxy-terminus 10-amino acid myc tag does not alter sorting and biological activity of the BDNFmyc protein (Moller et al., 1998). These transgenic mice overexpress BDNF in many tissues including several regions of the brain. Northern blots of the hippocampus from young adult BDNF transgenic mice indicated that the levels of BDNF mRNA increased by two- to three-fold in the hippocampus (Croll et al., 1999). In situ hybridizations of the hippocampus revealed increased expression levels of BDNF mRNA in the granule cell layer and in the hilus and CA3 areas, which are the areas of granule cell axon targets (Croll et al., 1999; Qiao et al., 2001). There was only a modest 32% increase in the level of BDNF protein in the hippocampus as determined by enzyme-linked immunoabsorbent assays (Croll et al., 1999). E¡ect of BDNF overexpression on TrkB receptors It has been previously reported that exposure to high doses of BDNF down-regulates protein levels of fulllength TrkB receptors both in vitro and in vivo (Frank et al., 1996; Knusel et al., 1997; Sommerfeld et al., 2000). In order to determine if such a down-regulation of TrkB receptor also occurred in our system, we analyzed the levels of truncated and full-length receptors. We examined two time points, one at postnatal day 2, when truncated TrkB receptor levels are starting to increase signi¢cantly, and the second at 8 weeks of age when the amounts of truncated TrkB receptors have reached their ¢nal adult levels (Allendoerfer et al., 1994; Escandon et al., 1994). The 8-week time point also represents the time of biocytin labeling and dendrite analysis.

Fig. 1. Western blot analysis of protein levels of full-length TrkB (TrkB FL) and truncated TrkB (TrkB T) receptors from hippocampi of BDNF transgenic and control mice at 2 days and 8 weeks of age. The levels of truncated TrkB increased signi¢cantly from day 2 to the 8-week time point. There were no signi¢cant changes in levels of full-length TrkB receptors in the BDNF transgenic mice at both 2 days and 8 weeks of age when compared to age-matched non-transgenic controls.

From day 2 to 8 weeks of age, the relative abundance of truncated compared to full-length TrkB receptors increased signi¢cantly, both in control and in BDNF transgenic mice. This di¡erence re£ects the increasing levels of truncated TrkB receptors during postnatal development (Allendoerfer et al., 1994). No obvious differences were observed in expression levels of both TrkB isoforms between BDNF transgenic and control animals at 2 days or 8 weeks of age (Fig. 1) (Escandon et al., 1994). In BDNF transgenic mice, the truncated to fulllength TrkB ratio was the same at 8 weeks of age (0.674 U 0.096 S.D. against 0.647 U 0.134 S.D. in controls) while only a non-signi¢cant slight increase of about 22% was observed in the 2-day-old animals (0.338 U 0.030 in BDNF transgenics and 0.276 U 0.052 in controls, t-test, P = 0.15). These results indicate that the moderate BDNF overexpression achieved in the BDNF transgenic mice did not induce signi¢cant down-regulation in full-length TrkB levels. TrkB downstream signaling cascade Two of the most relevant intracellular pathways that are implicated in BDNF-TrkB action are the ERK/ MAPK and PI-3K^Akt pathways (Atwal et al., 2000; Bartlett et al., 1997; Kaplan and Miller, 2000). In order to determine if the continued overexpression of BDNF in the hippocampus of the transgenic mice a¡ected the activity of the ERK/MAPK pathway, we performed western blot studies, with anti-phosphoERK-speci¢c antibodies, on hippocampal lysates from 2-day and 8-week-old BDNF transgenic and control animals (Fig. 2). As controls for ERK activation, wild-type PC12 cells (expressing TrkA but not TrkB receptors) and PC12-TrkB cells (a stably transfected PC12 cell line that expresses high levels of TrkB receptors) were used. The treatment of PC12 cells with NGF or the PC12-TrkB with BDNF produced a strong increase in the activation of the ERK/MAPK pathway as seen by an increase in phospho-ERK immunoreactivity (Fig. 2A). In the animals, which express similar amounts of ERK as PC12 cells, the level of ERK activation was similar to that observed in unstimulated PC12 cells. The levels of phospho-ERK normalized to the levels of total ERK protein were similar at 2 days of age (93 U 43% S.D. in BDNFoverexpressing mice and 100 U 18% S.D. in controls, t-test, P = 0.803) and at 8 weeks of age (99 U 28% S.D. in BDNF-overexpressing mice and 100 U 47% S.D. in

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Fig. 2. Western blot analysis of TrkB downstream signaling pathways. (A) Lysates from hippocampi of BDNF transgenic and control mice at 2 days and 8 weeks of age were probed with anti-phospho-ERK and anti-ERK antibodies. PC12 cells expressing TrkA only (lanes 1 and 2) and PC12-TrkB cells expressing both TrkA and TrkB (lanes 3 and 4) were stimulated for 10 min with medium alone (lanes 1 and 3), 200 ng/ml of NGF (lane 2) or 200 ng/ml of BDNF (lane 4) and used as controls. Cells were lysed immediately after stimulation and lysates separated electrophoretically and blotted together with the hippocampal lysates. Anti-phospho-ERK antibody detects two protein bands at 42 and 44 kDa representing phosphorylated ERK1 and ERK2 proteins. Bottom panel shows the same blot reprobed with anti-ERK2 antibody to detect the 42-kDa ERK2 protein in the cell lysates and hippocampus. (B) Activation of Akt was detected by probing with an anti-phospho (Ser-473)-speci¢c Akt antibody, detecting a band at 60 kDa. Bottom panel represents the same blot probed with anti-Akt antibody.

controls, t-test, P = 0.948). At both ages studied, therefore, the BDNF transgenic mice presented a similar level of ERK activity as wild-type mice. We also examined the PI-3K TrkB signaling pathway by measuring phosphorylated Akt (Kaplan and Miller, 2000), a Ser/Thr kinase that lies downstream in the PI-3K pathway (Crowder and Freeman, 1998). We evaluated Akt activation by immunoblotting hippocampal lysates from BDNF transgenic and control mice with a phosphorylation-speci¢c Akt antibody normalized against total Akt expression (Fig. 2). PC12 cells were used as controls of Akt activation. The anti-phosphoAkt and anti-Akt identi¢ed bands at 60 kDa. Akt total protein levels decreased over time and at 8 weeks of age

only represented 47% of the values found in 2-day-old mice, reaching a level similar to that found in PC12 cells. The levels of phospho-Akt were also lower in 8-week-old mice compared to 2-day-old mice, possibly due to decreased levels of Akt protein in the 8-week-old group. The levels of phospho-Akt normalized to the levels of total Akt were similar between BDNF and control mice at 2 days of age (105 U 40% in the BDNF transgenic and 100 U 26% in the controls, t-test, P = 0.923) and at 8 weeks of age (81 U 23% in the BDNF transgenic and 100 U 23% in the controls, t-test, P = 0.47). These results indicate that both ERK and Akt activity in the BDNF transgenic mice are comparable with the levels found in control animals.

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Fig. 3. Representative sections from biocytin-labeled granule cells from (A) control and (B) BDNF transgenic mice. The missing dendrites in these examples were present in adjacent serial sections. Scale bar = 25 Wm.

Biocytin labeling and reconstruction of hippocampal granule cells We intracellularly labeled granule cells of the hippocampal dentate gyrus from four 8-week-old BDNF transgenic and three non-transgenic littermate control mice. Thirty-¢ve granule cells from BDNF transgenic mice and 21 granule cells from non-transgenic control mice were labeled. Fifteen granule cells from BDNF transgenic mice and 19 granule cells from control mice met the criteria for analysis and were reconstructed (Figs. 3 and 4). BDNF increases dendrite length and complexity There were no gross observable abnormalities between granule cells from BDNF transgenic and control mice. All reconstructed granule cells projected their dendrites into the molecular layer and projected axons into the hilus and toward the CA3 ¢eld. A Sholl analysis of the reconstructed cells demonstrated a higher degree of complexity in the dendritic tree of granule cells from BDNF transgenic mice compared to control mice (Fig. 5). In order to determine which aspects were speci¢cally a¡ected by BDNF, we measured the following parameters of cell morphology and dendrite structure: number of ¢rst order dendrites per cell, total dendrite length per cell, number of branch points per cell and average length of branch segments per branch order of dendrite1 . The number of ¢rst order dendrites of BDNF transgenic and control mice di¡ered signi¢cantly (P 6 0.005, M2 test; Fig. 6). In addition to evaluating the recon-

1 Interested parties can contact the corresponding author for data ¢les of the dendritic reconstructions.

structed granule cells, we also evaluated additional biocytin-injected neurons that were not reconstructed because portions of the dendrite tree were missing. We were able to determine the number of ¢rst order dendrites from a total of 28 neurons from BDNF transgenic mice and 30 neurons from control mice. The average number of ¢rst order dendrites was 1.4 times higher in BDNF transgenic mice compared to controls. Total dendrite length per granule cell was measured from three-dimensional reconstructions of the dendritic tree. While the total dendrite length of reconstructed granule cells from control mice averaged 1148 U 381 Wm S.D. (ranging from 818 to 1966 Wm), the total dendrite length of reconstructed granule cells from BDNF transgenic mice was double that of controls (P 6 0.001, twotailed t-test), averaging 2366 U 544 Wm S.D. (ranging from 1087 to 3337 Wm) (Fig. 7A). The total number of branch points from BDNF transgenic mice averaged 18.5 per cell ( U 5.4 S.D.), double that of controls whose average number of branch points was 8.5 per cell ( U 3.2 S.D., P 6 0.001, t-test) (Fig. 7B). We wanted to determine whether the increase in number of ¢rst order dendrites was entirely responsible for the increased total dendrite length and number of branch points in granule cells of BDNF transgenic mice. In order to examine the in£uence of increased ¢rst order dendrites on total dendrite length and branching, we compared the dendrite length and the number of branch points per ¢rst order dendrite (instead of per cell) between BDNF transgenic and control mice. The total dendrite length per ¢rst order dendrite of granule cells from BDNF transgenic mice was 1.4 times higher than in control mice (P 6 0.01, t-test) (Fig. 7C). Similarly, the total number of branch points per ¢rst order dendrite in granule cells from BDNF transgenic mice was 1.6 times higher than in control mice (P 6 0.01, t-test) (Fig. 7D).

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BDNF transgenic mice, therefore, does not result from changes in length of individual dendrite segments per branch order but from the increase in total numbers of branch order dendrites due to increased branching. BDNF increases dendritic complexity independent of the location of the granule cells

Fig. 4. Two-dimensional representations of the three-dimensional reconstructions of dendritic arbors of granule cells from control mice (A^D) and BDNF transgenic mice (E^H). Granule cells from BDNF transgenic mice have increased dendritic complexity when compared to those of control mice. Reconstructed granule cell in D is the same cell illustrated in Fig. 3A and represents the granule cell with the greatest total dendrite length from the control group. Granule cell G is the same cell illustrated in Fig. 3B. Scale bar = 25 Wm, consistent for all neurons.

Because the total dendritic length and number of branch points were increased in granule cells from BDNF transgenic mice, we evaluated the dendrite segment length per branch order of the segment to determine whether BDNF increased the length of individual dendrite segments. There were no signi¢cant di¡erences in the average dendrite segment length with the ¢rst through the seventh order dendrite segments between control and BDNF transgenic mice as calculated by the Neurolucida software (t-test, data not shown). Very high order dendrites (eighth, ninth and 10th order), however, were present only in BDNF transgenic mice. The increase in total dendrite length in granule cells from

Previous detailed studies of granule cells in the rat dentate gyrus indicated that granule cells located on the suprapyramidal blade of the dentate gyrus had greater total dendritic length than granule cells located in the infrapyramidal blade. Additionally, granule cells located super¢cially in the stratum granulosum, or closer to the molecular layer, project a greater number of ¢rst order dendrites than cells positioned deeper in the dentate gyrus (Claiborne et al., 1990). Since the total dendrite length and number of ¢rst order dendrites is in£uenced by the location of granule cells, we examined the e¡ect of BDNF on di¡erent subpopulations of neurons based on their location within the dentate gyrus. In the suprapyramidal blade, total dendrite length of reconstructed granule cells from BDNF transgenic mice (n = 13) was signi¢cantly higher (average 2383 U 537 Wm S.D., P 6 0.001, t-test) than from control mice (average 1163 U 387 Wm S.D., n = 16). In the infrapyramidal blade, while the total dendrite length was increased in the cells from BDNF transgenic mice as compared to controls, the low numbers of neurons in this subgroup (n = 2 BDNF and n = 3 control mice) preclude an accurate statistical analysis of dendrite length comparisons. We also compared the number of ¢rst order dendrites of reconstructed and additional biocytin-labeled granule cells (n = 28 BDNF and n = 30 control) with relation to depth within the dentate. Within the super¢cial layer of the dentate gyrus, granule cells from BDNF transgenic mice had a 1.3 times higher number of ¢rst order dendrites than that from control mice (P 6 0.05, M2 test). Similarly, with granule cells located in the deep dentate layer, BDNF transgenic mice had a 1.6 times higher number of ¢rst order dendrites as compared to control mice (P 6 0.05, M2 test). While cell position in the dentate in£uences dendrite length and numbers of ¢rst order dendrites, our results clearly demonstrate BDNF overexpression increases these parameters in granule cells regardless of its relative position in the hippocampus.

DISCUSSION

While previous mammalian studies have demonstrated that BDNF and/or TrkB in£uence dendrite structure of neurons in the cortex (Cohen-Cory and Fraser, 1994), we chose to study the hippocampus since it is well known to play a fundamental role in learning and memory (Korte et al., 1995; Kang and Schuman, 1995a,b). Granule cells were examined because their dendritic structure is uniform and well characterized, and both BDNF and TrkB are expressed in granule cells (Maisonpierre et al., 1990; Merlio et al., 1993). Although modestly increased BDNF levels did not signi¢cantly alter expression or activation

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Fig. 5. Sholl analysis of dendritic arbors of granule cells from BDNF transgenic and control mice. The Sholl analysis illustrates the number of dendritic intersections at speci¢ed interval distances from the cell body, thereby depicting dendritic complexity. The number of intersections was calculated at intervals of 25-Wm radius from the cell body using the Neurolucida software system in three-dimensional dendritic tree reconstructions. BDNF-overexpressing mice had more dendrite intersections from 25 to 175 Wm from cell body (P 6 0.05, t-test), which indicates an increase in the proximal dendrite complexity. Error bars represent standard deviation.

of TrkB receptor signaling cascades, they did increase dendritic growth and dendritic complexity of dentate granule cells. BDNF overexpression does not signi¢cantly a¡ect TrkB protein levels or TrkB activity Previous studies reported that BDNF treatment decreases full-length TrkB receptor message and protein levels (Frank et al., 1996; Knusel et al., 1997; Sommerfeld et al., 2000). Since it is also known that

Fig. 6. Number of ¢rst order dendrites of granule cells, expressed in percent, from control and BDNF transgenic mice. Granule cells in BDNF transgenic mice have more ¢rst order dendrites than in controls. The distribution of ¢rst order dendrites was evaluated after adding additional neurons that were biocytin-labeled but not reconstructed ; this created a total pool of 28 neurons from the BDNF transgenic mice and 30 neurons from control mice. The distribution of ¢rst order dendrites in these pools of granule cells revealed that BDNF transgenic mice had a higher number of ¢rst order dendrites (P 6 0.005, M2 test).

TrkB isoforms regulate dendritic growth (Yacoubian and Lo, 2000; Xu et al., 2000a,b), we examined whether chronic exposure to BDNF could also induce an alteration of TrkB receptor isoform levels in a way that could in£uence TrkB signaling and the mode of dendritic growth of dentate granule cells. We examined both the e¡ects of chronic BDNF overexpression on the two TrkB receptor isoform levels and on the level of activation of several intracellular pathways involved in TrkB activity. The levels of full-length receptors in BDNF transgenic and control mice remained unchanged at both the 2-day and the 8-week time points. These results indicate that, while signi¢cant short-term elevations in BDNF downregulate full-length TrkB receptors (Frank et al., 1996), modest long-term increases in BDNF found in the transgenic animals used in this study fail to do this. The binding of BDNF to full-length TrkB leads to TrkB autophosphorylation and subsequent activation of a series of intracellular signaling pathways (Kaplan and Miller, 2000). Trk receptor signaling and PI-3K activity regulate small G proteins of the Cdc-42/Rac/ Rho family responsible for polymerization and turnover of F-actin and therefore controlling actin cytoskeleton formation (Bishop and Hall, 2000; Liu and Burridge, 2000). Out of the three major signal transduction pathways activated by Trk receptors, both the ERK/MAPK and the PI-3K^Akt signaling pathways have been implicated in the regulation of axonal growth and dendritic pruning (Huang and Reichardt, 2001; Atwal et al., 2000; Bartlett et al., 1997; Kaplan and Miller, 2000; Kuruvilla et al., 2000). The comparison of the extent of ERK and Akt protein phosphorylation in hippocampal tissue between BDNF transgenic and control mice failed to reveal any signi¢cant di¡erences at the time points used in this study. These results indicate that, while the BDNF transgenic mice have increased BDNF in the hippocampus, this increase is not su⁄cient to produce any detectable di¡erence in some of the major TrkB signaling pathways. Altogether, these results indicate that the in vivo BDNF transgenic animal model allows the analysis

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Fig. 7. Quantitative assessment of reconstructed granule cell dendrites from BDNF transgenic and control mice. Granule cells from BDNF transgenic mice had greater (A) total dendrite length per cell (P 6 0.001, two-tailed t-test) and (B) greater number of branch points per cell (P 6 0.001, t-test). To determine if increased total dendrite length and branching in cells from BDNF transgenic mice were entirely due to the increased number of ¢rst order dendrites we examined the total dendrite length and number of branch points per ¢rst order dendrite. Granule cells from BDNF transgenic mice had (C) increased total dendrite length per ¢rst order dendrite (P 6 0.01, two-tailed t-test) and (D) increased number of branch points per ¢rst order dendrite (P 6 0.01, two-tailed t-test), indicating that BDNF increases dendritic complexity of dentate granule cells by increasing branching and number of ¢rst order dendrites. Error bars represent standard error means.

of the e¡ects of BDNF on dendrite structure in an environment in which only subtle di¡erences in TrkB receptor levels and signaling are present. BDNF overexpression increases dendrite complexity in dentate granule cells Previous in vitro studies pointed out that signi¢cant increases in neurotrophin levels altered dendritic structure (McAllister et al., 1995, 1997). The BDNF transgenic mice with only a 32% increase in BDNF protein levels (Croll et al., 1999) within the hippocampus allowed us to study the e¡ects of modest but sustained increases in BDNF on dendrite structure. In situ hybridizations of the hippocampus revealed that BDNF message was overexpressed within the granule cell layer of the dentate gyrus, allowing for delivery of BDNF to granule cells by autocrine or paracrine modes, and within the hilus and CA3 area (Croll et al., 1999; Qiao et al., 2001), allowing for delivery of BDNF to granule cells by retrograde transport. Since BDNF overexpression occurs in many regions of the brain, however, the dendritic structure may have been in£uenced by BDNF delivered from other regions of the brain. The extent of dendritic arborization was profoundly increased in the transgenic mice when compared to their control littermates. Our results indicate that both total dendrite length and branching are doubled in the BDNF transgenic mice. Since BDNF did not increase the average dendrite segment length per branch order, the increase in total dendrite length in granule cells from

BDNF transgenic mice did not result from increased elongation of dendrites per se, but from increased dendrite number and complexity. The generation of the dendritic arbor can be divided in three related but independent processes: generation of primary dendrites, elongation and branching. Our results clearly demonstrate that BDNF is implicated in the generation and branching of dendrites because both the number of primary dendrites per neuron and the number of branching points per primary dendrite are increased, suggesting similar mechanisms of action. Dendritic branching, therefore, appears to be sensitive to modest increases in the levels of BDNF. However, the elongation phase is not regulated by BDNF because the length of the individual dendrite segments was not in£uenced by increasing BDNF levels. These results are consistent with a previous study where overexpression of full-length TrkB receptors in the ferret cortex slices increased branching but did not promote elongation of dendrite segments (Yacoubian and Lo, 2000). The increase in dendritic complexity observed in the transgenic mice is most likely due to a direct e¡ect of BDNF on the granule cell, most probably mediated via the TrkB receptor, even though we did not detect a signi¢cant change in TrkB signaling. In order to test this hypothesis additional studies could be performed in animals with decreased BDNF levels and/or TrkB signaling. Although both BDNF and TrkB knockout mice do not survive as homozygotes, the heterozygotes are viable and it could be of great interest to see if, as expected, these present a reduced dendritic arborization in the hippo-

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BDNF increases dendrite complexity

campus. Indeed, conditional deletion of TrkB receptors reduced the dendritic complexity of pyramidal cells and compressed cortical layers in vivo (Xu et al., 2000b). E¡ects of early and long-term BDNF exposure Using the BDNF transgenic mice we were able to evaluate the e¡ects of long-term increases in BDNF levels on dendritic morphology in contrast to previous studies evaluating the short-term in£uences of BDNF (McAllister et al., 1995, 1997; Horch et al., 1999). Long-term e¡ects of increased BDNF may di¡er from its short-term e¡ects because the longer time course of BDNF exposure may alter activation of TrkB receptors or in£uence changes in TrkB receptor isoforms (Frank et al., 1996; Knusel et al., 1997; Sommerfeld et al., 2000). Our results indicate that long-term BDNF overexpression increased dendrite complexity similarly to that seen in shorter-term studies. The time course of BDNF delivery also di¡ers from most previous studies. In our transgenic model, BDNF levels are increased from early development and sustained for long periods of time. Previous studies examined the e¡ects of BDNF during speci¢c early development stages when neurons are being established and elaborating their processes (McAllister et al., 1995, 1997; Horch et al., 1999; Fryer et al., 1996). In this study, we were able to examine the cumulative e¡ects of increased BDNF levels from early in development into the adult. Our dendrite analysis represents the actual dendrite structure of granule cells speci¢cally at 8 weeks of age, thus we are unable to determine when the dendrite structural changes occurred. The increased complexity in dendritic structure may result from the cumulative e¡ects of prolonged BDNF exposure on granule cells beginning during the neurogenesis of granule cells. Granule cell neurogenesis occurs fairly late in development with the majority being generated between postnatal days 5 and 7 (Schlessinger et al., 1975). At this time these immature granule cells have short simple processes that elongate and continue to mature. The granule cell dendritic processes continue to be regulated into later stages of postnatal development (Rihn and Claiborne, 1990). Increased BDNF gradients in early development may send appropriate signals to increase the number of ¢rst order dendrites and branching during the time when granule cells are establishing their dendritic processes. Additionally, since new granule cells are continually generated in the dentate gyrus during later stages and on into adulthood (Schlessinger et al., 1975; Kempermann et al., 1997), BDNF exposure may increase dendritic complexity in these newly forming granule cells. After the increased number of ¢rst order dendrites and initial proximal branching is established, the continued elevation in BDNF levels may maintain this increased arbor complexity until the mice reach adulthood when the analysis was performed. In this scenario, the changes in dendritic structure may have resulted from the cumulative e¡ects of increased BDNF levels from early development through maturity. Additionally or otherwise, the

803

increased dendritic complexity may result from BDNF decreasing the extent of dendritic ‘pruning’, or loss, of dendritic branches. Dendritic pruning has been reported to occur in several neuronal populations including granule cells of the dentate gyrus (Rihn and Claiborne, 1990). Alternatively, the increase in dendrite complexity may result from elevated BDNF levels during the time frame immediately preceding the analysis. This hypothesis is supported by the fact that the changes in dendrite structure are dynamic and in£uenced by various factors, including neurotrophins (McAllister et al., 1999; Horch et al., 1999). Physiological relevance of the BDNF transgenic mouse Previous studies demonstrated behavioral and physiological di¡erences in the BDNF transgenic mice used in this study. The BDNF transgenic mice developed a passive avoidance de¢cit dependent on continued BDNF overexpression. Additionally, BDNF transgenic mice were observed to have spontaneous seizures and had increased seizure severity following kainic acid administration (Croll et al., 1999). Behavior abnormalities in these mice could be due to many possible consequences of BDNF overexpression or release. One possibility is that changes in dendritic structure, like those found in granule cells, alter neuronal circuits that underlie the a¡ected behaviors. For example, a neuron with an expanded dendritic tree has the capacity to receive more synaptic input. However, although our conclusion that BDNF levels regulate dendritic complexity of granule cells is in agreement with previous in vitro studies (McAllister et al., 1995, 1997; Horch et al., 1999) we cannot completely exclude the possibility that the observed behavioral abnormalities of these mice are not a result but the cause of the increased dendritic complexity observed in the hippocampus. Interestingly, another recent study concluded that BDNF overexpression in the hippocampus does not obviously alter granule cell axon arborization (Qiao et al., 2001). These results suggest that BDNF may modulate dendritic and axonal complexity di¡erently even in the same neuronal population as previously demonstrated in the Xenopus tadpole (Lom and Cohen-Cory, 1999).

CONCLUSION

This is the ¢rst study evaluating the e¡ects of BDNF overexpression on dendritic morphology in the intact hippocampus. This in vivo study extends previous in vitro observations that BDNF may in£uence synaptic plasticity by changing the structure of dendritic arbors.

Acknowledgements*This research was supported by Grants K01-RR00129 (R.J.T.), NS40276 (P.S.B.) and NS04270 (E.M.S.) from the National Institutes of Health. We are grateful to Dr. Linda C. Cork for helpful discussion and critical comments on the manuscript.

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