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Regional- and Age-Dependent Reduction in trkB Receptor Expression in the Hippocampus Is Associated with Altered Spine Morphologies
Regional- and Age-Dependent Reduction in trkB Receptor Expression in the Hippocampus Is Associated with Altered Spine Morphologies Oliver von Bohlen und Halbach, Sonja Krause, Diego Medina, Carla Sciarretta, Liliana Minichiello, and Klaus Unsicker Background: Changes in densities and in the morphology of dendritic spines in the hippocampus are linked to hippocampal long-term potentiation (LTP), spatial learning, and depression. Decreased brain-derived neurotrophic factor (BDNF) levels seem to contribute to depression. Through its receptor trkB, BDNF is also involved in hippocampal LTP and hippocampus-dependent learning. Conditionally gene-targeted mice in which the ablation of trkB is restricted to the forebrain and occurs only during postnatal development display impaired learning and LTP. Methods: To examine whether there is a link among impaired hippocampal synaptic plasticity, altered spines, and trkB receptors, we performed a quantitative analysis of spine densities and spine length in the hippocampal area CA1 and the dentate gyrus in conditional mutant mice (trkBlox/loxCaMKII-CRE mice). TrkB protein and mRNA levels were assayed using Western blot and in situ hybridization analysis. Results: Fifteen-week-old mutant mice exhibit specific reductions in spine densities and a significant increase in spine length of apical and basal dendrites in area CA1. These alterations correlate with a time- and region-specific reduction in full-length trkB mRNA in the hippocampus. Conclusions: TrkB functions in structural remodeling of hippocampal dendritic spines, which in turn may affect synaptic transmission and plasticity. Key Words: Dendritic spine, hippocampus, trkB
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pines are small postsynaptic structures that protrude from the surface of dendrites of many neurons. Dendritic spines have been shown to be the predominant site of excitatory synapses on CA1 pyramidal neurons in the rat hippocampus (Megias et al 2001), where most spines are contacted by only one presynaptic bouton (Andersen 1990). Dendritic spines can undergo morphologic plastic changes. Thus, some forms of learning have been shown to increase the number of dendritic spines (Geinisman 2000; Leuner et al 2003; Nimchinsky et al 2002; Yuste and Bonhoeffer 2001). Moreover, structural remodeling of synapses and formation of new synaptic contacts have been postulated as a mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Indeed, LTP seems to be associated with increased spine densities (Muller et al 2000) and with the formation of new, mature, and probably functional synapses (Toni et al 1999). Thus, hippocampal LTP, induced in the perforant path/dentate granule cell synapse, leads to an increase in spine density (Trommald et al 1996), indicating that new spine synapses are formed following LTP. Using two-photon microscopy, it has been convincingly demonstrated that after induction of long-lasting functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite (Engert and Bonhoeffer 1999).
From the Department of Neuroanatomy (OvBuH, SK, KU), Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany; and Mouse Biology Unit (DM, CS, LM), European Molecular Biology Laboratory, Monterotondo, Italy. Address reprint requests to Oliver von Bohlen und Halbach, Interdisciplinary Center for Neurosciences (IZN), Department of Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany; E-mail: [email protected]. Received May 9, 2005; revised July 26, 2005; accepted August 31, 2005.
0006-3223/06/$32.00 doi:10.1016/j.biopsych.2005.08.025
Spine reductions in the hippocampus are associated with not only schizophrenia but also major depression (Law et al 2004). Antidepressant treatment has been shown to produce a significant increase in dendritic spine density in the hippocampal area CA1 of rats (Hajszan et al 2005; Norrholm and Ouimet 2000, 2001), and increase levels of brain-derived neurotrophic factor (BDNF) mRNA in the brain (Altar 1999). Brain-derived neurotrophic factor is a member of the neurotrophin (NT) family of neurotrophic factors (Barbacid 1994). Polymorphisms in the BDNF gene seem to be associated with major depression (Jiang et al 2005; Strauss et al 2004). It has been proposed that decreased BDNF levels may be implicated in the pathogenesis of depression (Altar 1999). Inappropriate neurotrophic support could lead to structural disorganization in the brain and ultimately to a decreased capacity of the brain to adapt to changes (Angelucci et al 2005). Under physiologic conditions, BDNF is known, in addition to its trophic functions, to be a key mediator of activity-dependent modifications of synaptic strength in the central nervous system (CNS). Thus, application of BDNF has been reported to produce a dramatic and sustained (2–3 hours) enhancement of synaptic strength at the Schaffer collateral CA1 synapses (Kang and Schuman 1995). Moreover, pretreatment of hippocampal slices with a trkB-IgG fusion protein, which scavenges endogenous BDNF, reduces the magnitude of LTP (Figurov et al 1996). In BDNF mutant mice, both hippocampal LTP and spatial learning are impaired (Korte et al 1995; Linnarsson et al 1997; Monteggia et al 2004). Interestingly, the cognate receptor for BDNF, trkB, has been localized immunocytochemically within dendritic spines of hippocampal neurons (Drake et al 1999). Moreover, cyclic adenosine monophosphate seems to facilitate trafficking of trkB to dendritic spines, possibly by promoting its interaction with the synaptic scaffolding protein PSD-95 (Ji et al 2005). Whether BDNF also affects spine morphology in the hippocampus has not been proven. Because BDNF, through activation of trkB, is able to induce hippocampal LTP, and because LTP induces spinogenesis, it may be speculated that trkB activation BIOL PSYCHIATRY 2006;59:793– 800 © 2005 Society of Biological Psychiatry
794 BIOL PSYCHIATRY 2006;59:793– 800 may increase spine densities. Cell culture studies addressing this issue have provided conflicting results, however. Using cultured hippocampal neurons, BDNF has been reported to reduce (Murphy et al 1998) or increase (Ji et al 2005) spine densities. Organotypic hippocampal slice cultures have revealed a BDNF mediated increase in dendritic spine densities in area CA1 (Alonso et al 2004). This study investigates the impact of trkB receptors on spine densities and spine morphology in the hippocampal formation. We evaluated this brain area because it is known to be involved in depression, spatial memory, LTP, and spinogenesis. Because mice with a targeted deletion of trkB or bdnf die shortly after birth, we examined mice in which trkB was conditionally deleted postnatally from the anterior forebrain (Minichiello et al 1999). These animals (termed “trkB-CRE”) survive into adulthood and have previously been shown to display deficits in hippocampal LTP and hippocampus-related behavior and represent an ideal model to investigate a possible role for trkB in the regulation of spine morphology and density in vivo.
Methods and Materials Animals The generation of conditionally gene-targeted mice (TrkBlox/ loxCaMKII–CRE⫹/⫺ mice or simply “trkB–CRE” mice), in which the knockout of the full-length trkB receptor is restricted to neurons of the anterior forebrain and occurs only during postnatal development has been previously described (Minichiello et al 1999). In these mice, a reduction in trkB protein in the hippocampus begins at about postnatal day 20 (Minichiello et al 1999). Age-matched mice with the genotypes wild-type, trkBlox/ lox, and trkBlox/⫹ did not differ in terms of the amounts of trkB they expressed and thus were grouped together and served as control animals. Two age groups of mice were analyzed: 8 weeks and 15 weeks of age. Golgi Method Mice were perfused with 4% paraformaldehyde (PFA) in phosphate buffer. Brains were dissected and postfixed in 4% PFA. Golgi-staining was performed according to the Golgi-Kopsch method, resulting in well-impregnated neurons (Rosoklija et al 2003). Blocks of about 2.5 mm thickness were prepared from each hemisphere, and brain pieces were soaked in a solution containing 2.5% potassium dichromate for 1 day at room temperature in the dark. This solution was then replaced by fresh solution of 2.5% potassium dichromate, and tissues were incubated for another 6 days. Tissues were then washed and transferred to a solution containing .75% AgNO3. After 5 to 6 days, brain pieces were removed, washed in 40% and 20% ethanol, and cut into 60-m coronal sections using a vibratom (Leica, Wetzlar, Germany). Sections were mounted on gelatin-coated slides and cover-slipped using Merkoglas (Merk, Darmstadt, Germany). Spine Analysis Spine analysis was conducted blind to the experimental conditions. As previously reported (Drake et al 1999), trkBimmunoreactivity accumulates at high levels in the dendritic spines within the stratum oriens and the stratum radiatum of area CA1 of the hippocampus and in the stratum moleculare of the dentate gyrus. Consequently, we examined spines on apical dendrites of CA1 pyramidal neurons in the stratum radiatum and on basal dendrites of CA1 pyramidal neurons in the stratum oriens, as well as on dendrites of the dorsal leaf of the dentate gyrus (DG; Figure 1). Spines were analyzed in the dorsal, but not www.sobp.org/journal
O. von Bohlen und Halbach et al ventral, leaf of the DG because spine densities are different in these locations (Desmond and Levy 1985). Analyses were conducted on Golgi-impregnated sections that were uniformly dark throughout the section. Only dendrites that displayed no breaks in their staining (Leuner et al 2003) and that were not obscured by other neurons or artifacts (Liu et al 2001) were evaluated. Only spines located on secondary or tertiary dendritic trees were evaluated. Only one segment per individual dendritic branch was chosen for the analysis. Quantitative three-dimensional analyses of dendritic fragments with their spines were conducted using a combined hardwaresoftware system (NeuroLucida, MicroBrightFields, Colchester, Vermont) controlling the x-y-z axis of the microscope (Axioscop Imaging, Zeiss, Germany) and a microscope-mounted video camera (Hitachi, Tokyo, Japan). The three-dimensional reconstruction was done using a 100⫻ objective (NA: 1.4; oil immersion) and the NeuroLucida system. Spine densities and mean spine length were calculated from the reconstructed dendrites with the help of NeuroExplorer (MicroBrightFields). Traditionally, spines have been classified into four major categories (filopodia, stubby, thin, and mushroom) based on their distinct morphologies (Parnass et al 2000); however, the recently discovered rapid morphologic plasticity of spines has suggested that these categories, rather than representing intrinsically different populations of spines, reflect temporal snapshots of a dynamic phenomenon (Parnass et al 2000). Therefore, no subdivision into different types of spines was made. For the analysis, at least three animals per age and genotype were analyzed. In these animals, individual dendrites were reconstructed. In case of basal dendrites of area CA1 between 46 and 55 individual dendritic segments per group (bearing between 529 and 779 individual spines), and in case of apical dendrites of area CA1 between 51 and 55 dendritic segments (bearing between 587 and 703 individual spines) were reconstructed and analyzed; in case of the dentate gyrus between 50 and 59 dendritic segments (bearing between 613 and 812 individual spines) per group were reconstructed. Determination of trkB Protein Levels Mice were killed by cervical dislocation, and hippocampi were quickly dissected and frozen in liquid nitrogen. Protein lysates were obtained by homogenizing the tissue in lysis buffer (20 mmol/mL Tris pH 7, 140 mmol/mL NaCl, 10% glycerol, 1% NP 40, 10 mg/mL leupeptin, 2 mg/mL aprotinin, 5 mmol/mL benzamidine, 1 mmol/mL PMSF, 10 mmol/mL NaF, 1 mmol/mL NaPP, 1 mmol/mL Na3VaO4), followed by centrifugation at 4°C to remove insoluble material (see also Minichiello et al 1999). Immunoprecipitations were performed using a pan-Trk antibody (C-14, Santa Cruz, California), and a TrkB polyclonal antibody, 113-5, was used for Western blots, as previously described (Minichiello et al 1999). To control for protein loading, Erk1 levels were probed on stripped blots using a monoclonal Erk1 antibody (Zymed, San Francisco, California). Comparison of trkB expression in hippocampal lysates of mutant and control mice was performed using densitometric analysis (NIH Image) of enhanced chemiluminescence (ECL)-stained Western blots. Density values were corrected by dividing them by the values obtained for Erk1. In Situ Hybridization Mice were perfused with 4% PFA. Brains were removed from the skull, and cryosections (25 m) were made and mounted on slides. A nonradioactive in situ hybridization (ISH) protocol was
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Figure 1. (A) Schematic drawing of the hippocampus. Area CA1 consists of the stratum oriens (Or), the pyramidal layer, the stratum radiatum (Rad), and the stratum lacunosum moleculare (LMol). Cell bodies of analyzed pyramidal cells are located in the pyramidal layer (indicated in gray). Basal dendrites within the stratum oriens were analyzed as well as apical dendrites within the stratum radiatum. Within the dentate gyrus, granule cells are located within the granular layer (indicated in gray). Dendrites within the molecular layer (Mol) of the dentate gyrus (DG) were analyzed. (B) Golgi-impregnated basal dendrite of area CA1 (control mouse; 8 weeks old). Image was made using a z-stack of seven individual images, generated by using a software-driven motorized stage (step width: .5 m) and microscope-mounted digital camera (Axiocam; Zeiss).
used. Dioxigenin-labeled riboprobes recognizing full-length trkB (trkB.fl, bp 2217–2776) were generated as follows: cDNA fragments were synthesized by polymerase chain reaction from murine-brain cDNA and cloned into a pGEM-Teasy vector (Promega, Madison, Wisconsin). The plasmids were appropriately linearized and transfected with SP6 or T7 for antisense and sense probes, respectively. Specificity of our riboprobes was confirmed by subsequent sequencing by MWG Biotech (Ebersberg, Germany). For ISH, slides were incubated overnight with hybridization buffer containing dioxigenin-labeled riboprobes. The slides were washed three times in a washing solution (50% formamide, .001% Tween and 5% sodium chloride, and 5% sodium citrate solution [SSC]) for 10 min, 30 min, and 45 min at 68°C and then twice with malein acid buffer and Tween (MABT) for 30 min at room temperature. After blocking in MABT containing 20% horse serum, sections were incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase (Roche, Switzerland) at room temperature. After three washes in MABT and two washes in prestaining buffer, the sections were developed using nitroblue tetrazolium salt/5=-bromo-4-chloro-idoylphosphate (NBT/BCIP) (Roche, Switzerland) and cover-slipped. For quantification of signal intensities, images were made using a software-controlled (Axiovision 3.0; Zeiss) digital camera
(Axiocam, Zeiss) attached to a microscope (Axioplan imaging; Zeiss) using a 20⫻ objective. For the process of image acquisition, the same parameters were used (e.g., illumination time, threshold). Images were stored onto hard disk and analyzed offline. Intensity measurements were done using ImageJ 1.31 (National Institute of Health). The color images were changed to 8-bit grayscale images that were represented using unsigned integers in the range 0 –255 (256 gray values coding for intensities). In each converted image, histograms of the distribution of pixel intensities were made. The mean intensity values were calculated for an area of interest (von Bohlen und Halbach et al 2002), which was defined to cover the area of either the pyramidal layer of the hippocampus or, in the case of the DG, the granular layer. For estimates of the relative cell densities of trkB mRNA– containing cells, a microscope (Axioplan 2 imaging; Zeiss) equipped with a computer-driven digital camera (Axiocam; Zeiss) was used. Positive cells were counted in a region of interest (ROI; representing a window of 10,000 m2), and data were expressed as cell densities per 10,000 m2(von Bohlen und Halbach et al 2004). To estimate cell densities within the CA1 area and within the dentate gyrus in the four mouse groups, three animals per group were used. In each animal, at least six sections were analyzed per brain area. Statistical analysis was performed using Prism (Graph Pad, www.sobp.org/journal
796 BIOL PSYCHIATRY 2006;59:793– 800 San Diego, California). Data sets were statistically compared using analysis of variance (ANOVA) with Student–Newmann– Keuls post hoc test. Data were presented as mean ⫾ SEM.
O. von Bohlen und Halbach et al Determinations of spine length revealed no age-related change in the trkB-CRE mice (Figure 3F); however, 15-week-old trkBCRE mice had significantly longer spines than their age-matched control counterparts.
Results Impact of Age on Spine Densities and Spine Length in Area CA1 and the Dentate Gyrus As shown in Figure 2A, mice at 15 weeks of age revealed a 17% increase in spine densities of basal dendrites in CA1 compared with 8-week-old control mice. Similar to CA1 basal dendrites, CA1 apical dendrites of 15-week, compared with 8-week-old control mice, exhibited an increase in spine densities (26%). As in the CA1 region of the hippocampus, densities of dendritic spines in the dorsal leaf of the dentate gyrus increased by 11% from 8 to 15 weeks of age (Figure 2A). Mice at 15 weeks of age displayed a 12% reduction in average spine length of basal dendrites in CA1, but showed no significant change in the mean length of spines, located on apical dendrites (Figure 2B). Concerning dendritic spines in the dorsal leaf of the dentate gyrus, a reduction of about 11% in mean spine length was noted by comparing 15-week-old animals with 8-week old mice (Figure 2B). Impact of Conditional Deletion of trkB on Spine Densities and Spine Length in Area CA1 and the Dentate Gyrus Basal Dendrites of Neurons Located in area CA1. As shown in Figure 3A, 8-week-old trkB-CRE mice did not differ in their spine densities of basal CA1 dendrites from age-matched control animals. Moreover, 15-week-old trkB-CRE mice did not reveal an increase in spine densities as compared to 8-week-old trkB-CRE mice. Thus, 15-week-old trkB-CRE mice displayed 11% less dendritic spines on basal dendrites of area CA1 than the agematched control mice. Eight-week-old trkB-CRE mice had significantly shorter spines (⫺7.5%) than the age-matched control mice, whereas spine length in 15-week-old trkB-CRE mice was increased by 16.8% compared with control mice (Figure 3B). Fifteen-week-old trkB-CRE mice had 13% longer spines than 8-week-old trkB-CRE mice. Apical Dendrites of Neurons Located in Area CA1. Spine densities did not significantly differ between 8-week-old control and trkB-CRE mice. In contrast to control mice, spine densities failed to increase in trkB-CRE mice between 8 and 15 weeks of age. Thus, compared with control mice, spine densities in 15-week-old mutant mice were reduced by 10% (Figure 3C). There was a significant 21% difference in spine length of 15-week-old mutant mice compared with control mice, however. Comparisons of 8and 15-week-old trkB-CRE mice revealed a 13% increase in spine length (Figure 3D). Dendrites of Neurons Located in the Dentate Gyrus. As in control mice, spine densities in the trkB-CRE mice increased significantly (⫹21%) from postnatal week 8 to 15 (Figure 3E).
TrkB-Protein Levels Hippocampal trkB protein levels were clearly reduced in trkB-CRE mutants as compared with control mice. Despite this obvious reduction in trkB-protein levels, these changes failed to reveal significant changes in trkB protein levels between 8- and 15-week-old trkB-CRE mutants (Figure 4). Expression of trkB mRNA in the Hippocampus Spine analyses had suggested both age-dependent and region-specific alterations in spine densities and length of trkB-CRE mice; however, these alterations were not paralleled by changes in levels of trkB protein. Because protein samples included the whole hippocampus, it was conceivable that region-specific changes in trkB expression might have escaped detection. We therefore used ISH to examine putative region-specific changes in trkB expression in area CA1 and the dentate gyrus. We used a probe that only identifies the mRNA of full-length trkB. Fulllength trkB expression in area CA1 was clearly reduced in trkB-CRE mice (Figures 5A–E). In these mice, full-length trkB mRNA expression decreases between postnatal weeks 8 and 15; however, trkB mRNA was still expressed in few CA1 neurons in the 15-week-old trkB-CRE mice (Figure 5E). In addition, fulllength trkB mRNA was reduced in the dentate gyrus at 15-weekof-age in the trkB-CRE mice (Figure 5F). Because many variables, in cases of nonisotopic hybridization histochemistry, influence the staining intensity, we also estimated the relative densities of full-length trkB mRNA expressing cells in the area CA1 and in the dentate gyrus. The density of full-length trkB mRNA expressing cells in area CA1 was not significantly increased from the 8th to the 15th postnatal week in control mice (Figure 5G). At 8 weeks, a significant 70% reduction in the density of full-length trkB mRNA expressing cells was noted in area CA1 by comparing the trkB-CRE mice with age-matched control mice. At 15 weeks, only some full-length trkB expressing cells remained in the area CA1 of trkB-CRE mice. Thus, the density of full-length trkB expressing cells in area CA1 was significantly reduced by approximately 95%, compared with age-matched control animals. Interestingly, by comparing 15-week-old with 8-week-old trkB-CRE mice, a significant reduction (⫺80%) in the density of full-length trkB-expressing cells was found (Figure 5G). Concerning the dentate gyrus, the density of full-length trkB mRNA expressing cells was not significantly altered in 15-weekold control mice compared with their 8-week-old littermates of the same genotype (Figure 5H). Comparing 8-week-old trkB-CRE mice with 8-week-old control mice, a significant reduction in the densities of positive cells was noted (⫺81%). By comparing 15-week-old trkB-CRE mice
Figure 2. Impact of age on spine densities (A) and mean spine length (B) in control mice. Asterisks represent significant changes (p ⱕ .01). CA1-bas, basal dendrites of area CA1; CA1-api, apical dendrites of area CA1; DG, dentate gyrus; con 8 weeks, control mice aged 8 weeks; con 15 weeks, control mice aged 15 weeks.
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Figure 3. Column bar graphs representing the results obtained in the evaluated brain areas. Spine densities (A) and spine lengths of basal dendrites (B) in area CA1, spine densities (C) and spine lengths of apical dendrites (D) in area CA1, and spine densities (E) and spine lengths of apical dendrites (F) in the dorsal leaf of the dentate gyrus. In these graphs, only statistical analyses between aged-matched groups as well as between 8-week and 15-week-old trkB-CRE mice are displayed. *Significant changes (p ⱕ .01). con 8 weeks, control mice aged 8 weeks; con 15 weeks, control mice aged 15 weeks; trkB 8 weeks, trkB-CRE mutant mice, aged 8 weeks; trkB 15 weeks, trkB-CRE mutant mice aged 15 weeks.
with control mice of the same age, a reduction in the density of full-length trkB expressing cells of about 90% was noted. However, we observed no significant difference in the densities of those cells by comparing 8-week-old and 15-week-old trkB-CRE mice (Figure 5H).
Discussion Dendritic morphology has a profound impact on neuronal information processing. Spines act as subcellular compartments that are crucially involved in receiving and processing synaptic information, and dendritic branching patterns determine the efficacy by which synaptic information is transmitted to the soma (Whitford et al 2002). Applied exogenously, BDNF has been found to increase dendritic length of hippocampal interneurons in organotypic slice cultures (Marty et al 1996). Moreover, exogenous application of both trkB ligands, BDNF and NT-4, has been found to increase dendritic length of pyramidal neurons in cortical organotypic cultures. Under these conditions, neutralization of endogenous NT-4 was found to cause shorter and less branched dendrites, whereas neutralization of endogenous BDNF was apparently not effective (Wirth et al 2003). Transfection of dentate gyrus granule cells in slice cultures with BDNF via
particle-mediated gene transfer was found to increase axonal branching and numbers of basal dendrites (Danzer et al 2002), whereas transfections of CA3 or CA1 pyramidal neurons were not efficient (Alonso et al 2004; Danzer et al 2002). These observations seem to indicate that in vitro trkB ligands modulate dendritic structure and spine densities in a cell-type-specific manner; however, interpretation of these data suggesting a potential link between trkB receptors and spine densities and morphology is seriously hampered by the fact that, at comparable ages, spine synapses in culture are more than threefold less abundant than in vivo (Boyer et al 1998). Concerning the control mice, we found an apparent increase in spine densities from 8 to 15 weeks of age, corroborating a previous report that spine densities increase with age in the hippocampus (van Praag et al 2002). Furthermore, spines on basal dendrites in CA1 and on dendrites in the dentate gyrus were substantially longer in 8 as compared to 15 week-old animals. This is consistent with the notion that not only spine densities but also spine morphology changes with postnatal development. Early spines are often very long, and over the next few weeks are reduced in length (Nimchinsky et al 2002). It has been argued that longer, thinner spines constitute a less efficacious postsynaptic phenotype (Matsuzaki et al www.sobp.org/journal
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Figure 4. Determination of trkB protein levels in whole hippocampi. (A) Immunoprecipitation (IP) and Western blot analysis of TrkB expression in protein lysates of hippocampal tissues from 8- or 15week-old trkBlox/lox mice, either in presence or absence of CamKII-CRE. (B) Densitometric analysis of TrkB protein in hippocampi of trkBlox/lox mice, in either the presence or absence of CamKII-CRE (n ⫽ 2– 8 animals per time point). Densitometric values for the control animals were averaged because they did not differ significantly (p ⫽ .8).
2001; Vanderklish and Edelman 2002) and that variations in spine length may even contribute to behavioral alterations. Along this line, the increased spine length that we found in trkB mutant mice may be indicative for a less efficacious phenotype. It has recently been reported that homer1a reduces spines sizes and inhibits Shankmediated spine enlargement in vitro (Sala et al 2003); BDNF has been found to upregulate homer 1a mRNA via the mitogenactivated protein kinase cascade in cultured cerebellar granule neurons (Sato et al 2001), suggesting that increased spine length in the conditional trkB mutant mice may be due to the downregulation of homer 1a. Spine density is thought to reflect excitatory input density (Konur et al 2003), and some forms of learning as well as LTP have been associated with increased spine densities in the hippocampus (Engert and Bonhoeffer 1999; Leuner and Shors 2004; Muller et al 2000). Moreover, BDNF signaling via trkB has been shown to be associated with synaptic plasticity, LTP, and hippocampal-dependent learning. A role for trkB ligands in local modulation of excitatory neurotransmission is suggested by the presence of trkB-immunoreactivity in dendritic spines, which are the targets of glutamatergic innervation in the trisynaptic circuit (Drake et al 1999). It is known that trkB receptors mediate some rapid effects of BDNF (Drake et al 1999), and application of the www.sobp.org/journal
trkB-ligand BDNF induces an enhancement of synaptic strength of CA1 synapses (Kang and Schuman 1995). Because disruption of the trkB-BDNF signaling pathway, through conditional deletion of trkB, results in altered spine densities and spine morphology, BDNF may be conceived to mediate both rapid effects and long-term changes in spine morphology, which interfere with synaptic strength. In a recent study (Luikart et al 2005), it has been suggested that postnatal trkB loss is not associated with the disassembly of existing synapses and that trkB is necessary to promote synapse formation during a period in which maturation of synaptic connectivities occurs. Consistent with this hypothesis, we do not observe a difference in spine densities in area CA1 by comparing the groups with an age of 8 weeks. At 15 weeks of age, trkB-CRE mice have a reduced spine density compared with age-matched control mice; however, the 15-week-old trkB-CRE mice did not display a reduced spine density compared with 8-week-old trkB-CRE or control mice. Thus, in support of the notion held by Luikart and colleagues, a disassembly of existing spines does not to occur. It is likely that during the time period from 8 weeks to 15 weeks of age, a maturation of synaptic connectivities together with an activity-dependent formation of new spines occurs in case of the control mice, but not in case of the trkB-CRE mice.
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Figure 5. In situ hybridization for full-length trkB mRNA. (A) Hippocampal area CA1 of a control animal (8 weeks old; in situ hybridization for full-length trkB mRNA). (B) Hippocampal area CA1 of a trkB-CRE mutant mouse (8 weeks old; in situ hybridization for full-length trkB mRNA). (C) Hippocampal area CA1 of a control animal (15 weeks old; in situ hybridization for full-length trkB mRNA). (D) Hippocampal area CA1 of a trkB-CRE mutant mouse (15 weeks old; in situ hybridization for full-length trkB mRNA). (E) Quantification of the in situ hybridization signals in area CA1 using the full-length trkB probe. (F) Quantification of the in situ hybridization signals in the dentate gyrus using the full-length trkB probe. *p ⱕ .05. ** p ⱕ .01. con 8 weeks, control mice aged 8 weeks; con 15 weeks, control mice aged 15 weeks; trkB 8 weeks, trkB-CRE mutant mice aged 8 weeks; trkB 15 weeks, trkB-CRE mutant mice aged 15 weeks.
Compared with age-matched control mice, spine densities were only reduced in 15-week, but not 8-week-old, trkB-CRE mice. Likewise, conditional deletion of the truncated together with the full-length form of trkB does not alter spine densities of CA1 dendrites in the stratum radiatum at postnatal day 60 (Luikart et al 2005). This suggests that at 8 weeks, the amounts of trkB mRNA and protein still present within the hippocampus may suffice to maintain regular spine morphologies. Hippocampal trkB protein levels did not parallel alterations in spine morphologies noted in conditional trkB mutants, possibly because samples taken from the whole hippocampus may not reveal distinct intrahippocampal regional differences. This notion is supported by the results obtained using in situ hybridization to examine full-length trkB mRNA expression in distinct subregions of the hippocampus. Both 8-week and 15-week-old mutant mice, compared with their control littermates, showed a significant decrease in full-length trkB mRNA in area CA1. Only at 15 weeks, however, was this decrease in trkB mRNA paralleled by a decrease in spine densities. In summary, our data have shown that a forebrain-specific, postnatal targeted deletion of the trkB gene affects hippocampal spines in an age- and region-specific manner. A marked reduction in trkB mRNA occurs in CA1 and is paralleled by a significant decrease in spine densities and an increase in mean spine length
at 15 weeks of age. This suggests a role for trkB-signaling in the structural remodeling of the CA1 excitatory spine synapses. The observed alterations in spine morphology and spine densities in area CA1 are likely to be linked to reduced synaptic plasticity and impaired learning in spatial memory tasks of these trkB-CRE mice at this age. This study was supported by the Deutsche Forschungsgemeinschaft (Grant No. SFB 636/A5). We thank Ulla Hinz for her excellent technical assistance. Alonso M, Medina JH, Pozzo-Miller L (2004): ERK1/2 activation is necessary for BDNF to increase dendritic spine density in hippocampal CA1 pyramidal neurons. Learn Mem 11:172–178. Altar CA (1999): Neurotrophins and depression. Trends Pharmacol Sci 20:59 – 61. Andersen P (1990): Synaptic integration in hippocampal CA1 pyramids. Prog Brain Res 83:215–222. Angelucci F, Brene S, Mathe AA (2005): BDNF in schizophrenia, depression and corresponding animal models. Mol Psychiatry 10:345–352. Barbacid M (1994): The Trk family of neurotrophin receptors. J Neurobiol 25:1386 –1403. Boyer C, Schikorski T, Stevens CF (1998): Comparison of hippocampal dendritic spines in culture and in brain. J Neurosci 18:5294 –5300. Danzer SC, Crooks KR, Lo DC, McNamara JO (2002): Increased expression of brain-derived neurotrophic factor induces formation of basal dendrites
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800 BIOL PSYCHIATRY 2006;59:793– 800 and axonal branching in dentate granule cells in hippocampal explant cultures. J Neurosci 22:9754 –9763. Desmond NL, Levy WB (1985): Granule cell dendritic spine density in the rat hippocampus varies with spine shape and location. Neurosci Lett 54: 219 –224. Drake CT, Milner TA, Patterson SL (1999): Ultrastructural localization of fulllength trkB immunoreactivity in rat hippocampus suggests multiple roles in modulating activity-dependent synaptic plasticity. J Neurosci 19:8009 – 8026. Engert F, Bonhoeffer T (1999): Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399:66 –70. Figurov A, Pozzo-Miller LD, Olafsson P, Wang T, Lu B (1996): Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381:706 –709. Geinisman Y (2000): Structural synaptic modifications associated with hippocampal LTP and behavioral learning. Cereb Cortex 10:952–962. Hajszan T, MacLusky NJ, Leranth C (2005): Short-term treatment with the antidepressant fluoxetine triggers pyramidal dendritic spine synapse formation in rat hippocampus. Eur J Neurosci 21:1299 –1303. Ji Y, Pang PT, Feng L, Lu B (2005): Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine formation in mature hippocampal neurons. Nat Neurosci 8:164 –172. Jiang X, Xu K, Hoberman J, Tian F, Marko AJ, Waheed JF, et al (2005): BDNF variation and mood disorders: A novel functional promoter polymorphism and Val66Met are associated with anxiety but have opposing effects. Neuropsychopharmacology 30:1353–1361. Kang H, Schuman EM (1995): Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science 267: 1658 –1662. Konur S, Rabinowitz D, Fenstermaker VL, Yuste R (2003): Systematic regulation of spine sizes and densities in pyramidal neurons. J Neurobiol 56: 95–112. Korte M, Carroll P, Wolf E, Brem G, Thoenen H, Bonhoeffer T (1995): Hippocampal long-term potentiation is impaired in mice lacking brainderived neurotrophic factor. Proc Natl Acad Sci U S A 92:8856 – 8860. Law AJ, Weickert CS, Hyde TM, Kleinman JE, Harrison PJ (2004): Reduced spinophilin but not microtubule-associated protein 2 expression in the hippocampal formation in schizophrenia and mood disorders: Molecular evidence for a pathology of dendritic spines. Am J Psychiatry 161: 1848 –1855. Leuner B, Falduto J, Shors TJ (2003): Associative memory formation increases the observation of dendritic spines in the hippocampus. J Neurosci 23:659 – 665. Leuner B, Shors TJ (2004): New spines, new memories. Mol Neurobiol 29: 117–130. Linnarsson S, Bjorklund A, Ernfors P (1997): Learning deficit in BDNF mutant mice. Eur J Neurosci 9:2581–2587. Liu WS, Pesold C, Rodriguez MA, Carboni G, Auta J, Lacor P et al (2001): Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse. Proc Natl Acad Sci U S A 98:3477–3482. Luikart BW, Nef S, Virmani T, Lush ME, Liu Y, Kavalali ET et al (2005): TrkB has a cell-autonomous role in the establishment of hippocampal Schaffer collateral synapses. J Neurosci 25:3774 –3786. Marty S, Carroll P, Cellerino A, Castren E, Staiger V, Thoenen H et al (1996): Brain-derived neurotrophic factor promotes the differentiation of various hippocampal nonpyramidal neurons, including Cajal-Retzius cells, in organotypic slice cultures. J Neurosci 16:675– 687. Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai H (2001): Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4:1086 –1092. Megias M, Emri Z, Freund TF, Gulyas AI (2001): Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102:527–540.
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O. von Bohlen und Halbach et al Minichiello L, Korte M, Wolfer D, Kuhn R, Unsicker K, Cestari V, et al (1999): Essential role for TrkB receptors in hippocampus-mediated learning. Neuron 24:401– 414. Monteggia LM, Barrot M, Powell CM, Berton O, Galanis V, Gemelli, T et al (2004): Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc Natl Acad Sci U S A 101:10827–10832. Muller D, Toni N, Buchs PA (2000): Spine changes associated with long-term potentiation. Hippocampus 10:596 – 604. Murphy DD, Cole NB, Segal M (1998): Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons. Proc Natl Acad Sci U S A 95:11412–11417. Nimchinsky EA, Sabatini BL, Svoboda K (2002): Structure and function of dendritic spines. Annu Rev Physiol 64:313–353. Norrholm SD, Ouimet CC (2000): Chronic fluoxetine administration to juvenile rats prevents age-associated dendritic spine proliferation in hippocampus. Brain Res 883:205–215. Norrholm SD, Ouimet CC (2001): Altered dendritic spine density in animal models of depression and in response to antidepressant treatment. Synapse 42:151–163. Parnass Z, Tashiro A, Yuste R (2000): Analysis of spine morphological plasticity in developing hippocampal pyramidal neurons. Hippocampus 10: 561–568. Rosoklija G, Mancevski B, Ilievski B, Perera T, Lisanby SH, Coplan JD, et al (2003): Optimization of Golgi methods for impregnation of brain tissue from humans and monkeys. J Neurosci Methods 131:1–7. Sala C, Futai K, Yamamoto K, Worley PF, Hayashi Y, Sheng M (2003): Inhibition of dendritic spine morphogenesis and synaptic transmission by activity-inducible protein Homer1a. J Neurosci 23:6327– 6337. Sato M, Suzuki K, Nakanishi S (2001): NMDA receptor stimulation and brainderived neurotrophic factor upregulate homer 1a mRNA via the mitogen-activated protein kinase cascade in cultured cerebellar granule cells. J Neurosci 21:3797–3805. Strauss J, Barr CL, George CJ, King N, Shaikh S, Devlin B, et al (2004): Association study of brain-derived neurotrophic factor in adults with a history of childhood onset mood disorder. Am J Med Genet B Neuropsychiatr Genet 131:16 –19. Toni N, Buchs PA, Nikonenko I, Bron CR, Muller D (1999): LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 402:421– 425. Trommald M, Hulleberg G, Andersen P (1996): Long-term potentiation is associated with new excitatory spine synapses on rat dentate granule cells. Learn Mem 3:218 –228. van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH (2002): Functional neurogenesis in the adult hippocampus. Nature 415:1030 – 1034. Vanderklish PW, Edelman GM (2002): Dendritic spines elongate after stimulation of group 1 metabotropic glutamate receptors in cultured hippocampal neurons. Proc Natl Acad Sci U S A 99:1639 –1644. von Bohlen und Halbach O, Albrecht D, Heinemann U, Schuchmann S (2002): Spatial nitric oxide imaging using 1,2-diaminoanthraquinone to investigate the involvement of nitric oxide in long-term potentiation in rat brain slices. Neuroimage 15:633– 639. von Bohlen und Halbach O, Schulze K, Albrecht D (2004): Amygdala-kindling induces alterations in neuronal densities and in densities of degenerated fibers. Hippocampus 14:311–318. Whitford KL, Dijkhuizen P, Polleux F, Ghosh A (2002): Molecular control of cortical dendrite development. Annu Rev Neurosci 25127– 49:127–149. Wirth MJ, Brun A, Grabert J, Patz S, Wahle P (2003): Accelerated dendritic development of rat cortical pyramidal cells and interneurons after biolistic transfection with BDNF and NT4/5. Development 130:5827–5838. Yuste R, Bonhoeffer T (2001): Morphological changes in dendritic spines associated with long-term synaptic plasticity. Annu Rev Neurosci 24: 1071–1089.
S
pines are small postsynaptic structures that protrude from the surface of dendrites of many neurons. Dendritic spines have been shown to be the predominant site of excitatory synapses on CA1 pyramidal neurons in the rat hippocampus (Megias et al 2001), where most spines are contacted by only one presynaptic bouton (Andersen 1990). Dendritic spines can undergo morphologic plastic changes. Thus, some forms of learning have been shown to increase the number of dendritic spines (Geinisman 2000; Leuner et al 2003; Nimchinsky et al 2002; Yuste and Bonhoeffer 2001). Moreover, structural remodeling of synapses and formation of new synaptic contacts have been postulated as a mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Indeed, LTP seems to be associated with increased spine densities (Muller et al 2000) and with the formation of new, mature, and probably functional synapses (Toni et al 1999). Thus, hippocampal LTP, induced in the perforant path/dentate granule cell synapse, leads to an increase in spine density (Trommald et al 1996), indicating that new spine synapses are formed following LTP. Using two-photon microscopy, it has been convincingly demonstrated that after induction of long-lasting functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite (Engert and Bonhoeffer 1999).
From the Department of Neuroanatomy (OvBuH, SK, KU), Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany; and Mouse Biology Unit (DM, CS, LM), European Molecular Biology Laboratory, Monterotondo, Italy. Address reprint requests to Oliver von Bohlen und Halbach, Interdisciplinary Center for Neurosciences (IZN), Department of Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany; E-mail: [email protected]. Received May 9, 2005; revised July 26, 2005; accepted August 31, 2005.
0006-3223/06/$32.00 doi:10.1016/j.biopsych.2005.08.025
Spine reductions in the hippocampus are associated with not only schizophrenia but also major depression (Law et al 2004). Antidepressant treatment has been shown to produce a significant increase in dendritic spine density in the hippocampal area CA1 of rats (Hajszan et al 2005; Norrholm and Ouimet 2000, 2001), and increase levels of brain-derived neurotrophic factor (BDNF) mRNA in the brain (Altar 1999). Brain-derived neurotrophic factor is a member of the neurotrophin (NT) family of neurotrophic factors (Barbacid 1994). Polymorphisms in the BDNF gene seem to be associated with major depression (Jiang et al 2005; Strauss et al 2004). It has been proposed that decreased BDNF levels may be implicated in the pathogenesis of depression (Altar 1999). Inappropriate neurotrophic support could lead to structural disorganization in the brain and ultimately to a decreased capacity of the brain to adapt to changes (Angelucci et al 2005). Under physiologic conditions, BDNF is known, in addition to its trophic functions, to be a key mediator of activity-dependent modifications of synaptic strength in the central nervous system (CNS). Thus, application of BDNF has been reported to produce a dramatic and sustained (2–3 hours) enhancement of synaptic strength at the Schaffer collateral CA1 synapses (Kang and Schuman 1995). Moreover, pretreatment of hippocampal slices with a trkB-IgG fusion protein, which scavenges endogenous BDNF, reduces the magnitude of LTP (Figurov et al 1996). In BDNF mutant mice, both hippocampal LTP and spatial learning are impaired (Korte et al 1995; Linnarsson et al 1997; Monteggia et al 2004). Interestingly, the cognate receptor for BDNF, trkB, has been localized immunocytochemically within dendritic spines of hippocampal neurons (Drake et al 1999). Moreover, cyclic adenosine monophosphate seems to facilitate trafficking of trkB to dendritic spines, possibly by promoting its interaction with the synaptic scaffolding protein PSD-95 (Ji et al 2005). Whether BDNF also affects spine morphology in the hippocampus has not been proven. Because BDNF, through activation of trkB, is able to induce hippocampal LTP, and because LTP induces spinogenesis, it may be speculated that trkB activation BIOL PSYCHIATRY 2006;59:793– 800 © 2005 Society of Biological Psychiatry
794 BIOL PSYCHIATRY 2006;59:793– 800 may increase spine densities. Cell culture studies addressing this issue have provided conflicting results, however. Using cultured hippocampal neurons, BDNF has been reported to reduce (Murphy et al 1998) or increase (Ji et al 2005) spine densities. Organotypic hippocampal slice cultures have revealed a BDNF mediated increase in dendritic spine densities in area CA1 (Alonso et al 2004). This study investigates the impact of trkB receptors on spine densities and spine morphology in the hippocampal formation. We evaluated this brain area because it is known to be involved in depression, spatial memory, LTP, and spinogenesis. Because mice with a targeted deletion of trkB or bdnf die shortly after birth, we examined mice in which trkB was conditionally deleted postnatally from the anterior forebrain (Minichiello et al 1999). These animals (termed “trkB-CRE”) survive into adulthood and have previously been shown to display deficits in hippocampal LTP and hippocampus-related behavior and represent an ideal model to investigate a possible role for trkB in the regulation of spine morphology and density in vivo.
Methods and Materials Animals The generation of conditionally gene-targeted mice (TrkBlox/ loxCaMKII–CRE⫹/⫺ mice or simply “trkB–CRE” mice), in which the knockout of the full-length trkB receptor is restricted to neurons of the anterior forebrain and occurs only during postnatal development has been previously described (Minichiello et al 1999). In these mice, a reduction in trkB protein in the hippocampus begins at about postnatal day 20 (Minichiello et al 1999). Age-matched mice with the genotypes wild-type, trkBlox/ lox, and trkBlox/⫹ did not differ in terms of the amounts of trkB they expressed and thus were grouped together and served as control animals. Two age groups of mice were analyzed: 8 weeks and 15 weeks of age. Golgi Method Mice were perfused with 4% paraformaldehyde (PFA) in phosphate buffer. Brains were dissected and postfixed in 4% PFA. Golgi-staining was performed according to the Golgi-Kopsch method, resulting in well-impregnated neurons (Rosoklija et al 2003). Blocks of about 2.5 mm thickness were prepared from each hemisphere, and brain pieces were soaked in a solution containing 2.5% potassium dichromate for 1 day at room temperature in the dark. This solution was then replaced by fresh solution of 2.5% potassium dichromate, and tissues were incubated for another 6 days. Tissues were then washed and transferred to a solution containing .75% AgNO3. After 5 to 6 days, brain pieces were removed, washed in 40% and 20% ethanol, and cut into 60-m coronal sections using a vibratom (Leica, Wetzlar, Germany). Sections were mounted on gelatin-coated slides and cover-slipped using Merkoglas (Merk, Darmstadt, Germany). Spine Analysis Spine analysis was conducted blind to the experimental conditions. As previously reported (Drake et al 1999), trkBimmunoreactivity accumulates at high levels in the dendritic spines within the stratum oriens and the stratum radiatum of area CA1 of the hippocampus and in the stratum moleculare of the dentate gyrus. Consequently, we examined spines on apical dendrites of CA1 pyramidal neurons in the stratum radiatum and on basal dendrites of CA1 pyramidal neurons in the stratum oriens, as well as on dendrites of the dorsal leaf of the dentate gyrus (DG; Figure 1). Spines were analyzed in the dorsal, but not www.sobp.org/journal
O. von Bohlen und Halbach et al ventral, leaf of the DG because spine densities are different in these locations (Desmond and Levy 1985). Analyses were conducted on Golgi-impregnated sections that were uniformly dark throughout the section. Only dendrites that displayed no breaks in their staining (Leuner et al 2003) and that were not obscured by other neurons or artifacts (Liu et al 2001) were evaluated. Only spines located on secondary or tertiary dendritic trees were evaluated. Only one segment per individual dendritic branch was chosen for the analysis. Quantitative three-dimensional analyses of dendritic fragments with their spines were conducted using a combined hardwaresoftware system (NeuroLucida, MicroBrightFields, Colchester, Vermont) controlling the x-y-z axis of the microscope (Axioscop Imaging, Zeiss, Germany) and a microscope-mounted video camera (Hitachi, Tokyo, Japan). The three-dimensional reconstruction was done using a 100⫻ objective (NA: 1.4; oil immersion) and the NeuroLucida system. Spine densities and mean spine length were calculated from the reconstructed dendrites with the help of NeuroExplorer (MicroBrightFields). Traditionally, spines have been classified into four major categories (filopodia, stubby, thin, and mushroom) based on their distinct morphologies (Parnass et al 2000); however, the recently discovered rapid morphologic plasticity of spines has suggested that these categories, rather than representing intrinsically different populations of spines, reflect temporal snapshots of a dynamic phenomenon (Parnass et al 2000). Therefore, no subdivision into different types of spines was made. For the analysis, at least three animals per age and genotype were analyzed. In these animals, individual dendrites were reconstructed. In case of basal dendrites of area CA1 between 46 and 55 individual dendritic segments per group (bearing between 529 and 779 individual spines), and in case of apical dendrites of area CA1 between 51 and 55 dendritic segments (bearing between 587 and 703 individual spines) were reconstructed and analyzed; in case of the dentate gyrus between 50 and 59 dendritic segments (bearing between 613 and 812 individual spines) per group were reconstructed. Determination of trkB Protein Levels Mice were killed by cervical dislocation, and hippocampi were quickly dissected and frozen in liquid nitrogen. Protein lysates were obtained by homogenizing the tissue in lysis buffer (20 mmol/mL Tris pH 7, 140 mmol/mL NaCl, 10% glycerol, 1% NP 40, 10 mg/mL leupeptin, 2 mg/mL aprotinin, 5 mmol/mL benzamidine, 1 mmol/mL PMSF, 10 mmol/mL NaF, 1 mmol/mL NaPP, 1 mmol/mL Na3VaO4), followed by centrifugation at 4°C to remove insoluble material (see also Minichiello et al 1999). Immunoprecipitations were performed using a pan-Trk antibody (C-14, Santa Cruz, California), and a TrkB polyclonal antibody, 113-5, was used for Western blots, as previously described (Minichiello et al 1999). To control for protein loading, Erk1 levels were probed on stripped blots using a monoclonal Erk1 antibody (Zymed, San Francisco, California). Comparison of trkB expression in hippocampal lysates of mutant and control mice was performed using densitometric analysis (NIH Image) of enhanced chemiluminescence (ECL)-stained Western blots. Density values were corrected by dividing them by the values obtained for Erk1. In Situ Hybridization Mice were perfused with 4% PFA. Brains were removed from the skull, and cryosections (25 m) were made and mounted on slides. A nonradioactive in situ hybridization (ISH) protocol was
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Figure 1. (A) Schematic drawing of the hippocampus. Area CA1 consists of the stratum oriens (Or), the pyramidal layer, the stratum radiatum (Rad), and the stratum lacunosum moleculare (LMol). Cell bodies of analyzed pyramidal cells are located in the pyramidal layer (indicated in gray). Basal dendrites within the stratum oriens were analyzed as well as apical dendrites within the stratum radiatum. Within the dentate gyrus, granule cells are located within the granular layer (indicated in gray). Dendrites within the molecular layer (Mol) of the dentate gyrus (DG) were analyzed. (B) Golgi-impregnated basal dendrite of area CA1 (control mouse; 8 weeks old). Image was made using a z-stack of seven individual images, generated by using a software-driven motorized stage (step width: .5 m) and microscope-mounted digital camera (Axiocam; Zeiss).
used. Dioxigenin-labeled riboprobes recognizing full-length trkB (trkB.fl, bp 2217–2776) were generated as follows: cDNA fragments were synthesized by polymerase chain reaction from murine-brain cDNA and cloned into a pGEM-Teasy vector (Promega, Madison, Wisconsin). The plasmids were appropriately linearized and transfected with SP6 or T7 for antisense and sense probes, respectively. Specificity of our riboprobes was confirmed by subsequent sequencing by MWG Biotech (Ebersberg, Germany). For ISH, slides were incubated overnight with hybridization buffer containing dioxigenin-labeled riboprobes. The slides were washed three times in a washing solution (50% formamide, .001% Tween and 5% sodium chloride, and 5% sodium citrate solution [SSC]) for 10 min, 30 min, and 45 min at 68°C and then twice with malein acid buffer and Tween (MABT) for 30 min at room temperature. After blocking in MABT containing 20% horse serum, sections were incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase (Roche, Switzerland) at room temperature. After three washes in MABT and two washes in prestaining buffer, the sections were developed using nitroblue tetrazolium salt/5=-bromo-4-chloro-idoylphosphate (NBT/BCIP) (Roche, Switzerland) and cover-slipped. For quantification of signal intensities, images were made using a software-controlled (Axiovision 3.0; Zeiss) digital camera
(Axiocam, Zeiss) attached to a microscope (Axioplan imaging; Zeiss) using a 20⫻ objective. For the process of image acquisition, the same parameters were used (e.g., illumination time, threshold). Images were stored onto hard disk and analyzed offline. Intensity measurements were done using ImageJ 1.31 (National Institute of Health). The color images were changed to 8-bit grayscale images that were represented using unsigned integers in the range 0 –255 (256 gray values coding for intensities). In each converted image, histograms of the distribution of pixel intensities were made. The mean intensity values were calculated for an area of interest (von Bohlen und Halbach et al 2002), which was defined to cover the area of either the pyramidal layer of the hippocampus or, in the case of the DG, the granular layer. For estimates of the relative cell densities of trkB mRNA– containing cells, a microscope (Axioplan 2 imaging; Zeiss) equipped with a computer-driven digital camera (Axiocam; Zeiss) was used. Positive cells were counted in a region of interest (ROI; representing a window of 10,000 m2), and data were expressed as cell densities per 10,000 m2(von Bohlen und Halbach et al 2004). To estimate cell densities within the CA1 area and within the dentate gyrus in the four mouse groups, three animals per group were used. In each animal, at least six sections were analyzed per brain area. Statistical analysis was performed using Prism (Graph Pad, www.sobp.org/journal
796 BIOL PSYCHIATRY 2006;59:793– 800 San Diego, California). Data sets were statistically compared using analysis of variance (ANOVA) with Student–Newmann– Keuls post hoc test. Data were presented as mean ⫾ SEM.
O. von Bohlen und Halbach et al Determinations of spine length revealed no age-related change in the trkB-CRE mice (Figure 3F); however, 15-week-old trkBCRE mice had significantly longer spines than their age-matched control counterparts.
Results Impact of Age on Spine Densities and Spine Length in Area CA1 and the Dentate Gyrus As shown in Figure 2A, mice at 15 weeks of age revealed a 17% increase in spine densities of basal dendrites in CA1 compared with 8-week-old control mice. Similar to CA1 basal dendrites, CA1 apical dendrites of 15-week, compared with 8-week-old control mice, exhibited an increase in spine densities (26%). As in the CA1 region of the hippocampus, densities of dendritic spines in the dorsal leaf of the dentate gyrus increased by 11% from 8 to 15 weeks of age (Figure 2A). Mice at 15 weeks of age displayed a 12% reduction in average spine length of basal dendrites in CA1, but showed no significant change in the mean length of spines, located on apical dendrites (Figure 2B). Concerning dendritic spines in the dorsal leaf of the dentate gyrus, a reduction of about 11% in mean spine length was noted by comparing 15-week-old animals with 8-week old mice (Figure 2B). Impact of Conditional Deletion of trkB on Spine Densities and Spine Length in Area CA1 and the Dentate Gyrus Basal Dendrites of Neurons Located in area CA1. As shown in Figure 3A, 8-week-old trkB-CRE mice did not differ in their spine densities of basal CA1 dendrites from age-matched control animals. Moreover, 15-week-old trkB-CRE mice did not reveal an increase in spine densities as compared to 8-week-old trkB-CRE mice. Thus, 15-week-old trkB-CRE mice displayed 11% less dendritic spines on basal dendrites of area CA1 than the agematched control mice. Eight-week-old trkB-CRE mice had significantly shorter spines (⫺7.5%) than the age-matched control mice, whereas spine length in 15-week-old trkB-CRE mice was increased by 16.8% compared with control mice (Figure 3B). Fifteen-week-old trkB-CRE mice had 13% longer spines than 8-week-old trkB-CRE mice. Apical Dendrites of Neurons Located in Area CA1. Spine densities did not significantly differ between 8-week-old control and trkB-CRE mice. In contrast to control mice, spine densities failed to increase in trkB-CRE mice between 8 and 15 weeks of age. Thus, compared with control mice, spine densities in 15-week-old mutant mice were reduced by 10% (Figure 3C). There was a significant 21% difference in spine length of 15-week-old mutant mice compared with control mice, however. Comparisons of 8and 15-week-old trkB-CRE mice revealed a 13% increase in spine length (Figure 3D). Dendrites of Neurons Located in the Dentate Gyrus. As in control mice, spine densities in the trkB-CRE mice increased significantly (⫹21%) from postnatal week 8 to 15 (Figure 3E).
TrkB-Protein Levels Hippocampal trkB protein levels were clearly reduced in trkB-CRE mutants as compared with control mice. Despite this obvious reduction in trkB-protein levels, these changes failed to reveal significant changes in trkB protein levels between 8- and 15-week-old trkB-CRE mutants (Figure 4). Expression of trkB mRNA in the Hippocampus Spine analyses had suggested both age-dependent and region-specific alterations in spine densities and length of trkB-CRE mice; however, these alterations were not paralleled by changes in levels of trkB protein. Because protein samples included the whole hippocampus, it was conceivable that region-specific changes in trkB expression might have escaped detection. We therefore used ISH to examine putative region-specific changes in trkB expression in area CA1 and the dentate gyrus. We used a probe that only identifies the mRNA of full-length trkB. Fulllength trkB expression in area CA1 was clearly reduced in trkB-CRE mice (Figures 5A–E). In these mice, full-length trkB mRNA expression decreases between postnatal weeks 8 and 15; however, trkB mRNA was still expressed in few CA1 neurons in the 15-week-old trkB-CRE mice (Figure 5E). In addition, fulllength trkB mRNA was reduced in the dentate gyrus at 15-weekof-age in the trkB-CRE mice (Figure 5F). Because many variables, in cases of nonisotopic hybridization histochemistry, influence the staining intensity, we also estimated the relative densities of full-length trkB mRNA expressing cells in the area CA1 and in the dentate gyrus. The density of full-length trkB mRNA expressing cells in area CA1 was not significantly increased from the 8th to the 15th postnatal week in control mice (Figure 5G). At 8 weeks, a significant 70% reduction in the density of full-length trkB mRNA expressing cells was noted in area CA1 by comparing the trkB-CRE mice with age-matched control mice. At 15 weeks, only some full-length trkB expressing cells remained in the area CA1 of trkB-CRE mice. Thus, the density of full-length trkB expressing cells in area CA1 was significantly reduced by approximately 95%, compared with age-matched control animals. Interestingly, by comparing 15-week-old with 8-week-old trkB-CRE mice, a significant reduction (⫺80%) in the density of full-length trkB-expressing cells was found (Figure 5G). Concerning the dentate gyrus, the density of full-length trkB mRNA expressing cells was not significantly altered in 15-weekold control mice compared with their 8-week-old littermates of the same genotype (Figure 5H). Comparing 8-week-old trkB-CRE mice with 8-week-old control mice, a significant reduction in the densities of positive cells was noted (⫺81%). By comparing 15-week-old trkB-CRE mice
Figure 2. Impact of age on spine densities (A) and mean spine length (B) in control mice. Asterisks represent significant changes (p ⱕ .01). CA1-bas, basal dendrites of area CA1; CA1-api, apical dendrites of area CA1; DG, dentate gyrus; con 8 weeks, control mice aged 8 weeks; con 15 weeks, control mice aged 15 weeks.
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Figure 3. Column bar graphs representing the results obtained in the evaluated brain areas. Spine densities (A) and spine lengths of basal dendrites (B) in area CA1, spine densities (C) and spine lengths of apical dendrites (D) in area CA1, and spine densities (E) and spine lengths of apical dendrites (F) in the dorsal leaf of the dentate gyrus. In these graphs, only statistical analyses between aged-matched groups as well as between 8-week and 15-week-old trkB-CRE mice are displayed. *Significant changes (p ⱕ .01). con 8 weeks, control mice aged 8 weeks; con 15 weeks, control mice aged 15 weeks; trkB 8 weeks, trkB-CRE mutant mice, aged 8 weeks; trkB 15 weeks, trkB-CRE mutant mice aged 15 weeks.
with control mice of the same age, a reduction in the density of full-length trkB expressing cells of about 90% was noted. However, we observed no significant difference in the densities of those cells by comparing 8-week-old and 15-week-old trkB-CRE mice (Figure 5H).
Discussion Dendritic morphology has a profound impact on neuronal information processing. Spines act as subcellular compartments that are crucially involved in receiving and processing synaptic information, and dendritic branching patterns determine the efficacy by which synaptic information is transmitted to the soma (Whitford et al 2002). Applied exogenously, BDNF has been found to increase dendritic length of hippocampal interneurons in organotypic slice cultures (Marty et al 1996). Moreover, exogenous application of both trkB ligands, BDNF and NT-4, has been found to increase dendritic length of pyramidal neurons in cortical organotypic cultures. Under these conditions, neutralization of endogenous NT-4 was found to cause shorter and less branched dendrites, whereas neutralization of endogenous BDNF was apparently not effective (Wirth et al 2003). Transfection of dentate gyrus granule cells in slice cultures with BDNF via
particle-mediated gene transfer was found to increase axonal branching and numbers of basal dendrites (Danzer et al 2002), whereas transfections of CA3 or CA1 pyramidal neurons were not efficient (Alonso et al 2004; Danzer et al 2002). These observations seem to indicate that in vitro trkB ligands modulate dendritic structure and spine densities in a cell-type-specific manner; however, interpretation of these data suggesting a potential link between trkB receptors and spine densities and morphology is seriously hampered by the fact that, at comparable ages, spine synapses in culture are more than threefold less abundant than in vivo (Boyer et al 1998). Concerning the control mice, we found an apparent increase in spine densities from 8 to 15 weeks of age, corroborating a previous report that spine densities increase with age in the hippocampus (van Praag et al 2002). Furthermore, spines on basal dendrites in CA1 and on dendrites in the dentate gyrus were substantially longer in 8 as compared to 15 week-old animals. This is consistent with the notion that not only spine densities but also spine morphology changes with postnatal development. Early spines are often very long, and over the next few weeks are reduced in length (Nimchinsky et al 2002). It has been argued that longer, thinner spines constitute a less efficacious postsynaptic phenotype (Matsuzaki et al www.sobp.org/journal
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Figure 4. Determination of trkB protein levels in whole hippocampi. (A) Immunoprecipitation (IP) and Western blot analysis of TrkB expression in protein lysates of hippocampal tissues from 8- or 15week-old trkBlox/lox mice, either in presence or absence of CamKII-CRE. (B) Densitometric analysis of TrkB protein in hippocampi of trkBlox/lox mice, in either the presence or absence of CamKII-CRE (n ⫽ 2– 8 animals per time point). Densitometric values for the control animals were averaged because they did not differ significantly (p ⫽ .8).
2001; Vanderklish and Edelman 2002) and that variations in spine length may even contribute to behavioral alterations. Along this line, the increased spine length that we found in trkB mutant mice may be indicative for a less efficacious phenotype. It has recently been reported that homer1a reduces spines sizes and inhibits Shankmediated spine enlargement in vitro (Sala et al 2003); BDNF has been found to upregulate homer 1a mRNA via the mitogenactivated protein kinase cascade in cultured cerebellar granule neurons (Sato et al 2001), suggesting that increased spine length in the conditional trkB mutant mice may be due to the downregulation of homer 1a. Spine density is thought to reflect excitatory input density (Konur et al 2003), and some forms of learning as well as LTP have been associated with increased spine densities in the hippocampus (Engert and Bonhoeffer 1999; Leuner and Shors 2004; Muller et al 2000). Moreover, BDNF signaling via trkB has been shown to be associated with synaptic plasticity, LTP, and hippocampal-dependent learning. A role for trkB ligands in local modulation of excitatory neurotransmission is suggested by the presence of trkB-immunoreactivity in dendritic spines, which are the targets of glutamatergic innervation in the trisynaptic circuit (Drake et al 1999). It is known that trkB receptors mediate some rapid effects of BDNF (Drake et al 1999), and application of the www.sobp.org/journal
trkB-ligand BDNF induces an enhancement of synaptic strength of CA1 synapses (Kang and Schuman 1995). Because disruption of the trkB-BDNF signaling pathway, through conditional deletion of trkB, results in altered spine densities and spine morphology, BDNF may be conceived to mediate both rapid effects and long-term changes in spine morphology, which interfere with synaptic strength. In a recent study (Luikart et al 2005), it has been suggested that postnatal trkB loss is not associated with the disassembly of existing synapses and that trkB is necessary to promote synapse formation during a period in which maturation of synaptic connectivities occurs. Consistent with this hypothesis, we do not observe a difference in spine densities in area CA1 by comparing the groups with an age of 8 weeks. At 15 weeks of age, trkB-CRE mice have a reduced spine density compared with age-matched control mice; however, the 15-week-old trkB-CRE mice did not display a reduced spine density compared with 8-week-old trkB-CRE or control mice. Thus, in support of the notion held by Luikart and colleagues, a disassembly of existing spines does not to occur. It is likely that during the time period from 8 weeks to 15 weeks of age, a maturation of synaptic connectivities together with an activity-dependent formation of new spines occurs in case of the control mice, but not in case of the trkB-CRE mice.
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Figure 5. In situ hybridization for full-length trkB mRNA. (A) Hippocampal area CA1 of a control animal (8 weeks old; in situ hybridization for full-length trkB mRNA). (B) Hippocampal area CA1 of a trkB-CRE mutant mouse (8 weeks old; in situ hybridization for full-length trkB mRNA). (C) Hippocampal area CA1 of a control animal (15 weeks old; in situ hybridization for full-length trkB mRNA). (D) Hippocampal area CA1 of a trkB-CRE mutant mouse (15 weeks old; in situ hybridization for full-length trkB mRNA). (E) Quantification of the in situ hybridization signals in area CA1 using the full-length trkB probe. (F) Quantification of the in situ hybridization signals in the dentate gyrus using the full-length trkB probe. *p ⱕ .05. ** p ⱕ .01. con 8 weeks, control mice aged 8 weeks; con 15 weeks, control mice aged 15 weeks; trkB 8 weeks, trkB-CRE mutant mice aged 8 weeks; trkB 15 weeks, trkB-CRE mutant mice aged 15 weeks.
Compared with age-matched control mice, spine densities were only reduced in 15-week, but not 8-week-old, trkB-CRE mice. Likewise, conditional deletion of the truncated together with the full-length form of trkB does not alter spine densities of CA1 dendrites in the stratum radiatum at postnatal day 60 (Luikart et al 2005). This suggests that at 8 weeks, the amounts of trkB mRNA and protein still present within the hippocampus may suffice to maintain regular spine morphologies. Hippocampal trkB protein levels did not parallel alterations in spine morphologies noted in conditional trkB mutants, possibly because samples taken from the whole hippocampus may not reveal distinct intrahippocampal regional differences. This notion is supported by the results obtained using in situ hybridization to examine full-length trkB mRNA expression in distinct subregions of the hippocampus. Both 8-week and 15-week-old mutant mice, compared with their control littermates, showed a significant decrease in full-length trkB mRNA in area CA1. Only at 15 weeks, however, was this decrease in trkB mRNA paralleled by a decrease in spine densities. In summary, our data have shown that a forebrain-specific, postnatal targeted deletion of the trkB gene affects hippocampal spines in an age- and region-specific manner. A marked reduction in trkB mRNA occurs in CA1 and is paralleled by a significant decrease in spine densities and an increase in mean spine length
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