Cell, Vol. 112, 257–269, January 24, 2003, Copyright 2003 by Cell Press

The BDNF val66met Polymorphism Affects
Activity-Dependent Secretion of BDNF
and Human Memory and Hippocampal Function
Michael F. Egan,1 Masami Kojima,4,5,6,7
Joseph H. Callicott,1,7 Terry E. Goldberg,1,7
Bhaskar S. Kolachana,1 Alessandro Bertolino,1
Eugene Zaitsev,4 Bert Gold,3 David Goldman,2Michael Dean,3 Bai Lu,4,*and Daniel R. Weinberger1,*
1
Clinical Brain Disorders Branch
National Institute of Mental HealthRoom 4s-235
10 Center Drive
Bethesda, Maryland 20892
2
Laboratory of Neurogenetics
National Institute on Alcohol Abuse and Alcoholism
Park Building
12420 Parklawn Drive
Rockville, Maryland 20857
3
National Cancer Institute-Frederick
Building 560
Room 21-18
NCI-FCRDC
Frederick, Maryland 21702
4
Section on Neural Development and Plasticity
National Institute of Child Health and Human
Development
National Institutes of Health, DHHS
Building 49/6A67
Bethesda, Maryland 20892
5
Cell Dynamics Research Group
National Institute of AIST
Midorigaoka, Ikeda
Osaka 563-8577
Japan
6
CREST
Japan Science and Technology Corporation
1-8, Honcho 4-chome
Kawaguchi 332-0012
Japan

Summary
Brain-derived neurotrophic factor (BDNF) modulates
hippocampal plasticity and hippocampal-dependent
memory in cell models and in animals. We examinedthe effects of a valine (val) to methionine (met) substitution in the 5ⴕ pro-region of the human BDNF protein.
In human subjects, the met allele was associated withpoorer episodic memory, abnormal hippocampal activation assayed with fMRI, and lower hippocampal
n-acetyl aspartate (NAA), assayed with MRI spectroscopy. Neurons transfected with met-BDNF-GFP showed
lower depolarization-induced secretion, while constitutive secretion was unchanged. Furthermore, metBDNF-GFP failed to localize to secretory granules orsynapses. These results demonstrate a role for BDNF
*Correspondence: weinberd@intra.nimh.nih.gov (D.R.W); bailu@
mail.nih.gov (B.L.)
7
These authors contributed equally to this work.

and its val/met polymorphism in human memory andhippocampal function and suggest val/met exerts
these effects by impacting intracellular trafficking andactivity-dependent secretion of BDNF.
Introduction
A remarkable feature of the human brain is its ability tostore and recall a seemingly endless series of experiences many of which occur only once. Clinical studies
suggest that this capacity, referred to as episodic memory, is critically dependent on the hippocampus andrelated mesial temporal lobe structures (Milner et al.,1998). Molecular studies of hippocampal synapses haverevealed that memory-related modifications in synaptictransmission occur in stages. The first stage, known
as early phase long-term potentiation (E-LTP), involves
rapid increases of intracellular calcium concentrations
and subsequent activation of protein kinases (Bliss andCollingridge, 1993). A second stage, referred to as latephase LTP (L-LTP), recruits the cAMP and CREB signaling pathway to direct protein synthesis-dependentchanges in structure and function of hippocampal synapses(Kandel, 2001). One protein in particular, brain derived
neurotrophic factor (BDNF), appears to play an important role in both (Lu and Gottschalk, 2000; Poo, 2001).
Evidence that BDNF is involved in hippocampal LTP,
learning, and memory in nonhuman species is substantial (Lu and Gottschalk, 2000; Poo, 2001). BDNF gene
expression is markedly enhanced by tetanic stimulationthat induces LTP (Patterson et al., 1992) and during
spatial memory tasks (Hall et al., 2000; Mizuno et al.,2000). BDNF application facilitates LTP while reductionof BDNF levels attenuates LTP (Figurov et al., 1996;
Korte et al., 1995; Patterson et al., 1996). Studies using avariety of approaches demonstrate that BDNF enhanceshigh frequency synaptic transmission by facilitating synaptic vesicle docking (Gottschalk et al., 1998, 1999; Jovanovic et al., 2000; Pozzo-Miller et al., 1999; Xu et al.,2000). BDNF also elicits rapid postsynaptic effects on
ion channels and NMDA receptors (Levine et al., 1995).
In addition to acute modulation of synaptic transmission
and E-LTP, recent studies have demonstrated a role of
BDNF in long-term changes in hippocampal synapses
and L-LTP (Korte et al., 1998; Tartaglia et al., 2001).
Inhibition of BDNF signaling in rodents by gene knockout
or infusion of antisense BDNF impairs spatial learningand memory (Linnarsson et al., 1997; Minichiello et al.,
1999; Mizuno et al., 2000). Despite substantial progressin animal studies, BDNF’s relevance in human memory
and hippocampal function has not been examined directly.
Unlike other growth factors, which are secreted primarily via a constitutive pathway, at least in hippocampal neurons, BDNF appears to be sorted into a regulated
pathway that secretes BDNF in response to neuronal
activity (Farhadi et al., 2000; Goodman et al., 1996;
Mowla et al., 2001). Transfection experiments using
BDNF-GFP (green fluorescence protein) fusion con-

Cell
258

structs demonstrate that BDNF is packaged into secretory vesicles, which are transported to somatodendritic
compartments (Kojima et al., 2001). Activity-dependent
BDNF secretion from postsynaptic dendrites may serve
as a retrograde or paracrine messenger that regulates
hippocampal LTP (Poo, 2001). Indeed, electric stimulation of hippocampal neurons elicits BDNF-GFP secretion from dendrites (Hartmann et al., 2001). Taken together, these results strongly argue for a critical role ofactivity-dependent secretion of BDNF in hippocampusbased synaptic plasticity and learning and memory.
Based on these experimental data, one might predictthat genetic interference of BDNF secretion would leadto deficits in hippocampal function, learning, and memory. The BDNF gene, like other peptide growth factors,
encodes a precursor peptide (proBDNF), which is proteolytically cleaved to form the mature protein (Seidah
et al., 1996). Only one frequent, nonconservative polymorphism in the human BDNF gene (dbSNP number
rs6265) has been identified, a single nucleotide polymorphism (SNP) at nucleotide 196 (G/A) producing an aminoacid substitution (valine to methionine) at codon 66 (val66met). We hypothesized that this SNP, though locatedin the 5⬘ pro-BDNF sequence, and thus unlikely to alter
the intrinsic biological activity of the mature protein,would affect intracellular processing and secretion of
BDNF, leading to impairments in hippocampal function
in humans. Because hippocampal dysfunction is associated with genetic risk for schizophrenia (Callicott et al.,
1998; Egan et al., 2001a), we also hypothesized that
BDNF genotype might be associated with genetic risk
for schizophrenia. To test these hypotheses, we examined first the effects of the BDNF val66met substitution ina cohort of normal controls, patients with schizophrenia
and their unaffected siblings, using three in vivo assaysof human hippocampal function: (1) cognitive measuresof episodic memory; (2) a neurophysiological measureof hippocampal activation during a memory task using
fMRI; and (3) an intracellular neurochemical measure ofhippocampal neuronal integrity and synaptic abundance
assayed with proton magnetic resonance spectroscopic
imaging (1H-MRSI). To investigate underlying molecularmechanisms, we transfected rodent hippocampal neurons with val- or met-BDNF fused with GFP and examined trafficking of val- and met-BDNF proteins underfluorescence microscopy. We also determined constitutive and regulated (i.e., depolarization-induced) BNDF
secretion in these neuronal cultures. Our analyses revealed striking deficits in the cellular distribution andregulated secretion of BDNF caused by this SNP andcorresponding alterations of human hippocampal function in vivo. While we found no relationship between
BDNF genotype and schizophrenia, our results demonstrate that BDNF plays a role in hippocampal functionand episodic memory in humans and provide insights
into the mechanisms for intracellular trafficking and secretion of BDNF.
Results
Effect of BDNF Genotype on Episodic Memory
In the first experiment, we examined the effects of BDNF
genotype on measures of episodic memory in a cohortof 641 subjects, including normal controls, patients withschizophrenia, and their unaffected siblings, using measures from the Wechsler Memory Scale, revised version(WMS-R), a test of verbal episodic memory. Delayedrecall scores from the WMS-R reflect the amount of
information from two stories with 50 total elements that
subjects are able to recall following a 0.5 hr delay. Patients with schizophrenia had substantially lower scorescompared to controls, while siblings were intermediate
between these groups, consistent with earlier reports(Egan et al., 2001b; Figure 1A). In the entire sample,
BDNF genotype had a significant effect on these memory scores (F ⫽ 3.89, df ⫽ 2, 591, p ⫽ .02). In the groupof 133 controls alone, BDNF genotype also had a significant effect on memory scores (F ⫽ 5.04, df ⫽ 2,130,
p ⫽ .008). While including patient and sibling groups
did not substantially add to the results, both groups
showed the same effect seen in controls with met/met
subjects tending to score lower than other genotype
groups (Figure 1A). Post hoc comparisons in the normal
subjects alone showed that met/met homozygotes had
lower scores compared to val/val (p ⬍ .005) and val/met (p ⫽ .052), while in the entire cohort, met/met homozygotes had lower scores compared to the other two
genotype groups (p ⫽ .007). Within each group (controls,
siblings, patients), each genotype group was wellmatched on a variety of demographic parameters (seeFigure 1B), suggesting they did not account for the effectof BDNF genotype. Similar effects of BDNF genotype
were seen with immediate recall scores (of the stories
described above) from the WMS-R (e.g., normal group,F ⫽ 3.65, df ⫽ 2,130, p ⬍ .03). Post hoc comparisons
again showed that met/met homozygotes had lower
scores compared to val/val (p ⬍ .01) and val/met (p ⫽.07) groups.
BDNF genotype had no significant effect in either thenormal subjects or the entire cohort on a second memory test which required recall of word lists, the California
Verbal Learning Test (CVLT), immediately after hearing
the list (1–5 summary scores, F ⫽ 0.79, df ⫽ 2, 560; p ⫽.45; post hoc comparisons p ⬎ .14) and again 20 min
later (long delay free recall scores, F ⫽ 0.23, df ⫽ 2,560; p ⫽ .79; post hoc comparisons p ⬎ .37). The CVLT
may have a larger “prefrontal component” (Kopelman
et al., 1998) compared to the WMS-R, possibly reducing
hippocampal and val66met related variance. No effectof BDNF genotype was seen on other types of memory,
such as semantic memory (F ⫽ 0.02, df ⫽ 2, 591; p ⫽98) or working memory/executive function (WCST, F ⫽0.33, df ⫽ 2, 590; p ⫽ .71) (Figure 1B). These results
suggest that the val66met polymorphism exerts its mostrobust effects on episodic memory.
Effect of BDNF Genotype
on Hippocampal Activation
To further characterize the effects of BDNF genotype,we performed an in vivo assay of hippocampal physiology using the blood oxygenation level dependent(BOLD) fMRI technique (Ogawa et al., 1992) in subjects
performing the N-back working memory task. The brain
network subserving the N-back task (which is not atest of episodic memory per se) is thought to involve
primarily neocortical regions, particularly dorsolateral

BDNF and Human Hippocampal Function
259

Figure 1. Effect of BDNF val66met Genotype on Episodic Memory(A) BDNF val66met genotype and episodic memory scores (⫾ SE) from the WMS-R (delayed recall) in three samples studied.
(B) Demographic and cognitive data by genotype. Means (⫾ SD) are presented. Genotype groups are well matched except for ap ⬍ .05, val/
val versus val/met.

prefrontal cortex (Callicott et al., 1999). However, thesetasks also produce a robust and reliable deactivation ordisengagement of the hippocampus (Meyer-Lindenberg
et al., 2001). Hippocampal deactivation has been reported to be disrupted in clinical pathological states(Callicott et al., 2000). We hypothesized, therefore, thatthe BDNF met allele would disrupt the normal hippocampal fMRI disengagement pattern during performance of
the N-back working memory task.
We studied two independent cohorts of healthy subjects (patients were not included), each comprised ofval/met and val/val genotype subgroups; no met/met
subjects were included due to their low population frequency (⬍ 5%). The members of the cohorts were selected so that N-back performance, age, and gender
did not differ between the genotype subgroups. In thefirst cohort, val/met individuals had an abnormal patternof increased activation of bilateral caudal hippocampus.
In contrast, val/val subjects showed the characteristic
hippocampal deactivation pattern. Directly comparing
val/val and val/met subjects showed this difference to bestatistically significant (see Figure 2). More specifically,
restricting the analysis to the medial temporal lobes
revealed two clusters of activation showing the inappro-

priate overactivation of val/met subjects in bilateral hippocampus that were significant at the set level (p ⬍0.05, c ⫽ 2) (Figure 2A). To replicate this unusual finding,we examined a second cohort of healthy subjects (patients not included). Val/met subjects again showed anabnormal pattern of increased bilateral hippocampal activation compared to baseline while val/val subjects
again showed deactivation. Direct comparison of thetwo genotype groups again showed significant differences in hippocampal locales (Figure 2B).
Because these healthy subjects were matched prior
to the experiment on task performance to remove thisas a potential confounder, it cannot account for thesefindings (Figure 2C). Within each cohort, genotype
groups were also matched on demographic variables,
suggesting they did not account for the BDNF effects.
One exception was a slight difference in full scale IQ
and WMS-R scores in the second cohort (Figure 2C).
However, adding IQ as a covariate again revealed significant bilateral clusters of differential caudal hippocampal
activation. To ensure the differences between groups
were not due to uneven sample sizes, all analyses werealso performed using subsets with the same numberof subjects in each genotype group. The results were

Cell
260

Figure 2. Effect of BDNF val66met Genotype on In Vivo Hippocampal fMRI Response(A) Brain map showing locales where BDNF genotype groups differed in blood oxygenation, an indirect measure of neuronal activity, measured
with fMRI during a working memory task. Regions marked in red are groups of voxels (“activation clusters”) where subjects with the val/met
genotype showed abnormal hippocampal activation and were significantly different when compared with val/val subjects (see Experimental
Procedures for statistical model). The statistical results are rendered on a canonical averaged T1 brain image and localized according to thestandard 3D stereotactic space of Talairach and Tournoux. The maximally activated voxels are: right hippocampus (t ⫽ 3.77, p ⬍ 0.01, cluster
size (k) ⫽ 25, 3D coordinates: 26 ⫺22 –12); left hippocampus (t ⫽ 2.39, p ⫽ 0.02, k ⫽ 10, ⫺38 ⫺15 ⫺12). Inset: these activation clusters are
rendered on a canonical averaged smoothed 3D rendered brain. Color bar ⫽ T values. R ⫽ right hemisphere.
(B) BDNF genotype effect on hippocampal fMRI response in a second independent cohort. The activation clusters are those areas where
BDNF val/met subjects again showed abnormal activation of bilateral hippocampi locales during the 2 back working memory task and weresignificantly different compared to val/val subjects. fMRI data rendered in the same manner as in (A) (p ⬍ 0.05, cluster size ⬎ 8.) Two clusters
were identified in the left hippocampus (⫺30 ⫺35 ⫺16, t ⫽ 2.49, p ⬍ 0.01, k ⫽ 40 and ⫺30 ⫺14 ⫺13, t ⫽ 2.45, p ⫽ 0.01, k ⫽ 13) and one in
the right hippocampus (28 ⫺31 ⫺2, t ⫽ 2.55, p ⫽ 0.01, k ⫽ 37).
(C) Demographic and cognitive data for cohorts from (A) and (B) by genotype. Means (⫾ SD) are presented. Genotype groups are well matched
except for ap ⬍ .05 and bp ⫽ .07 (covariance analyses revealed no effects of these differences on hippocampal activation patterns).

essentially identical. Scatter plots did not reveal outliers.
In summary, in two cohorts we found abnormal activation of hippocampus in val/met subjects compared to
the normal pattern of deactivation in val/val subjects.
Although this was seen in the context of a working memory task, which is typically more dependent on prefrontal
function, and although the mechanism by which val66met might produce this effect is uncertain, the data
indicate that the BDNF met allele relative to val is associated with a qualitatively altered hippocampal response.
Effects of BNDF Genotype on In Vivon-acetyl-aspartate Measures
We next examined the effects of val66met on an in vivo
measure of neuronal/synaptic activity in the human hippocampal formation. NAA is an intracellular marker ofneuronal function, related to mitochondrial oxidative
phosphorylation (Jenkins et al., 2000) and appears to
be an indirect measure of neuronal integrity and synaptic

abundance (Maier et al., 1995). Patients and siblings
both had lower NAA measures compared to controls,consistent with earlier studies (Callicott et al., 1998) (Figure 3A). A significant effect of BDNF val66met genotype
was found in the entire cohort (F ⫽ 3.19, df ⫽ 2, 280,
p ⫽ .04), accounting for 1.9% of the overall variance(Figure 3A). Post hoc comparisons indicated that heterozygotes (val/met) had lower NAA levels compared to
val/val subjects (p ⬍ .02). Using multiple regression analysis, a significant linear reduction in NAA levels withincreasing number of met alleles was observed, indicating a possible allele dose effect (t(299) ⫽ ⫺2.29, p ⫽.02). BDNF genotype did not significantly affect righthippocampal NAA measures, although, again individuals with the met allele tended to have lower NAA signals(val/val versus val/met, t ⫽ 1.38, p ⫽ .17). ROI analysesof other brain regions (see Experimental Procedures)showed no effect of BDNF genotype on NAA measures(data not shown). Within each genotype group, subjects
were well matched on demographic parameters (Figure

BDNF and Human Hippocampal Function
261

Figure 3. Effect of BDNF val66met Genotype on In Vivo Hippocampal NAA Measures(A) BDNF val66met genotype and NAA measures in the left hippocampal formation (⫾ standard error, SE) in three samples studied.
(B) Demographic data (means ⫾ SD) by genotype. Groups are well matched on all variables.

3B), suggesting they do not account for the effect of
BDNF genotype. The results were similar in magnitude(i.e., effect size) but statistically slightly weaker (F ⫽2.35, df ⫽ 2,254, p ⫽ .097; val/val versus val/met p ⫽.04) in more restricted analyses of one ethnic groupalone (European Americans). Taken together, these results suggest that the val66met polymorphism elicits a
specific reduction in hippocampal neuronal integrity/
synaptic activity.
Val- and Met-BDNF Expression in Cultured
Hippocampal Neurons
To understand how val66met elicits the in vivo phenotypes, we examined its impact on intracellular distribution, processing, and secretion of BDNF. We expressed
constructs containing either vBDNF or mBDNF, followedby the green fluorescence protein (GFP), in cultured
neurons using the sindbis virus expression system (Figure 4A). This approach allowed analysis of the expression, processing, biological activity, and release properties of the BDNF isoforms and to visualize theirdistribution in living neurons (Hartmann et al., 2001; Kojima et al., 2001; Mowla et al., 1999). The viral titer wascontrolled to 10% to avoid problems associated withoverexpression of exogenous proteins (Figure 6A). Fluorescence microscopy revealed that vBDNF-GFP was
localized to the cell body and distal processes (Figure
4B). Double staining using an antibody against the dendritic marker MAP2 showed that vBDNF-GFP fluorescence was mostly localized in dendrites (Figure 4B). In
contrast, mBDNF-GFP was primarily localized in cellbodies (Figure 4B). The green fluorescence extended,at best, to a few proximal dendrites. Quantitatively, thenumber of GFP-bearing varicosities on distal dendrites(within a 100 ␮m diameter circle centered in the cellbody) in mBDNF-expressing cells was dramatically reduced, compared to vBDNF-expressing cells (Figure 4C).
Confocal microscopy showed that vBDNF-GFP exhibited a striking punctate fluorescence, distributed
throughout the cell body and extending to processes.
mBDNF-GFP neurons, however, exhibited a diffuse fluorescence pattern mainly in cell bodies (Figure 5A). Greenfluorescence appeared to accumulate as a large patchin the peri-nuclear region. Quantitative analysis showeda markedly reduced number of small fluorescence granules, suggesting an impairment in regulated secretionof BDNF protein in mBDNF cells.
Secretion of val BDNF and met BDNF
We used a modified two-site immunoenzyme assay tomeasure vBDNF-GFP or mBDNF-GFP secretion fromvirus-infected hippocampal neurons. To ensure thatsimilar levels of vBDNF and mBDNF are expressed, we
performed Northern blot using a GFP cDNA probe. No
differences in levels of BDNF-GFP mRNAs were detected in hippocampal neurons infected with vBDNFGFP and mBDNF-GFP (Figure 6D). Moreover, Western
blot using an anti-GFP antibody indicated that levels of
mBDNF proteins in mBDNF-GFP transfected cells (boththe pro- and mature forms) were not different from thoseof vBDNF proteins in vBDNF-GFP transfected cells (Figure 6E).

Cell
262

Figure 4. Abnormal Distribution of met BDNF
in Dendrites of Hippocampal Neurons(A) Diagram of BDNF constructs. A cDNA
fragment for the entire coding sequence isfused with cDNA for GFP. Arrow ⫽ amino acid66, which is val (vBDNF) or met (mBDNF).
(B) Representative low-power images of cultured hippocampal neurons expressing
vBDNF-GFP or mBDNF-GFP. Twenty-four
hrs after transfection, neurons were processed for MAP2 immunocytochemistry (red)and GFP imaging (green). GFP images were
overexposed to improve visualization of GFP
in dendrites. vBDNF-expressing neurons exhibit multiple labeled long processes while
mBDNF-expressing cells have fewer andshorter labeled processes. Scale bar equals
10 ␮m.
(C) Quantitation of BDNF dendritic expression. GFP-bearing varicosities (arrowheads inB) on dendrites. N ⫽ 50 neurons/genotype
group. In this and all remaining figures, data
are represented as means ⫾SE *: p ⬍ 0.001,
student t test.

Conditioned media containing vBDNF-GFP or mBDNFGFP were incubated in culture dishes precoated with
anti-GFP monoclonal antibody. After extensive washes,levels of secreted BDNF in the media were determinedby ELISA using an anti-BDNF antibody. vBDNF-GFP ormBDNF-GFP were virtually undetectable in a 10 min
incubation period (Figure 6B). Neuronal depolarization
by high concentration of KCl (55 mM) for 10 min induced
a marked increase in vBDNF-GFP secretion (Figure 6B).
The concentration of vBDNF-GFP reached approximately 30 ng/ml in the conditioned medium. In contrast,
the activity-dependent release of mBDNF-GFP was severely reduced and sometimes not detectable. Of thoseexperiments in which high K⫹ did induce detectablerelease, the average concentration reached only 19 ng/
ml. We next investigated constitutive secretion in neurons infected by vBDNF-GFP or mBDNF-GFP. Cultures

were washed, and the conditioned media were collected
2 hr later without high K⫹. As shown in Figure 6C, secretion of BDNF was similar in cells infected with either
vBDNF-GFP or mBDNF-GFP. These data indicate thatthe val66met polymorphism in the pro-region significantly attenuated the activity-dependent form of BDNF
secretion without affecting its constitutive secretion.
Finally, we examined whether the properties of secreted mBDNF had been changed due to some unforeseen consequence of the variation in the pro-region.
COS cells were transfected with vBDNF-GFP ormBDNF-GFP. The sizes of the precursor as well as mature proteins did not differ, and the proBDNF-GFP/
BDNF-GFP ratios were similar in the two groups (Figure
7A). Moreover, the magnitude of induced TrkB phosphorylation, as detected by Western blot using antibody
against phosphorylated TrkB (pTrkB, Figure 7B), did notBDNF and Human Hippocampal Function
263

Figure 5. Abnormal Distribution of met BDNF
in Cell Body of Hippocampal Neurons(A) Higher power images (3 per construct) ofcell body regions using confocal microscopy.
Signals for vBDNF are punctate and extend
into dendrites while those for mBDNF are diffuse and concentrated in the peri-nuclear
region.
(B) Quantitation of fluorescent granules on
the cell body of transfected neurons. N ⫽ 50
neurons in each genotype.

differ between vBDNF-GFP and mBDNF-GFP proteins.
Further, the two proteins elicited comparable biological
function, as measured by the neurite outgrowth assay
using PC12 cells expressing TrkB receptors. Approximately 90% of the cells extended neurites longer thantheir cell bodies after treatment with BDNF or NGF, but
not conditioned media from mocked transfected cells.
In cultures treated with vBDNF-GFP or mBDNF-GFP
proteins, similar percentage of the cells (90%) extended
neurites (Figure 7C). Taken together, these results

strongly suggest that met substitution for val in the proregion does not affect the structural and functional properties of the mature protein.
Subcellular Distribution of valBDNF-GFP
and metBDNF-GFP
Differences in the distribution of vBDNF-GFP and
mBDNF-GFP and in the depolarization-induced secretion of the proteins further suggest that this mutation may
lead to errors in BDNF sorting into different secretory
Figure 6. Activity-Dependent and Constitutive Secretion of vBDNF and mBDNF(A) Infection rate, as the percentage of MAP2positive neurons showing GFP fluorescence(approximately 10% in both).
(B) Activity-dependent secretion is markedly
reduced in mBDNF-GFP neurons.
(C) Constitutive secretion. Amounts ofvBDNF-GFP or mBDNF-GFP secreted into
the media during 2 hr is similar. In (A–C), N ⫽4 independent experiments.
(D) Expression of vBDNF-GFP or mBDNFGFP mRNAs. Northern blot was performed
using total RNAs from neurons transfectedwith vBDNF-GFP or mBDNF-GFP. Control:
RNA from untransfected cultures of the samedensity. Equal amounts of RNAs (10 ␮g) were
loaded in each lane. A GFP cDNA probe wasused to detect BDNF-GFP transcript (2 kb,
upper image), and the expression of NADPH
mRNA (1.7 kb, lower image) was used as theloading control.
(E) Expression of vBDNF-GFP or mBDNFGFP proteins. Western blot probed with ananti-GFP antibody using proteins from transfected neurons. Equal amounts of proteins(10 ␮g) were loaded in each lane. Note thatvBDNF and mBDNF proteins are expressedat the same level and processed in the same
way.

Cell
264

Figure 7. Characterization of Secreted vBDNF and mBDNF(A) Secretion of BDNF in COS cells expressing BDNF-GFP isoforms. Conditioned media from COS-7 cells transfected with vBDNF-GFP ormBDNF-GFP genes were collected and concentrated. Note that the molecular weights of both pro- and mature proteins are similar in thetwo groups. Moreover, the ProBDNF-GFP/BDNF-GFP ratios are similar in both groups, suggesting that mBDNF-GFP is successfully processedand secreted.
(B) Activation of TrkB receptor by vBDNF-GFP and mBDNF-GFP. Cultured hippocampal neurons were treated for 5 min with the conditioned
media (100 ng/ml) derived from vBDNF-GFP, mBDNF-GFP, or mock-transfected COS-7 cells. Recombinant BDNF and BSA (50 ng/ml) wereused as positive and negative controls, respectively. Western blots were performed using an antibody against phospho-TrkB (pTrkB; upper
image) or TrkB (lower image).
(C) Effects of vBDNF-GFP and mBDNF-GFP on neurite outgrowth. PC12 cells expressing TrkB (PC12-pAB1) were cultured for three days in
the absence or presence of various factors. Upper image: examples of treated PC12-pAB1 cells. Lower image: quantitation of percentage ofneurite-bearing cells. The cells were treated with nothing (A), recombinant BDNF (B, 50 ng/ml), recombinant NGF (C, 50 ng/ml). Conditioned
media from mock transfected cells (D), or cells transfected with either vBDNF-GFP (E, 100 ng/ml) or mBDNF-GFP (F, 100 ng/ml) were also
used. Note that mBDNF-GFP exhibits the same biological activity as vBDNF-GFP.

pathways. To test this, we attempted to determine thesubcellular localization of vBDNF-GFP and mBDNF-GFP
by double-staining techniques. Hippocampal neurons
expressing vBDNF-GFP or mBDNF-GFP were stainedusing antibodies for markers of intracellular organelles.
Some vBDNF-GFP and mBDNF-GFP signals colocalized
well with the Golgi marker TGN38, suggesting that bothare successfully targeted into Golgi (Figure 8A). Manyfluorescent vBDNF-GFP particles, but not mBDNF-GFP
particles, were associated with the secretory vesicle
marker secretegranin II (SecII) (Figure 8B). This strongly
implies that met BDNF is not sorted from Golgi to secretory granules. Both val BDNF and met BDNF failed tocolocalize with the large dense core marker chromogranin A (CGA) (Figure 8C), indicating that BDNFcontaining particles are not large dense core vesicles.
In almost all cases, mBDNF-GFP formed large clusters
in the peri-nuclear region (Figures 8A–8C). While it isunclear to what subcellular constituents these clusters
belong, failures in sorting met BDNF into the appropriate
secretory granules may explain the deficits in activitydependent secretion of met BDNF. Finally, it appearedthat vBDNF-containing particles often superimposedwith punctate staining for the synapse marker synaptophysin (Figure 8D). Although we cannot resolve whether
vBDNF is located pre- or postsynaptically at the lightmicroscopic levels, the synaptic localization of vBDNF
obviously would help its secretion locally at synapses.
In marked contrast, mBDNF showed no colocalization
with synaptophysin (Figure 8D). Failure to localize at
synapses may contribute to the in vivo phenotypes associated with the val66met polymorphism.
Discussion
This study examines the role of BDNF in hippocampal
function and episodic memory in humans. Our strategy
was to study the functional consequences of the only
reported common, single nucleotide polymorphism thatalters amino acid sequence in the pro-region of thehuman BDNF gene. We have employed several in vivo
approaches, including cognitive testing, fMRI, and 1HMRSI to demonstrate the impact of this polymorphismon human brain function. We have also investigatedBDNF and Human Hippocampal Function
265

Figure 8. Subcellular Localization of val BDNF and met BDNF in Hippocampal Neurons
Neurons infected with either vBDNF-GFP or mBDNF-GFP (green) were fixed and stained with various antibodies (red). The cell body regionof pyramidal neurons is shown in (A), (B), and (C). Scale bar equals 10 ␮m.
(A) Golgi marker TGN38. Note that some vBDNF-GFP and mBDNF-GFP signals (yellow on Merged) are colocalized with TGN38.
(B) Secretory vesicle marker secretegranin II (SecII). Note the colocalization of vBDNF-GFP, but not mBDNF-GFP with SecII.
(C) Large dense core marker chromogranin A (CGA). Both vBDNF and mBDNF fail to colocalize with CGA.
(D) Synapse marker synaptophysin. Neuronal processes of a pyramidal cell are shown. Note that vBDNF punctates often colocalize withsynaptophysin while mBDNF does not colocalize at all.

potential molecular and cellular mechanisms underlying
the biological effects of this polymorphism, using virusmediated transfection of val BDNF or met BDNF into
cultured hippocampal neurons. The in vivo experiments
provide converging evidence that BDNF plays a role inhuman verbal episodic memory as well as hippocampal
neuronal integrity and physiological activation. The invitro data suggest that the val66met substitution impacts activity-dependent secretion of BDNF. These results are important for several reasons. First, they demonstrate the feasibility of studying, in vivo, the effectsof specific genes on hippocampal biology and memory.
Second, they show that variation within the normal rangeof human verbal episodic memory involves BDNF signaling, suggesting that the basic molecular mechanismsof LTP and spatial memory in lower species have beenconserved in the more highly evolved verbal episodic
memory of humans. Third, the results implicate a specific genetic polymorphism as a factor in altered humanhippocampal function, which is therefore likely to havean impact on susceptibility to or expression of illnesses
that involve hippocampal neuronal integrity. Finally, thepresent data also reveal the importance of the proregion of the BDNF polypeptide, particular val66 andperhaps the nearby sequence, in intracellular trafficking
and secretion of BDNF.
Cognitive aspects of human brain function have been
largely inaccessible to direct molecular experimental
approaches. Limited data have come from pharmacological challenge studies (Mattay et al., 1996), but thisapproach has been constrained by the relative paucityof drugs and their regional and molecular nonspecificity,
compared with the number of genes likely involved incognition. More recently, genetic studies have identified

polymorphisms related to neurological disorders that
produce severe cognitive deficits, but their relevancefor normal human cognition is unclear (de Geus et al.,2001). One exception is the ApoE gene, a variant of
which (ApoE4) increases risk for Alzheimer’s disease(Roses, 1998) and may exert a negative effect on cognition and physiology even in unaffected subjects (Bookheimer et al., 2000).
The current study has several unique features. First,
we focus on the normal range of cognition in a cohort
including healthy, middle aged subjects. Second, a specific polymorphism in a promising candidate gene isselected and examined using multiple clinical neurobiological methods. The results provide converging evidence for a specific genetic effect on hippocampal neurochemistry, physiology, and cognition. Third, cellularand molecular biological approaches are used to address the underlying mechanisms. This combined approach provides direct in vivo data that the molecularmechanisms related to activity dependent BDNF secretion and signaling, such as LTP and synaptic plasticity,
may underlie the human ability to form memories. Thisapproach is probably generalizable to many other genes
and cognitive processes. It is also worth emphasizingthat these results demonstrate the remarkable power offMRI methods to detect specific genetic effects with
substantially lower numbers of subjects than are required for more complex human phenotypic measures(see also Egan et al., 2001b; Hariri et al., 2002).
Our results also speak to a current debate about thegenetics of human intelligence. Specific cognitive abilities are partially correlated, and the extent of this correlation is referred to as “g”, synonymous with IQ. Much
genetic epidemiological data suggest that the genetic

Cell
266

determinants of different cognitive abilities largely overlap with each other and with IQ. In other words, genes
influence specific cognitive traits by virtue of their “topdown” effects on g (Plomin, 1999). The alternative “bottom up” view that genes contribute to IQ primarily
through their unique and more direct effects on specificcognitive modules has found little support. The present
data are consistent with the latter formulation. We did
not detect an effect of BDNF on IQ or any other cognitive
ability (although we did not examine some other typesof memory, such as priming, skill and habit acquisition,and fear conditioning). A recent study of the role of the
COMT gene on working memory and prefrontal physiology, but not IQ, provides further evidence for this view
(Egan et al., 2001b). While COMT and BDNF may ultimately be shown to exert small effects on IQ, our current
data suggest that their effects are somewhat specificand mediated through “modular” cognitive elements.
Cellular Processing of BDNF
An unusual aspect of BDNF as a neurotrophic molecule
is its secretion via both constitutive and regulated pathways (Goodman et al., 1996; Mowla et al., 1999). Activitydependent secretion of BDNF makes it an attractive
candidate for mediating various forms of synaptic plasticity, such as hippocampal LTP (Katz and Shatz, 1996).
BDNF secreted in response to transient or sustained
neuronal activity is likely to participate in acute modulation of synaptic transmission or long-term regulationof synaptic structures and functions, respectively. It istherefore critical to understand the molecular mechanisms that control activity-dependent BDNF secretion.
We show that depolarization-dependent secretion of
BDNF in hippocampal neurons is significantly impaired
when val66 in the proBDNF sequence is replaced witha met. This might be due to the inability of met BDNF to
be sorted from Golgi to appropriate secretory granules.
Thus, our study identifies the pro-region of BDNF, particularly the val66 and adjacent regions, as an important
molecular determinant for intracellular sorting and activity-dependent secretion of this molecule. Finally, using double-staining techniques, we demonstrated val
BDNF-containing secretory granules are colocalizedwith synaptophysin, a marker for synapses. In contrast,
met BDNF aggregates are accumulated in the cell bodyand rarely colocalize with synaptophysin. This result
suggests that even if it can be secreted in small amounts
near the cell body through the constitutive pathway, themet BDNF protein cannot be secreted at synapses. Thismay hamper local and synapse-specific regulation by
BDNF, leading to impairments in hippocampal functionand episodic memory in humans. Of course, the proposed cellular mechanism is based on transfection of
artificial constructs alone and the effects of val66met on
in vivo hippocampal BDNF targeting/secretion remain
uncertain. Thus, our conclusion that these defects contribute to impairments in human hippocampal activity
and memory must be viewed as tentative.
Clinical Caveats and Implications
Several caveats about our clinical data are worth emphasizing. First, the magnitude of the effects of BDNF
genotype on in vivo measures of hippocampal function

is small. This is expected, given the likely polygenic
genetic architecture of these functions. However, theconvergence of data using three in vivo methods and thereplication of fMRI findings in two independent cohorts
argues strongly that the BDNF effect is at the level ofhippocampal function. Similarly, studies of heterozygote
BDNF knockout rodents, who presumably have intermediate BDNF levels, demonstrate clear physiological(Korte et al., 1995) and behavioral (Lyons et al., 1999)
abnormalities, suggesting that secretion levels are critical. A second caveat applies to genetic studies in general whereby spurious results can arise when ethnic
groups are mixed (“population stratification”). Because
we see identical effects in one ethnic group alone (European Americans) and because similar effects are seen inboth the normal controls and our schizophrenia families,
stratification is unlikely to explain our results (Wacholder
et al., 2000). Third, genetic association per se cannot
exclude a potential effect of another nearby polymorphism in linkage disequilibrium with val66met. We resequenced the BDNF coding region in 16 subjects (32chromosomes) and scanned for other polymorphisms
in 66 subjects. Only one rare synonymous change wasobserved, suggesting linkage disequilibrium to a nearby
locus does not account for these results. The demonstration that the met allele is both associated with relatively poorer human hippocampal function and impaired
intracellular trafficking and regulated secretion argues
strongly that the effects in humans are due specifically tothe met allele. Finally, the BDNF val66met polymorphism
may have important implications for clinical medicine.
While we did not find evidence that BDNF was associated with increased risk for schizophrenia, the met allele
may affect other human illnesses. Impaired memory andhippocampal function is present in many human disorders, including Alzheimer’s disease (Morris et al., 2001),
head trauma, various psychiatric conditions, and evennormal aging. Because the distribution of hippocampal
function in met/met subjects is shifted downward, suchpatients may suffer greater functional impairment.
In conclusion, we have demonstrated that an aminoacid altering polymorphism at codon 66 in the BDNF
gene affects intracellular distribution, packaging, andrelease of the BDNF protein in vitro. Furthermore, inhumans, this polymorphism has significant effects on
verbal episodic memory, hippocampal physiological activation, and measures of hippocampal neuronal integrity and synaptic abundance. These convergent datasuggest that, similar to animal models, BDNF plays arole in human episodic memory by virtue of its effects
on hippocampal neuronal function. Inheritance of the
BDNF met allele might be expected to confer susceptibility to or modify the expression of conditions that impact adversely on hippocampal function. Finally, because the relatively deleterious met allele, which has
not been observed in lower primates, remains in thehuman genome, it may confer some compensatory advantage in other biological processes.
Experimental Procedures
Human Subjects
Subjects were recruited from the Clinical Brain Disorders Branch“sibling study” of schizophrenia and included healthy controls,

BDNF and Human Hippocampal Function
267

schizophrenic probands, and their mostly unaffected siblings (age18–60; ethnicity was 6.3% African American, 89.8% European American, and 3.9% other) (Egan et al., 2000). Applicants were screened
for factors that could impact brain function, such as head trauma,
etc. Availability of the MRI scanner was the only factor determining
whether MRSI and fMRI were performed.

were reported as ratios of the area under the peaks for NAA/CRE,
NAA/CHO, and CHO/CRE. Hippocampal formation (inclusive of
amygdala, hippocampus, entorhinal cortex, and parahippocampal
gyrus) and other regions of interest (dorsolateral prefrontal cortex,
putamen, caudate, cingulate, thalamus, and superior temporal gyrus) were examined (Bertolino et al., 1996).

Cognitive Testing
Subjects performed an extensive cognitive test battery (Egan etal., 2001a). Two tests dependent on episodic memory included theWechsler Memory Scale, revised version (WMS-R) and the California
Verbal Learning Test (CVLT), standard tests of medial temporal lobe
function. To reduce comparisons, WMS-R delayed recall scores
were chosen as the primary outcome measure, as these scores
were most strongly related to genetic risk for schizophrenia in thiscohort. Data from Logical Memory I, CVLT 1-5 summary scores,
and long delay free recall scores are also described. IQ and reading
comprehension (using the WRAT) were also included to control for
general intelligence. Exploratory analyses of other types of memory
were performed using the Wisconsin Card Sorting Test (WCST), a
test of working memory/executive function (perseverative errors Tscores) and verbal fluency for categories, a measure of semantic
memory (Egan et al., 2001a).

Genetic Analysis
BDNF val66met genotype was determined using the Taqman 5⬘exonuclease assay (details available on request). To validate genotyping results, relevant amplicons from 75 subjects were sequenced
and were identical to those generated from Taqman. The frequencies of the two BDNF alleles were 0.81 for val and 0.19 for met.
Genotype frequencies (val/val ⫽ 0.67, val/met ⫽ 0.28, met/met ⫽0.05) were in Hardy Weinberg equilibrium. There were no differences
between individual groups in allele or genotype frequencies. In thecohort with schizophrenia, no overtransmission of either allele fromparents to affected offspring was seen in a family based transmission disequilibrium analysis. HPLC variant scanning of exons andsplice sites in 66 subjects of various val66met genotypes and resequencing of the coding open reading frame in 16 subjects revealed
no other sequence variants, except a rare synonymous F63F.

Functional Magnetic Resonance Imaging
We examined hippocampal function using fMRI in two cohorts ofhealthy right-handed subjects (#1:8 val/vals and 5 val/mets; #2: 12
val/vals and 5 val/mets, including 5 unrelated healthy subjects fromthe family cohort). The N-back was used as a cognitive challenge;
normal subjects show robust hippocampal deactivation (Callicott
et al., 2000). We used a block design, alternating between two back(“2B”; subjects must recall a number [1, 2, 3, or 4] seen two stimuli
previously) and a control condition (“0B”; subjects identified thenumber currently seen) (Callicott et al., 2000). Within each cohort,
subjects for each genotype group were matched on N back performance to remove possible variance due to performance. Thus, differences in the fMRI data presumably reflect variation in how the
information is processed in brain, not in overt behavior.
For the first cohort, whole brain BOLD fMRI data were collected
on a 3 Tesla GE scanner using a GE-EPI RT pulse sequence (GE,
Milwaukee, WI). For the second cohort, whole brain BOLD fMRI data
were collected at 1.5 Tesla (GE, Milwaukee, WI) using a gradientecho fast spiral acquisition. All fMRI data were processed as described previously (Callicott et al., 2000). Single subject contrast
maps were created by contrasting 2B and 0B using a 1 sample
T-test (p ⬍ 0.05, corrected). All individual maps showed activationof the typical WM network.
To determine the effect of BDNF genotype, group data were analyzed in two steps. First, a within genotype group analysis tested
for voxels showing significant changes (p ⬍ 0.05, cluster size ⬎ 8voxels, uncorrected) in BOLD signal in the two versus zero backcondition for each subject. The use of a cluster size ⬎ 8 increases
the statistical threshold for significance to p ⬍ 0.0001. These voxels
were pooled for each genotype group. Second, pooled voxels wereused for between group comparisons. A bounding box created apriori (details on request) was used to select only voxels within
the hippocampal ROI (identical to a region of interest approach).
Between group comparisons were performed as a second level(random effects) one-way ANOVA within SPM 99 (p ⬍ 0.05 uncorrected, cluster size [k] ⬎ 8) (Figure 2A). Coordinates for statisticallysignificant activation clusters were converted back to the standard
space of Talairach and Tournoux and reported as the local maxima.
1

H Magnetic Resonance Spectroscopic Imaging (MRSI)H-MRSI was performed on a GE-SIGNA 1.5 Tesla MR scanner as
in earlier reports (Bertolino et al., 1996). The 1H-MRSI slices werepositioned parallel to the sylvian fissure and encompassing the hippocampal formation as visualized on T1 weighted MRI scans. Eachvolume element (“voxel”) had nominal dimensions of 7.5 mm ⫻ 7.5mm ⫻ 15 mm (0.84 mL). To produce metabolite maps, location andintegration of the signal strength of N-acetyl-aspartate compounds(NAA), creatine plus phosphocreatine (CRE), and choline-containing
compounds (CHO) was automatically computed. Metabolite signals
1

Statistical Analyses
Effects of genotype on cognitive measures and NAA were assessedusing ANOVA. For analyses that included families, we used mixed
effects ANOVA. Effects of demographic factors were examined using ANOVA or regression and included as main effects or covariates
in BDNF analyses when significant. To avoid population stratification, all analyses were performed on the entire cohort and subjects
from one ethnic group (the largest, European Americans); the latterare reported only if different from the former.

Cell Cultures and Neurite Growth Assay
Dissociated cultures of hippocampal neurons were prepared using
embryonic day 20 rats (E20, Wistar ST) as described (Kojima etal., 2001). Neurons were maintained in Neurobasal medium (Gibco)containing 0.1% horse serum, 2% B27 supplement (Gibco), and 0.5
mM L-glutamine (Sigma) for 6–8 days. Polyethyleneimine-coatedglass-bottomed chambers (Matsunami, surface area, 1.5 cm2 ) wereused for imaging, and 12-well culture plates (Coster) were used for
BDNF immunoassay, Northern, and Western blots. Stable TrkBexpressing PC12 cells, PC12-pAB1 (Nakatani et al., 1998) were cultured in collagen-coated glass-bottomed chambers in Neurobasal
medium containing 2% B27 and 0.5 mM L-glutamine. Cells weretreated with conditioned media or 50 ng/ml BDNF for 4 days. Neurite
outgrowth was assayed by calculating the percentage of cells bearing neurites longer than the cell body (Kojima et al., 2001).

Sindbis Virus Construction and Infection
vBDNF was cloned in pEGFP-N1 plasmid (Clontech) between EcoRI
and BamHI sites. The val to met mutation in the met BDNF construct
was generated by replacing the BstBI and SapI fragment containing
the val66 sequence with a mutant dsDNA cassette (CGAACACATtop and ATCACGTGTT-bottom strands) containing the met66 sequence. Recombinant sindbis virus was constructed according to
the Invitrogen manual. Each BDNF-GFP construct was inserted into
the multiple cloning site of sindbis vector pSinRep5. The RNAs ofpSinRep5-BDNF-GFP and helper DH (26S) were electroporated into
BHK cells. The pseudovirions-containing medium (55DME) was collected 36 hr later. Sindbis virus infection of hippocampal neurons
was performed as described (Osten et al., 2000) with minor modifications. The titer of the infection was determined by the percentage
of cells expressing GFP fluorescence. Cultured neurons were infected at a titer of 10% for five hours, followed by a 24 hr incubationin B27-containing Neurobasal medium. COS-7 cells were infectedwith the sindbis virus containing the vBDNF-GFP or mBDNF-GFP
gene for 24 hr at a titer of 100%, followed by a 24 hr incubation in
DMEM containing L-glutamine (500 ␮M). The conditioned media
were collected and concentrated by Centriplus (Millipore).

Cell
268

Northern and Western Blots
RNAs were prepared from infected hippocampal cultures, using thetotal RNA isolation reagent ISOGEN (NIPPON GENE), separated on
agarose gels, and transferred to a Hybond N⫹ nylon membrane(Amersham). The BDNF-GFP transcripts were detected by a GFP
cDNA probe (Clontech). Hybridization and detection were carriedout using AlkPhos direct labeling system, CDP Star, and Hyperfilm
ECL (Amersham).
Cell lysates were prepared from infected hippocampal cultures.
Conditioned media from infected COS-7 cells were collected andconcentrated. The proteins in the lysates or media were separated
on SDS-PAGE, transferred to Immobilon P membrane (Millipore),and probed on Western blot replica with an anti-GFP or an antiBDNF antibody.
To detect TrkB phosphorylation, we treated the hippocampal cultures for 5 min with various factors. These included the conditioned
media collected in 12 hr period from non-infected, vBDNF-GFP infected, or mBDNF-GFP infected COS-7 cells. Recombinant BDNF
was used as a positive control and BSA as a negative control. The
cells were lysed and processed for Western blot using a polyclonal
antibody for phospho-Trks (Tyr490; Cell Signaling) and anti-TrkBspecific monoclonal antibody (kindly provided by Dr. S. Koizumi,
Novartis Pharma).
Immunofluorescence Staining and Imaging
Infected neurons were fixed in 4% paraformaldehyde in PBS [pH7.4] and permeabilized in 0.2% Triton-X. Cells were incubated withthe following primary antibodies at 4⬚C overnight: TGN38 (monoclonal, 1:500; BD Biosciences), synaptophysin (Boehringer Mannheim), chromogranin A (CGA, DAKO), and secretegranin II (SecII,
polyclonal, 1:250; kindly provided by T. Watanabe) and then incubated with the appropriate TRITC-conjugated secondary antibodies(1: 300; Jackson Labs) for 1 hr at room temperature. The fluorescence images were captured by either a CoolSNAP CCD camera(Photometrics) mounted on an Olympus RX60 microscope or a BioRad RTS2000 laser confocal microscope. GFP-bearing varicosities
within a circle (100 ␮m centered on cell body) were identified andcounted using a quantitation menu of the MetaMorph software (Integrated Morphometric Analysis, or IMA,). GFP fluorescent granules
in the cell body were first visualized using Z-axis confocal scanningwith a pinhole size 0.4 ␮m. The granules were counted at the middle
confocal plane, which has the maximal number of granules.
Immuno-Enzyme Assays
BDNF-GFP protein was measured using the BDNF Emax Immunoassay System (Promega) (Kojima et al., 2001). To reduce the background immunoreactivity of endogenous BDNF, culture dishes wereprecoated with anti-GFP monoclonal antibody (0.5 ␮g/well, JL-8,
Clontech), instead of anti-BDNF antibody. Standards and samples
were incubated in ELISA plates in duplicate, and data obtained from
4 cultures were averaged. Before experiments, cells were washed
3 times with Krebs Ringer. The conditioned media collected at 37⬚C
in a 2 hr period after washes were used as a measure of constitutive
secretion. To determine regulated secretion, cells were washed 3times with Krebs, followed by a 10 min incubation in Krebs at 37⬚C.
This medium was collected and replaced with Krebs containing highK⫹ (80 mM NaCl, 55 mM KCl, and 5 mM CaCl2) and the cells wereincubated for an additional 10 min at 37⬚C. Conditioned media before and after high K⫹ depolarization were used for measurementof regulated secretion.
Acknowledgments
This work was supported in part by grants from PRESTO and NEDO,
Japan; from the NIMH and NICHD intramural programs; from the
National Alliance for Research on Schizophrenia and Affective Disorders Young Investigator Award (to J.H.C.); and from the Essel Foundation through the National Alliance for Research on Schizophrenia
and Affective Disorders (to D.R.W., an Essel Investigator).
Received: May 8, 2002
Revised: January 6, 2003

References
Bertolino, A., Nawroz, S., Mattay, V.S., Barnett, A.S., Duyn, J.H.,
Moonen, C.T., Frank, J.A., Tedeschi, G., and Weinberger, D.R.
(1996). Regionally specific pattern of neurochemical pathology inschizophrenia as assessed by multislice proton magnetic resonancespectroscopic imaging. Am. J. Psychiatry 153, 1554–1563.
Bliss, T.V., and Collingridge, G.L. (1993). A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39.
Bookheimer, S.Y., Strojwas, M.H., Cohen, M.S., Saunders, A.M.,
Pericak-Vance, M.A., Mazziotta, J.C., and Small, G.W. (2000). Patterns of brain activation in people at risk for Alzheimer’s disease.
N. Engl. J. Med. 343, 450–456.
Callicott, J.H., Egan, M.F., Bertolino, A., Mattay, V.S., Langheim,
F.J., Frank, J.A., and Weinberger, D.R. (1998). Hippocampal N-acetyl
aspartate in unaffected siblings of patients with schizophrenia: apossible intermediate neurobiological phenotype. Biol. Psychiatry44, 941–950.
Callicott, J.H., Mattay, V.S., Bertolino, A., Finn, K., Coppola, R.,
Frank, J.A., Goldberg, T.E., and Weinberger, D.R. (1999). Physiological characteristics of capacity constraints in working memory as
revealed by functional MRI. Cereb. Cortex 9, 20–26.
Callicott, J.H., Bertolino, A., Mattay, V.S., Langheim, F.J.P., Duyn,J., Coppola, R., Goldberg, T.E., and Weinberger, D.R. (2000). Physiological dysfunction of the dorsolateral prefrontal cortex in schizophrenia revisited. Cereb. Cortex 10, 1078–1092.
de Geus, E.J., Wright, M.J., Martin, N.G., and Boomsma, D.I. (2001).
Genetics of brain function and cognition. Behav. Genet. 31, 489–495.
Egan, M., Goldberg, T., Gscheidle, T., Weirick, M., Bigelow, L., andWeinberger, D. (2000). Relative risk of attention deficits in siblingsof patients with schizophrenia. Am. J. Psychiatry 157, 1309–1316.
Egan, M.F., Goldberg, T.E., Gscheidle, T., Weirich, M., Rawlings, R.,
Hyde, T.M., Bigelow, L., and Weinberger, D.R. (2001a). Relative risk
for cognitive impairments in siblings of patients with schizophrenia.
Biol. Psychiatry 50, 98–107.
Egan, M.F., Goldberg, T.E., Kolachana, B.S., Callicott, J.H., Mazzanti, C.M., Straub, R.E., Goldman, D., and Weinberger, D.R. (2001b).
Effect of COMT Val108/158 Met genotype on frontal lobe function
and risk for schizophrenia. Proc. Natl. Acad. Sci. USA 98, 6917–6922.
Farhadi, H.F., Mowla, S.J., Petrecca, K., Morris, S.J., Seidah, N.G.,
and Murphy, R.A. (2000). Neurotrophin-3 sorts to the constitutive
secretory pathway of hippocampal neurons and is diverted to theregulated secretory pathway by coexpression with brain-derived
neurotrophic factor. J. Neurosci. 20, 4059–4068.
Figurov, A., Pozzo-Miller, L.D., Olafsson, P., Wang, T., and Lu, B.
(1996). Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature, 381,
706–709.
Goodman, L.J., Valverde, J., Lim, F., Geschwind, M.D., Federoff,
H.J., Geller, A.I., and Hefti, F. (1996). Regulated release and polarized
localization of brain-derived neurotrophic factor in hippocampal
neurons. Mol. Cell. Neurosci. 7, 223–228.
Gottschalk, W., Pozzo-Miller, L.D., Figurov, A., and Lu, B. (1998).
Presynaptic modulation of synaptic transmission and plasticity bybrain-derived neurotrophic factor in the developing hippocampus.
J. Neurosci. 18, 6830–6839.
Gottschalk, W.A., Jiang, H., Tartaglia, N., Feng, L., Figurov, A., andLu, B. (1999). Signaling mechanisms mediating BDNF modulationof synaptic plasticity in the hippocampus. Learn. Mem. 6, 243–256.
Hall, J., Thomas, K.L., and Everitt, B.J. (2000). Rapid and selective
induction of BDNF expression in the hippocampus during contextual
learning. Nat. Neurosci. 3, 533–535.
Hariri, A.R., Mattay, V.S., Tessitore, A., Kolachana, B., Fera, F., Goldman, D., Egan, M.F., and Weinberger, D.R. (2002). Serotonin transporter genetic variation and the response of the human amygdala.
Science 297, 400–403.
Hartmann, M., Heumann, R., and Lessmann, V. (2001). Synaptic
secretion of BDNF after high-frequency stimulation of glutamatergic
synapses. EMBO J. 20, 5887–5897.

BDNF and Human Hippocampal Function
269

Jenkins, B.G., Klivenyi, P., Kustermann, E., Andreassen, O.A., Ferrante, R.J., Rosen, B.R., and Beal, M.F. (2000). Nonlinear decreaseover time in N-acetyl aspartate levels in the absence of neuronal loss
and increases in glutamine and glucose in transgenic Huntington’sdisease mice. J. Neurochem. 74, 2108–2119.
Jovanovic, J.N., Czernik, A.J., Fienberg, A.A., Greengard, P., andSihra, T.S. (2000). Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat. Neurosci. 3, 323–329.
Kandel, E.R. (2001). The molecular biology of memory storage: a
dialogue between genes and synapses. Science 294, 1030–1038.
Katz, L.C., and Shatz, C.J. (1996). Synaptic activity and the construction of cortical circuits. Science 274, 1133–1138.
Kojima, M., Takei, N., Numakawa, T., Ishikawa, Y., Suzuki, S., Matsumoto, T., Katoh-Semba, R., Nawa, H., and Hatanaka, H. (2001).
Biological characterization and optical imaging of brain-derived
neurotrophic factor-green fluorescent protein suggest an activitydependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons. J. Neurosci. Res. 64, 1–10.
Kopelman, M.D., Stevens, T.G., Foli, S., and Grasby, P. (1998). PET
activation of the medial temporal lobe in learning. Brain 121,
875–887.
Korte, M., Carroll, P., Wolf, E., Brem, G., Thoenen, H., and Bonhoeffer, T. (1995). Hippocampal long-term potentiation is impaired inmice lacking brain-derived neurotrophic factor. Proc. Natl. Acad.
Sci. USA 92, 8856–8860.
Korte, M., Kang, H., Bonhoeffer, T., and Schuman, E. (1998). A rolefor BDNF in the late-phase of hippocampal long-term potentiation.
Neuropharmacology 37, 553–559.
Levine, E.S., Dreyfus, C.F., Black, I.B., and Plummer, M.R. (1995).
Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase
receptors. Proc. Natl. Acad. Sci. USA 92, 8074–8077.
Linnarsson, S., Bjorklund, A., and Ernfors, P. (1997). Learning deficitin BDNF mutant mice. Eur. J. Neurosci. 9, 2581–2587.
Lu, B., and Gottschalk, W. (2000). Modulation of hippocampal synaptic transmission and plasticity by neurotrophins. Prog. Brain Res.
128, 231–241.
Lyons, W.E., Mamounas, L.A., Ricaurte, G.A., Coppola, V., Reid,
S.W., Bora, S.H., Wihler, C., Koliatsos, V.E., and Tessarollo, L. (1999).
Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic
abnormalities. Proc. Natl. Acad. Sci. USA 96, 15239–15244.
Maier, M., Ron, M.A., Barker, G.J., and Tofts, P.S. (1995). Proton
magnetic resonance spectroscopy: an in vivo method of estimating
hippocampal neuronal depletion in schizophrenia. Psychol. Med.
25, 1201–1209.
Mattay, V.S., Berman, K.F., Ostrem, J.L., Esposito, G., Van Horn,
J.D., Bigelow, L.B., and Weinberger, D.R. (1996). Dextroamphetamine enhances “neural network-specific” physiological signals: apositron-emission tomography rCBF study. J. Neurosci. 16, 4816–
4822.
Meyer-Lindenberg, A., Poline, J.B., Kohn, P.D., Holt, J.L., Egan, M.F.,
Weinberger, D.R., and Berman, K.F. (2001). Evidence for abnormal
cortical functional connectivity during working memory in schizophrenia. Am. J. Psychiatry 158, 1809–1817.
Milner, B., Squire, L.R., and Kandel, E.R. (1998). Cognitive neuroscience and the study of memory. Neuron 20, 445–468.
Minichiello, L., Korte, M., Wolfer, D., Kuhn, R., Unsicker, K., Cestari,V., Rossi-Arnaud, C., Lipp, H.P., Bonhoeffer, T., and Klein, R. (1999).
Essential role for TrkB receptors in hippocampus-mediated learning.
Neuron 24, 401–414.
Mizuno, M., Yamada, K., Olariu, A., Nawa, H., and Nabeshima, T.
(2000). Involvement of brain-derived neurotrophic factor in spatial
memory formation and maintenance in a radial arm maze test in
rats. J. Neurosci. 20, 7116–7121.
Morris, J.C., Storandt, M., Miller, J.P., McKeel, D.W., Price, J.L.,
Rubin, E.H., and Berg, L. (2001). Mild cognitive impairment represents early-stage Alzheimer disease. Arch. Neurol. 58, 397–405.
Mowla, S.J., Pareek, S., Farhadi, H.F., Petrecca, K., Fawcett, J.P.,

Seidah, N.G., Morris, S.J., Sossin, W.S., and Murphy, R.A. (1999).
Differential sorting of nerve growth factor and brain-derived neurotrophic factor in hippocampal neurons. J. Neurosci. 19, 2069–2080.
Mowla, S.J., Farhadi, H.F., Pareek, S., Atwal, J.K., Morris, S.J., Seidah, N.G., and Murphy, R.A. (2001). Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic
factor. J. Biol. Chem. 276, 12660–12666.
Nakatani, A., Yamada, M., Asada, A., Okada, M., Ikeuchi, T., andHatanaka, H. (1998). Comparison of survival-promoting effects ofbrain-derived neurotrophic factor and neurotrophin-3 on PC12h
cells stably expressing TrkB receptor. J. Biochem. (Tokyo) 123,
707–714.
Ogawa, S., Tank, D.W., Menon, R., Ellermann, J.M., Kim, S.G., Merkle, H., and Ugurbil, K. (1992). Intrinsic signal changes accompanying sensory stimulation: functional brain mapping with magnetic
resonance imaging. Proc. Natl. Acad. Sci. USA 89, 5951–5955.
Osten, P., Khatri, L., Perez, J.L., Kohr, G., Giese, G., Daly, C., Schulz,
T.W., Wensky, A., Lee, L.M., and Ziff, E.B. (2000). Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface
accumulation of the AMPA receptor. Neuron 27, 313–325.
Patterson, S., Grover, L.M., Schwartzkroin, P.A., and Bothwell, M.
(1992). Neurotrophin expression in rat hippocampal slices: a stimulus paradigm inducing LTP in CA1 evokes increases in BDNF andNT-3 mRNAs. Neuron 9, 1081–1088.
Patterson, S.L., Abel, T., Deuel, T.A., Martin, K.C., Rose, J.C., andKandel, E.R. (1996). Recombinant BDNF rescues deficits in basal
synaptic transmission and hippocampal LTP in BDNF knockout
mice. Neuron 16, 1137–1145.
Plomin, R. (1999). Genetics and general cognitive ability. Nature 402,
C25–C29.
Poo, M.M. (2001). Neurotrophins as synaptic modulators. Nat. Rev.
Neurosci. 2, 24–32.
Pozzo-Miller, L., Gottschalk, W.A., Zhang, L., McDermott, K., Du, J.,
Gopalakrishnan, R., Oho, C., Shen, Z., and Lu, B. (1999). Impairments
in high frequency transmission, synaptic vesicle docking and synaptic protein distribution in the hippocampus of BDNF knockout mice.
J. Neurosci. 19, 4972–4983.
Roses, A.D. (1998). Apolipoprotein E and Alzheimer’s disease. The
tip of the susceptibility iceberg. Ann. N Y Acad. Sci. 855, 738–743.
Seidah, N.G., Benjannet, S., Pareek, S., Chretien, M., and Murphy,
R.A. (1996). Cellular processing of the neurotrophin precursors of
NT3 and BDNF by the mammalian proprotein convertases. FEBS
Lett. 379, 247–250.
Tartaglia, N., Du, J., Neale, E., Tyler, W.J., Pozzo-Miller, L., and Lu,
B. (2001). Protein synthesis dependent and independent regulationof hippocampal synapses by brain-derived neurotrophic factor. J.
Biol. Chem. 276, 37585–37593.
Wacholder, S., Rothman, N., and Caporaso, N. (2000). Population
stratification in epidemiologic studies of common genetic variants
and cancer: quantification of bias. J. Natl. Cancer Inst. 92, 1151–
1158.
Xu, B., Gottschalk, W., Chow, A., Wilson, R.I., Schnell, E., Zang, K.,
Wang, D., Nicoll, R.A., Lu, B., and Reichardt, L.F. (2000). The roleof brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic
mechanism involving TrkB. J. Neurosci. 20, 6888–6897.