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16. The plasmid pFL-EBOVe
⫹
was designed to place the T7
promoter adjacent to the viral leader region, and the
viral trailer region was constructed to be adjacent to a
ribozyme sequence followed by tandem terminators of
T7 transcription. In this case, the correct 3⬘end of the
transcribed EBOV antigenome, free of additional nucle-
otides, was generated by self-cleavage of the ribozyme
(24). To increase the transcriptional activity of the T7
RNA polymerase, an additional guanosine residue was
introduced between the promoter of the T7 polymerase
and the first residue of the EBOV genome (25). The
length of the encoded antigenome (FL-EBOVe
⫹
)is
therefore increased by 1 nt compared to that of
the wild-type virus (GenBank accession number,
AF086833). The size of the full-length antigenome en-
coded by the plasmid pFL-EBOVe
⫺
was 1 nt longer than
that encoded by pFL-EBOVe
⫹
and 2 nt longer than the
size of the genome of the wild-type virus because of the
insertion of additional adenosine residues at the editing
site. Mutations at the GP gene editing site were intro-
duced into the plasmid pKSS25 by site-directed mu-
tagenesis using the primers 5⬘-GG GAAACTAAGAA-
GAAACCTCACTAG and 5⬘-CTAGTGAGGTT TCTTCT-
TAGTTTCCC.
17. The Sal I restriction site (GTCGAC) located in the GP
gene at position 6180 was mutated by site-directed
mutagenesis using the pair primers 5⬘-GGTTAGTGAT-
GTAGATAA ACTAGTTTG and 5⬘-CAAACTAGTTTATC
TACATCACTAACC. This mutation is silent. In addition,
an accidental mutation in a nontranslated region of the
L gene (A 3U at position 18227) was found after
complete sequence analysis of the final plasmid clones.
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20. BSR T7/5 cells were grown overnight to about 60 to
80% confluency in 25-cm
2
flasks in 1⫻Glasgow
medium supplemented with 10% NCS (newborn calf
serum). One hour before transfection, cells were
washed twice with medium without NCS. Cells were
transfected with a plasmid mixture containing 2 g
of full-length plasmid (pFL-EBOVe⫹), 0.2 gofpT/
VP30EBOV, 0.5 g of pT/VP35EBOV, 0.5 gofpT/
NPEBOV, and 1 g of pGEM-LEBOV (14, 19). Trans-
fection experiments were carried out with a Fusion 6
reagent (transfection protocol supplied by Roche).
The transfection medium was removed at 8 hours
after transfection; cells were washed and maintained
in Glasgow medium containing 2.5% NCS for 6 to 9
days after transfection. On average, in each rescue
experiment, approximately 100 foci of rounded cells
were observed in the cell culture flask (about 1 ⫻10
5
to2⫻10
5
cells). That means that one in approxi-
mately 10
3
cells allowed the formation of viral par-
ticles. However, virus release from BHK cells was
extremely low, and amplification of recombinant
EBOV on Vero cells was necessary before further
analysis. Determination of the virus titers by plaque
formation showed that about 200 infectious particles
were recovered from the average rescue experiment.
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22. For RT-PCR, RNA from culture supernatants of Vero E6
cells infected with individual plaques of recEBOV-e
⫹
,
recEBOV-e
⫺
, or wild-type EBOV was purified (with the
Rneasy Kit, Qiagen) when an extensive CPE was ob-
served. To verify the identity of the recombinant virus-
es, the region containing the marker restriction site Sal
I shown in Fig. 1 was amplified by RT-PCR using primers
5⬘-AGTCATCCACAATAGCACAT and 5⬘-TCGTGGCA-
GAGGGAGTGT. The PCR products were only seen when
the RT step was performed, which confirms that they
were derived solely from viral RNA and not from con-
taminating cDNA. PCR products were consistent with
the predicted size of 1298 bp. Demonstration of the
presence or absence of a Sal I site in authentic EBOV
and recEBOV was performed on 1% agarose gel. Sal I
digestion products were consistent with the predicted
sizes of 1130 and 168 bp. In addition, the sequences at
the restriction site marker and at the GP gene editing
site were confirmed by partial nucleotide sequencing of
RT-PCR products.
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27. For electron microscopy, 72 hours after infection,
control and virus-infected cells were fixed with
Hanks’ balanced salt solution (HBSS) containing 2.5%
glutaraldehyde, postfixed with HBSS containing 1%
osmium tetraoxide, dehydrated, and embedded in
Epon. Ultrathin sections were cut, placed on 200-
mesh copper electron microscopy grids, stained with
uranyl acetate and lead citrate, and examined with a
Zeiss 109 transmission electron microscope at 80 kV.
28. See Web Fig. 2 on Science Online at www.sciencemag.
org/cgi/content/full/1057269/DC1.
29. For immunofluorescent staining, Vero E6 cells were
infected at a multiplicity of infection (MOI) of 10
⫺2
and were processed 3 days later for indirect immu-
nofluorescence analysis. Cells grown on coverslips
were washed with phosphate-buffered saline (PBS)
solution, fixed with 4% paraformaldehyde at 4°C for
24 hours, and permeabilized with 0.1% Triton X-100
in PBS for 5 min. The nonspecific binding was blocked
by blocking buffer (2% bovine serum albumin, 5%
glycerol, and 0.2% Tween-20 in PBS), and cells were
then incubated with 100 mM glycine for 10 min,
washed with PBS, and incubated at 4°C for 18 hours
with the respective antibodies [human monoclonal
antibody (mAb) KZ52, which is specific for the EBOV
GP (dilution 1:100 in blocking buffer); and mouse
mAb B6C5, which is specific to EBOV NP (dilution
1:10 in blocking buffer)]. Subsequently, cells were
washed three times with PBS and stained with fluo-
rescein isothiocyanate–conjugated goat anti-human
immunoglobulin G (IgG) (diluted 1: 50 in blocking
buffer) or with rhodamine-conjugated goat anti-
mouse IgG (1:100 in blocking buffer) for 1 hour at
room temperature. Nuclear counterstaining was per-
formed with 4⬘,6⬘-diamidino-2-phenylindole (DAPI)
(0.1 g/ml). Finally, cells were washed three times
with PBS, dipped into dH2O, covered with mounting
medium, coverslipped, and examined with a fluores-
cence microscope (Axiomat, Zeiss) with digital pho-
tographic equipment for taking images (Spot camera
system, version 2.1.2, Diagnostic Instruments).
30. See Web Fig. 3 on Science Online at www.sciencemag.
org/cgi/content/full/1057269/DC1.
31. All experiments involving infectious EBOV were car-
ried out in biosafety level 4 (BSL4) laboratories at the
Institute of Virology in Marburg, Germany, and at the
Jean Merieux P4 Research Center in Lyon, France. We
thank D. Burton and P. Parren for providing mAb
KZ52A; S. Becker for mAb B6C5; K.-K. Conzelmann for
the BSR T7/5 cell line; A. Sergeant and E. Derrington
for critical reading of the manuscript; and C. Laukel
and M. Rossi for expert technical help. M.W. was
supported as a recipient of a fellowship from the
Boehringer Ingelheim Fonds. This work was supported
in part by the Deutsche Forschungsgemeinschaft
(SFB 286), the Fonds der Chemischen Industrie, and
INSERM; and by a grant from the Fondation pour la
Recherche Medicale to V.E.V.
8 November 2000; accepted 22 January 2001
Published online 1 February 2001;
10.1126/science.1057269
Include this information when citing this paper.
Genetic Correlates of Musical
Pitch Recognition in Humans
Dennis Drayna,
1
* Ani Manichaikul,
1
Marlies de Lange,
2
Harold Snieder,
2
†Tim Spector
2
We used a twin study to investigate the genetic and environmental contribu-
tions to differences in musical pitch perception abilities in humans. We ad-
ministered a Distorted Tunes Test (DTT), which requires subjects to judge
whether simple popular melodies contain notes with incorrect pitch, to 136
monozygotic twin pairs and 148 dizygotic twin pairs. The correlation of DTT
scores between twins was estimated at 0.67 for monozygotic pairs and 0.44 for
dizygotic pairs. Genetic model-fitting techniques supported an additive genetic
model, with heritability estimated at 0.71 to 0.80, depending on how subjects
were categorized, and with no effect of shared environment. DTT scores were
only weakly correlated with measures of peripheral hearing. This suggests that
variation in musical pitch recognition is primarily due to highly heritable dif-
ferences in auditory functions not tested by conventional audiologic methods.
The perception of pitch requires both the ear,
which receives auditory signals, and the
brain, which performs substantial processing
of auditory signals to produce a perceived
pitch (1–3). Although the general features of
human pitch processing have been well de-
scribed, the precise cellular and molecular
mechanisms involved remain largely ob-
scure. One approach to understanding the
mechanisms of pitch perception is to use
genetic methods that exploit naturally occur-
ring variation in pitch perception ability (4).
If such variability is due to genetic factors,
linkage and positional cloning studies could
identify genes that encode the components of
the pitch perception apparatus (5). To exam-
ine the genetic contributions to musical pitch
1
National Institute on Deafness and Other Commu-
nication Disorders, National Institutes of Health, 5
Research Court, Rockville, MD 20850, USA.
2
Twin
Research and Genetic Epidemiology Unit, St. Thomas’
Hospital, London, SE1 7EH, UK.
*To whom correspondence should be addressed.
†Present address: Georgia Prevention Institute, Med-
ical College of Georgia, Building HS-1640, Augusta,
GA 30912, USA.
REPORTS
www.sciencemag.org SCIENCE VOL 291 9 MARCH 2001 1969
recognition ability in humans, we performed
a twin study (6) using the Distorted Tunes
Test (DTT), which requires subjects to rec-
ognize notes with incorrect pitch in simple
popular melodies (7).
The original DTT was developed in the
1940s (8) and used in large studies in the
British population. These studies suggested
that cultural biases and the effects of musical
experience could be minimized by the appro-
priate choice of melodies, and that test scores
in the same individual were stable across
decades. They also revealed that a small but
significant portion of the population (about
5%) scored no better than chance in their
ability to distinguish correct from incorrect
melodies. These individuals were classified
as “tune deaf.”
For our study, we created a similar, up-
dated DTT and validated it for use in the
current U.S. and British populations (9). The
updated DTT was recorded on a compact disk
and presented to all subjects in the same
setting. Briefly, subjects were presented with
26 short popular melodies, ranging in length
from 12 to 26 notes. Tunes were presented
once, and after each presentation, subjects
were asked to score whether the melody was
correct or incorrect, and whether they were
familiar or unfamiliar with that melody. We
first measured the performance of 50 unrelat-
ed males and 50 unrelated females on the
updated DTT. The distribution of scores in
males and females did not differ (Kolmog-
orov-Smirnov ⫽0.94, Mann-Whitney test ⫽
0.78). Test-retest scores in the same subject
were highly correlated (n⫽40, r⫽0.77),
confirming that like the original DTT, the
updated DTT is reproducible in individuals.
In contrast to results obtained by Kalmus and
Fry with the original DTT, we did not ob-
serve a clear distinction between tune deaf
and normal individuals.
Because our goal was to determine the
extent to which genes and/or environment
influence musical pitch-recognition ability,
we chose a twin study, which can discrimi-
nate between the effect of the shared envi-
ronment and that of shared genes. The study
was approved by the St. Thomas’ Hospital
Research Ethics Committee (EC95/041, mod-
ification approved 29 September 1999), and
informed consent was obtained from all sub-
jects. A total of 284 female Caucasian twin
pairs [136 identical (monozygotic, MZ) and
148 nonidentical (dizygotic, DZ)] aged 18 to
74 years from the St. Thomas’ UK Adult
Twin Registry (10) participated in the study.
Subjects in this registry were ascertained
from the general population through national
media campaigns in the United Kingdom.
Participating twins were part of an ongoing
study into the genetics of common complex
diseases (10–13). Twins were unaware of the
specific hypotheses tested and were not
screened for IQ, musical training, or musical
experience.
The median ages of the MZ group and the
DZ group were 50.7 and 47.9 years, respec-
tively. Zygosity was determined by standard-
ized questionnaire, with DNA fingerprinting
used for confirmation (10). Each subject was
administered the DTT and the 5 Minute Hear-
ing Test (FMHT) to help identify subjects
with potentially confounding hearing loss.
The FMHT, promulgated by the American
Academy of Otolaryngology, has been wide-
ly used for initial screening for hearing loss,
and high correlations have been reported with
a wide range of hearing measures, including
pure-tone audiometry (14).
Using the DTT, we measured musical
pitch recognition ability on an ordinal scale,
scored as the number of correctly classified
tunes. The scores of the subjects ranged from
26 (a perfect score) to 9. The distribution of
scores in the MZ and DZ pairs is shown in
Fig. 1, along with the overall score distribu-
tion of the entire sample. Although there was
a trend for the MZ twins to have lower
scores, this difference was not statistically sig-
nificant (chi-square, P⫽0.12; Kolmogorov-
Smirnov, P⫽0.26).
The statistical package STATA (15) was
used to analyze the data. Spearman correla-
tions were used to describe the associations
between the scores on the DTT and the
FMHT. A slight negative correlation between
the DTT and the FMHT was observed
(Spearman’s r⫽⫺0.10, P⫽0.01). Despite
having statistical significance, this small neg-
ative correlation may or may not have func-
tional significance. To test if this affected
scores on the DTT, we divided subjects into a
hearing and a hearing-impaired group on the
basis of their FMHT scores (hearing im-
paired ⫽FMHT score ⬎15). No difference in
DTT score was found between the groups as
determined with statistical methods that ac-
count for the nonindependence of the twin
pairs (generalized estimating equation, P⫽
0.45). There was no correlation between the
DTT and age (Spearman’s r⫽⫺0.01, P⫽
0.87). Probandwise concordance rates for the
data divided into the two original categories
described by Kalmus and Fry were 0.75 for
MZ and 0.57 for DZ twin pairs, and a test for
the difference was significant (P⫽0.01)
(16), indicating a genetic influence.
We applied genetic model-fitting tech-
niques using the structural equation modeling
package Mx (17) to obtain estimates of the
genetic and environmental factors. Model fit-
ting allows separation of the observed phe-
notypic variance into additive (A) or domi-
nant (D) genetic components and common
(C) and unique (E) environment. E also con-
tains measurement error. The heritability,
which estimates the extent to which variation
in liability to disease in a population can be
explained by genetic variation, can be de-
fined as the ratio of genetic variance (A ⫹D)
Fig. 1. Distribution of
twin scores on the Dis-
torted Tunes Test. Bars
indicate the number of
subjects attaining each
score on the DTT. MZ,
scores of monozygotic
twins; DZ, scores of dizy-
gotic twins.
REPORTS
9 MARCH 2001 VOL 291 SCIENCE www.sciencemag.org1970
to total phenotypic variance (A ⫹C⫹D⫹
E). We tested the significance of A, C, and D
by removing them sequentially in specific
submodels and testing the deterioration in
model fit after each component was dropped
from the full model. This leads to a model
explaining the data with as few parameters as
possible. Standard hierarchical chi-squared
tests were used to select the best-fitting mod-
el (18).
The genetic and environmental contribu-
tions for categorical traits can be quantified
by assuming a continuous underlying normal
liability distribution with multiple thresholds
that discriminate between the categories (18).
We estimated the correlation in liability with-
in the twin pairs by deriving polychoric cor-
relations from the pairwise categorical distri-
bution. The model-fitting approach compares
the size of the polychoric correlations in MZ
and DZ twins and provides estimates of the
relative contribution of genetic and environ-
mental factors to the liability distribution un-
derlying musical pitch perception ability (18,
20). Using the original definition of Kalmus
and Fry (tune deaf ⫽a score ⱕ23) on our
data resulted in a large percentage of tune
deaf individuals (39.6%). Using the updated
DTT, we also did not observe a clear distinc-
tion between tune deaf and normal individu-
als. We therefore also fitted the data without
imposing any arbitrary cut-off points and as-
signed categories as the raw scores them-
selves, with the exception of the few lowest
scorers (scores between 9 and 15) that were
assigned to one category, resulting in 12 cat-
egories. Contingency tables for the 12 cate-
gories were produced for the MZ and DZ
twins and used as input data for Mx.
Analysis of the complete data (with no
arbitrary cut-off point) showed a correlation
in liability within the twin pairs of 0.67 and
0.44 for MZ and DZ twins, respectively (see
Table 1, Model 1 for twin correlations in
liability on the basis of two category data).
Across the analyses, the model providing
the best fit to the data included an additive
genetic and a unique environmental compo-
nent. The heritability as estimated by genetic
model-fitting with all of the available data
was 71% [95% confidence interval (CI): 61
to 78%). Using the original cut-off value of
Kalmus and Fry (ⱕ23) to define two classes
corresponds to a simplified model that con-
tains only two groups: those with normal
pitch recognition and those with some deficit
in pitch recognition, regardless of severity.
Using this model, we estimated heritability at
80% (95% CI: 65 to 90%). In both analyses,
no dominant genetic effects and no signifi-
cant effect of shared environment were de-
tected (Table 1).
Despite the major role of genetic factors
underlying DTT scores, a certain amount of
musical experience is nevertheless required
to perform well on the DTT, and the original
results of Kalmus and Fry provided evidence
for the effects of such experience (7, 19).
Any effects of culture or musical experience
are likely to be the same for both MZ and DZ
twins and thus should have no effect on the
heritability estimates that we have found. Our
data indicate that individual differences in
musical experience may be at least partly
responsible for the fraction of variance in
DTT scores attributable to unique environ-
ment (E), 20 to 29%. Because of the DTT’s
requirement for musical experience, it may be
a conservative measure of the heritability of
variation in pitch perception in isolation, that
is, measured in a way that does not require
such experience.
Because the FMHT serves as a rough
measure of peripheral hearing, its poor cor-
relation with the DTT suggests that musical
pitch perception is largely independent of
peripheral hearing and that variation in pitch
perception originates in portions of the audi-
tory system that are independent of this
function.
Melodies consist of a series of tones pre-
sented in a specific order and rhythm, in
which successive tones differ by specific in-
tervals. In the DTT, the note order and
rhythm remain unchanged, and only the in-
terval between successive tones is altered.
Because variation in long-term tonal memory
(9) and musical experience appear to have
modest effects, the DTT primarily determines
a subject’s ability to measure successive pitch
intervals. Although the DTT has a number of
characteristics that make it ideal for a large-
scale twin study, it will be important to use
other measures of pitch recognition to con-
firm the results obtained with the DTT. Sim-
ilarly, indistinguishable distributions of DTT
scores in males and females suggest that re-
sults from female subjects can be generalized
to the whole population, but it will be impor-
tant to confirm this with male subjects.
The original DTT was designed to identi-
fy individuals with severe deficits in pitch
recognition. Our results with the modified
DTT indicate a high heritability for perfor-
mance across the full spectrum of pitch rec-
ognition abilities in the general population.
Studies elsewhere have demonstrated a sig-
nificant genetic contribution to absolute pitch
(AP), a relatively rare phenomenon in which
individuals are capable of identifying a par-
ticular tone without the use of a reference
tone. However, AP individuals do not neces-
sarily have superior pitch acuity compared to
those without AP (20). In addition, the full
development of AP apparently has a strong
environmental component, with specific mu-
sical training ( perhaps during a critical devel-
opmental time period) being required for ex-
pression (21). Thus, AP stands in contrast to
the ability measured by the DTT, and it is not
clear if the genetic factors in AP have any
relation to those that underlie DTT scores.
The heritability estimates we observe for
this measure of deficits in pitch recognition
are very substantial and are as high as or
higher than those for many common complex
traits in humans (22). However, these high
heritability estimates do not address several
important issues, including the number of
genes involved and their relative effects. Our
results indicate that genetic approaches,
which are ideal for studying traits with un-
known biochemical or cellular mechanisms,
are likely to be fruitful in efforts to under-
stand this neural function.
References and Notes
1. B. C. J. Moore, in Springer Handbook of Auditory
Research: Human Psychophysics, W. A. Yost, A. N.
Popper, R. R. Fay, Eds. (Springer-Verlag, New York,
1993), pp. 56–115.
2. A. J. M. Houtsma, in Handbook of Perception and
Cognition: Hearing, B. C. J. Moore, Ed. (Academic
Press, New York, 1995), pp. 267–295.
3. W. M. Hartmann, Signals,Sound and Sensation
(American Institute of Physics, Woodbury, NY, 1997),
pp. 117–145.
4. D. Drayna, Nature Genet. 18, 96 (1998).
5. F. Collins, Nature Genet. 9, 347 (1995).
6. T. D. Spector, H. Snieder, A. J. MacGregor, Advances in
Twin and Sib-Pair Analysis (Greenwich Medical Media,
London, 2000).
7. H. Kalmus, D. Fry, Ann. Hum. Genet. 43, 369 (1980).
8. D. B. Fry, Speech (March 1948), p. 1.
9. The original DT T consisted of 26 short melodies,
ranging in length from 12 to 26 notes. Ten of these
melodies were played normally, whereas 16 con-
tained tonal errors (7). Our updated test contained
8 of the original DTT melodies verbatim, plus 18
new melodies, for a total of 26, which ranged from
12 to 26 notes in length. Of these, 9 were played
correctly, whereas 17 were distorted to produce
errors. The errors were introduced according to the
general rules used by Kalmus and Fry in their
original DTT, changing the pitch of two to nine
notes, generally within one or two semitones of
the correct note and following the overall rise and
fall of the normal melody. As in the original DTT,
all melodies were unaltered in rhythm and note
order. A list of the melodies and the errors intro-
duced is available at www.nidcd.nih.gov/intram/
scientists/draynad.htm. The melodies were pro-
duced in pure tones with Mozart version 3.2 soft-
ware (D. Webber, Mozart Music Software, War-
rington, UK) on a Macintosh G3, rendered more
natural by the addition of overtones with Arnold’s
MIDI (Musical Instrumentation Digital Interface)
player software. Ultra Recorder version 2.4 (E. J.
Campbell, EJ Enterprises, http://members.aol.com/
EJC3/) was used to convert the MIDI files to AIFF
(Audio Interchange File Format) and, after joining
voice-over spoken instructions with SndSampler,
was recorded along with spoken instructions on a
Table 1. Twin correlations and heritabilities of
best-fitting models for the DTT data with two
categories (Model I) or the actual scores (Model II).
rMZ, monozygotic twin correlation; rDZ, dizygotic
twin correlation; h
2
, heritability; 95% CI, 95%
confidence interval.
Model Thresholds rMZ rDZ h
2
(95% CI)
Iⱕ23 0.79 0.46 80% (65–90%)
II ⱕ15,
actual
score
0.67 0.44 71% (61–78%)
REPORTS
www.sciencemag.org SCIENCE VOL 291 9 MARCH 2001 1971
compact disk with Adaptec Toast 3.5.4. Copies of
the updated DTT on a compact disk are available
from the corresponding author. The updated DTT
contained a preponderance of tunes played incor-
rectly (17/26), but our data suggest that whether a
tune was played correctly or incorrectly did not
significantly bias subjects’ answers. Four tunes
were played twice in the updated DTT; each was
played correctly once and incorrectly once. Wrong
answers on these eight questions were evenly dis-
tributed; 51% were correct melodies identified as
incorrect, and 49% were incorrect melodies iden-
tified as correct. Two methods were used to deter-
mine the role of long-term memory and cultural
experience in the performance on the updated
DTT. First, subjects were asked whether they were
familiar with each tune presented, and familiarity
was positively correlated with correct answers. We
also developed a second test, termed the Interna-
tional Tunes Test, that consisted of 18 short mel-
odies chosen to be unfamiliar to all subjects and
that used both Western and non-Western tonal
systems. Subjects were presented with each mel-
ody played correctly twice in succession and were
then asked whether a third rendition played imme-
diately thereafter was the same as or different
than the first two playings. Six of the International
Tunes were played correctly the third time, where-
as 12 were played incorrectly the third time. The
distribution of scores on the International Test
were indistinguishable from those on the updated
DTT, with no significant differences between males
and females. Individuals’ scores on the DTT and the
International Test were highly correlated (r⫽
0.71). These results suggest that performance on
the updated DTT is not highly dependent on long-
term musical memory.
10. T. Spector et al.,Br. Med. J. 312, 940 (1996); C. J.
Hammond et al.,N. Engl. J. Med. 342, 1786 (2000).
11. H. Snieder et al.,Hypertension 35, 574 (2000).
12. V. Bataille, H. Snieder, A. J. MacGregor, P. Sasieni,
T. D. Spector. J. Natl. Cancer Inst. 92, 457 (2000).
13. M. E. de Lange, H. Snieder, R. A. S Arie¨ns, T. D. Spector,
P. J. Grant. Lancet 357, 101 (2001).
14. K. Koike et al.,Otolaryngol. Head Neck Surg. 111,
625 (1994).
15. StataCorp 1997 Stata Statistical Software, Release
5.0 (Stata Corporation, College Station, TX ).
16. J. S. Witte, J. B. Carlin, J. L. Hopper, Genet. Epidemiol.
16, 290 (1999).
17. Mx, version 4; M. C. Neale, Medical College of Vir-
ginia, Virginia Commonwealth University.
18. M. C. Neale, L. Cardon, Methodology for Genetic
Studies of Twins and Families (Kluwer, Dordrecht,
Netherlands, 1992). The chi-square for the best-
fitting model (additive genetic component and
unique environmental component same thresh-
olds) ⫽256.810 (df ⫽274), with P⫽0.765, indi-
cating an excellent fit of the model to the data.
19. D. Levitin. Percept. Psychophys. 56, 414 (1994).
20. J. Profita, G. Bidder, Am. J. Med. Genet. 29, 763
(1988); A. H. Takeuichi, S. H. Hulse, Psychol. Bull.
113, 345 (1993).
21. S. Baharloo, P. A. Johnston, S. K. Service, J. Gitschier,
N. B. Freimer, Am. J. Hum. Genet. 62, 224 (1998);
P. K. Gergesen et al.,Am. J. Hum. Genet. 65, 911
(1999).
22. A. J. MacGregor, H. Snieder, N. J. Schork, T. D. Spector,
Trends Genet. 16, 131 (2000).
23. We thank the research nurses for skillful data
collection and especially the twin volunteers who
participated in this study. We also thank E. Balaban
and the anonymous reviewers for their insightful
comments. Supported by NIH grant Z01-DC-
00043-03 from the National Institute on Deafness
and Other Communication Disorders (D.D. and
A.M.). M.d.L. and H.S. are sponsored by the Brit-
ish Heart Foundation (grants FS/99010 and
FS/99050). The Twins Research Unit gratefully ac-
knowledges support from the Arthritis and Rheu-
matism Council, Wellcome Trust, British Heart
Foundation, and Gemini Genomics.
1 September 2000; accepted 26 January 2001
Presynaptic Kainate Receptor
Mediation of Frequency
Facilitation at Hippocampal
Mossy Fiber Synapses
Dietmar Schmitz, Jack Mellor, Roger A. Nicoll*
Inhibition of transmitter release by presynaptic receptors is widespread in the
central nervous system and is typically mediated via metabotropic receptors.
In contrast, very little is known about facilitatory receptors, and synaptic
activation of a facilitatory autoreceptor has not been established. Here we
show that activation of presynaptic kainate receptors can facilitate trans-
mitter release from hippocampal mossy fiber synapses. Synaptic activation
of these presumed ionotropic kainate receptors is very fast (⬍10 ms) and lasts
for seconds. Thus, these presynaptic kainate receptors contribute to the short-
term plasticity characteristics of mossy fiber synapses, which were previously
thought to be an intrinsic property of the synapse.
Neurotransmitter receptors are located on the
presynaptic, as well as the postsynaptic, side
of the synapse. In vertebrates these presynap-
tic receptors are typically metabotropic re-
ceptors and inhibit transmitter release (1,2),
although in invertebrates facilitatory metabo-
tropic actions have also been described (3,4).
Ionotropic receptors are also present on pre-
synaptic terminals (5), and their activation
generally inhibits synaptic transmission (6–
11). Although facilitation has been observed,
there is no evidence that synaptically released
transmitter could have access to these recep-
tors (5). Kainate receptors (KARs) have re-
cently been shown to exert presynaptic ef-
fects on glutamatergic (10,12) as well as
GABAergic terminals (9,13). However, at
both types of terminals activation of kainate
receptors causes an inhibition of release. Here
we report that activation of presynaptic kai-
nate receptors by low levels of synaptically
released glutamate enhances transmitter re-
lease at hippocampal mossy fiber synapses.
Previous studies found that application of
kainate inhibits synaptic transmission at hip-
pocampal mossy fiber synapses (14–16).
However, kainate applied at concentrations
considerably below those used in these pre-
vious studies strongly facilitates synaptic
transmission. Kainate (50 nM) produced a
robust and reversible enhancement of ␣-ami-
no-3-hydroxy-5-methyl-4-isoxazolepropri-
onic acid receptor (AMPAR) mediated exci-
tatory postsynaptic currents (EPSCs) (Fig.
1A) (17), and this enhancement was associ-
ated with a decrease in paired pulse facilita-
tion (PPF) (P2/P1: control, 2.95 ⫾0.15;
kainate, 1.9 ⫾0.16) (Fig. 1A), suggesting
that the enhancement is mediated by an in-
crease in the probability of transmitter release
(18). Kainate (50 nM) also produced a robust
and reversible enhancement in the synaptic
field potential responses (170 ⫾9%), and
this enhancement was also associated with a
decrease in PPF (control, 2.7 ⫾0.16; kainate,
1.9 ⫾0.17) (19). To study the mechanism
involved in this facilitation, AMPA and
GABA
A
receptors were selectively blocked
by GYKI 53655 and picrotoxin, respectively,
and N-methyl-D-aspartate (NMDA) receptor
(NMDAR)–mediated EPSCs were recorded
at positive holding potentials. We used the
non-NMDAR antagonists CNQX or NBQX,
which block KARs, to identify effects of
bath-applied kainate and synaptically re-
leased glutamate that are due to KAR activa-
tion. Low concentrations of kainate (50 nM)
also reversibly enhanced NMDAR EPSCs,
and this effect was completely blocked by
CNQX (10 M), indicating that the enhance-
ment was mediated by high-affinity KARs
(Fig. 1B). The enhancement of AMPAR- and
NMDAR-mediated EPSCs occurred in the
absence of any change in the rise time,
indicating that no polysynaptic inputs were
recruited (Fig. 1, A and B, insets). This same
concentration of kainate also enhanced the
size of the presynaptic fiber volley, and this
effect was also blocked by CNQX (see Fig.
1E).
The enhancing action of kainate, both on
the NMDAR EPSC (Fig. 1, A and B) and on
the fiber volley, was mimicked by the addi-
tion of 4 mM K
⫹
to the superfusing medium
(Fig. 1C), suggesting that the effect of kainate
was mediated by a depolarizing action on the
terminal. Neither kainate nor K
⫹
had any
effect on the holding current, making a
postsynaptic site of action most unlikely. In
addition, kainate (50 nM) had no effect on
Departments of Cellular and Molecular Pharmacology
and Physiology, University of California, San Fran-
cisco, CA 94143, USA.
*To whom correspondence should be addressed. E-
mail: nicoll@phy.ucsf.edu
REPORTS
9 MARCH 2001 VOL 291 SCIENCE www.sciencemag.org1972
