Metabotropic Glutamate Receptor–Mediated Use–Dependent
Down-Regulation of Synaptic Excitability Involves the Fragile X Mental
Sarah Repicky and Kendal Broadie
Program in Developmental Biology, Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee
Submitted 22 August 2008; accepted in final form 24 November 2008
Repicky S, Broadie K. Metabotropic glutamate receptor–mediated use–
dependent down-regulation of synaptic excitability involves the fragile X
mental retardation protein. J Neurophysiol 101: 672–687, 2009. First
published November 26, 2008; doi:10.1152/jn.90953.2008. Loss of the
mRNA-binding protein FMRP results in the most common inherited
form of both mental retardation and autism spectrum disorders: fragile
X syndrome (FXS). The leading FXS hypothesis proposes that
metabotropic glutamate receptor (mGluR) signaling at the synapse
controls FMRP function in the regulation of local protein translation
to modulate synaptic transmission strength. In this study, we use the
Drosophila FXS disease model to test the relationship between
Drosophila FMRP (dFMRP) and the sole Drosophila mGluR (dmGluRA)
in regulation of synaptic function, using two-electrode voltage-clamp
recording at the glutamatergic neuromuscular junction (NMJ). Null
dmGluRA mutants show minimal changes in basal synapse properties
but pronounced defects during sustained high-frequency stimulation
(HFS). The double null dfmr1;dmGluRA mutant shows repression of
enhanced augmentation and delayed onset of premature long-term
facilitation (LTF) and strongly reduces grossly elevated post-tetanic
potentiation (PTP) phenotypes present in dmGluRA-null animals.
Null dfmr1 mutants show features of synaptic hyperexcitability, in-
cluding multiple transmission events in response to a single stimulus
and cyclic modulation of transmission amplitude during prolonged
HFS. The double null dfmr1;dmGluRA mutant shows amelioration of
these defects but does not fully restore wildtype properties in dfmr1-
null animals. These data suggest that dmGluRA functions in a nega-
tive feedback loop in which excess glutamate released during high-
frequency transmission binds the glutamate receptor to dampen syn-
aptic excitability, and dFMRP functions to suppress the translation of
proteins regulating this synaptic excitability. Removal of the transla-
tional regulator partially compensates for loss of the receptor and,
similarly, loss of the receptor weakly compensates for loss of the
I N T R O D U C T I O N
Fragile X syndrome (FXS) is a broad-spectrum neurological
disease with symptoms including hyperactivity, hypersensitiv-
ity to sensory stimuli, and seizures (Koukoui and Chaudhuri
2007; Visootsak et al. 2005). FXS is commonly caused by an
expanded 5?-CGG repeat region upstream of the fragile X
mental retardation 1 (fmr1) gene, which causes hypermethyl-
ation and transcriptional silencing. The fmr1 product, FMRP, is
an mRNA-binding protein that associates with polyribosomes
and the RNA-induced silencing complex (RISC) and functions as a
negative translational regulator (Garber et al. 2006). In mice,
fmr1 knockout causes enhanced hippocampal long-term de-
pression (LTD) (Huber et al. 2002), dependent on group I class
5 metabotropic glutamate receptor (mGluR5) signaling, which
is sensitive to translational inhibitors (Huber et al. 2000;
Koekkoek et al. 2005; Nosyreva and Huber 2006). Knockout
(KO) mice also display depressed long-term potentiation (LTP)
in the visual neocortex, dependent on mGluR5activation (Wil-
son and Cox 2007). These results have given rise to the
hypothesis that FMRP modulates synaptic transmission strength by
regulating local protein synthesis downstream of mGluR sig-
naling (Bear et al. 2004; Dolen et al. 2007; Pfeiffer and Huber
2006). This theory has been bolstered by findings of increased
protein synthesis with mGluR stimulation in the absence of
FMRP (Chuang et al. 2005; Hou et al. 2006; Koekkoek et al.
2005). Recently, rescue of several mouse fmr1 null phenotypes
has been achieved by reducing mGluR signaling in the mGluR5
heterozygote background (Dolen et al. 2007), although without
any assessment of neurotransmission itself. Here, we test this
mGluR hypothesis by evaluating synaptic function in genetic
mutants that completely eliminate mGluR signaling, FMRP
function, or both in double homozygous null mutants.
Drosophila provides a powerful, simplified genetic model to
test the mGluR theory of FXS, because the fly genome encodes
a single fmr1 family member (dfmr1) and single mGluR
(dmGluRA) (Bogdanik et al. 2004; Parmentier et al. 1996; Wan
et al. 2000; Zhang et al. 2001). As in mice, dfmr1 mutants
display synaptic structure defects and mistrafficking of iono-
tropic glutamate receptors (Gatto and Broadie 2008; Pan and
Broadie 2007; Pan et al. 2004, 2008; Tessier and Broadie 2008;
Zhang et al. 2001). Similarly, dmGluRA mutants display de-
fective synaptic architecture, glutamate receptor trafficking,
and activity-dependent synaptic plasticity (Bogdanik et al.
2004; Pan and Broadie 2007). In dfmr1;dmGluRA double
mutants, synergisitic effects occur in the regulation of coordi-
nated movement, sculpting of synaptic structure, and control of
glutamate receptor trafficking (Pan and Broadie 2007; Pan
et al. 2008). However, we have not yet addressed the hypoth-
esized intersecting roles of dmGluRA signaling and dFMRP
function in the modulation of synaptic functional plasticity.
In this study, we examine the relationship between dFMRP
and dmGluRA in regulating neurotransmission at the glutama-
tergic neuromuscular junction (NMJ), a long-established sys-
tem for studying the genetic foundations of synaptic plasticity.
Null dfmr1 and dmGluRA mutants display largely normal basal
transmission and short-term facilitation, but dfmr1;dmGluRA
Address for reprint requests and other correspondence: K. S. Broadie, Dept.
of Biological Sciences, Vanderbilt Univ., VU Station B, Box 351634, Nash-
ville, TN 37235-1634 (E-mail: email@example.com).
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of page charges. The article must therefore be hereby marked “advertisement”
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J Neurophysiol 101: 672–687, 2009.
First published November 26, 2008; doi:10.1152/jn.90953.2008.
672 0022-3077/09 $8.00 Copyright © 2009 The American Physiological Societywww.jn.org
double mutants show increased facilitation. Single null mutant
phenotypes are manifest during prolonged high-frequency
stimulation (HFS). Null dmGluRA mutants display heightened
augmentation, a dramatically lowered threshold for long-term
facilitation, and greatly elevated post-tetanic potentiation. Im-
portantly, removing dFMRP in dfmr1;dmGluRA double mu-
tants partially alleviates all these defects, albeit with very weak
reduction of the facilitation defects and strong suppression of
the potentiation defect. Null dfmr1 mutants display an intrigu-
ing cyclic transmission amplitude periodicity during HFS trains
and synaptic hyperexcitability during and following HFS. Impor-
tantly, removing dmGluRA in dfmr1;dmGluRA double mutants
strongly reduces these defects. Taken together, these data suggest
that loss of dFMRP translational inhibition partially alleviates the
phenotypes resulting from loss of dmGluRA signaling and that
loss of the receptor similarly partially corrects defects caused
by impaired translation regulation. However, reduction of mu-
tant phenotypes in both directions is mostly quite weak, show-
ing that the convergence of dmGluRA and dFMRP function is
limited and that other signaling pathways must interact with
both glutamate receptor and translation regulator.
M E T H O D S
All Drosophila stocks were maintained at 25°C on standard food
under standard conditions. The P-element imprecise excision deletion
dmGluRA112b(hereafter called dmGluRA) is a null mutation of the
sole functional Drosophila mGluR; the precise excision line from the same
screen, dmGluRA2b(hereafter called control), was used as the genetic
background control (Bogdanik et al. 2004; Pan and Broadie 2007).
The P-element imprecise excision deletion dfmr13(hereafter called
dfmr1) is a dfmr1 null mutation (Dockendorff et al. 2002; Pan et al.
2008). The dfmr13allele was backcrossed for six generations into the
dmGluRA2bgenetic background, so that this single genetic back-
ground control could be used for all conditions (Pan and Broadie
2007; Pan et al. 2008). A dfmr13;dmGluRA112bdouble homozygous
null mutant (hereafter called dfmr1;dmGluRA) was generated using
standard genetic techniques. Both single null mutants and the double
null mutant are adult viable, and all genotypes have been repeatedly
confirmed by sequencing, anti-dmGluRA/dFMRP immunocytochem-
istry and Western blot analyses (Pan and Broadie 2007; Pan et al.
Two-electrode voltage-clamp (TEVC) recordings were made from
the wandering third-instar NMJ synapse as described previously
(Long et al. 2008; Rohrbough et al. 1999; Trotta et al. 2004). Animals
were dissected at 18°C in standard saline (in mM: 128 NaCl, 2 KCl,
4 MgCl2, 70 sucrose, 5 HEPES, pH 7.2) containing 0.15 mM Ca2?
unless otherwise indicated in individual experiments. All TEVC
recordings were done at 18°C in anterior abdominal segments A3-4 at
the muscle 6/7 NMJ with the muscle held at ?60 mV using an
Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Intra-
cellular recording electrodes were filled with 3 M KCl and typically
had a resistance of 8–12 MOhms. Excitatory junction current (EJC)
responses were evoked using a glass suction electrode on the severed
motor nerve by application of brief stimuli (0.5 ms) from a S88
stimulator (Grass Instruments, Quincy, MA). Muscle 6 is innervated
by two motor axons of differing physiological properties. In all
experiments, both axons were stimulated in unison (suprathreshold)
with stimulations at 150% of threshold. Acceptable recordings re-
quired a resting leak current of ?25 nA for the entirety of the
recording session, with no significant shifts in the leak current. Data
acquisition was performed using pClamp8.0 (Axon Instruments) and
Clampfit9.1 (Axon Instruments) software in all experiments.
Data analyses were performed using Prism software 4.0 (Graphpad,
San Diego, CA). Appropriate statistical analyses were used as noted
and included both one-way and two-way ANOVAs. Statistical signif-
icance was determined with appropriate posttests, most commonly a
Bonferroni posttest, as indicated. Mean EJC amplitudes were deter-
mined by averaging all the stimulated responses that occurred within
the designated period and stimulation condition, with the mean re-
sponses for each animal then averaged for all animals of each genetic
line. For normalized comparisons of EJC amplitudes, the 0.5-Hz
response amplitudes were first averaged before the high-frequency
stimulation (HFS) condition. The response amplitudes from each
genotype during the HFS period (At) and the basal 0.5-Hz stimulation
period following HFS (potentiation period) were divided by the
average of the initial basal amplitude (Ai) to determine the normalized
value for each animal. The normalized values (At/Ai) were averaged
to determine the mean normalized values for each genotype.
Hyperpotentiation was defined as a post-HFS response that was at
least five times greater than the mean response amplitude (Ai) before
the HFS train. The percentage of animals with hyperpotentiation was
determined by identifying animals with at least one post-HFS stimu-
lation normalized amplitude greater than five, and dividing this
number by the n for that genetic condition. To determine the number
of post-HFS stimulations defined as hyperpotentiated, the number of
response amplitudes greater than five were counted for each animal
within a given strain and averaged with all the other animals within
that strain. Statistical significance for the average number of response
amplitudes for each genotype was determined using one-way ANOVA and
a Bonferroni post-test.
R E S U L T S
Basal Ca2?-dependent function largely normal in dfmr1
and dmGluRA mutants
Our previous work has shown that basal synaptic transmis-
sion amplitude is unaltered compared with genetic background
control in the dmGluRA null mutant (Bogdanik et al. 2004);
however, this finding has recently been challenged by a report
of significantly elevated basal transmission amplitude in this
mutant (Howlett et al. 2008). Our previous work has shown
elevated presynaptic vesicle cycling and heightened transmis-
sion amplitude in the dfmr1 null mutant (Gatto and Broadie
2008; Zhang et al. 2001); however, this work was not pursued
in the Ca2?concentration range used in functional plasticity
assays. In addition, we particularly wanted to assay the dfmr1;
dmGluRA double null mutant, which has never before been
subject to functional analyses. We therefore first performed
assays of synaptic transmission strength at the wandering
third-instar NMJ synapse over a range of external Ca2?con-
centrations while suprathreshold stimulating the motor nerve at
a basal frequency of 0.5 Hz. EJC recordings were made in the
muscle clamped at ?60 mV. The results of these basal anal-
yses are shown in Fig. 1.
During low-frequency stimulation, all genotypes display
robust, high fidelity neurotransmission. Figure 1A shows rep-
resentative traces of 20 superimposed EJC responses from the
genetic background control (dmGluRA2b; see METHODS), single
null mutant alleles of dfmr1 and dmGluRA, and the double null
mutant dfmr1;dmGluRA. Both single mutants, dfmr1 and
673dFMRP AND dmGluRA REGULATE SYNAPTIC EXCITABILITY
J Neurophysiol • VOL 101 • FEBRUARY 2009 • www.jn.org
dmGluRA, showed a tendency toward elevated response ampli-
tudes compared with control and especially compared with the
dfmr1;dmGluRA double null (Fig. 1A). At 0.3 mM [Ca2?],
mean amplitudes were 40.78 ? 7.54 (control), 54.2 ? 4.95
(dfmr1), 50.98 ? 11.53 nA (dmGluRA), and 31.01 ? 6.44 nA
(dfmr1;dmGluRA). However, under these conditions, there
were no statistically significant differences in EJC amplitude
between any of the genotypes (one-way ANOVA, Kruskal-
control dfmr1 dmGluRA dfmr1;dmGluRA
Mean Amplitude (nA)
674 S. REPICKY AND K. BROADIE
J Neurophysiol • VOL 101 • FEBRUARY 2009 • www.jn.org
dfmr1 mutants, the hyperexcitable responsiveness persists fol-
lowing the HFS train during basal stimulation, but post-HFS
hyperexcitability was never observed in dfmr1;dmGluRA ani-
mals. Thus co-removal of dmGluRA does indeed diminish the
consequences of loss of dFMRP, only partially in the case of
the cyclic transmission defect, but quite strongly to block
dfmr1 hyperexcitability. Together, these data support the con-
clusion of a partial co-dependency of dmGluRA receptor
signaling on dFMRP regulative function, and vice versa in a
feedback loop, to modulate synapse properties critical for the
maintenance of transmission fidelity and activity-dependent
Our laboratory has previously shown both rescue and syn-
ergistic interactions of dfmr1 and dmGluRA null mutations in a
range of synaptic mechanisms (Pan and Broadie 2007; Pan
et al. 2008). However, a major, persistent limitation has been
the lack of any functional data on synaptic transmission, a
primary focus of FXS dysfunction. This crucial question has
similarly not as yet been addressed in the mouse fmr1 KO
model, despite evidence of rescue in other fmr1 defects (Dolen
et al. 2007). Here we show that activity-dependent synaptic
plasticity defects in dmGluRA nulls, including elevated aug-
mentation, potentiation, and premature LTF, are each reduced
by the co-removal of dFMRP. Similarly, the synaptic defects in
dfmr1 nulls, including transmission amplitude cycling during
HFS and multiple EJCs in response to a single stimulus, are
decreased by the co-removal of dmGluRA, and hence loss of
all mGluR signaling at the synapse. The striking exception to
this trend is STF, which is somehow enhanced in the dfmr1;
dmGluRA double null compared with both single mutants.
These interactions clearly support the conclusion of a relation-
ship between dFMRP function and dmGluRA signaling, but
argue against a simple direct signaling cascade. Rather,
dFMRP function is likely controlled by several converging
signaling pathways, of which dmGluRA-mediated glutamater-
gic synaptic signaling is only one.
A C K N O W L E D G M E N T S
We thank Dr. Jeff Rohrbough for advice on TEVC electrophysiology
techniques and L. Coffee, C. Gatto, A. Long, J. Rohrbough, and C. Tessier for
critical feedback on this manuscript.
G R A N T S
S. Repicky was supported by a postdoctoral training grant for the Program
in Developmental Biology at Vanderbilt University. This work was supported
by National Institute of General Medical Sciences Grant GM-54544 to K.
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