ORIGINAL ARTICLE
Phosphorylation of Delta2 Glutamate Receptors at Serine 945
is Not Required for Cerebellar Long-term Depression
Ryoichi Nakagami,1, 2 Kazuhisa Kohda,1 Wataru Kakegawa,1 Tetsuro Kondo,1Nobumasa Kato2 and Michisuke Yuzaki1

Department of Physiology, School of Medicine, Keio University, Tokyo, Japan2
Department of Psychiatry, University of Tokyo, Tokyo, Japan

1(Received for publication on February 1, 2008)(Revised for publication on March 17, 2008)(Accepted for publication on April 17, 2008)

Abstract: Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell synapses is thought to regulate motor learning and memory formation in the cerebellum. Neuronal activity-evoked protein kinase C (PKC) activation is required for the induction of LTD. In addition, theδ
δ2 glutamate receptor (GluRδ
δ2), which is predominantly
expressed at PF-Purkinje cell synapses, is indispensable for the induction of LTD; however,
the mechanisms by which GluRδδ2 regulates LTD and its relationship with PKC activation
remain elusive. Interestingly, GluRδδ2 is phosphorylated by PKC on serine 945 (Ser945)
near its C-terminus and a postsynaptic protein S-SCAM, which could potentially regulate
glutamate receptor trafficking and synaptic plasticity, binds to the extreme C-terminus ofGluRδδ2 in a phosphorylation-dependent manner on Ser945. Here, using a Sindbis-based virus expression approach, we show that a mutant GluRδδ2, in which alanine replaced Ser945
and did not undergo PKC phosphorylation, was normally localized at the postsynaptic sitesof PF-Purkinje cell synapses. In addition, like wild-type GluRδδ2, the phosphorylation-disrupted GluRδδ2 successfully rescued abrogated LTD in GluRδ2-null Purkinje cells. These results indicate that Ser945, a major PKC phosphorylation site of of GluRδδ2, may not play a
crucial role in induction of LTD in the cerebellum. (Keio J Med 57 (2) : 105－110, June 2008)
Key words: cerebellum, Purkinje cell, long-term depression, δ2 glutamate receptor, phosphorylation

Introduction
Information storage in neural circuits depends on neuronal activity-dependent persistent changes in neurotransmission efficiency, such as long-term potentiationand long-term depression (LTD). Experimental studies
have established that LTD in the cerebellum is a majormechanism responsible for motor learning.1 Cerebellar
Purkinje cells receive two excitatory inputs from parallel
fibers (PFs; axons of granule cells) and climbing fibers(CFs; axons of inferior olivary neurons); according to
the LTD theory, PF responses are persistently depressed
when they are repeatedly evoked together with performance error-encoding CF activities. Since LTD is observed in various brain regions, an understanding of cer-ebellar LTD is not only essential for an appreciation of
normal and abnormal cerebellar functions, but also provides key insights into basic mechanisms underlying
synaptic plasticity.
Like LTD in other brain regions, cerebellar LTD isthought to be caused by the selective endocytosis of thepostsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole(AMPA)-subtype of glutamate receptors that normallymediate synaptic transmission.2, 3 Nevertheless, cerebellar LTD is unique in that it absolutely requires the presence of another class of glutamate receptor, the δ2 glutamate receptor (GluRδ2), which is predominantly expressed at PF-Purkinje cell synapses.4 Indeed, LTD iscompletely absent in GluRδ2-null mice5, 6 and the application of an antibody against GluRδ2’s extracellular

Corresponding Author: Michisuke Yuzaki, M.D., Ph.D., Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan, E-mail : myuzaki@a5.keio.jp105Nakagami R, et al: Long-term Depression and Receptor Phosphorylation

106

N-terminal region to wild-type Purkinje cells rapidly induced the endocytosis of AMPA receptors and abrogated
the further induction of LTD.7 Interestingly, although
GluRδ2 belongs to the ionotropic glutamate receptor
family, a GluRδ2 transgene, in which the putative channel pore domain was disrupted, rescued all abnormal
phenotypes of GluRδ2-null mice.8, 9 In contrast, the extreme C-terminal end of GluRδ2, which binds to various
intracellular proteins like PSD-93, 1 0 PTPMEG, 1 1
S-SCAM/MAGI-2,1 2 and delphilin,1 3 was indispensablefor the induction of LTD and motor learning. These results indicate that GluRδ2 may function as a nonionotropic receptor of which C-terminus controls the numberof postsynaptic AMPA receptors and regulates LTD;
however, the precise mechanisms by which GluRδ2 regulates LTD remain elusive.
LTD can be induced by direct protein kinase C (PKC)
activation in Purkinje cells.14, 15 In addition, conjunctive
stimulation of PFs and CFs could not induce LTD when
PKC activities were blocked in Purkinje cells,16 indicating that PKC activation in Purkinje cells is necessary andsufficient for LTD induction. Indeed, recent imaging
studies indicated that PKCα is activated during LTD induction in cultured Purkinje cells.1 7 One of the crucial
substrates of PKC in Purkinje cells during LTD is the
C-terminus of the AMPA receptor subunit GluR2.2, 18 In
addition, we previously demonstrated that GluRδ2 was
phosphorylated at Ser945 by PKC in vitro and by an LTDinducing stimulus in slice preparations.1 9 Interestingly,
the binding of a synaptic scaffolding molecule, S-SCAM,
to the extreme C-terminus of GluRδ2 was shown to besignificantly enhanced by the phosphorylation of
Ser945.12 Since S-SCAM is associated with several synaptic proteins including transmembrane AMPA receptor
regulatory proteins (TARPs),20 which regulate AMPA receptor localization at synapses,2 1 we hypothesized that
the S-SCAM signaling pathway, which could be controlled by the PKC phosphorylation status of GluRδ2’s
C-terminus, may be involved in the regulation of LTD
induction. To test this hypothesis, we employed a virusmediated “rescue” approach22 using a mutant GluRδ2 inwhich alanine replaced Ser945 (GluRδ2S945A). We demonstrated that like wild-type GluRδ2, GluRδ2S945A wasexpressed at postsynaptic sites and rescued abrogated
LTD in GluRδ2-null Purkinje cells. These results indicate that the PKC phosphorylation site Ser945 does notplay a major role in the induction of LTD.
Materials and Methods
Vector constructs and virus production
A modified Sindbis virus vector was used, as previously described.2 2 The second subgenomic promoter wasinserted into pSinRep5 (Invitrogen, Carlsbad, CA) to ex-press a yellow fluorescent protein (YFP; Invitrogen) for
the identification of the infected cells.
In vivo microinjection
GluRδ2-null mice were anesthetized using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (20mg/kg; Sigma, St. Louis, MO). A small hole was madein the occipital bone with a dental drill, and the dura mater was ablated. A glass pipette (30 µm in diameter) was
inserted into the cerebellum 900 µm from the surface,and 250 nL of virus solution was injected using a Nanoliter (World Precision Instruments, Sarasota, FL). The
mice were sacrificed 24-48 hours after injection. All theexperimental procedures involving the use of animals
were approved by the Animal Resource Committee of
the School of Medicine, Keio University.
Immunohistochemistry
The immunohistochemical preparations were performed as described previously.22 Under deep anesthesia
with an intraperitoneal injection of pentobarbital, the virus-injected mice were fixed by cardiac perfusion with
4% paraformaldehyde in 0.1 M sodium phosphate buffer(PBS, pH 7.4), and the cerebellum was removed andpost-fixed by 4% paraformaldehyde in PBS overnight.
Parasagittal slices (150 µm thick) were prepared using a
microslicer (Dosaka EM, Kyoto, Japan). Then, the slices
were treated with 1 mg/mL of pepsin (DAKO) in 0.2 N
HCl at 37°C for 20 min. After being permeabilized by
0.4% Triton X-100 in PBS with 0.2% normal goat serum
and 0.2% bovine serum albumin at 4°C overnight, thesections were treated with antibodies against GluRδ2(kindly donated by Dr. Watanabe, Hokkaido Univ., Japan) and YFP (Invitrogen). For visualization, secondary
antibodies conjugated with Alexa 594 and 488 (Molecular Probes, Eugene, OR) were used.
Electrophysiology
Parasagittal cerebellar slices (200 µm thick) were prepared from GluRδ2-null mice (postnatal day 17-28) using a linear slicer (Dosaka EM). Whole-cell voltageclamp recordings from YFP-positive Purkinje cells were
performed as described previously.7 Excitatory postsynaptic currents (EPSCs) at PF-Purkinje cell synapses (PFEPSCs) were evoked by stimulating the molecular layer(pulse width, 200 µsec) with a glass pipette filled with
140 mM NaCl and 10 mM HEPES. Stimulation of thePFs was confirmed by the paired-pulse facilitation of PFEPSCs with a 50 msec inter-stimulus interval. Purkinjecells were voltage-clamped at -80 mV, and PF-EPSCs
evoked at 0.1 Hz were monitored. After obtaining stable
recordings of the PF-EPSCs for at least 10 min, the Pur-

Keio J Med 2008; 57 (2): 105－110

Fig. 1 Distribution patterns of virally transduced wild-type
GluRδ2 (GluRδ2WT) and GluRδ2S945A in GluRδ2-null cerebellar
slices. GluRδ2 immunoreactivities were visualized using Alexa
594 (red); immunoreactivities against YFP, visualized with Alexa
488 (green), were overlaid. Scale bars, 20 µm.

kinje cells were depolarized to 10 mV for 200 msec at 1
Hz 30 times in conjunction with evoking the PF-EPSCs.
The measurements were discarded when the series resistance changed by 20% of its original value. The current
traces were filtered at 2 kHz and digitized at 10 kHz.
For the statistical analysis, the EPSC amplitudes wereaveraged every 1 min. These averaged EPSCs were normalized by those for 1 min before the conjunctive stimuli.
Results and Discussion
We employed a Sindbis virus vector to express wildtype GluRδ2 and YFP (Sin-GluRδ2wt-Y), GluRδ2S945A in which alanine had replaced the PKC phosphorylationsite Ser945 - and YFP (Sin-GluRδ2 S945A-Y), or YFP
alone (Sin-Y). Twenty-four to 48 hours after injection
into lobule VI or VII of the cerebellar vermis of
GluRδ2-null mice, we observed YFP signals and GluRδ2
immunoreactivities mainly in Purkinje cells in acutely
prepared cerebellar slices, as previously reported (Fig.
1).22 Although many neurotransmitter receptors are anchored at postsynaptic membranes via an interaction
with scaffolding proteins at their C-termini, we previously demonstrated that a virally expressed mutant GluRδ2lacking the C-terminal ends normally localized at the107

postsynaptic membranes.22 To confirm that GluRδ2S945A
was also expressed at postsynaptic sites, we performed
an immunohistochemical analysis of GluRδ2-null Purkinje cells infected with GluRδ2wt or GluRδ2S945A. Confocal microscopic studies showed no difference in localization patterns within dendrites and spines between
GluRδ2wt and GluRδ2S945A (Fig. 1). Therefore, the PKC
phosphorylation site of the C-terminus of GluRδ2 is unlikely to be essential for localizing GluRδ2 at synapses.
To examine whether GluRδ2S945A, like GluRδ2wt, could
rescue abrogated LTD at PF-Purkinje cell synapses in
GluRδ2-null cerebellum, we performed whole-cell voltage-clamp recordings in virally transduced acute cerebellar slices. Sindbis virus infection itself did not significantly affect the basic membrane properties of the Purkinje cells or the kinetics of the PF-evoked EPSCs in the
Purkinje cells (Table 1). In addition, the probability ofpresynaptic transmitter release, as assessed by measuring
the paired-pulse facilitation of PF-EPSCs,23 was similar
among GluRδ2-null Purkinje cells infected with SinGluRδ2wt-Y, Sin-GluRδ2S945A-Y, or Sin-Y (Table 1). Finally, we examined whether LTD could be restored in
GluRδ2-null Purkinje cells infected with Sin-GluRδ
2S945A-Y. To exclude any effect from abnormal CF innervation in GluRδ2-null Purkinje cells, we replaced CF
stimulation with direct depolarization of the Purkinjecells.24 In GluRδ2-null Purkinje cells infected with SinGluRδ2wt-Y (Fig. 2A) or Sin-GluRδ2S945A-Y (Fig. 2B),
this protocol successfully induced LTD in PF-EPSCs,
but it did not induce LTD in Purkinje cells expressing
YFP (Fig. 2C). The amplitude of PF-EPSCs 26 - 30 min
after conjunctive stimuli was 64 ± 4% (n = 5 from 3mice) of the control responses in Purkinje cells infectedwith Sin-GluRδ2wt-Y and 66 ± 8% (n = 5 from 4 mice)in cells infected with Sin-GluRδ2S945A-Y, whereas that ofcells treated with Sin-Y was 96 ± 8% (n = 5 from 4 mice;Student t-test, p < 0.001 vs. Sin-GluRδ2wt-Y or Sin-GluR
δ2S945A-Y; Fig. 2D). In contrast, we previously showedthat Sindbis virus-based expression of GluRδ2 that had amutation in the channel pore domain (GluRδ2-VR) or
GluRδ2 that lacked the C-terminal seven amino acids(GluRδ2-∆CT7) could not rescue the LTD phenotype in
GluRδ2-null Purkinje cells in acute slice preparations,8, 9
indicating that LTD could not be rescued by overexpression of mutant GluRδ2 proteins. These results suggestthat PKC phosphorylation site Ser945 is not essential for
GluRδ2 to regulate LTD induction in acute slice preparations.
Although GluRδ2 is crucial for LTD and motor learningin the cerebellum, the mechanisms by which GluRδ2 participates in cerebellar functions have been elusive.4 We previously demonstrated that abrogated LTD was restored in
GluRδ2-null Purkinje cells by introducing wild-type GluRδ
2, but not a mutant GluRδ2-∆CT7, to which PSD-93, PTPMEG, delphilin and S-SCAM cannot bind. 2 2 , 2 5

Nakagami R, et al: Long-term Depression and Receptor Phosphorylation

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Table 1 Electrophysiological properties of virus-infected Purkinje cells*
Construct/ Background(number)
Membrane
resistance (MΩ)
Membrane
capacitance (pF)
PF-EPSC
Rise time** (ms)PF-EPSC
Decay time*** (ms)PF-EPSCPPF ratio****Sin-δ2Wt-Y/ GluRδ2-null(n = 13)Sin-δ2S945A-Y/ GluRδ2-null(n = 11)

Sin-Y/ GluRδ2-null(n = 12)80 ± 588 ± 577 ± 6594 ± 43581 ± 32596 ± 48

3.6 ± 0.2

3.2 ± 0.2

3.4 ± 0.4

22 ± 225 ± 222 ± 22.2 ± 0.2

2.1 ± 0.1

1.9 ± 0.1

*Data represent mean ± SEM. There were no statistically significant differences among groups.
**The time required for the synaptic current to increase from 10% to 90%.
***Current decay was fitted to single exponential curves.
****Paired-pulse facilitation (PPF) ratio of PF-EPSCs was defined as the ratio of the amplitude of the second EPSC to that of the first
EPSC, when the two stimuli were given at 50 ms intervals.

Fig. 2 Virally introduced GluRδ2S945A rescued LTD induction in GluRδ2-null Purkinje cells. (A-C) LTD induction in GluRδ2-nullPurkinje cells infected with various vectors. LTD could not be induced in GluRδ2-null Purkinje cells by the expression of YFP (cont;A), but it could be induced by the expression of wild-type GluRδ2 (δ2wt; B) and a mutant GluRδ2 in which Ala replaced Ser 945(δ2S945A; C). LTD-inducing conjunctive stimuli were applied at the time point indicated by the vertical arrows. The sweeps in theinset indicate the EPSCs at t = 0 (1), t = 30 min (2), and the merged traces (1+2). Scale bars: 100 pA, 25 ms. (D) Averaged EPSCs in
GluRδ2-null Purkinje cells infected with various vectors. EPSCs were normalized by the averaged EPSCs from -1 to 0 min prior to the
conjunctive stimuli. Each point represents the mean and standard errors of normalized EPSC (GluRδ2-null Purkinje cells expressing
control YFP (◊, n = 5), δ2wt (□, n = 5) and GluRδ2S945A (▲, n=5). The LTD-inducing conjunctive stimulus was applied at the time
point indicated by a vertical arrow.

Keio J Med 2008; 57 (2): 105－110

We also previously indicated that PSD-93 and delphilin
were dispensable for LTD induction.22 In this study, wedemonstrated that GluRδ2 S945A, which could not be
phosphorylated by PKC19 and could not strongly interact
with S-SCAM,12 successfully rescued abrogated LTD at
PF-Purkinje cell synapses in GluRδ2-null cerebellum.
Therefore, although we cannot completely rule out thepossibility that overexpression of a mutant GluRδ2S945A
protein may have compensated its reduced binding to
S-SCAM, GluRδ2-S-SCAM interaction is unlikely toplay a major role in LTD induction. Instead, we proposethat other molecules that bind to the C-terminal end of
GluRδ2, such as PTPMEG, may be crucial for the induction of LTD.
Even if the GluRδ2-S-SCAM interaction may not playa major role in LTD induction, it may serve for otherfunctions of GluRδ2. For example, presynaptic elements
are often misaligned with, or detached from, their opposing postsynaptic counterparts at PF-Purkinje cell synapses in GluRδ2-null cerebella.2 6 As S-SCAM assembles
with the adhesion molecule neuroligin-1, which induces
presynaptic differentiation by binding to a presynaptic
protein neurexin,27 GluRδ2 may regulate the adhesion ofpre- and post-synaptic elements at PF-Purkinje cell synapses via S-SCAM. Because of the cytotoxicity of theSindbis virus, we could not observe the longer-term effects of GluRδ2 transduction on these morphologicalphenotypes.22 Thus, further studies utilizing other virus
vectors with attenuated toxicity or a transgenic micebased rescue approach will be required to elucidate thelonger-term functions of GluRδ2-S-SCAM interaction
and its possible regulation by PKC.
Acknowledgements
This work was supported by a grant-in-aid for Grantin-Aid for Scientific Research (A) (to M. Y.), Grant-inAid for Scientific Research on Priority Areas (to M. Y.),and Keio Gijuku Academic Development Funds (to K.
K.). We thank J. Motohashi and S. Narumi for theirtechnical assistance.
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