Eye opening and PSD95 are required for long-term
potentiation in developing superior colliculus
The MIT Faculty has made this article openly available. Please share
how this access benefits you. Your story matters.
Citation
Zhao, J.-P., Y. Murata, and M. Constantine-Paton. Eye Opening
and PSD95 Are Required for Long-term Potentiation in
Developing Superior Colliculus. Proceedings of the National
Academy of Sciences 110, no. 2 (January 8, 2013): 707-712.
As Published
http://dx.doi.org/10.1073/pnas.1215854110
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Thu May 26 08:55:51 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/79769
Terms of Use
Article is made available in accordance with the publisher's policy
and may be subject to US copyright law. Please refer to the
publisher's site for terms of use.
Detailed Terms
Eye opening and PSD95 are required for long-term
potentiation in developing superior colliculus
Jian-Ping Zhao1, Yasunobu Murata, and Martha Constantine-Paton1
McGovern Institute for Brain Research and Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139
The only major glutamate receptor membrane-associated guanylate
kinase scaffolds expressed in the young superficial superior colliculus
(SC) are synapse-associated protein 102 (SAP102) and postsynaptic
density protein 95 (PSD95). In this, as in all visual brain regions examined, synaptic PSD95 increases rapidly following simultaneous
eyelid opening (EO). We show that EO and PSD95 are necessary for
SC NMDA receptor (NMDAR)-dependent long-term potentiation
(LTP) and this LTP is eliminated or reinstated by manipulating EO.
PSD95 knockdown (KD) in vivo blocks this LTP, but not long-term
depression, and reduces frequencies of miniature AMPA receptor
and NMDAR currents with no change in presynaptic release. Furthermore, miniature NMDAR currents after PSD95 KD show an activitytriggered calcineurin sensitivity that is normally only found in the
pre-EO period when SAP102 binds mixed GluN2A/GluN2B NMDARs.
These data indicate that young SC LTP arises from PSD95 unsilencing
of silent synapses, that unsilencing is labile in young brain, and
that even though SAP102 and PSD95 can bind the same NMDARs,
only PSD95 enables SC synaptic maturation.
experience-dependent synaptogenesis
| pattern vision
G
enetic knockout (KO) and knockdown (KD) of the dominant
postsynaptic density (PSD) glutamate receptor scaffold,
membrane-associated guanylate kinase (MAGUK) postsynaptic
density protein 95 (PSD95), have been examined intensively for
synaptic effects in area CA1 of rodent hippocampus and mostly in
older animals or with cultured hippocampal slices. In the hippocampus, the synaptic MAGUKs synapse-associated protein 102
(SAP102), PSD95, and postsynaptic density protein 93 (PSD93)
bind ionotropic glutamate receptors and many molecules through
which the receptors signal (1–3). A fourth MAGUK, synapseassociated protein 97 (SAP97), binds selectively to AMPA receptor
(AMPAR) subunit GluR1 helping to deliver it to, but not remain
at the PSD (4).
Although the hippocampus is a highly evolved and important
brain region, it is unlikely to reflect properties of all other regions
that are critical to brain function. Synaptic development has been
studied much more intensively in the visual pathway where it
differs significantly from the hippocampus because the onset of
pattern vision is necessary for completion of its synaptic connectivity (5–7) and it has a critical period in which considerable visual
plasticity disappears (8). Moreover, the superficial superior colliculus (SC), central to eye movements and integration of multiple
sensory pathways for orientation in space, is the only brainstem
region where synaptic MAGUK function has been studied at all,
and it is a region where SAP102 and PSD95 are the only significant
MAGUK glutamate receptor scaffolds.
In rodent visual pathways, SAP102 is the dominant scaffold at
the PSD until 2–3 h after controlled eyelid opening (EO) and the
onset of pattern vision. At this time, PSD95 in visual synapses
increases twofold to threefold (9, 10). This is followed, in the SC
where retinal, visual cortical, and thalamic inputs converge, by an
increase in excitatory synapses (7); a functional refinement of
innervating axons (11); an anatomical refinement of the corticocollicular projection (7); and maturation of SC inhibition (12, 13).
Furthermore, in PSD95 KO mice (14), the normal increase in
synapse number after EO fails to occur in dorsally oriented vertical neurons of the SC (7).
www.pnas.org/cgi/doi/10.1073/pnas.1215854110
It is significant that MAGUKs show different expression patterns at different ages because there is increasing evidence that
each may bind different signaling molecules at glutamate synapses
(15). For example, in rodent hippocampus, SAP102 is present in
the early postnatal PSD, it binds NMDA receptors (NMDARs)
via GluN2B, AMPARs via stargazin (16), and a complex containing
SynGAP (17, 18). In visual cortex, SAP102 is replaced at the PSD
by the PSD95 complex containing GluN2A and TrkB upon EO
(9, 18). At this stage, SAP102 and GluN2B-rich NMDARs remain in extrasynaptic regions (4, 13) where they mediate mostly
evoked currents, whereas the PSD95–GluN2A complex is responsible for miniature NMDAR currents (mNMDARcs). This
scenario, first suggested in the hippocampus (19), was documented
in the SC of the developing GluN2A KO mouse (20). Extrasynaptic NMDARs with functions that differ from PSD NMDARs
have been identified in several brain regions (13, 21–24) and
PSD95 selectivity for the GluN2A tail has now been verified in
the developing hippocampus (25).
For NMDARs in forebrain and dorsal midbrain regions,
the change from binding MAGUKs via GluN2B to binding via
GluN2A has frequently been interpreted as a switch from GluN1/
GluN2B diheteromeric receptor currents to short decay-time
currents characteristic of GluN1/GluN2A diheteromeric receptors. However, in the SC of rats and mice when SAP102 is still
the major PSD MAGUK but GluN2A subunits are increasing,
NMDARc decay times decrease abruptly. This is mediated via a
calcineurin (CaN)-dependent dephosphorylation of the GluN2A
tail (26, 27). Therefore, NMDARc decay time cannot reveal when
a subunit change at the PSD from GluN2B/GluN2A to all GluN2A
occurs or whether SAP102 or PSD95 scaffolds the receptor.
Here, we used short hairpin RNA (shRNA) to KD PSD95 in
single cells of the neonatal SC and studied their synaptic currents
and NMDAR-dependent plasticity at intervals after EO. Our
findings differ from those expected from PSD95 KD experiments
in hippocampus CA1 (25, 28–31) in three fundamental respects:
In SC, long-term potentiation (LTP) does not survive PSD95
KD; in SC, long-term depression (LTD) does survive PSD95 KD;
in SC, reductions in AMPAR and NMDAR synaptic responses
occur with PSD95 KD without any change in presynaptic release.
In addition, in young SC, the ability to induce LTP is not stable;
eyelid reclosure (ERC) for several days causes SC LTP to disappear and eye reopening (ERO) following ERC reintroduces
LTP. Finally, the CaN-mediated decrease in NMDARc decay
times, observed in normal SC neurons only as activity increases
before EO (26), is present after EO in PSD95 KD neurons. This
suggests that SAP102 binding triheteromeric GluN1/GluN2B/
GluN2A NMDARs remains at the PSD in these cells.
Author contributions: J.-P.Z. and M.C.-P. designed research; J.-P.Z. and Y.M. performed
research; J.-P.Z. and Y.M. analyzed data; and J.-P.Z. and M.C.-P. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1
To whom correspondence may be addressed. E-mail: jpzhao@mit.edu or mcpaton@mit.
edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1215854110/-/DCSupplemental.
PNAS | January 8, 2013 | vol. 110 | no. 2 | 707–712
NEUROSCIENCE
Edited* by Charles F. Stevens, The Salk Institute for Biological Studies, La Jolla, CA, and approved November 28, 2012 (received for review September 11, 2012)
Results
LTP Is Labile in the Young SC. In SC, acute slices from postnatal day
15 (P15) to P17 rat pups with EO at P13 or P14, stimulating the
stratum opticum at 20 Hz for 20 s produces an NMDAR- and
L-type Ca2+ channel-dependent LTP in a major excitatory SC
neuron population, narrow field vertical (NFV) neurons: a group
we have focused LTP studies on because they are visually driven,
abundant, and of relatively uniform size (32). This same stimulation applied to slices from pups of the same age with eye closure
(EC) showed no LTP in NFV neurons. However, if similarly
deprived pups received 4–5 h of visual experience after simultaneous EO, LTP could be induced (Fig. 1A). The same EOdependent LTP was induced in slices from pups killed 2–4 d after
EO, but not in pups with EC during the same period (Fig. 1B).
Also when only one eye remained shut and the other was opened
at P13 with patterned visual experience for 3–4 h before killing,
LTP was induced only in neurons that had received input from
the contralateral open eye (Fig. 1C). In a final paradigm, glued
eyelids were opened at P13, and then reclosed on P16. At P20,
one-half of the animals had their eyelids reopened for 4–5 h
before killing and recording. SC LTP was obtained in slices from
these eye-reopened animals. However, LTP was not present in
the reclosed animals whose eyes were not reopened before P20
A
P0
B
P13
P13 P15-17
P0
P15-17
EO
EO
P0
P0
P11-12
EC
EC
EPSP slope (%)
1.5
1.5
EO
1
EO
1
0.5
0.5
EC
0
0
10
20
30
Time (min)
C
40
50
0
D
P13
0
10
20
30
Time (min)
40
P0
P13 P16 P20
P0
P13 P16 P20-21
EO
EPSP slope (%)
1.5
50
ERO
EC
P0
EC
ERC
1.5
EO
ERO
1
1
0.5
0.5
EC
0
0
10
20
30
Time (min)
40
50
0
ERC
0
10
20
30
Time (min)
40
50
Fig. 1. SC LTP induction depends on pattern vision. (A) LTP is induced in SC
slices from P13 rats, killed 4–5 h after EO (1.22 ± 0.05, n = 11/4, P < 0.01). LTP
could not be induced in P11–P12 slices from rats with EC (0.91 ± 0.07, n = 8/3,
P = 0.17). In this and subsequent LTP/LTD figures, the inset traces represent
averages of 30 consecutive responses obtained during baseline (thin traces)
and 30–40 min following completion of induction (thick traces) (scale bars:
5 mV, 20 ms); the arrow indicates induction onset; the bars and diagrams
above graphs indicate ages at manipulation or analysis. (B) LTP is present in
SC slices from P15–P17 rats, 2–4 d after EO (1.25 ± 0.06, n = 15/4, P < 0.01), but
absent in age-matched littermates with EC (1.02 ± 0.08, n = 9/3, P = 0.59). (C)
LTP was induced in P13 rat slices from the SC contralateral to the single eye
that was open for 4–5 h before killing (1.24 ± 0.07, n = 8/3, P < 0.01), but LTP
could not be induced in age-matched littermate slices from the SC contralateral to the closed eye (0.95 ± 0.05, n = 9/3, P = 0.27). (D) In SC slices of P20–
P21 rats 4–5 d after ERC at P16 following normal EO at P13, LTP disappeared
(0.98 ± 0.06, n = 9/3, P = 0.74); in contrast, LTP could be reinstated in littermates killed 4–5 h after ERO at P20 (1.2 ± 0.03, n = 9/3, P < 0.01).
708 | www.pnas.org/cgi/doi/10.1073/pnas.1215854110
recording (Fig. 1D). Therefore, young SC LTP had a pronounced dependence on pattern vision as well as a pronounced
lability to loss of pattern vision. Importantly, these EC and EO
regimes were identical to ones used in the initial study of ECand EO-associated changes in synaptic levels of PSD95 in visual
cortex and SC synapses (9). This tight correlation suggested that
PSD95, or molecules in a complex with PSD95 at the synapse
were necessary for induction of SC LTP.
SAP102 and PSD95 Are the Only MAGUKs in Young SC. We documented the MAGUKs present in the SC with quantitative Western
blotting of PSD95, PSD93, and SAP102. SAP97 immunoreactivity
is not present in the SC (33). Homogenates from the SC at P15 and
hippocampus at P19, two roughly corresponding stages of synapse
maturity (4, 9, 25), were analyzed. Identical protein concentrations
from SC and hippocampus were run in adjacent lanes (Fig. 2A),
blotted, and probed with antibodies for PSD95, SAP102, and
PSD93. SAP102 and PSD95 bands were present in both lanes,
with the expected, higher levels in the hippocampus. However,
PSD93 was present as an intense band only in hippocampal lanes
and barely detectable SC lanes (Fig. 2A).
PSD95 KD Decreases mAMPARc and mNMDARc Frequency. We
designed two KD shRNAs (KD I and II) against PSD95, and
two scrambled shRNAs (Scr I and II) as negative controls.
These shRNAs were inserted into the lentiviral plasmid carrying GFP, and lentiviruses were produced. Efficacy and
specificity of these shRNAs were documented in HEK cells
and cultured occipital cortical neurons (Fig. 2 B and C). The
lentiviruses were injected into the SC of neonates (P1–P3).
The eyes of these pups were opened at P14 and they were
killed between P15 and P17 for slice physiology with the investigator blind to the shRNA lentiviruses injected. In acute
SC slices, infected neurons were identified by their GFP fluorescence followed by infrared differential interference contrast for
whole-cell patch electrode placement on NFV neurons, and
mAMPARcs and mNMDARcs were isolated with appropriate
antagonists. All neurons expressing the PSD95 KD shRNAs
showed significantly lower mAMPARc frequencies than the
corresponding Scr controls (Fig. 3), and neither KD I nor KD
II had an effect on mAMPARc amplitude (Fig. S1). Frequencies and amplitudes in uninfected NFV neurons in the
same slices were not significantly different from those recorded in neurons from the same animal infected with the Scr
control lentiviruses (Fig. S2 A–C and F–H). Rise and decay
times of average mAMPARcs were also unchanged between
PSD95 KD, Scr, and uninfected neurons (Fig. S2 D and E, and I
and J). Decreases in mAMPARc frequency have been documented in virtually all PSD95 KD or KO studies in CA1 pyramids
where they were examined (28–30); however, none of these
studies reported changes in mNMDARcs. In the relatively small
NFV neurons, it was possible to record mNMDARcs, and
similar to the mAMPARcs, their frequencies (Fig. 4 A–D) but not
their amplitudes (Fig. S3) were significantly reduced in all PSD95
KD cells. To assay for an effect of PSD95 KD shRNA on presynaptic release (34) that might cause a change in both mAMPARc and mNMDARc frequencies, we examined paired-pulse
ratios (PPRs). In all cases, PPRs were identical for PSD95 KD and
Scr neurons (Fig. 4 E and F). Also consistent with decreases in
both mAMPARc and mNMDARc frequencies, we found no
differences in evoked AMPAR current (eAMPARc)/evoked
NMDAR current (eNMDARc) ratios between neurons expressing
KD I or II and their corresponding Scr controls (Fig. 4 G and H).
In Vivo PSD95 KD Eliminates NMDAR-Dependent LTP. Characterization of SC LTP in whole-cell patch-clamped NFV neurons
expressing the PSD95 KD or the corresponding Scr shRNA
revealed that LTP was absent in all PSD95 KD neurons but
present in all Scr-expressing neurons (Fig. 5 A and B). In addition, because the mAMPARc and mNMDARc recordings indicated reduced numbers of AMPAR- and NMDAR-containing
Zhao et al.
PSD-95 scrambled II (Scr II)
PSD-95 shRNA II (KD II)
PSD-95 scrambled II (Scr II)
HEK cell lysate
Hip
PSD-95 shRNA II (KD II)
Cortical culture
PSD-95 scrambled I (Scr I)
PSD-95 shRNA I (KD I)
Cortical culture
C
SA
P1
PS 02
D
PS -95
D
β -93
ac
tin
GFP-only plasmid
B
GFP-only plasmid
A
PSD-95 shRNA I (KD I)
PSD-95 scrambled I (Scr I)
HEK cell lysate
SC
Fig. 2. PSD95 and SAP102 dominate in the young
SC, and shRNA against PSD95 causes effective KD of
this MAGUK in HEK cells and cultured cortical
SAP102
neurons. (A) Homogenates from rat SC and hippoPSD-95
campus (Hip) at P15 and P19, respectively, analyzed
by Western blotting (20 μg/lane). SAP102 and PSD95,
PSD-93
but not PSD93, are the two dominant MAGUKs
β actin
expressed in the young SC. Compared with corresponding protein levels in Hip, SAP102 and PSD95
Hip
SC
were significantly lower and PSD93 was very signifi1.2
*
* ***
cantly lower in SC. (B) HEK 293 cells were cotransfected with PSD95-GFP and either a PSD95 KD shRNA
0.8
plasmid or a scrambled (Scr) shRNA plasmid. After
PSD-9
PSD-95
0.4
48 h of cotransfection, cell lysates were analyzed by
Western blotting. Both PSD95 shRNA KD I and KD II
0.0
Tubulin
SAP10
eliminated detectable PSD95, whereas shRNA Scr I
and Scr II had no effect on PSD95 expression. (C)
Tubulin
Cultured occipital cortical neurons were infected
with PSD95 KD or scrambled shRNA lentiviruses at
day in vitro (DIV) 2 and analyzed by Western blotting at DIV 21. Endogenous protein expression level of PSD95 was reduced by PSD95 shRNA KD I or KD II, whereas
shRNA Scr I or Scr II did not change PSD95 expression.
LTD Is Normal in SC PSD95 KD Neurons. Colledge et al. (35) pro-
posed that PSD95 removal could be causative in NMDAR-dependent LTD. Also, LTD is absent in the hippocampus of PSD95
KO mice (14, 15, 28) and impaired in PSD95 KD neurons in
cultured hippocampal slices (30, 31). However, there is evidence
that only extrasynaptic NMDARs are coupled to LTD generation
(36, 37), and in the SC, normal NMDAR-dependent LTD is
present in young GluN2A KO mice (38). These mice have no
mNMDARcs and spontaneous NMDAR currents (sNMDARcs)
probably because once PSD95 is at the PSD, it requires the
GluN2A tail to bind NMDARs (13, 20). Moreover, in both WT
and GluN2A KO mice, SC LTD can be eliminated by blockade of
A
Scr I
KD I
0
0
p<0.01
0.6
0
Scr I KD I
10
20
30
mAMPARc IEI (sec)
40
1
0.5
Scr II
KD II
0
0
Frequency (Hz)
0.5
1.2
Cumulative probability
D
1
Frequency (Hz)
B
Cumulative probability
KD I
KD II
Scr I
Scr II
C
1.6 p=0.01
0.8
0
Scr II KD II
10
20
30
mAMPARc IEI (sec)
40
Fig. 3. PSD95 KD reduces mAMPARc frequency. (A and C) Sample traces
recorded at −70 mV of mAMPARcs from shRNA Scr I and KD I-expressing
neurons and from shRNA Scr II and KD II-expressing neurons. (Scale bars: 10
pA, 128 ms.) (B and D) Cumulative distributions of mAMPARc interevent
intervals (IEI) from all sets of neurons. (Insets) Bar graphs showing significant
differences in mAMPARc frequency [(B) Scr I: 0.75 ± 0.09 Hz, n = 15/4, vs. KD I:
0.31 ± 0.05 Hz, n = 16/3, P < 0.01; (D) Scr II: 1.29 ± 0.2 Hz, n = 7/2, vs. KD II:
0.39 ± 0.08 Hz, n = 8/2, P = 0.01].
Zhao et al.
either GluN2B receptors or L-type Ca2+ channels (38). Consequently, we hypothesized that NMDAR-dependent LTD in the
PSD95 KD SC neurons would not show disrupted LTD. Indeed,
we found that NMDAR-dependent LTD remained in these neurons and was identical in amplitude to the LTD induced in neurons expressing the corresponding Scr shRNA (Fig. 5 C and D).
FK506 Increases sNMDARc Decay Time in PSD95 KD Neurons. After
PSD95 KD, the only other significant MAGUK in the young SC,
SAP102, was expected to be the remaining scaffold at the PSD.
However, in all of the PSD95 KD cells we recorded sNMDARcs
with the short decay-time characteristic of the diheteromeric
GluN1/GluN2A NMDARs usually bound by PSD95 after EO (9)
in both the PSD95 KD and Scr neurons (Fig. 6 A, C, E, and G,
upper three tracesfigE). In normal SC neurons, the effect of the
CaN blockade disappears before EO as the level of PSD95 and
GluN2A increase (9, 12). However, the GluN2A subunit protein,
first detectable in the SC at ∼P7, is significantly increased in the
P15–P17 EO pups studied here (12). It is also likely that these
increases are independent of PSD95. Consequently, we tested the
hypothesis that the pre-EO decrease in NMDARc decay times
that were observed with GluN1/GluN2A/GluN2B triheteromeric
NMDARs bound by SAP102 had reappeared in PSD95 KD
neurons by applying the membrane-permeable CaN antagonist
FK506. In the study by Shi et al. (26), both FK506 and the CaN
inhibitory peptide effectively eliminated the decay-time decrease. We bath-applied FK506 and within 12 min PSD95 KD
neurons developed long sNMDARc decay times (Fig. 6 C and G,
lower three traces, and D and H) typical of GluN2A/GluN2Brich NMDARcs in younger animals with SAP102 as the PSD
MAGUK FK506 had no effect on neurons carrying the corresponding Scr shRNA (Fig. 6 A and E, lower three traces, and B
and F). The results indicate that SAP102 binding NMDARs
by the GluN2B C-terminal (16) supported GluN2B/GluN2A
NMDARs at the PSD despite EO, and that the generally higher
levels of activity after EO when it impinges on the PSD95 KD
neurons triggered the same activity-dependent CaN response
documented by Shi et al. (26). Thus, the normal NMDAR activitydependent CaN shortening of NMDARc decay times found before EO could remain active on triheteromeric GluN1/GluN2A/
GluN2B NMDARs at older SC synapses because activity was
sufficiently high and because SAP102 binding NMDARs by the
GluN2B C-terminal remained at the PSD.
Discussion
Despite many reports describing the functions of PSD95 at glutamate synapses, identification of specific roles for synaptic
MAGUKs has been difficult because the KD and KO work has
been focused on the hippocampus where four MAGUK family
PNAS | January 8, 2013 | vol. 110 | no. 2 | 709
NEUROSCIENCE
synapses in PSD95 KD cells, we compared the amplitude and
frequency of responses to inducing stimuli that were determined
for each neuron as the intensity producing a response of halfmaximal size. We found no differences in the amplitudes of the
evoked responses or in their ability to follow each stimulating
pulse between neurons expressing the PSD95 KD or corresponding Scr shRNAs (Fig. S4).
and mNMDARc frequency with no change in presynaptic release.
In addition, we show that young SC LTP in vivo is critically linked
to pattern vision. This LTP is not stable at least within the week
after EO when ERC for several days causes the LTP to disappear
and ERO reintroduces LTP. Lability of LTP on the order of
minutes to hours has been previously noted in the optic tectum
and hippocampus upon changes in activity (43), but to date there
have been no attempts to abnormally reduce activity for days in
other young brain regions where PSD95 has recently appeared.
C
p=0.56
2
1
0
Frequency(Hz)
Fig. 4. PSD95 KD reduces mNMDARc frequency, but not PPR or evoked
AMPARc/NMDARc ratio. (A and C) Sample traces recorded at −70 mV of
mNMDARcs from shRNA Scr I and KD I-expressing neurons and from shRNA
Scr II and KD II-expressing neurons. (Scale bars: 10 pA, 200 ms.) (B and D)
Cumulative distributions of mNMDARc interevent intervals (IEI) from the all
sets of neurons. (Insets) The bar graphs showing significant differences in
mNMDARc frequency [(B) Scr I: 0.17 ± 0.03 Hz, n = 9/3, vs. KD I: 0.1 ± 0.009
Hz, n = 9/3, P = 0.01; (D) Scr II: 0.23 ± 0.04 Hz, n = 9/3, vs. KD II: 0.12 ± 0.009
Hz, n = 8/3, P = 0.02]. (E and F) (Upper traces) Samples of average pairedpulse evoked AMPARcs from Scr I and KD I neurons, and from Scr II and KD II
neurons. (Scale bars: 20 pA, 50 ms.) (Lower) Bar graphs showing no significant differences in PPR [(E) Scr I: 1.08 ± 0.13, n = 14/3, vs. KD I: 1.3 ± 0.18, n =
11/3, P = 0.35; (F) Scr II: 1.13 ± 0.26, n = 7/2, vs. KD II: 0.93 ± 0.13, n = 11/3, P =
0.42]. (G and H) (Upper traces) Samples of averaged eAMPARc and eNMDARc from Scr I and KD I neurons and from Scr II and KD II neurons. (Scale
bars: 20 pA, 100 ms.) (Lower) Bar graphs showing no significant differences
in eAMPARc/eNMDARc ratio [(G) Scr I: 1.91 ± 0.18, n = 11/4, vs. KD I: 2.29 ±
0.13, n = 9/3, P = 0.21; (H) Scr II: 1.83 ± 0.18, n = 10/3, vs. KD II: 1.73 ± 0.12, n =
12/4, P = 0.56].
members can scaffold glutamate receptors and several can
compensate for each other (15, 29). The hippocampus is one of
the most highly evolved structures in the mammalian brain necessary for many forms of learning and memory and therefore
equipped with many compensating mechanisms to maintain its
critical functions with the specialized mammalian neocortex. By
contrast, the SC continues to subserve most of the functions of
localization in space, persistent activity, and initiation of single or
multisensory motor output that has been crucial to the survival of
the vertebrate line throughout evolution (39, 40). However, the SC
and its nonmammalian homolog the optic tectum, like most other
central nervous system regions, maintain activity-dependent interactions critical to adaptive circuitry during development (41,
42). In this report, we document effects of SC PSD95 KD during
development that are unexpected from the MAGUK manipulations performed in hippocampal CA1. We show here that PSD95
KD in the developing SC eliminates NMDAR-dependent LTP,
and in CA1 hippocampus it does not. PSD95 KD in SC does not
eliminate NMDAR-dependent LTD, and in the hippocampus it
does. PSD95 KD in SC also causes reductions in both mAMPARc
710 | www.pnas.org/cgi/doi/10.1073/pnas.1215854110
A
B
Scr I
1.5
EPSP slope (%)
KD II
n=10/3
p=0.21
H Scr II
60
n=12/4
KD I
2
0
0
Scr II KD II
20
40
mNMDARc IEI (sec)
Scr I
1
0.1
In acute SC slices, robust LTP requires NMDAR and L-type Ca2+
channel activity (32) as well as GluN2A subunits at the PSD
(20, 38), and, as shown here (Fig. 1), SC LTP in the superficial
visual layers only occurs after EO. This potentiation results
almost entirely from unsilencing of silent synapses (32). Fig. 5 A
and B demonstrates that PSD95 KD eliminates SC LTP; therefore,
both high levels of synaptic PSD95 and its unique ability to bind
NMDARs having two GluN2A subunits at the center of SC synapses are necessary for NMDAR-dependent LTP in the developing
SC. Furthermore, the finding that mixed triheteromeric NMDARs
reappear at the PSD of PSD95 KD cells supports our previous
proposal that the insertion of PSD95 actively displaces SAP102
bound receptors from the center to the extrasynaptic region of
synapse (13). The current evidence for a crucial function of PSD95
in SC synaptic increases is fully consistent with the study by Phillips
et al. (7) where another type of SC neurons, dorsally oriented
vertical neurons, in the PSD95 KO mouse (14) fail to show the
significant increase in synapse number found in WT SC neurons
upon EO and the onset of pattern vision. These new synapses are
from the cortico-collicular projection, which develops later than
retinal inputs, and, without EO, this set of converging inputs is not
only functionally but also structurally withdrawn (7). The data of
Phillips et al. therefore reinforce the present results by showing
that new SC synapses resulting from activity increases cannot be
stabilized unless PSD95 is present. This report shows that SC LTP
disappears with several days of ERC after EO, and reappears with
ERO. This is completely consistent with the data of Yoshii et al.
(9) showing corresponding decreases and reincreases of PSD95
levels in visual synapses using the same EO, EC paradigms used
Scr II
1.5
1
1
0.5
0.5
KD I
0
KD II
0
0
10
C 1.5
EPSP slope (%)
0
G
0.2
eAMPARc/eNMDARc
0
n=9/3
p=0.42
1
0.5
0
0.3 p=0.02
Sc
rI
I
KD
II
Sc
rI
KD
I
0
KD II
Scr II
KD II
n=11/4
1
0.5
1.5
0.5
rI
KD
I
p=0.35
F Scr II
Functional Effects of EO and PSD95 on SC LTP and Synapse Stabilization.
1
60
eAMPARc/eNMDARc
1.5
KD I
n=14/3
Paired-pulse ratio
Scr I
0
Scr I KD I
20
40
mNMDARc IEI (sec)
n=11/3
E
0.1
n=7/2
0
0.2
Sc
rI
I
KD
II
0
p=0.01
n=11/3
Scr I
KD I
Frequency(Hz)
0.5
0.3
Cumulative probability
D
1
Paired-pulse ratio
Cumulative probability
B
Sc
KD I
KD II
Scr I
Scr II
A
20 30
40
Time (min)
50
0
60
10
D
20 30
40
Time (min)
50
60
1.5
Scr II
Scr I
1
1
0.5
0.5
KD II
KD I
0
0
0
10
20
30 40 50
Time (min)
60
70
0
10
20
30 40 50
Time (min)
60
70
Fig. 5. In vivo PSD95 KD affects LTP, but not LTD. (A and B) LTP was normal
in shRNA Scr I and Scr II-expressing neurons but absent in shRNA KD I and KD
II-expressing neurons [(A) Scr I: 1.21 ± 0.04, n = 6/2, P < 0.01; KD I: 0.99 ± 0.04,
n = 7/2, P = 0.73; (B) Scr II: 1.24 ± 0.06, n = 7/2, P < 0.01; KD II: 0.99 ± 0.04, n =
7/2, P = 0.94]. (C and D) LTD was induced in both Scr I and KD I and Scr II and
KD II neurons [(C) Scr I: 0.78 ± 0.07, n = 7/3, P < 0.03; KD I: 0.74 ± 0.03, n = 11/
3, P < 0.01; Scr I vs. KD I, P = 0.59; (D) Scr II: 0.76 ± 0.04, n = 11/4, P < 0.01; KD
II: 0.81 ± 0.05, n = 13/5, P < 0.01; Scr II vs. KD II, P = 0.43]. The black bars
indicate application of LTD induction stimulation.
Zhao et al.
After
FK506
Before
FK506
E
80
p=0.06
D
After
FK506
60
40
Before
FK506
20
Be
FK for
50 e
6
Af
FK te
50 r
6
B
G
Scr II
Before
FK506
After
FK506
After
FK506
After
FK506
Before
FK506
80
p=0.64
60
20
80
p<0.01
60
40
20
KD II
H
80
After
FK506
40
Be
FK for
50 e
6
Af
FK ter
50
6
F
Decay time (ms)
Before
FK506
Decay time (ms)
After
FK506
Be
FK for
50 e
6
A
FK fte
50 r
6
After
FK506
Decay time (ms)
Before
FK506
Decay time (ms)
Before
FK506
KD I
p<0.01
60
40
Before
FK506
20
Fig. 6. CaN inhibition increases sNMDARc decay time in PSD95 KD neurons.
(A, C, E, and G) Sample traces recorded at +40 mV of sNMDARcs from shRNA
Scr I and KD I and from shRNA Scr II and KD II-expressing neurons before and
after bath application of the CaN inhibitor FK506. (Scale bars: 10 pA, 200
ms.) (B, F) (Left) Superposition of average scaled sNMDARcs obtained before
and 12–15 min after FK506 application from the same Scr I and Scr II neuron.
(Right) Pooled data showing no significant differences in the decay times of
average sNMDARcs before and after FK506 application in Scr I or Scr II
neurons [(B) Scr I, n = 7/2, P = 0.06; (F) Scr II, n = 6/2, P = 0.64]. (D and H) (Left)
Superposition of average scaled sNMDARcs obtained from the same PSD95
KD I and KD II neurons before and 12–15 min after FK506 application. (Right)
Pooled data showing significant lengthening of sNMDARc decay times 12–15
min after FK506 application [(D) KD I, n = 8/2, P < 0.01; (G) KD II, n = 8/3, P <
0.01]. Paired t tests were used, the open diamonds represent individual
experiments, and the filled diamonds are means of all of the experiments in
groups (B, D, F, and H).
here. It remains to be seen whether this highly labile form of
synapse potentiation and stabilization is also present in older brains
and other visual centers after prolonged pattern vision deprivation.
LTD Survives PSD95 KD in SC. This finding also differs from those in
the hippocampus where the KD or KO of PSD95 eliminates
NMDAR-dependent LTD. In the hippocampus where PSD93
remains to compensate for PSD95 KO or KD (14, 15, 30, 31), LTD
but not LTP disappears. Carlisle et al. (15) suggest that PSD93
may bind the signaling complex necessary for LTP while PSD95 is
involved in LTD and normally mitigates the potentiating effects of
LTP resulting in the enhanced LTP seen in the hippocampus of
genetic PSD95 KOs (14, 28). However, the lack of PSD93 in the
young SC is not consistent either with the loss of LTP or the
maintenance of LTD in this structure when PSD95 is depleted. Xu
et al. (31) also provided evidence for PSD95 involvement in LTD.
With a series of deletion constructs and point mutations in the
PSD95 C terminus following WT PSD95 depletion by shRNA,
Zhao et al.
they showed that the C-terminal domain of PSD95 normally
scaffolds the signaling complex necessary for CA1 LTD. This explanation is also not consistent with our finding that NMDARdependent LTD is maintained in SC neurons after PSD95 KD.
However, CaN activity is believed to be critical to NMDAR-dependent LTD at least in the hippocampus (44, 45), and, as noted
above, both GluN2B and L-type Ca2+ channels are necessary for
SC LTD. In addition, this study demonstrates CaN involvement in
decreasing sNMDARc decay time when SAP102 is the only
remaining major MAGUK in SC neurons. These findings suggest
that the early appearing MAGUK SAP102 that is still highly
expressed in the young SC after EO can also scaffold the complex
for inducing LTD.
CaN-Mediated Decrease of sNMDARc Decay Time. A final finding in
this study is that the CaN-mediated decrease in sNMDARc decay
times (26) observed in normal SC neurons can be retained after
EO in SC PSD95 KD neurons. This CaN effect involves a dephosphorylation of at least one protein kinase A (PKA) site,
serine 900, on the GluN2A cytoplasmic tail (27). Similar CaN
activity was described by Lieberman and Moody (46) as a CaNdependent change in NMDAR channel open time, in analyses of
single channel currents. Krupp et al. (47) found that the same
decrease in NMDARc decay time resulted from CaN dephosphorylation of two PKA sites on the GluN2A cytoplasmic
tail. The present data show that this NMDARc decay-time
shortening can reappear when PSD95 KD causes the GluN2B
subunit composition of NMDARs at PSDs to be unusually high
and when input activity is increased due to pattern vision. The
finding is potentially significant for understanding differences
between the subunit makeup of NMDARs throughout the brain
and the activity-dependent control of the receptor’s currents. For
example, Flint et al. (48) documented short NMDARc decay
times characteristic of GluN1/GluN2A diheteromeric NMDAR
in young somatosensory cortex neurons when measured levels of
GluN2A were still extremely low. They concluded that just one
GluN2A subunit in a triheteromeric receptor with GluN2B was
sufficient to shorten the decay times of the NMDARcs. It is
likely that the CaN-mediated dephosphorylation of GluN2A is
responsible for the short NMDARc decay times reported by
Flint et al. (48). The same CaN mechanism may also explain why
recent biochemical analyses of synaptic NMDAR composition
report a prevalence of GluN1/GluN2A/GluN2B triheteromers at
mature glutamate synapses even though sNMDARc decay times
are short in the mature brain (49, 50). Finally, the reappearance
of this CaN effect in older SC neurons when levels of triheteromeric NMDARs are abnormally present and when converging
glutamatergic activity is high suggests that it may represent yet one
more mechanism for homeostatically regulating cytoplasmic Ca2+
concentrations in the brain (51).
Conclusion
We document a requirement for PSD95 for the maintenance of
the normal levels of mAMPARc and mNMDARc frequency,
normal synaptic input, and for the appearance of NMDARdependent LTP in the SC where only SAP102 and PSD95
MAGUKs are normally prominent. Unlike normal SC neurons
where diheteromeric GluN1/GluN2A NMDARs are at the PSD,
in PSD95 KD SC neurons triheteromeric NMDARs bound by
SAP102 are at the PSD. Nevertheless, in the P15–P17 rats
studied, the SAP102 MAGUK cannot replace the PSD95 function of binding GluN2A diheteromeric receptors or facilitating
NMDAR-dependent LTP. Most significantly, this study suggests
that much of the intensive and sophisticated work on MAGUK
function in hippocampal CA1, may not generalize to synaptogenesis and developmental plasticity in many other regions of the
vertebrate central nervous system.
Methods
Animals. Sprague Dawley rat pups were treated with synchronized EC, EO,
ERC, and ERO procedures as described previously (9). All experiments were
PNAS | January 8, 2013 | vol. 110 | no. 2 | 711
NEUROSCIENCE
C
Scr I
Be
FK for
50 e
6
Af
FK te
50 r
6
A
performed in accord with the guidelines of the Massachusetts Institute of
Technology Institutional Animal Care and Use Committee.
Construction of Lentiviral Vectors. Two shRNAs (shRNA KD I and II) against
mRNA sequences unique to PSD95 and also two similar but scrambled shRNAs
(shRNA Scr I and II) as negative controls were designed. These were inserted
into the lentiviral plasmid carrying GFP, and lentiviruses were produced in HEK
cells (see SI Methods for shRNA sequences and further details).
Expression Analysis of MAGUKs in Developing SC and Hippocampus. Homogenates from rat SC and hippocampus at P15 and P19, respectively, were
analyzed by Western blotting for evaluation of PSD95, SAP102, and PSD93
protein levels (SI Methods).
Electrophysiological Recordings in Mouse Acute Slices. Acute parasagittal SC
slices were prepared as previously described (32). Whole-cell recordings were
made in NFV neurons in the mid-stratum griseum superficiale of the SC. Detailed conditions for recordings of excitatory postsynaptic potential, excitatory
postsynaptic current, LTP/LTD induction, measurement, and chemicals are
described in SI Methods.
Statistics. Data are shown as mean ± SEM, and n is given as the number of
neurons recorded/the number of animals used. Statistical significance was
determined using the two-tail unpaired Student t test unless otherwise stated
following an F test indicating a normal distribution.
Injection of Lentivirus into the SC. P1–P3 rat pups were cold-anesthetized, and
lentiviral solution (0.1 μL) was injected into the SC (SI Methods).
ACKNOWLEDGMENTS. We thank Johannes Hell for providing the antiSAP102 antibody, Morgan Sheng for providing the anti-SAP97 antibody, and
Carlos Lois for providing the lentivirus plasmids. This work was supported by
National Institutes of Health Grant EY014074 (to M.C.-P.).
1. Elias GM, Nicoll RA (2007) Synaptic trafficking of glutamate receptors by MAGUK
scaffolding proteins. Trends Cell Biol 17(7):343–352.
2. Sheng M, Hoogenraad CC (2007) The postsynaptic architecture of excitatory synapses:
A more quantitative view. Annu Rev Biochem 76:823–847.
3. Zheng C-Y, Seabold GK, Horak M, Petralia RS (2011) MAGUKs, synaptic development,
and synaptic plasticity. Neuroscientist 17(5):493–512.
4. Sans N, et al. (2000) A developmental change in NMDA receptor-associated proteins
at hippocampal synapses. J Neurosci 20(3):1260–1271.
5. White LE, Fitzpatrick D (2007) Vision and cortical map development. Neuron 56(2):
327–338.
6. Prusky GT, Silver BD, Tschetter WW, Alam NM, Douglas RM (2008) Experience-dependent plasticity from eye opening enables lasting, visual cortex-dependent enhancement of motion vision. J Neurosci 28(39):9817–9827.
7. Phillips MA, et al. (2011) A synaptic strategy for consolidation of convergent visuotopic maps. Neuron 71(4):710–724.
8. Rittenhouse CD, Majewska AK (2009) Synaptic mechanisms of activity-dependent
remodeling in visual cortex during monocular deprivation. J Exp Neurosci 2:32–41.
9. Yoshii A, Sheng MH, Constantine-Paton M (2003) Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons. Proc Natl Acad Sci USA 100(3):
1334–1339.
10. Yoshii A, et al. (2011) TrkB and protein kinase Mζ regulate synaptic localization of
PSD-95 in developing cortex. J Neurosci 31(33):11894–11904.
11. Lu W, Constantine-Paton M (2004) Eye opening rapidly induces synaptic potentiation
and refinement. Neuron 43(2):237–249.
12. Shi J, Aamodt SM, Constantine-Paton M (1997) Temporal correlations between
functional and molecular changes in NMDA receptors and GABA neurotransmission
in the superior colliculus. J Neurosci 17(16):6264–6276.
13. van Zundert B, Yoshii A, Constantine-Paton M (2004) Receptor compartmentalization
and trafficking at glutamate synapses: A developmental proposal. Trends Neurosci
27(7):428–437.
14. Migaud M, et al. (1998) Enhanced long-term potentiation and impaired learning in
mice with mutant postsynaptic density-95 protein. Nature 396(6710):433–439.
15. Carlisle HJ, Fink AE, Grant SGN, O’Dell TJ (2008) Opposing effects of PSD-93 and PSD95 on long-term potentiation and spike timing-dependent plasticity. J Physiol 586(Pt
24):5885–5900.
16. Chen B-S, Thomas EV, Sanz-Clemente A, Roche KW (2011) NMDA receptor-dependent
regulation of dendritic spine morphology by SAP102 splice variants. J Neurosci 31(1):89–96.
17. Kim MJ, Dunah AW, Wang YT, Sheng M (2005) Differential roles of NR2A- and NR2Bcontaining NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking.
Neuron 46(5):745–760.
18. Yoshii A, Constantine-Paton M (2007) BDNF induces transport of PSD-95 to dendrites
through PI3K-AKT signaling after NMDA receptor activation. Nat Neurosci 10(6):702–711.
19. Steigerwald F, et al. (2000) C-Terminal truncation of NR2A subunits impairs synaptic
but not extrasynaptic localization of NMDA receptors. J Neurosci 20(12):4573–4581.
20. Townsend M, Yoshii A, Mishina M, Constantine-Paton M (2003) Developmental loss of
miniature N-methyl-D-aspartate receptor currents in NR2A knockout mice. Proc Natl
Acad Sci USA 100(3):1340–1345.
21. Stocca G, Vicini S (1998) Increased contribution of NR2A subunit to synaptic NMDA
receptors in developing rat cortical neurons. J Physiol 507(Pt 1):13–24.
22. Hardingham GE, Fukunaga Y, Bading H (2002) Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci
5(5):405–414.
23. Atasoy D, et al. (2008) Spontaneous and evoked glutamate release activates two
populations of NMDA receptors with limited overlap. J Neurosci 28(40):10151–10166.
24. Zhang J, Diamond JS (2009) Subunit- and pathway-specific localization of NMDA
receptors and scaffolding proteins at ganglion cell synapses in rat retina. J Neurosci
29(13):4274–4286.
25. Elias GM, Elias LAB, Apostolides PF, Kriegstein AR, Nicoll RA (2008) Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development. Proc Natl Acad Sci USA 105(52):20953–20958.
26. Shi J, Townsend M, Constantine-Paton M (2000) Activity-dependent induction of tonic
calcineurin activity mediates a rapid developmental downregulation of NMDA receptor currents. Neuron 28(1):103–114.
27. Townsend M, Liu Y, Constantine-Paton M (2004) Retina-driven dephosphorylation of
the NR2A subunit correlates with faster NMDA receptor kinetics at developing retinocollicular synapses. J Neurosci 24(49):11098–11107.
28. Béïque J-C, et al. (2006) Synapse-specific regulation of AMPA receptor function by
PSD-95. Proc Natl Acad Sci USA 103(51):19535–19540.
29. Elias GM, et al. (2006) Synapse-specific and developmentally regulated targeting of
AMPA receptors by a family of MAGUK scaffolding proteins. Neuron 52(2):307–320.
30. Ehrlich I, Klein M, Rumpel S, Malinow R (2007) PSD-95 is required for activity-driven
synapse stabilization. Proc Natl Acad Sci USA 104(10):4176–4181.
31. Xu W, et al. (2008) Molecular dissociation of the role of PSD-95 in regulating synaptic
strength and LTD. Neuron 57(2):248–262.
32. Zhao J-P, Phillips MA, Constantine-Paton M (2006) Long-term potentiation in the
juvenile superior colliculus requires simultaneous activation of NMDA receptors and
L-type Ca2+ channels and reflects addition of newly functional synapses. J Neurosci
26(49):12647–12655.
33. Müller BM, et al. (1995) Molecular characterization and spatial distribution of SAP97,
a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large
tumor suppressor protein. J Neurosci 15(3 Pt 2):2354–2366.
34. Futai K, et al. (2007) Retrograde modulation of presynaptic release probability
through signaling mediated by PSD-95-neuroligin. Nat Neurosci 10(2):186–195.
35. Colledge M, et al. (2003) Ubiquitination regulates PSD-95 degradation and AMPA
receptor surface expression. Neuron 40(3):595–607.
36. Lu W, et al. (2001) Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron
29(1):243–254.
37. Massey PV, et al. (2004) Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression. J Neurosci
24(36):7821–7828.
38. Zhao J-P, Constantine-Paton M (2007) NR2A−/− mice lack long-term potentiation but
retain NMDA receptor and L-type Ca2+ channel-dependent long-term depression in
the juvenile superior colliculus. J Neurosci 27(50):13649–13654.
39. Nevin LM, Robles E, Baier H, Scott EK (2010) Focusing on optic tectum circuitry
through the lens of genetics. BMC Biol 8:126.
40. Stein BE, Rowland BA (2011) Organization and plasticity in multisensory integration:
Early and late experience affects its governing principles. Prog Brain Res 191:145–163.
41. Constantine-Paton M, Cline HT, Debski E (1990) Patterned activity, synaptic convergence, and the NMDA receptor in developing visual pathways. Annu Rev Neurosci 13:
129–154.
42. Ruthazer ES, Cline HT (2004) Insights into activity-dependent map formation from the
retinotectal system: A middle-of-the-brain perspective. J Neurobiol 59(1):134–146.
43. Zhou Q, Poo MM (2004) Reversal and consolidation of activity-induced synaptic
modifications. Trends Neurosci 27(7):378–383.
44. Mulkey RM, Endo S, Shenolikar S, Malenka RC (1994) Involvement of a calcineurin/
inhibitor-1 phosphatase cascade in hippocampal long-term depression. Nature 369
(6480):486–488.
45. Kameyama K, Lee HK, Bear MF, Huganir RL (1998) Involvement of a postsynaptic
protein kinase A substrate in the expression of homosynaptic long-term depression.
Neuron 21(5):1163–1175.
46. Lieberman DN, Mody I (1994) Regulation of NMDA channel function by endogenous
Ca2+-dependent phosphatase. Nature 369(6477):235–239.
47. Krupp JJ, Vissel B, Thomas CG, Heinemann SF, Westbrook GL (2002) Calcineurin acts
via the C-terminus of NR2A to modulate desensitization of NMDA receptors. Neuropharmacology 42(5):593–602.
48. Flint AC, Maisch US, Weishaupt JH, Kriegstein AR, Monyer H (1997) NR2A subunit
expression shortens NMDA receptor synaptic currents in developing neocortex. J
Neurosci 17(7):2469–2476.
49. Luo J, Wang Y, Yasuda RP, Dunah AW, Wolfe BB (1997) The majority of N-methyl-Daspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). Mol Pharmacol 51(1):79–86.
50. Dunah AW, Standaert DG (2003) Subcellular segregation of distinct heteromeric
NMDA glutamate receptors in the striatum. J Neurochem 85(4):935–943.
51. Turrigiano G (2011) Too many cooks? Intrinsic and synaptic homeostatic mechanisms
in cortical circuit refinement. Annu Rev Neurosci 34:89–103.
712 | www.pnas.org/cgi/doi/10.1073/pnas.1215854110
Zhao et al.