Reduced release probability prevents vesicle depletion
and transmission failure at dynamin mutant synapses
Xuelin Loua,b,1, Fan Fana,b, Mirko Messaa, Andrea Raimondia,2, Yumei Wua, Loren L. Loogerc, Shawn M. Fergusona,
and Pietro De Camillia,1
aDepartment of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Kavli Institute for
Neuroscience, Yale University School of Medicine, New Haven, CT 06510;bDepartment of Neuroscience, School of Medicine and Public Health, University of
Wisconsin, Madison, WI 53706; andcHoward Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147
Contributed by Pietro De Camilli, January 4, 2012 (sent for review November 19, 2011)
Endocytic recycling of synaptic vesicles after exocytosis is critical for
nervous system function. At synapses of cultured neurons that lack
the two “neuronal” dynamins, dynamin 1 and 3, smaller excitatory
postsynaptic currents are observed due to an impairment of the
fission reaction of endocytosis that results in an accumulation of
arrested clathrin-coated pits and a greatly reduced synaptic vesicle
number. Surprisingly, despite a smaller readily releasable vesicle
pool and fewer docked vesicles, a strong facilitation, which corre-
lated with lower vesicle release probability, was observed upon ac-
tion potential stimulation at such synapses. Furthermore, although
network activity in mutant cultures was lower, Ca2+/calmodulin-de-
pendent protein kinase II (CaMKII) activity was unexpectedly in-
creased, consistent with the previous report of an enhanced state
of synapsin 1 phosphorylation at CaMKII-dependent sites in such
neurons. Thesechanges were partially reversed byovernight silenc-
ing of synaptic activity with tetrodotoxin, a treatment that allows
progression of arrested endocytic pits to synaptic vesicles. Facilita-
tion was also counteracted by CaMKII inhibition. These findings re-
veal a mechanism aimed at preventing synaptic transmission failure
a defect in dynamin-dependent endocytosis and provide new in-
sight into the coupling between endocytosis and exocytosis.
active zone|short-term synaptic plasticity|syndapin|membrane fission
(1, 2). The interplay of multiple forms of short-term plasticity,
including facilitation and depression, leads to bidirectional
changes of synaptic strength. Whereas both pre- and postsynaptic
factors contribute tosynaptic plasticity, short-termplasticityis due
primarily to regulatory mechanisms occurring in the presynaptic
compartment. Synaptic vesicle availability and release probability
number of vesicles in the readily releasable pool (RRP), the
availability of synaptic vesicles for exocytosis rapidly becomes the
limiting step during high-frequency action potential firing, leading
least partially dependent on the endocytic recycling of synaptic
vesicle membranes (9, 10).
During a brief stimulation, synapses do not rely on endocytosis
to support secretion because of the existence of reserve pools of
vesicles in addition to the RRP. In contrast, during sustained
becomes essential for continuous synaptic transmission (11–14).
For example, in model organisms, genetic or biochemical per-
turbations of synaptic vesicle endocytosis have typically minimal
effects on the initial synaptic transmission, although they strongly
impact the release of neurotransmitter in response to prolonged
stimulatory trains (15–26).
Recent studies also suggest that a feedback between endocy-
tosis and exocytosis may occur over very short timescales (milli-
seconds to seconds) (1, 7, 27, 28). For example, the efficient
ynapses undergo several forms of short-term plasticity with
clearing of fusion sites via endocytosis may affect availability of
vesicle fusion sites for transmitter release. Proteins with a dual
role in exo- and endocytosis have been identified (such as syn-
aptotagmin, synaptobrevin, and endophilin) (29–34), and signal-
ing pathways that produce interrelated effects on exo- and
endocytosis have been described (28).
A protein that plays a key role in endocytic membrane recycling
at synapses is the GTPase dynamin (9, 22, 35–38). Dynamin
assembles around the necks of endocytic pits and participates in
their fission via a GTP-hydrolysis–dependent conformational
change leading to their constriction (36, 39–46). Three dynamin
genes are present in mammals, which encode dynamin 1, 2, and 3,
respectively. Dynamin 2, which is ubiquitously expressed, has
a housekeeping endocyticfunctionin allcells (47). Dynamin1 and
3 are expressed primarily in neurons, where they are concentrated
in nerve terminals and account for the majority of the total neu-
ronal dynamin, with dynamin 1 being by far the predominant
species (9, 48, 49). In recent gene knockout (KO) studies in mice,
we have found that whereas both dynamin 1 and dynamin 3 are
concentrated in nerve terminals, neither isoform is essential for
synaptic vesicle recycling (9, 35). A main role of dynamin 1 is to
allow synapses to scale up the speed of endocytosis when strong
and prolonged stimuli result in massive exocytosis (38). This
function is likely to be dependent, at least in part, on its very high
level of expression. The absence of dynamin 3 alone has little im-
pact on synaptic function, but worsens the presynaptic defect
produced by the lack of dynamin 1 (35).
In neuronal cultures derived from dynamin 1 and 3 double-KO
(DKO) mice, synaptic vesicles are greatly reduced in number and
much of the synaptic vesicle membrane material is sequestered in
clathrin-coated pits. Accordingly, as we have shown, excitatory
postsynaptic currents (EPSCs) are severely decreased in size at
such synapses. However, the impact of the lack of two neuronal
dynamins (dynamin 1 and 3) on the efficiency of neurotransmitter
release in response to continuous action potential firing and on
the synaptic plasticity has not been investigated. Given the pro-
found impact of synaptic plasticity on neuronal communication
and on information processing in neural circuits, we have now
further analyzed such an impact.
Author contributions: X.L. and P.D.C. designed research; X.L., F.F., M.M., A.R., Y.W., and
S.M.F. performed research; L.L.L. contributed new reagents/analytic tools; X.L., F.F., M.M.,
A.R., Y.W., and S.M.F. analyzed data; and X.L., S.M.F., and P.D.C. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: Pietro.email@example.com or
2Present address: Department of Neuroscience and Brain Technologies, Istituto Italiano di
Tecnologia, via Morego, 30 16163 Genoa, Italy.
See Author Summary on page 2711 (volume 109, number 8).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online January 30, 2012
Decreased but Still Synchronized Synaptic Network Activity in the
Absence of Dynamin 1 and 3. We have shown previously that al-
though DKO mice for both dynamin 1 and 3 die perinatally, brain
cells derived from newborn DKO mice differentiate in vitro and
form synaptic networks that are morphologically similar to those
of control mice (35). A preliminary analysis of synaptic activity in
these cultures revealed slightly reduced frequency of miniature
excitatory postsynaptic currents (mEPSCs) and a severe reduction
of evoked EPSCs (35).
We have now compared overall neuronal network activity in
cultures from control and DKO mouse brains, using ratio-metric
Ca2+imaging (Fig.1). Twotypes of Ca2+signalswere observed in
individual cells: fast Ca2+spikes and slow Ca2+waves with large
amplitude, arising from neurons and surrounding glia cells, re-
spectively (Fig. S1).Fast Ca2+spikescan be selectively blocked by
1 μM tetrodotoxin (TTX), indicating occurrence of action po-
tential firing in neurons (Fig. S1A) (50, 51). The number and
amplitude of these spikes were further drastically enhanced by
administration of high extracellular K+solution, a global stimulus
of neuron depolarization, and synaptic transmission (Fig. S1B).
The slow Ca2+waves were insensitive to both TTX and high K+
(Fig. S1 A and B).
The abundance of fast, in many cases synchronous, spikes in
control cultures (Fig. 1 A and C) demonstrated the occurrence of
vigorous synaptic transmission and often synchronized network
activity (51). In DKO cultures (Fig. 1 B and D), fast spikes oc-
curred at much lower frequency, but with similar amplitude (Fig.
1 E and F). However, in these cultures as well there were
examples of synchronous events, reflecting network activity me-
diated by synaptic transmission (Fig. 1D). A small population of
neurons did not show obvious Ca2+spikes in either culture and
this population was greater in DKO (31% of all of the tested
neurons) than in controls (19% of neurons tested). Basal Ca2+
levels were very slightly higher in DKO neurons (Fig. 1G).
Furthermore, the lack of dynamin 1 and 3 had little effect on the
slow Ca2+waves of glial cells (Fig. S1C).
These data demonstrate that absence of dynamin 1 and 3
dramatically scales down, but does not abolish, the network ac-
tivity in these neuronal circuits, consistent with residual synaptic
vesicle recycling function in DKO neurons.
Strong Synaptic Facilitation at Dynamin 1 and 3 DKO Synapses.
DKO synapses have a lower content of synaptic vesicles and, ac-
cordingly, smaller evoked EPSCs (35). Here we examined short-
term plasticity changes at excitatory DKO synapses, using whole-
cell patch clamp recordings. According to the vesicle pool
depletion hypothesis (2, 4), one would expect enhanced synaptic
depression and faster vesicle pool depletion during continuous
stimulation in DKO synapses. To our surprise, we detected strong
facilitation, which is in strong contrast to the enhanced synaptic
depression observed at synapses of mice (16, 17, 52), Caeno-
rhabditis elegans (20), and Drosophila (10, 15, 21, 53) harboring
inactivating mutations of endocytic proteins. We conclude that
the impact of the lack of dynamin 1 and 3 on exo–endocytosis
coupling at active zones, and thus on short-term plasticity, is dif-
ferent from that of other perturbations that affect the endocytic
recycling of synaptic vesicles.
EPSCs evoked by a train of 30 action potentials (APs) at 1 Hz
in a control and a DKO neuron are shown in Fig. 2 A and B,
respectively. The first EPSC was much smaller in DKO neurons
(Fig. 2A), as expected (35). However, in contrast to the de-
pression observed at the control neuron, a strong facilitation was
observed at the DKO neuron. Such facilitation during the
derived from dynamin 1 and 3 double-KO (DKO) mice. (A and B) Differential
interference contrast images and fluorescence images excited at 380 nm
(F380) from a control (A) and a DKO (B) culture preloaded with Fura-2 AM
under a 20× water immersion objective (NA = 0.6). (C and D) Intracellular Ca2+
responses (F340/F380 fluorescence) of individual neurons from control and
DKO cultures shown in A and B. Arrows indicate synchronized Ca2+spikes. (E–
G) Ca2+spike frequency (E), amplitude (F), and resting Ca2+levels (G) in
control and DKO cultures (control, n = 99 neurons; DKO, n = 85 DKO neurons;
*P < 0.05, **P < 0.01).
Reduced spontaneous network activity in cortical neuronal cultures
quency stimulation at DKO synapses. (A) Superimposed traces of EPSCs eli-
cited by 30 APs at 1 Hz in a control and a DKO neuron. The first EPSC is
shown as a solid trace. Note the different EPSC scales for the neurons of the
two genotypes. (B) Average normalized EPSCs elicited by 30 APs at 1 Hz from
control (n = 21) and DKO neurons (n = 18). EPSCs were normalized to the
peak amplitude of the first EPSC in each AP train. (C) EPSCs recorded from
a control and a DKO neuron in response to 30 APs at 20 Hz. (D) Average
normalized EPSCs elicited by 30 APs at 20 Hz from control (n = 18) and DKO
(n = 16) neurons. EPSCs were normalized to the peak amplitude of the first
EPSC in each AP train. Note the depression at control synapses and facilita-
tion at DKO synapses.
Strong synaptic facilitation in response to both low- and high-fre-
| www.pnas.org/cgi/doi/10.1073/pnas.1121626109 Lou et al.
30-APs (at 1 Hz) stimulation was consistently observed, as
demonstrated in Fig. 2B where the average EPSC values from
different recordings were normalized to the first EPSC peak
amplitude. The synaptic facilitation was even stronger when the
same number of APs was delivered at higher frequency (20 Hz)
(see Fig. 2C for a representative control and DKO synapses and
Fig. 2D for the average responses). The transient enhancement
of the EPSCs at both frequencies was followed by depression,
likely reflecting a gradual depletion of the RRP of vesicles.
However, the normalized DKO curve never crossed the control
curve despite the smaller RRP, indicating persistence of facili-
tation throughout the stimulatory train.
A similar persistent facilitation was also observed after a pro-
longed high-frequency stimulus (300 APs at 10 Hz; Fig. 3 A–C).
Fig. 3A shows individual EPSCs from a control and a DKO
neuron recorded at different time points during such stimulus.
The time courses of average EPSCs (Fig. 3B) as well as their
normalized values (normalized to the size of the first EPSC) (Fig.
3C)demonstrate thatonlya fewquantalevents weredetectable at
the end of the stimulation in both control and DKO neurons,
likely reflecting dramatic vesicle depletion. However, in contrast
to the fast depression observed in control synapses, DKO syn-
apses showed an initial strong facilitation (note in Fig. 3A that the
10th EPSC was approximately twofold bigger than the initial
EPSC), which was followed by a slow depression. Because of the
strong facilitation in DKO synapses, the normalized EPSC curves
from control and DKO synapses clearly separated at the begin-
ning of the stimulation and then tended to merge at the end of
is close to the depletion state (Fig. 3 B and C). These results
demonstrate reluctance to vesicle depletion at DKO synapses.
We next examined the time course of synaptic transmission
recovery as tested by APs delivered at 0.2 Hz, starting immedi-
ately after the end of the 300-AP/10-Hz stimuli. As shown by the
analysis of individual EPSCs (Fig. 3D) and by curves reporting
either absolute EPSC amplitude (Fig. 3E) or normalized EPSC
(relative to its prestimulus value) (Fig. 3F), EPSCs recovered up
to ∼75% of the prestimulus amplitude in 5 min in control cul-
tures, with a biphasic time course (fast and slow time constants of
8.5 s and 150 s, respectively). In contrast, only the fast recovery
(time constant of 7.5 s) was observed in DKO cultures, followed
with a steady state at ∼50% of the prestimulus amplitude.
No Major Postsynaptic Contribution to the Observed Synaptic Facil-
itation. Short-term synaptic plasticity can have presynaptic and
5-methyl-4-isoxazole (AMPA) receptors (AMPARs) in dendritic
are believed to underlie long-term plasticity and memory. Because
dynamin-dependent AMPAR endocytosisisthought tocontrolthe
levels of a pool of mobile surface-exposed AMPARs (55–58),
a postsynaptic contribution to the facilitation observed at DKO
synapses should be considered. We investigated therefore whether
dynamin function may modulate synaptic strength in spines by
disrupting the AMPAR trafficking.
The relative ratio of synaptic currents through AMPARs and
N-methyl-D-aspartate (NMDA) receptors at individual synapses
has been widely used to study postsynaptic AMPA trafficking,
because levels of surface exposed NMDA receptors are relative
stable (59). Thus, if fast AMPAR trafficking contributes signifi-
cantly to the facilitation at DKO synapses, a change of AMPA/
NMDA ratio is expected. Fig. 4A shows AMPA and NMDA
currents recorded at a control and a DKO neuron. Because the
kinetics of AMPA and NMDA current differ dramatically, they
can be clearly separated temporally without using specific
pharmacological blockers (60, 61). NMDA current amplitudes
were extracted at 100 ms (+60 mV) after stimulation, a time
point at which the AMPA current had completely decayed (61).
Although both AMPA and NMDA currents were much smaller
at DKO than at control synapses (Fig. 4 A and B), no significant
difference was observed in the ratio of AMPA/NMDA currents
between the two groups (Fig. 4 B and C). These data suggest that
postsynaptic mechanisms contribute little, if at all, to the changes
of short-term plasticity observed at DKO synapses.
Another possibility is that some silent synapses could be acti-
vated after the first AP by the fast recruiting of AMPA receptors
to previously silent synapses, thus contributing to the subsequent
synaptic enhancement. However, the very fast timescale of facil-
itation (it occurs within subseconds after the first AP) seems in-
compatible with the timescale of AMPAR trafficking (seconds to
minutes). This possibility was further tested by comparing the
paired-pulse ratio (PPR) of AMPA and NMDA currents (61)
after two closely spaced stimuli. Upon a paired-pulse (2 APs with
was similar for AMPA currents and for NMDA currents (Fig.
4D), speaking against a role of quick activation of silent synapses
in the facilitation observed at DKO synapses. We conclude that
postsynaptic mechanisms do not play a major role in the synaptic
facilitation produced by the lack of dynamin 1 and 3.
Decreased Readily Releasable Vesicle Pool Size and Synaptic Release
Probability. We next used two different approaches to determine
the changes of RRP and release probability at DKO synapses.
First, we used stimulation by acute high osmolarity (62). Upon
locally puffing 500 mM sucrose for 5 s onto the recorded neuron,
and 3. (A) Individual EPSCs recorded during a 300-APs train at 10 Hz (i.e.,
a stimulus leading to a near depletion of releasable synaptic vesicles) in
a control and a DKO neuron. The AP number is indicated at the top. (B and C)
Average EPSC amplitudes (B) and normalized EPSCs (C) from control (n = 14)
and DKO neurons (n = 14). (D) Recovery of synaptic transmission immediately
after the vesicle depletion induced by 300 APs at 10 Hz in a control neuron
and a DKO neuron. Recovery was estimated by EPSCs induced with AP stim-
recovery. The AP number is indicated at the top. (E and F) Time course ofEPSC
recovery after vesicle depletion in control (n = 12) and DKO neurons (n = 15).
EPSCs were normalized to the first EPSC amplitude of the depletion train (F).
Synaptic vesicle depletion and recovery in the absence of dynamin 1
Lou et al.PNAS
| Published online January 30, 2012
the surrounding synapses, was observed in control neurons (Fig. 5
A and B). The response to this stimulus was less robust at DKO
synapses (∼50% decrease in peak amplitude and 70% decrease in
smaller than the reduction in the amplitude of AP-evoked EPSCs
(∼90%) (Fig. 2 A and C). Paired stimuli of hypertonic sucrose
further revealed a decreased refilling rate of the RRP at DKO
synapses (Fig. S2), most likely reflecting the strongly reduced
vesicle number within terminals.
Second, we estimated RRP size within these synapses using
a train of APs (30 APs, 20 Hz, Fig. 5 C1–D3) in high extracellular
Ca2+(10 mM) (63, 64). The high Ca2+condition maximizes the
release probability and minimizes the submaximum transmitter
release during the stimulation (65); whereas this condition leads
to faster depression than at physiological Ca2+concentration (2
mM), the kinetics of depression were still much slower at DKO
synapses relative to controls (Fig. 5C2), demonstrating a re-
luctance of DKO synapse to deplete the RRP vesicles. The RRP
size, derived from linear back extrapolation of the cumulative
peak EPSCs to time 0 (Fig. 5C3), is strongly decreased at DKO
synapses (Fig. 5D2).
On the basis of the EPSC size triggered by the first AP (Fig.
5D1) and the corresponding RRP size at individual synapses
(Fig. 5D2), we found that DKO synapses had a significantly lower
release probability (defined by the ratio between EPSC ampli-
tude and RRP size) than control synapses (Fig. 5D3). Of note,
the size of the RRP estimated by sucrose (Fig. 5 A and B) was
bigger than the one estimated by action potential stimulation
(Fig. 5D2), indicating that RRP vesicles at DKO synapses are less
responsive to physiological stimulation (APs) and are reluctant
to undergo exocytosis in response to this stimulus.
Release probability was further analyzed with a paired-pulses
test with variable interpulse intervals between the two APs. In
contrast to the synaptic depression at control synapses, DKO
synapses showed significant facilitation at most of the time
intervals tested (Fig. 5E), so that the curve of paired-pulse ratios
5F). These data supports a significant decrease of release prob-
ability in DKO synapses compared with controls.
One of the key factors affecting release probability is Ca2+
influx. To determine potential differences in Ca2+influx be-
tween DKO and control synapses, we used the fluorescence of
SyGCaMP3, a fusion protein compromising the high-affinity,
genetically encoded Ca2+probe GCaMP3 (66) and synapto-
physin for selective presynaptic targeting. A similar method using
SyGCaMP2 was recently applied to monitor synaptic Ca2+sig-
nals (67, 68). No significant differences of the SyGCaMP3 signals
were observed in our experiments between control and DKO
synapses (Fig. S3), which speaks against a major impact of the
lack of dynamin 1 and dynamin 3 on Ca2+dynamics, although
potential effects of an abnormal localization of SyGCaMP3 in
DKO nerve terminals cannot be ruled out.
Collectively, these findings demonstrate that the two neuronal
dynamins are needed to maintain a normal RRP of vesicles and
that in their absence a lower release probability counteracts the
rapid depletion of this pool.
Facilitation at DKO Synapses Is Activity Dependent and Reversible.
We showed previously that TTX treatment largely reverses at least
some of the structural and biochemical changes produced by the
coated pits, the accompanying decrease of the number of synaptic
at Ca2+/calmodulin-dependent protein kinases II (CaMKII) de-
pendent sites (sites 2 and 3) and its dispersed distribution (9, 35).
We have now examined whether TTX also reverses electrophysi-
ological parameters. As shown in Fig. 6A, following an overnight
incubation of the neuronal cultures with 1 μM TTX, the EPSC
amplitude increased in both control and DKO synapses, in
agreement with the homeostatic up-regulation of synaptic strength
known to be produced by such treatment (69). Importantly, how-
at DKO synapses (327 ± 92.8% of untreated control, n = 10) than
at control synapses (139 ± 21.7%, n = 14, P < 0.05), although it
remained lower than in controls (Fig. 6A, Right). Strikingly, in
DKO synapses under these conditions, low-frequency AP stimu-
than facilitation, despite its much smaller initial EPSC (Fig. 6B).
Upon high-frequency stimulation, synaptic facilitation also dis-
appeared and converted to depression, but such depression
remained much weaker than at control synapses (Fig. 6C). The
reversal of facilitation following TTX treatment speaks against the
possibility that the change of short-term plasticity observed in
DKO neurons may result from chronic developmental or other
long-term compensatory changes.
We next used electron microscopy to determine whether the
reversibility of the electrophysiological changes upon TTX
treatment correlated with reversible changes in the number of
synaptic vesicles docked at active zones, i.e., the vesicles that are
thought to represent the RRP. In DKO nerve terminals, not only
was the synaptic vesicle cluster smaller than in controls (35), but
also the numbers both of vesicles within 1 μm of the active zones
and of the docked vesicles were fewer (Fig. 7 A and B), in
agreement with the smaller RRP detected by electrophysiologi-
cal recordings. Notably, TTX treatment resulted in an increase in
the number of docked vesicles both at control and DKO synapses
(Fig. 7C), but this increase was much more robust at DKO
AMPA and NMDA currents triggered by single APs. The amplitude of NMDA
currents recorded at +60 mV (holding potential) was measured at a time
point of 100 ms (arrowhead) after the AP stimulation (arrow), the same as
for B–D. (B and C) Smaller AMPA and NMDA current amplitude (B) but
similar AMPA/NMDA current ratio (C) at control (n = 11) and DKO neurons
(n = 9). (D) AMPA and NMDA currents induced by a paired pulse (at 100-ms
intervals). The second NMDA current amplitude (second arrow) was mea-
sured at 100 ms after the second AP but subtracting the residual component
of NMDA current from the first AP (gray trace, obtained by a single AP at the
same neuron). (E) Paired-pulse ratio of AMPA/NMDA currents in both con-
trol and DKO cultures (n = 5 for each).
Presynaptic origin of the facilitation observed at DKO synapses. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1121626109Lou et al.
synapses (Fig. 7D), where a partial recovery of the global number
of synaptic vesicles was also observed (35). Although after TTX
treatment the number of docked vesicles at active zones of DKO
synapses was restored to the level of TTX-untreated control
synapses (Fig. 7C), the EPSC amplitude was still smaller (<50%
of control), indicating that additional mechanisms, besides vesi-
cle availability, affect the probability of release at DKO synapses.
Increased CaMKII Activity Contributes to Synaptic Facilitation at DKO
Synapses. We explored biochemical changes at DKO synapses
that could provide insight into mechanisms underlying synaptic
facilitation. As we have reported previously (35), a markedly
increased phosphorylation state of synapsin 1 at sites 2 and 3, i.e.,
the Ca2+-dependent (CaMKII) regulated sites (70, 71), occurs in
DKO cultures under conditions of spontaneous network activity.
To determine whether this change reflects an increased activity
state of CaMKII, despite the lower activity state of synapses, we
have now assessed by immunoblotting the phosphorylation of
βCaMKII at threonine 286, a site that undergoes autophos-
phorylation leading to autoactivation. As shown in Fig. 8A, an
increase of the signal in DKO cultures relative to controls was
observed, although the total CaMKII level (as revealed by a pan-
CaMKII antibody) was the same in DKO and controls (Fig. 8A).
These findings raise the possibility that the increased activity
state of CaMKII could contribute to the short-term plasticity
observed at DKO synapses. We tested this possibility by per-
forming experiments in the presence of Ant-AIP-II, a cell-per-
meable version (due to a fusion to the Antennapedia transport
peptide) of the autocamtide-2 related inhibitory peptide II (AIP-
II), a specific, noncompetitive CaMKII inhibitor. Ant-AIP-II has
a potent inhibitory effect on CaMKII activity when added to the
extracellular medium of living neurons (72, 73). Accordingly, as
shown in Fig. 8B, addition of Ant-AIP-II to control cultures re-
duced the phosphorylation of threonine 286 in CaMKII and of
and a DKO neuron. (B) The RRP, as estimated by both EPSC amplitude and total charge transfer, is smaller in DKO synapses than in control (n = 21, control; n =
18, DKO; *P < 0.05, **P < 0.01). (C1–C3) Estimation of the RRP by a high-frequency AP train in 10 mM extracellular CaCl2. (C1) EPSCs evoked by 30 APs at 20 Hz.
(C2) EPSC peaks (solid circles, left axis) and synaptic depression (open circles, right axis) from the data shown in C1. (C3) Linear regression of the EPSC steady
state and back extrapolation (to time 0) from control and DKO synapses. (D1–D3) Average initial EPSC amplitude (D1), RRP (D2), and vesicle release probability
(D3) from control (n = 9) and DKO neurons (n = 11; *P < 0.05, **P < 0.01). (E) Representative EPSCs induced by paired-pulse stimuli at variable interpulse
intervals from a control and a DKO neuron. (Inset) Synaptic responses at an expanded timescale. Note the strong facilitation at the DKO neuron. (F) Average
paired-pulse ratios in control (n = 16) and DKO (n = 17) neurons show that the ratios from DKO neurons are higher at all time intervals tested.
Readily releasable pool (RRP) and vesicle release probability are reduced in DKO synapses. (A) A total of 500 mM sucrose evoked EPSCs from a control
Lou et al.PNAS
| Published online January 30, 2012
sites 2 and 3 in its substrate synapsin 1. The inhibition also oc-
curred and was much stronger, in the DKO neurons, consistent
with an activated state of the kinase in these neurons (Fig. 8B).
Ant-AIP-II also slightly enhanced the synaptic depression
observed at control synapses both at low- (1 Hz) and high (20
Hz)-frequency stimulation (Fig. 8 C and D). Importantly, it
blocked the facilitation elicited by low-frequency stimulation at
DKO synapses (Fig. 8C) and also strongly, although not com-
pletely, inhibited the facilitation produced by high-frequency
stimulation (20 Hz) (Fig. 8D), thus implicating CaMKII signaling
in these changes. However, because Ant-AIP-II has little effect
on the early phase of synaptic facilitation elicited by high-fre-
quency stimulation in DKO synapses (Fig. 8D), additional
mechanisms should contribute to the synaptic facilitation.
This study demonstrates that the endocytic defect resulting from
the combined absence of dynamin 1 and 3 has a major and un-
expected impact on short-term synaptic plasticity at excitatory
synapses. EPSCs evoked at DKO synapses are smaller due to the
smaller RRP of synaptic vesicles. However, DKO synapses, in-
stead of displaying an accelerated synaptic depression upon high-
frequency AP stimulation, exhibit decreased release probability
and strong facilitation, indicating the occurrence of mechanisms
that preserve synaptic transmission in face of the dramatic re-
duction of the overall synaptic vesicle abundance. Surprisingly,
given the overall reduction in the state of neuronal network ac-
tivity of DKO cultures and the lower probability of release, these
changes correlate with an increased activation state of CaMKII in
DKO neurons and are reversed by inhibition of CaMKII. All
these changes, including CaMKII activation, are to a large extent
reversed by previous silencing of electrical activity, pointing to
their dependence on a backup of endocytic vesicle traffic in
stimulated nerve terminals. Collectively, our results demonstrate
that the backup of synaptic vesicle recycling traffic produced by
the lack of the two neuronal dynamins inhibits the probability of
release, thus revealing an interesting aspect of the coupling be-
tween endocytosis and exocytosis at central excitatory synapses.
Despite the absence of the great majority of dynamin (the
dynamin 2 isoform contributes minimally to the total level of
dynamin in neurons) (35), synaptic transmission in DKO neurons
still occurs and is strong enough to fire APs in postsynaptic neu-
mice in the minutes/hours after birth. However, the properties
of synaptic transmission are different in DKO neuronal cultures.
The size of the RRP, a parameter that has a key role in de-
fining synaptic strength and plasticity (4), is clearly decreased, as
demonstrated both by electrophysiological data and by ultra-
structural analyses of synaptic active zones. On the basis of these
observations and the very strong decrease in the overall number
of synaptic vesicles at DKO synapses, one would have expected
faster depression and stronger vesicle depletion at mutant syn-
apses upon prolonged stimulation. In striking contrast, two sur-
prising results, opposite to these predictions, were obtained.
First, synaptic facilitation in response to both low- and high-
frequency AP stimulation was observed. Second, even at late
tially reversible. (A) EPSC amplitude increases after silencing spontaneous
network activity with TTX in control and DKO neurons (n = 14 for control, n =
10 for DKO; *P < 0.05, **P < 0.01). (B) TTX exposure completely reversed the
synaptic facilitation observed in DKO neuronal cultures upon stimulation at
1 HZ, so that a similar depression was observed in DKO and controls. Repre-
sentative recording (Left) and normalized EPSCs (Right) from control (n = 16)
and DKO neurons (n = 14) subjected to 1 Hz stimulation after overnight TTX
treatment are shown. (C) TTX exposure also abolished the facilitation ob-
served in DKO neurons upon high-frequency stimulation (20 Hz). In this case,
a depression was observed in both DKO and controls, although this de-
pression was smaller than controls. Representative recording (Left) and nor-
malized EPSCs (Right) from control (n = 13) and DKO neurons (n = 12)
subjected to 20 Hz stimulation after overnight TTX treatment are shown.
Synaptic facilitation at DKO synapses is activity dependent and par-
zone of the synapse, which is surrounded by a large number of coated ve-
sicular profiles (arrows), some of which have tubular necks in the plane of the
section. As described previously (35), most clathrin-coated vesicular profiles
are pits and not vesicles. White asterisks label the vesicle cluster in both
micrographs. (Scale bar, 200 nm.) (B) Spatial distribution of synaptic vesicles
around active zones in control (n = 19) and DKO synapses (n = 80). (C) Number
of vesicles docked at the presynaptic plasma membrane of active zones be-
fore (n = 19, control; n = 80, DKO; P < 0.01) and after an overnight TTX
treatment (n = 86, control; n = 63, DKO; P < 0.01). (D) Relative increase of the
docked vesicle numbers per active zone after TTX treatment at control and
DKO synapses. The data after TTX (shown in C) were normalized to the av-
erage number of docked vesicles before TTX was added. Note the stronger
increase of the number of docked vesicles at DKO synapses.
Active zone ultrastructure and docked vesicles at DKO synapses. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1121626109 Lou et al.
stages of a stimulatory train, the normalized depression curve in
DKO cultures never exceeded the depression observed in control
neurons (although the two curves merged eventually). These
results unmask a mechanism aimed at preserving the property to
release neurotransmitters in the face of severely reduced vesicle
availability in dynamin DKO synapses.
The facilitation observed in dynamin DKO mouse neurons is in
contrast to the enhanced depression observed in mouse neurons
that lack other abundant endocytic proteins and whose absence
produces a delay in the clathrin-dependent reformation of syn-
aptic vesicles, such as neurons from synaptojanin 1 KO mice and
from endophilin triple-KO mice (16, 52, 74). The only mouse
mutant synapses exhibiting a weaker depression than in control
neurons are those lacking syndapin 1, a protein that has a major
role in recruiting dynamin to synaptic membranes (75). A stronger
depression than in controls was observed in flies and worms after
genetic perturbation of endocytosis genes (other than dynamin,
see below) that results in defects of synaptic vesicle recycling and
smaller vesicle pools (10, 15, 18, 20, 21, 53, 76). Thus, it appears
trigger facilitation and that some specific features of the endocytic
block produced by dynamin are implicated. It will be of interest to
determine whether the same phenomenon occurs at inhibitory
synapses, because a previous study of the postsynaptic response at
inhibitory synapses that lack dynamin 1 only did not reveal facili-
Thefacilitation observed hereisalsoincontrast withthelackof
facilitation observed at the neuromuscular junction of dynamin
(shibire) mutants of Drosophila when stimulated at the restrictive
temperature (18). However, there are some key differences in the
experimental conditions under which these contrasting results
were obtained. First, in our experiments we start from a baseline
condition that is already highly compromised due to the smaller
number of synaptic vesicles, the decreased size of the RRP, and
the accumulation of endocytic intermediates. In fact, silencing of
the cultures with an overnight exposure to TTX before our
experiments, a condition that we have found to partially reverse
many of the changes observed in DKO synapses relative to con-
trols, abolished the facilitation, although a slower depression
relative to controls was still observed. Second, the Drosophila
shibire mutation is a dominant mutation (77, 78) whose effect on
nerve terminal physiology can be accounted for not only by tem-
perature-induced changes in dynamin itself, but also by the se-
questration of dynamin binding partners, a scenario quite
different from the near-lack of dynamin. The persistence of
neurotransmitter release in the near absence ofthe overwhelming
majority of dynamin is also in contrast to the block of action po-
tential triggered endocytosis (79) and more pronounced de-
pression (80) observed in neuronal cultures following exposure to
dynasore, a compound that inhibits dynamin 1 and dynamin 2
(dynasore was not tested on dynamin 3, but the stronger similarity
of dynamin 3 than of dynamin 2 to dynamin 1 makes it likely that
may be at least partially explained by the acute nature of experi-
ments with dynasore. Moreover, the off-targets effect of dynasore
have some effect on the dynamin-related protein 1 (DRP1) (81), to
change Ca2+signaling (82, 83), and to affect actin dynamics (84).
One potential explanation for the functional changes reported
here at DKO synapses is that they result from the dramatic
backup of the endocytic traffic at the cell surface, where synaptic
vesicle membranes (lipids and proteins) are stranded in clathrin-
coated pits. The massive increase in the cell surface area and the
corresponding structural changes of nerve terminals (35) may
impact on the functional and biochemical parameters of the
active zone and thus on release probability. Feedback mecha-
nisms between membrane traffic and phosphorylation–de-
phosphorylation cascades may mediate the increase in CaMKII
activity and synapsin phosphorylation (35). Supporting this pos-
sibility is the major reversal of facilitation and lowered release
probability after TTX treatment, a treatment that silences the
spontaneous network activity and results in the disappearance of
the massive coated pit accumulation at DKO synapses. Another
potential explanation is that the absence of the neuronal dyna-
mins may result in a major perturbation of signaling. Dynamin
binds many signaling proteins and may affect signaling via direct
interactions with such proteins.
Whereas the precise mechanisms triggering facilitation remain
to be elucidated, our results strongly suggest that the CaMKII
activation via its phosphorylation at threonine 286 is an impor-
tant mediator of these changes. Both the state of phosphoryla-
tion of synapsin 1 at CaMKII-dependent sites (sites 2 and 3) and
autophosphorylation of βCaMKII at threonine 286 (85) are in-
creased in DKO synapses. All these changes are reversed by
inhibition of CaMKII activity or by TTX-dependent silencing of
the cultures. It is interesting to note that synapsin 1 KO synapses
have enhanced paired-pulse facilitation reminiscent of what we
have observed in dynamin DKO synapses (86), raising the pos-
sibility that a chronic dissociation of synapsin 1 from the vesicles
may play a role in such facilitation at DKO synapses. Whether
the Ca2+-dependent phosphatase calcineurin, which plays an
important role in the Ca2+-dependent dephosphorylation of
several endocytic proteins including dynamin and directly binds
the dynamin 1b splice variant (87, 88), is implicated in these
changes remains an interesting question to address in future
studies. Neither sites 1 and 2 of synapsin 1 nor threonine 286 of
CaMKII are dephosphorylated by calcineurin, but the phos-
phorylation state of these sites could be controlled by de-
phosphorylation cascades triggered by calcineurin (89, 90).
synapses. (A) (Left) immunoblotting analysis revealing that levels of phos-
pho-βCaMKII (threonine 286) and phospho-synapsin I (sites 2 and 3, serines
566 and 603) are increased in DKO neuronal cultures relative to control.
Total levels of these proteins, as well as of synaptophysin, used as a loading
control, are unchanged. The genotype of the DKO was confirmed by the
pan-dynamin signal. (Right) Quantification (percentage of control) of the
phospho-βCaMKII (threonine 286) signal in control and DKO cultures (n = 4).
(B) Effect of the Ant-AIP-II peptide on the phosphorylation of βCaMKII
(threonine 286) and synapsin I (sites 2 and 3). (C and D) Effect of the Ant-AIP-
II peptide on short-term synaptic plasticity in response to low- (1 Hz) (C) and
high (20 Hz) (D)-frequency stimulation in control (n = 12 and 11, respectively)
and DKO synapses (n = 10 and 12, respectively).
Up-regulation of CaMKII activity contributes to facilitation at DKO
Lou et al.PNAS
| Published online January 30, 2012
Interestingly, syndapin 1, the only endocytic protein whose ab-
sence reduces, rather than enhances, synaptic depression (75), is
the major protein whose interaction with dynamin is triggered
by calcineurin-dependent phosphorylation (91).
In conclusion, our study demonstrates the occurrence of
a feedback mechanism through which impaired dynamin-de-
pendent endocytosis reduces the probability of release. This
mechanism ensures persistence of neurosecretion even in the
face of a profound reduction of synaptic vesicle number.
Neuronal Culture. Primary cortical neuron cultures were prepared from
neonatal mouse brain as described previously (9). Dynamin 1 and 3 DKO
(dynamin 1−/−; dynamin 3−/−) mice were generated from the mating of
dynamin 1+/−; dynamin 3−/−mice, and their littermates were used as controls
to minimize variability. Such littermates controls (KO for dynamin 3 and
either wild type or heterozygous for dynamin 1) did not differ in a signifi-
cant way from wild-type newborn mice (35). In our experimental condition,
the synaptic density between control and DKO cultures is comparable as
demonstrated previously (35).
Fluorescent Ca2+Imaging in Live Neurons. Two different Ca2+imaging
methods were used. Ca2+imaging to monitor spontaneous network activity
(Fig. 1 and Fig. S1) was performed by Fura2-AM fluorescence (92). Imaging of
presynaptic Ca2+dynamics was carried out by the fluorescent imaging of
SyGCaMP3 that was expressed by adeno-associated virus-mediated gene
transduction (Fig. S3).
Patch-Clamp Recording. Whole-cell patch clamp recordings were performed
using the cultured neurons after 10–14 days in vitro differentiation (DIV) (35,
93). EPSCs were elicited by a local stimulation electrode connected with an
isolated pulse stimulator. The holding potential was −70 mV unless other-
wise noted and no liquid-junction potential correction was applied. Data
were analyzed with Igor Pro (Wavemetrics). Cell-Permeable Autocamtide-2
Related Inhibitory Peptide II (Ant-AIP-II; Calbiochem) was added to the
cultures (1 μM final concentration) 1 h before the recording and was con-
tinuously present during the recording.
Biochemistry of Neuronal Cultures. Determination of protein concentration,
SDS/PAGE, and Western blotting were carried out by standard procedures (SI
Experimental Procedures). For the analysis of the effect of Ant-AIP-II on
protein phosphorylation, neuronal cultures were incubated with the drug as
described above for electrophysiology.
Electron Microscopy and Morphological Analysis. Primary cortical cultures (18–
22 DIV) were fixed with glutaraldehyde in sodium cacodylate buffer, post-
fixed in OsO4/K4Fe(CN)6, embedded in plastic, and stained with uranyl ace-
tate following ultrathin sectioning (35). Images were taken with a Philips
CM10 microscope and further analyzed with iTEM (Olympus) and Igor Pro
software as described in SI Experimental Procedures.
Statistics. Unless otherwise specified, data were presented as mean ± SEM;
statistical significance was determined using Student’s t tests; P < 0.05 was
taken as the level of significance; *P < 0.05, **P < 0.01.
For other details, see SI Experimental Procedures.
ACKNOWLEDGMENTS. We thank Frank Wilson, Lijuan Liu, and Louise Lucast
for technical assistance. This work was supported in part by the G. Harold and
Leila Y. Mathers Charitable Foundation, National Institutes of Health Grants
R37NS036251 and DA018343, a National Alliance for Research on Schizo-
phrenia and Depression distinguished Investigator Award (to P.D.C.), and
a pilot grant from the Yale Diabetes Endocrinology Research Center (to X.L.).
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Lou et al. PNAS
| Published online January 30, 2012