Preserved morphology and physiology of excitatory synapses in profilin1-deficient mice.
ABSTRACT Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity.
- SourceAvailable from: Marco B Rust[show abstract] [hide abstract]
ABSTRACT: Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell-cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN-BG interaction. Here we demonstrate that the actin-binding protein profilin1 is essential for CGN-glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.EMBO Reports 11/2011; 13(1):75-82. · 7.19 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1(ko)) allele in mice. Homozygous pfn1(ko/ko) mice are not viable. Pfn1(ko/ko) embryos died as early as the two-cell stage, and no pfn1(ko/ko) blastocysts were detectable. Adult pfn1(ko/wt) mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1(ko/wt) embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.Proceedings of the National Academy of Sciences 04/2001; 98(7):3832-6. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.Current Opinion in Neurobiology 03/2006; 16(1):95-101. · 7.34 Impact Factor
Preserved Morphology and Physiology of Excitatory
Synapses in Profilin1-Deficient Mice
Andreas Go ¨rlich1., Anika-Maria Zimmermann1., Doreen Schober1, Ralph T. Bo ¨ttcher2, Marco Sassoe `-
Pognetto3, Eckhard Friauf4, Walter Witke5,6, Marco B. Rust1,5*
1Neurobiology/Neurophysiology Group, University of Kaiserslautern, Kaiserslautern, Germany, 2Max Planck Institute of Biochemistry, Martinsried, Germany,
3Department of Anatomy, Pharmacology and Forensic Medicine and National Institute of Neuroscience-Italy, University of Turin, Turin, Italy, 4Animal Physiology
Group, University of Kaiserslautern, Kaiserslautern, Germany, 5Mouse Biology Unit, European Molecular Biology Laboratory, Monterotondo, Italy, 6Institute of Genetics,
University of Bonn, Bonn, Germany
Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes
of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of
glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity
were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic
mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic
function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout
mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron
micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse
ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic
transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss
of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with
two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent
morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions
particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether
profilins are relevant for dendritic spine morphology and synaptic plasticity.
Citation: Go ¨rlich A, Zimmermann A-M, Schober D, Bo ¨ttcher RT, Sassoe `-Pognetto M, et al. (2012) Preserved Morphology and Physiology of Excitatory Synapses in
Profilin1-Deficient Mice. PLoS ONE 7(1): e30068. doi:10.1371/journal.pone.0030068
Editor: Fabien Tell, The Research Center of Neurobiology-Neurophysiology of Marseille, France
Received November 4, 2011; Accepted December 9, 2011; Published January 11, 2012
Copyright: ? 2012 Go ¨rlich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Research Initiative Membrane Transport RIMB of the University of Kaiserslautern and by the Vigoni-Program of the
Deutscher Akademischer Austausch-Dienst (DAAD; 50756644). MBR was supported by a post-doctoral fellowship from the Deutsche Forschungsgemeinschaft
(DFG; RU-1232/1-1) and by the Stiftung Rheinland-Pfalz (961-386261/877). MS-P was supported by a grant from Compagnia di San Paolo (2007). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Dendritic spines are highly dynamic protrusions that form the
postsynaptic part of most excitatory synapses. Changes in spine
number and shape influence the strength of excitatory synaptic
transmission and are thought to be the basis for learning and
memory [1–3]. Actin is highly enriched in dendritic spines and is
essential for their morphological changes (reviews: [4–5]). In fact,
actin dynamics appear to be crucially important for structural
adaptations of neuronal circuits associated with learning and
memory formation [6–10]. Hence, it is important to understand
the detailed mechanisms that link actin dynamics and synaptic
Actin dynamics critically depend on the activity of profilins that
make actin monomers available for the incorporation into actin
filaments and direct them to the site of actin polymerization .
Of the four identified profilin isoforms, only profilin1 and profilin2
are expressed in the mouse central nervous system . Both
proteins are located in synaptic structures  and show an
activity-dependent recruitment to dendritic spines in neuronal
cultures [14–15]. Moreover, analysis of organotypic hippocampal
cultures suggests a role for profilin1 and profilin2 in dendritic spine
morphology . Based on these studies, it was proposed that
profilins have an important role in activity-driven actin dynamics
in dendritic spines and synaptic plasticity . Accordingly, a
learning-dependent recruitment of profilins into dendritic spines
was observed in fear-conditioned rats . However, these data
are difficult to reconcile with the phenotype of profilin2-deficient
mice as these mutants display normal synaptic plasticity, learning,
and memory . Instead, they show increased neurotransmitter
release, pointing to a critical role of profilin2 in presynaptic
excitability. Therefore, our aim was to investigate the discrepancy
of in vitro and in vivo findings and, for the first time, to elucidate the
isoform-specific synaptic function of profilin1 in vivo.
Profilin1-deficient embryos die during early development 
and profilin1 inactivation during brain development interferes
with neuronal migration and brain development . Thus,
analysis of profilin1 in synaptic plasticity requires the deletion of
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e30068
profilin1 specifically in the adult forebrain. To do so, we crossed
conditional profilin1 mutants (Pfn1flx/flx) with a transgenic line
expressing cre under the control of the Ca2+/calmodulin-
dependent protein kinase II a subunit (CaMKII-cre) [20–21].
Our analysis revealed normal synapse density in profilin1 mutant
mice and virtually no defect in synapse morphology, with the
exception of a slight increase in the neck length of mushroom-like
spines. Moreover, basal synaptic transmission, presynaptic phys-
iology, as well as postsynaptic plasticity were independent of
profilin1 activity. Hence, our data demonstrate that profilin1
inactivation has no adverse effects on excitatory synapses. We
suggest that profilin1 and profilin2 have the capacity to
compensate each other in postsynaptic structures. Analyses of
double mutant mice are required to ultimately unravel the
postsynaptic function of profilins in vivo.
Materials and Methods
Treatment of mice was in accordance with the German law for
conducting animal experiments and followed the NIH guide for
the care and use of laboratory animals. Killing of mice for tissue
analysis was approved by the Landesuntersuchungsamt Rhein-
breeding was approved by the City of Kaiserslautern – Referat
Forebrain-specific deletion of profilin1 was achieved by crossing
the conditional profilin1 allele (Pfn1flx/flx)  with a transgenic
cre-expressing line, driven by Ca2+/calmodulin-depedent protein
kinase II a subunit (CaMKII-cre) .
homogenizing fresh tissue in ice-cold lysis buffer (in mM): 20
Tris-HCl (pH 8.0), 100 NaCl, 5 EGTA, 2 EDTA, supplemented
with 0.5% TritonX-100 and EDTA-free complete protease
inhibitor mix (Roche) using a tightly fitting douncer.
Preparation of hippocampal synaptosomes was essentially performed as
described before . Briefly, tissue was homogenized in homog-
enization solution containing (pH 7.4, in mM): 320 sucrose, 1
EDTA, 5 HEPES, supplemented with 0.1% bovine serum
albumin and EDTA-free Complete protease inhibitor mix (Roche)
using a tight fitting douncer. After removing nuclei and cell debris,
material containing synaptosomes was resuspended in Krebs-
Ringer solution (pH 7.4) containing (in mM): 140 NaCl, 5 KCl, 1
EDTA, 10 HEPES, 5 glucose. Synaptosomes were enriched on a
floatation gradient consisting of 35% Percoll. Anti-b tubulin
antibody was purchased from Sigma-Aldrich (clone TUB 2.1,
#T5201; 1:5,000). Antibodies that specifically recognize profilin1
and profilin2 were used as described before [12–13].
Brain extractswere preparedby
cervical dislocation, and their brains were rapidly removed and
dissected in chilled solution (4uC) containing (in mM): 87 NaCl, 2
KCl, 0.5 CaCl2, 7 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 25
glucose, 75 sucrose, bubbled with a mixture of 95% O2/5% CO2,
leading to a pH of 7.4. 300–370 mm-thick horizontal hippocampal
slices were cut with a VT1200S vibratome (Leica), preincubated
for 30 min at 37uC, and then transferred to recording solution
(room temperature) containing in (mM): 125 NaCl, 2.5 KCl, 2
CaCl2, 1.3 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, 2
4–6 week-old mice were sacrificed by
sodium pyruvate, 3 myo-inositol, 0.44 ascorbic acid, bubbled with
a mixture of 95% O2/5% CO2, leading to a pH of 7.4. Slices
rested in this solution for at least one hour before recordings
Single cell recordings.
Patch pipettes had resistances of 4–
8 MV when filled with a solution containing (in mM): 117.5
CsMeSO4, 2.5 CsCl, 8 NaCl, 10 HEPES, 10 TEA, 0.2 EGTA,
4 Na2ATP, 0.6 Na2GTP, 5 QX-314 pH was adjusted to 7.2
with CsOH. Slices were transferred into the recording chamber,
which was continuously perfused at a rate of 1.5–2 ml/min with
recording solution at room temperature. CA1 hippocampal
neurons were visualized with DIC-infrared optics using a 606/
1.0 water immersion objective on an upright Eclipe E600-FN
PatchMaster and FitMaster software (HEKA Elektronik). For
measurements of miniature EPSCs (mEPSCs), the bath solution
contained 4 mM CaCl2and 4 mM MgSO4. During recordings,
0.5 mMtetrodotoxin (TTX;
picrotoxin (Ascent Scientific), and 250 mM trichlormethiazide
(TCM; Sigma-Aldrich) were added to the recording solution.
CA1 hippocampal neurons were voltage clamped at 270 mV,
and spontaneous mEPSCs were recorded for five minutes.
Amplitude and inter-event intervals (IEI) were analyzed with
miniAnalysis (Synaptosoft), with an amplitude threshold of 3.5
Field potential recordings.
collaterals, monopolar glass electrodes, filled with recording
solution, were placed in the stratum radiatum of the CA1 region.
For field potential experiments, pipettes were filled with 3 M NaCl,
and fEPSPs were measured at a stimulus intensity that elicited
amplitudes that were ,30–50% of the maximum. Input-output
curves were built by measuring the fiber volley and fEPSP responses
evoked by stimulating afferent fibers with current intensities ranging
from 20 to 300 mA. Paired-pulse ratio (PPR) was analyzed by
single one-second 100 Hz train or by 10 bursts of four pulses at
100 Hz, separated by 200 ms (theta-burst stimulation). For the
measurement of long-term depression (LTD) experiments, mice
LTD, a low-frequency stimulation (LFS) was used, consisting of 900
pairs of stimuli (distance 50 ms) at 1 Hz.
Ascent Scientific),100 mM
For stimulation of Schaffer
Rapid GolgiStainTMkit (FD Neurotechniques) was used for Golgi
staining; tissue impregnation and tissue section staining were
performed according to the manufacturer’s data sheet. Briefly,
mice were perfused with 4% formaldehyde and brains were
quickly removed from the skull and postfixed in the same fixative
overnight. After incubation in impregnation solution and solution
C, brains were imbedded in gelatin-albumin and cut into 100 mm
coronal sections usingavibrating
Instruments Ltd.). Sections were mounted on gelatinized glass
slides, further processed for the Golgi staining procedure, and
finally mounted in Entellan (Merck). High magnification images
of 2ndorder dendritic branches in the hippocampal CA1 stratum
radiatum were generated with an Axioskop microscope and a Plan-
Neofluar 1006/1.30 oil immersion objective (Carl Zeiss). Spine
density and morphology were measured using ImageJ 1.42q
imaging software (NIH). Image acquisition and morphometric
analyses were performed by an experimenter blind to the
genotype of the mice. Electron microscopy: 10–12 week-old mice
Mice aged 10–12 weeks were used. The FD
Profilin1 Is Dispensable for Excitatory Synapses
PLoS ONE | www.plosone.org2January 2012 | Volume 7 | Issue 1 | e30068
were perfused with 1% formaldehyde/1% glutaraldehyde in
phosphate buffer (0.1 M PB, pH 7.4). Their brains were postfixed
in the same fixative overnight, and small specimens taken from
the dorsal hippocampus were postfixed in 1% OsO4 in 0.1 M
cacodylate buffer, dehydrated, and embedded in epoxy resin.
Ultrathin sections were stained with uranyl acetate and lead
citrate and observed in a JEM-1010 transmission electron
microscope (Jeol) equipped with a side-mounted CCD camera
(Mega View III, Soft Imaging System). Spine density was assessed
by analyzing 192 digitized images from four mice of each group.
Images (30,0006 magnification, area size 14.66 mm2) were
captured in the proximal part of CA1 stratum radiatum.
Morphometric analysis was done on electron micrographs taken
at 75,0006 using ImageJ 1.42q imaging software. Synaptic
structures were identified by presynaptic terminals with at least
three synaptic vesicles, a visible synaptic cleft and a well-defined
postsynaptic density. Image acquisition and morphometric
analysis were performed by an experimenter blind to the
genotype of the mice.
The unpaired two-tailed t-Student’s test was used for statistical
Forebrain-specific deletion of profilin1 and unaltered
profilin2 expression levels
To investigate the role of profilin1 in synapse physiology and
plasticity, we generated conditional mutants with a selective
Figure 1. Deletion of profilin1 in Pfn1flx/flx,CaMKII-cremice. (A) Immunoblot analysis in different brain regions from an adult Pfn1flx/flxcontrol
and an adult Pfn1flx/flx,CaMKII-cremutant (P70), revealing efficient deletion of profilin1 in the forebrain of mutants. In all three forebrain tissues (cortex
(CX), striatum (STR), hippocampus (HIP)), profilin1 expression was almost undetectable in mutants. In contrast, profilin1 expression level was
unchanged in the cerebellum (CB), in which cre is not expressed. Identical results were obtained when investigating profilin1 expression levels in two
other Pfn1flx/flx,CaMKII-cremice. (B) Immunoblot analysis of hippocampal synaptosomes, demonstrating the absence of profilin1 from synaptic
structures in mutants. (C) No changes in profilin2 expression were found in the cortex, hippocampus, or striatum of three individual profilin1-
deficient mice. (D) Normal profilin2 content in hippocampal synaptosomes from two individual Pfn1flx/flx,CaMKII-cremice. Expression of b tubulin was
examined to control protein load in A–D.
Profilin1 Is Dispensable for Excitatory Synapses
PLoS ONE | www.plosone.org3 January 2012 | Volume 7 | Issue 1 | e30068
deletion of the profilin1 gene in principal neurons of the adult
forebrain (Pfn1flx/flx,CaMKII-cre). Immunoblot analysis of protein
lysates from various brain regions of Pfn1flx/flx,CaMKII-cremice at
P70 confirmed the efficient deletion of profilin1 in all three
forebrain structures examined (cortex, hippocampus, striatum;
Fig. 1A). As expected, no changes in profilin1 expression levels
were detectable in lysates from the cerebellum, where cre is not
expressed . Notably, no profilin1 immunoreactivity was
Figure 2. Unaltered spine density and morphology in hippocampal CA1 region of Pfn1flx/flx,CaMKII-cremice. (A) Representative images of
2ndorder dendritic branches of Golgi-stained pyramidal cells in the hippocampal CA1 stratum radiatum. Scale bar: 2 mm. (B) Unaltered spine density
in Pfn1flx/flx,CaMKII-cremice. Spines were morphologically categorized into mushroom-like, stubby, and thin spines (.1,000 mm length of dendritic
branches for both groups, four mice per group). Representative electron micrographs of CA1 stratum radiatum of (C) Pfn1flx/flxcontrols and (D)
Pfn1flx/flx,CaMKII-cremice. Scale bar in C: 200 nm. b: presynaptic bouton, sp: dendritic spine, *: postsynaptic density. Unaltered spine area (E) and PSD
length (F) in Pfn1flx/flx,CaMKII-cremice as deduced from cumulative distributions and mean values (insets in E and F).
Profilin1 Is Dispensable for Excitatory Synapses
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detectable in hippocampal synaptosomes of Pfn1flx/flx,CaMKII-cre
mice, demonstrating the absence of any profilin1 from synaptic
structures (Fig. 1B). In none of the three Pfn1flx/flx,CaMKII-cre
forebrain regions (Fig. 1C), nor in hippocampal synaptosomes
(Fig. 1D), did we find evidence for different expression levels of
Spine density and synapse ultrastructure are normal in
The activity-dependent recruitment to dendritic spines in
dissociated hippocampal neurons suggests that profilin1 is
important for the morphology of postsynaptic compartments
. To address this point, we visualized dendritic spines in
Golgi-stained neurons from coronal sections of control and
mice at P70–P80. For the analysis of
dendritic spine density and morphology, we chose 2ndorder
dendritic branches in the CA1 stratum radiatum (Fig. 2A), in which
profilin1 immunoreactivity is reportedly particularly pronounced
. The density of spines was similar in controls (23.960.9
spines/20 mm dendrite) and Pfn1flx/flx,CaMKII-cremice (25.660.3
Table 1. Dendritic spine morphology.
mushroom-like head perimeter (mm) 2.3160.082.2160.09
neck length (mm)0.4660.02 * 0.5260.01
Stubby perimeter (mm) 2.5360.052.4660.04
Thin length (mm)1.1960.051.1660.05
Shown are the mean values (6SEM).
Figure 3. Normal presynaptic function of hippocampal CA3-CA1 synapses in Pfn1flx/flx,CaMKII-cremice. (A) Basal synaptic transmission, as
deduced from input-output curves, was normal in Schaffer-collateral-CA1 synapses of Pfn1flx/flx,CaMKII-cremice (n=15 for controls and 10 for mutants).
(B) In Pfn1flx/flx,CaMKII-cremice, no changes were found in paired-pulse ratios (PPR; n=14 for controls, n=17 for mutants) at various inter-stimulus
intervals (ISI; 10–200 ms). Cumulative curves of amplitudes (C) and inter-event intervals (IEI) of mEPSCs (D) were virtually equal between genotypes
(n=8 in each group). Insets in C and D depict mean values.
Profilin1 Is Dispensable for Excitatory Synapses
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spines/20 mm dendrite; Fig. 2B). Moreover, there was no
difference in the density of mushroom-like, stubby, or thin spines
between the two groups. Likewise, morphometric analyses
revealed virtually no difference in spine morphology between
controls and mutants, except for a slight increase in the neck
length of mushroom-like spines (controls: 0.4660.02 mm, mu-
tants: 0.5260.01; P,0.05; Table 1). Electron microscopic
analysis in CA1 stratum radiatum (Fig. 2C–D) confirmed that the
density of excitatory synapses was unchanged in the absence of
profilin1 (controls: 4.7160.46 synapses/10 mm2, n=3386 mm2
fromfour mice; mutants:
n=2829 mm2/4 mice). Moreover, spine area and length of the
postsynaptic density (PSD) were indistinguishable between
controls and mutants (Fig. 2E–F).
Profilin2 has been implicated in the organization of synaptic
vesicles , and we wanted to know whether profilin1 fulfills a
similar function. We therefore analyzed the synaptic vesicle
organization in Pfn1flx/flx,CaMKII-cremice and found no changes
in the vesicle density (control: 178.9968.25 vesicles/mm2, n=182
presynaptic terminals/4 mice; mutant: 160.4468.75 vesicles/mm2,
n=177 presynaptic terminals/4 mice) or in the density of docked
vesicles (control: 12.9261.02 docked vesicles/mm of active zone,
n=129 presynaptic terminals/4 mice; mutant: 12.5660.66
docked vesicles/mm, n=141 presynaptic terminals/4 mice). In
summary, synapse density, spine morphology, as well as synap-
tic vesicle density and organization were all unchanged in
4.6660.35 synapses/10 mm2,
Profilin1 is dispensable for pre- and postsynaptic
Profilin2 is required for presynaptic function, yet not for
postsynaptic plasticity . We next set out to test whether
profilin1 plays a similar or complementary role in pre- and
postsynaptic physiology. To do so, we first assessed whether
general synaptic transmission and synaptic efficiency were affected
in the absence of profilin1. We recorded extracellular field
potentials in the CA1 region in acute hippocampal slices upon
stimulation of the Schaffer collateral pathway with intensities
ranging from 20–300 mA. The resulting input-output curves
revealed no differences in presynaptic fiber volley amplitude or
postsynaptic fEPSP slope between the genotypes (Fig. 3A). To
elucidate a potential involvement of profilin1 in presynaptic
physiology, we determined the paired-pulse ratio (PPR) at various
inter-stimulus intervals (ISI; 10–200 ms) and again found no
(Fig. 3B). Moreover, the amplitudes and the inter-event intervals
(IEI) of miniature excitatory postsynaptic currents (mEPSCs)
obtained from patch-clamped CA1 pyramidal neurons were not
changed in profilin1-deficient mice (Fig. 3C–D). Together our
data demonstrate that presynaptic vesicle loading, vesicle release
probability, and the vesicle release machinery are not altered in
Localization experiments have suggested a potential role of
profilin1 in postsynaptic physiology . We tested this hypothesis
Figure 4. Unimpaired synaptic plasticity in the absence of
profilin1. (A) In Pfn1flx/flx,CaMKII-cremice, no difference was found in LTD
induced by low frequency stimulation (1 Hz) of 15 min duration (n=9
for controls, n=12 for mutants) when analyzing the last 10 min of the
recordings. LTP induced by either a single 100 Hz tetanus of 1 s
duration (B) or by theta-burst stimulation (TBS) (C) was also not
different between genotypes (16100 Hz: n=10 for controls and 8 for
mutants; TBS: n=10 for both groups). ns: not significant.
Profilin1 Is Dispensable for Excitatory Synapses
PLoS ONE | www.plosone.org6January 2012 | Volume 7 | Issue 1 | e30068
in our genetic model by measuring synaptic strength modulation
during LTD and LTP. When LTD was evoked through paired
stimulation at 1 Hz for 15 min, we did not see a significant
difference in the induced steady state (45–85 min of the recording)
between controls and profilin1 mutants (Fig. 4A; P.0.05,
considering the last 10 min of the recordings for statistical
analysis). Also, when we induced LTP by a single 100 Hz tetanus
of 1 s duration (16100 Hz) or by theta-burst stimulation (TBS), we
did not find any significant differences in the resulting steady states
(25–40 min of the recording) between controls and profilin1
mutants (Fig. 4B–C; P.0.05 in both experiments considering the
last 10 min of the recordings for statistical analysis). Hence, our
data demonstrate unchanged synaptic plasticity of hippocampal
CA3-CA1 synapses in the absence of profilin1.
A depolarization- and NMDAR-driven recruitment of profilin1
and profilin2 to postsynaptic sites of excitatory synapses has been
demonstrated in studies on dissociated hippocampal neurons [14–
15]. Accordingly, analysis of organotypic hippocampal cultures
suggests a role of profilin1 and profilin2 in dendritic spine
morphology . Based on these experiments, it was suggested
that profilins are involved in actin turnover in postsynaptic
compartments, in activity-dependent morphological changes of
dendritic spines, and in postsynaptic plasticity [4–5,22]. These
ideas were supported by results obtained from fear-conditioned
rats which showed a learning-induced translocation of profilins
into dendritic spines of lateral amygdala neurons . However,
as the antibody used in this study recognizes both profilin isoforms,
the relative contribution of profilin1 and profilin2 to postsynaptic
mechanisms remained unclear. For example, profilin2 is present in
a much larger fraction of dendritic spines than profilin1 .
Thus, a predominant contribution of profilin2 to postsynaptic
plasticity was postulated [4,23–24], which, however, could not be
confirmed in vivo in profilin2-mutant mice . Moreover, various
forms of synaptic plasticity (LTP, LTD), as well as learning and
memory, were normal in this mouse model . Thus, the
discrepancy between the results obtained from in vitro experiments
and those of profilin2-mutant mice raised two important questions:
First, do profilins indeed play a role in dendritic spines in vivo?
Second, which profilin isoform does then contribute to postsyn-
To address these questions, we chose a genetic approach: we
deleted profilin1 specifically in principal neurons of the mouse
forebrain by using a conditional knockout mouse model and a
As profilin1 expression levels are particularly high in hippocampal
neurons , we chose CA1 hippocampal pyramidal cells for
morphometric analysis and hippocampal CA3-CA1 projections for
the characterization of profilin1 function in excitatory synapses.
By two independent approaches (Golgi-staining and electron
microscopy), we found that inactivation of profilin1 has no
effect on the organization of synaptic vesicles or on the density
and morphology of excitatory synapses, except for a slight
increase in the neck length of mushroom-like spines. Moreover,
our extensive electrophysiological analyses revealed that basal
synaptic transmission, presynaptic mechanisms (vesicle loading,
vesicle release probability), and postsynaptic plasticity (LTP,
LTD) are fully preserved in the absence of profilin1. Together,
these data indicate that inactivation of profilin1 has no adverse
effects on the structure and function of excitatory synapses.
Thus, in contrast to previous suggestions, we show that profilin1
is not essential for dendritic spine morphology and synaptic
Our data are in agreement with two possible scenarios. First:
profilins are not relevant for actin regulation in postsynaptic
structures, activity-dependent morphological changes of dendritic
spines, and synaptic plasticity. Second: profilin1 and profilin2 have
the capacity to compensate each other in postsynaptic structures.
In agreement with the latter suggestion, down-regulation of
profilin2 is functionally compensated by profilin1, specifically in
dendritic spines . Whether this also occurs in vivo still needs to
be addressed experimentally. Future analyses of double-mutant
mice are therefore needed for a comprehensive understanding of
profilin function in synaptic physiology and will ultimately unravel
whether profilin activity is relevant for dendritic spine morphology
and postsynaptic plasticity.
We thank Dr. R. Fa ¨ssler for conditional profilin1 mutants, K. Ociepka for
excellent technical support, Dr. L. Viltono for technical assistance with
electron microscopy, and Dr. P. Pilo Boyl for critical reading an earlier
version of the manuscript.
Conceived and designed the experiments: MBR WW EF MS-P. Performed
the experiments: AG A-MZ DS. Analyzed the data: AG A-MZ DS.
Contributed reagents/materials/analysis tools: RTB. Wrote the paper:
MBR EF WW MS-P.
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