Neurobiology of Learning and Memory xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Neurobiology of Learning and Memory

journal homepage: www.elsevier.com/locate/ynlme

The memory gene KIBRA is a bidirectional regulator of synaptic

and structural plasticity in the adult brain
Fabrice D. Heitz a, Mélissa Farinelli a, Safa Mohanna a, Martin Kahn a, Kerstin Duning b, Marco C. Frey c,
Hermann Pavenstädt d, Isabelle M. Mansuy a,⇑
a
Laboratory of Neuroepigenetics, Medical Faculty of the University of Zürich and Department of Health Science and Technology of the Swiss Federal Institute of Technology
Zürich, Brain Research Institute, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
b
Internal Medicine D, Molecular Nephrology, University Hospital Münster, Domagkstrasse 3a, D-48149 Münster, Germany
c
Division of Psychiatry Research, Medical Faculty of the University of Zürich, August Forel Strasse 1, CH-8008 Zürich, Switzerland
d
Medizinische Klinik und Poliklinik D, Molecular Nephrology, University of Münster, Domagkstrasse 3a, D-48149 Münster, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Memory formation is associated with activity-dependent changes in synaptic plasticity. The mechanisms
Received 29 May 2016 underlying these processes are complex and involve multiple components. Recent work has implicated
Revised 23 July 2016 the protein KIBRA in human memory, but its molecular functions in memory processes remain not fully
Accepted 28 July 2016
understood. Here, we show that a selective overexpression of KIBRA in neurons increases hippocampal
Available online xxxx
long-term potentiation (LTP) but prevents the induction of long-term depression (LTD), and impairs spa-
tial long-term memory in adult mice. KIBRA overexpression increases the constitutive recycling of AMPA
Keywords:
receptors containing GluA1 (GluA1-AMPARs), and favors their activity-dependent surface expression. It
Neurons
LTP
also results in dramatic dendritic rearrangements in pyramidal neurons both in vitro and in vivo.
GluA1 KIBRA knockdown in contrast, abolishes LTP, decreases GluA1-AMPARs recycling and reduces dendritic
arborization. These results establish KIBRA as a novel bidirectional regulator of synaptic and structural
plasticity in hippocampal neurons, and of long-term memory, highly relevant to cognitive processes
and their pathologies.
Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction as WWC1) has been newly associated with episodic memory in

Episodic memory in mammals is a complex form of information
storage primarily processed in the hippocampus. It recruits multi-
ple molecular cascades and components which to date, remain
only partially understood. Recently, the gene KIBRA (also known
Abbreviations: AMPAR, a-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionate
receptor; BiTetO, rtTA2-responsive Tet-On bidirectional promoter; CaMKIIa,
calcium/calmodulin-dependent protein kinase alpha; CNQX, 6-cyano-7-nitroqui
noxaline-2,3-dione; C-term, cytoplasmic C-terminus domain; dox, doxycycline;
EGFP, enhanced green fluorescent protein; EPSC, excitatory postsynaptic current;
f-EPSP, field excitatory postsynaptic potential; acGFP, acetylated green fluorescent
protein; HFS, high frequency stimulation; LFS, low frequency stimulation; LTD,
long-term depression; LTP, long-term potentiation; cLTP, chemical long-term
potentiation; MAP2, microtubule-associated protein 2; NMDAR, N-methyl-D-
Aspartate receptor; PKMf, protein kinase M zeta; PSD, postsynaptic density;
PSD-95, postsynaptic density protein 95; shRNA, small hairpin RNA; SNP, single
nucleotide polymorphism; SNX4, sorting nexin-4; rtTA2, reverse tetracycline-
controlled transactivator.
⇑ Corresponding author.
E-mail address: mansuy@hifo.uzh.ch (I.M. Mansuy).
human. A genetic variant of KIBRA with a single nucleotide poly-
morphism (SNP) leading to T-C exchange (rs17070145) in the
ninth intron of the KIBRA gene was found to correlate with a vari-
ability in memory performance in healthy subjects (Almeida et al.,
2008; Bates et al., 2009; Burgess et al., 2010; Papassotiropoulos
et al., 2006; Preuschhof et al., 2009; Schaper, Kolsch, Popp,
Wagner, & Jessen, 2008; Schwab, Luo, Clarke, & Nathan, 2014).
KIBRA is a large protein that is highly expressed in the brain, in
particular in structures important for memory including the hip-
pocampus and cortex (Johannsen, Duning, Pavenstadt,
Kremerskothen, & Boeckers, 2008; Kremerskothen et al., 2003;
Papassotiropoulos et al., 2006). KIBRA has a somatodendritic distri-
bution in neuronal cells and is enriched in the postsynaptic density
(PSD). Recent studies have shown that it interacts with the
cytoskeleton-associated proteins dendrin and synaptopodin, which
are both involved in synaptic plasticity and higher-order brain
functions (Buther, Plaas, Barnekow, & Kremerskothen, 2004;
Duning et al., 2008; Johannsen et al., 2008; Kremerskothen et al.,
2003). Importantly, KIBRA also binds to PKMf, a protein kinase

http://dx.doi.org/10.1016/j.nlm.2016.07.028
1074-7427/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
2 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx
involved in AMPARs trafficking (Yao et al., 2008) that is critical for
the maintenance of hippocampal LTP and for memory persistence
(Pastalkova et al., 2006; Shema, Sacktor, & Dudai, 2007;
Yoshihama, Hirai, Ohtsuka, & Chida, 2009). Owing to its cellular
distribution and its link with synaptic proteins, KIBRA appears as
a potentially important regulator of synaptic plasticity.
In the hippocampus, the forms of activity-dependent plasticity
thought to be implicated in memory formation involve the
strengthening or weakening of synaptic connections between neu-
rons (Bliss & Collingridge, 1993). Such adaptive changes occur in
part, through the activation of a-amino-3-hydroxyl-5-methyl-4-i
soxazole-proprionate (AMPARs) and N-methyl-D-Aspartate recep-
tors (NMDARs) in response to glutamate release, and through the
recruitment of downstream intracellular signaling pathways.
AMPARs are tetrameric receptors composed of GluA1-4 subunits
that mediate fast synaptic transmission at most excitatory
synapses. The combination of these subunits determines the func-
tional properties of the receptor subtypes (Dingledine, Borges,
Bowie, & Traynelis, 1999). In the hippocampus, AMPARs are mostly
composed of tetramers of GluA1A2 and GluA2A3 (Wenthold,
Petralia, Blahos, & Niedzielski, 1996) with GluA1A2 heteromers at
CA1 pyramidal neuron synapses (Lu et al., 2009). They are mini-
mally present at synapses in basal conditions, and are recruited
by constitutive vesicle transport mechanisms for synaptic trans-
mission and plasticity. Upon neuronal activity, new AMPARs are
inserted to the plasma membrane by exocytosis and lateral diffu-
sion. They are then endocytosed and recycled back to the mem-
brane (Hanley, 2008; Kessels & Malinow, 2009). AMPAR
trafficking and recycling are critical for synaptic plasticity, in par-
ticular for long-term potentiation (LTP) and long-term depression
(LTD) (Park, Penick, Edwards, Kauer, & Ehlers, 2004; Shepherd &
Huganir, 2007). Here, we investigated the potential contribution
of KIBRA to synaptic plasticity and to memory processes using
in vitro and in vivo models.
2. Methods
2.1. Ethical approval
Animal experiments were approved by Zürich cantonal author-
ities (License No. 205/2007) and performed as recommended by
the Federal Veterinary Office (FVO).
2.2. Transgenic mice
2.2.1. Generation
Double transgenic mice for inducible and forebrain-specific
KIBRA expression were generated by crossing CaMKIIa promoter-
rtTA2 mice (Michalon, Koshibu, Baumgartel, Spirig, & Mansuy,
2005) with pTRE-Tight-BI-AcGFP1-KIBRA mice carrying mouse
KIBRA cDNA (GenBank acc. No. NM_170779) and an acGFP reporter
driven by a rtTA2-responsive Tet-On bidirectional promoter. pTRE-
Tight-BI-AcGFP1-KIBRA transgenic mice were generated as fol-
lows: KIBRA cDNA was excised from the vector pBSK(-)-KIBRA by
EcoRI/XhoI digestion, blunt-ended with T4 polymerase (New Eng-
land Biolabs), and reinserted into the EcoRV site of pNN265. A NotI
fragment was excised (5.2 kb) and introduced in the NotI site of
pTRE-Tight-BI-AcGFP1 (Clontech). XmnI/EcoRV digest resulted in
a 7 kb fragment that was microinjected into fertilized oocytes with
a mixed C57Bl/6J and DBA2 background. Integration of the KIBRA
construct occurred in 4 founders that transmitted the transgene
to the F1 generation, giving rise to 4 independent lines. The 4 lines
were backcrossed to CaMKIIa promoter-rtTA2 mice with a
C57Bl/6J background. For the experiments, 3–4 months old trans-
genic males from the line 2 F4 generation (backcrossed for 3
Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
generations) and heterozygous for either one (control mice) or
both transgenes (double positive mice, ‘‘KIBRA overexpressing”
mice, OE) were used. Mice were housed under constant tempera-
ture and humidity in a reversed 12 h light-dark cycle (dark
8 am–8 pm). Food and water were available at libitum. Genotyping.
Transgenic mouse identification was performed by polymerase
chain reaction (PCR) on isolated genomic DNA for acGFP and rtTA2
transgenes. Pups were ear clipped at weaning. Genomic DNA was
extracted from ear biopsies by alkaline lysis (HotSHOT method)
prior to PCR. The PCR reaction was performed under standard con-
ditions, using the following forward and reverse primers for acGFP
and rtTA2 transgenes detection: acGFP for.: 50 -GTAGATCATGT
GATCGCGC-30 , acGFP rev.: 50 -GTGAATCGCATCGAGCTGAC-30 ,
F1: 50 -TGCCTTTCTCTCCACAGGTGTCC-30 , rtTA2-278-R: 50 -GAGAGCA
CAGCGGAATGAC-30 . PCR products were separated by electrophoresis
on a 2% agarose gel and visualized with GelRed staining
(5 ll GelRed/100 ml) under UV light. Doxycycline (dox) administration.
Double positive mice and control littermates were fed with dox-
containing food for 10 days. Briefly, food pellets (Alleinfuttermittel
für Versuchstiere, KLIBA NAFAG) were mixed with water at a ratio
1:1 for a few hours to overnight. 6 mg dox (Pelodis-doxycyline
hyclate capsules; Apical Pharmaceutical Corp.) were added per gram
of food. Medicated food was prepared freshly daily.
2.2.2. RT-PCR
Prefrontal cortex, insular cortex, and hippocampus were dis-
sected and total RNA was extracted using the NucleoSpin Kit II
(Macherey-Nagel), purified with RQ1 DNase (Promega) and
reverse-transcribed using the SuperScript First-Strand Synthesis
System for RT-PCR II (Invitrogen). PCR was performed on 2 ll
cDNA, using the primers acGFP for. (50 -GTAGATCATGT
GATCGCGC-30 ) and acGFP rev. (50 -GTGAATCGCATCGAGCTGAC-30 ).
PCR products were resolved on a 2% agarose gel. b-actin was used
as an internal control for normalization.
2.3. Generation of a monoclonal anti-KIBRA antibody
Mouse monoclonal KIBRA antibodies were raised against a puri-
fied recombinant human KIBRA fragment (aa 923-1113) fused to
GST. Antibodies from mouse hybridoma cell culture supernatants
were affinity-purified using a GST-KIBRA 923-1113 affinity column
and were tested by Western blot analysis with cell lysates from
KIBRA overexpressing cells.
2.4. Western blot analysis
The mouse hippocampus was dissected out and homogenized
using a 26G syringe in 10 mM HEPES, 1 mM MgCl2, 5 mM EDTA,
0.2% (v/v) Triton X-100 (Sigma), 10% (v/v) glycerol (Sigma), pro-
tease inhibitor cocktail (Sigma), 250 lM PMSF (Sigma), and
15 mM b-mercaptoethanol (Sigma). About 20 lg total protein
was resolved on 10% SDS-PAGE and transferred onto a nitrocellu-
lose membrane (BioRad). Membranes were blocked (Rockland IR
blocking buffer, Rockland), and incubated in monoclonal anti-
mouse KIBRA (1:1000) and mouse anti-mouse b-actin (1:2000;
AC-15, Sigma) primary antibodies. Membranes were then incu-
bated in goat anti-rabbit IRDye 800 and goat anti-mouse IRDye
680 secondary antibodies (1:5000, Li-Cor Biosciences). Band inten-
sity was determined and quantified using an Odyssey IR scanner
(Li-Cor Biosciences). The protein signal was normalized to b-actin.
2.5. Immunohistochemistry
Mice were sacrificed and transcardially perfused with Ringer
solution followed by 4% paraformaldehyde (Sigma) in phosphate
buffer (0.1 M, pH7.4; flow: 20 ml/min). Brains were quickly
 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx
removed, post-fixed in the same solution and transferred into a
30% sucrose solution. Coronal sections (40 lm thick) were cut with
a cryostat. Free-floating brain sections were washed in 0.1 M PB,
blocked and permeabilized in 0.1 M PB, 0.4% Triton X-100 (Sigma)
and 10% heat-inactivated horse serum (HS; Sigma) for 12 h at 4 °C.
Slices were then incubated with a primary rabbit polyclonal anti-
KIBRA antibody (V-13, Santa Cruz Biotechnology) with or without
KIBRA blocking peptide (sc-133374 P, Santa Cruz Biotechnology)
and a primary mouse monoclonal anti-NeuN antibody (A60, Milli-
pore) for 12 h at 4 °C in 0.1 M PB, 0.4% Triton X-100 and 5% HS.
Slices were washed in 0.1 M PB, 0.4% Triton X-100 and incubated
overnight at 4 °C with a goat anti-rabbit FITC and a donkey anti-
mouse TRITC fluorescence-conjugated secondary antibody (Jack-
son ImmunoResearch). DAPI (Invitrogen) was used for the staining
of neuronal nuclei. After washing in 0.1 M PB, slices were mounted
using Moviol (Molecular Probes) and stored in the dark at 4 °C. Low
magnification fluorescence images were acquired at 20 with a
CoolSNAPK4 digital camera (Roper Scientific) mounted on an Axio-
phot microscope (Zeiss) and analyzed using MCID Elite 7.0 soft-
ware (MCID). High magnification images were acquired at 80
and 160 with a Zeiss LSM 410 confocal laser-scanning micro-
scope equipped with a 40 oil-immersion objective (N.A. = 1.25)
and laser excitation at 460 nm (DAPI), 488 nm (FITC) and 570 nm
(TRITC). Each image was a z series projection of 10 stacks taken
at 1 lm depth intervals.
2.6. Behavioral testing
Morris water maze testing was carried out using a white round
pool (150 cm in diameter and 50 cm high) filled with opaque water
maintained at 21–22 °C. The pool was in a dimly lit room with var-
ious distal cues on white walls. Each animal was tracked using a
video tracking system (EthoVision, Noldus). Visible platform train-
ing (non-spatial or cued learning): Mice were trained to reach a ran-
domly located visible (flagged) platform with four trials per day
(max 90 s) for two days with 15 min inter-trial intervals (ITIs).
Mice were left on the platform for 20 s after each trial. This task
was conducted before the spatial version as a control condition
to test the ability to learn to swim towards a cued goal. Hidden plat-
form training (spatial learning): Mice were trained to find an hidden
platform with four trials per day (max 90 s), 15 min ITIs for six
days. Mice were split into subgroups, each trained with a different
platform location to counterbalance potential quadrant effects.
Mice were left on the platform for 15 s after each trial. Memory test
(probe trial): 24 h and 28 days after the last training trial, mice
were tested for their memory for the platform location after the
platform was removed from the water (60 s). Percent time spent
in each quadrant and number of platform crossings were
measured.
2.7. Electrophysiological recordings
Mice were anesthetized with isoflurane then decapitated. The
brain was quickly removed and immediately immersed in ice-
cold freshly prepared artificial CSF (aCSF; field recording:
119 mM NaCl, 26 mM NaHCO3, 2.5 mM KCl, 1.3 mM NaH2PO4,
1.3 mM MgCl2, 11 mM glucose, 2.5 mM CaCl2, gassed with 95%
O2/5% CO2; whole-cell recording: 125 mM NaCl, 25 mM NaHCO3,
2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgCl2, 25 mM glucose,
2 mM CaCl2, gassed with 95% O2/5% CO2.
2.7.1. Field recording
Acute slices (400 lm thick) were prepared with a vibratome (VT
1000S; Leica Microsystems, Bannockburn, IL) in ice-cold gassed
aCSF. Sections were incubated in aCSF at 34 °C for 20 min then kept
at room temperature for at least 1 h. Recording was performed in
Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
3
an interface chamber continuously flowed with aCSF at 1–2 ml/
min. A monopolar electrode was placed in the Schaffer collateral
fibers, and stimulation pulses applied at 0.033 Hz with stimulus
intensity ranging from 20 to 80 lA, yielding evoked field EPSPs
(f-EPSPs) of 0.2–0.5 V. f-EPSPs were recorded in the stratum radia-
tum using a borosilicate micropipette filled with aCSF. The signal
was amplified with an Axopatch 200A amplifier (Molecular
Devices, Union City, CA), digitized by a Digidata 1200 interface
(Molecular Devices) and sampled at 10 kHz with Clampex 8.2
(Molecular Devices). Baseline was recorded for a minimum of
20 min or until stable. Plasticity was then induced by stimulation
with either 1 Hz for 15 min (900 pulses), 100 Hz for one train of
1 s tetanus, or 100 Hz for three trains of 1 s tetanus separated by
20 s. Data were analyzed by measuring the slope of individual
f-EPSPs at 1–1.5 ms after the stimulus pulse by linear fitting using
Clampfit (Molecular Devices).
2.7.2. Whole-cell recording
Transversal slices (250 lm thickness) were cut using a tissue
vibratome (VT 1000S; Leica Microsystems, Bannockburn, IL). Slices
were kept in aCSF at room temperature before recording. Then,
slices were transferred to a submerged recording chamber and per-
fused with aCSF containing 2.5 lM bicuculline and bubbled with
95% O2/5% CO2. All experiments were performed at room temper-
ature. Patch pipettes (2–5 MX) for whole-cell voltage recordings
were filled with intracellular solution containing (in mM) 120
Cs-gluconate, 10 CsCl, 10 HEPES, 8 NaCl, 0.2 EGTA, 2 MgATP, 0.3
Na3GTP, and 10 phosphocreatine (pH 7.3 with CsOH, osmolarity
290–300 mOsm). In some experiments 100 lM spermine was
added to the intracellular solution. Liquid junction potentials were
not corrected. Pyramidal cells in the CA1 region of the hippocam-
pus were identified by infrared differential interference contrast
video microscopy with an Olympus BX51 microscope. Whole-cell
voltage clamp recordings were made with a MultiClamp 700A
(Molecular Devices) amplifier. Signals were filtered at 2 kHz and
digitized at 10 kHz by an analog-to-digital converter (Digidata
1322A, Molecular Devices). To evoke synaptic potentials and cur-
rents from two independent pathways, two low-resistance
(2 MX) glass electrodes filled with standard ACSF were placed at
a distance of 100 lm from the soma. Excitatory synaptic potentials
were evoked by extracellular field stimulation (100 ls) at 0.125 Hz
with a delay of 4 s between control and paired pathway. Stimulus
intensity was adjusted to produce a single excitatory postsynaptic
current (EPSC) with an average amplitude of 50–150 pA. For anal-
ysis of synaptic AMPARs, the AMPA component of the mixed
AMPAR and NMDAR-mediated EPSC at a holding potential of
+40 mV was measured at the beginning of the EPSC peak between
0 and 70 ms. Series and input resistance was continuously moni-
tored during the recording in response to a small, hyperpolarizing
current or voltage step (10 mV; 250 ms). Cells were clamped at
70 mV during EPSC recording and series resistance was compen-
sated 80–90%. LTP experiments were performed in a modified aCSF
containing 4 mM MgCl2, 4 mM CaCl2 and 10 lM Glycine. Extracel-
lular stimulation was adjusted until a stable baseline of EPSCs
could be recorded for 10 min (75 sweeps). The peak EPSC ampli-
tude of the synaptic response was calculated as the difference
between maximum current in a time window of 25 ms after extra-
cellular stimulation and the average current during the 50 ms pre-
ceding extracellular stimulation. EPSC amplitudes were
normalized to the average amplitude before LTP induction. EPSC
decays were fitted with a single exponential from 90% to 10% of
the EPSC peak. A postsynaptic depolarization pairing protocol
was used for LTP induction in voltage clamp experiments. For
depolarization pairing 180 paired pathway EPSCs were evoked at
1 Hz and were paired with continuous postsynaptic depolarization
at 0 mV. After LTP induction, EPSC sampling was continued for up
4 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx
to 30 min. Data analysis was performed using custom written soft-
ware in IGOR (Wavemetrics, Lake Oswego, OR). Bath application of
AMPA was performed as previously described {Schnell, 2002
#142}. Values are expressed as mean ± SEM. Two-tailed Student’s
t-tests were used for the calculation of statistical significance.
2.8. Hippocampal slice culture and viral infection
Organotypic hippocampal slices from 5 to 7 day old postnatal
wild-type C57/BL6 mice were prepared and cultured for 2 weeks
using the roller-tube technique (Gahwiler, Capogna, Debanne,
McKinney, & Thompson, 1997). Slices were then transferred to an
electrophysiological recording chamber at room temperature and
the CA1 pyramidal cell layer was injected with 2 ll of lentiviral
particles (5  106 infectious units/ml) containing several expression
constructs, each encoding a 19–25 nucleotide shRNA designed to
specifically knockdown kibra expression (sc-146464-V, Santa Cruz
Biotechnology). Lentiviral particles containing constructs encoding
a scrambled shRNA sequence were used as control (sc-108080,
Santa Cruz Biotechnology). Slices were returned to roller tubes
after injection, and cultured for 1 week in medium supplemented
with penicillin/streptomycin (1:500; Sigma) until recorded.
2.9. Quantitative real-time RT-PCR
Quantitative PCR was performed on 0.5 ll cDNA using specific
Taqman probes for the mouse KIBRA full-length mRNA
(Mm01325387_m1; Applied Biosystems) and an Applied Biosys-
tems 7500 Thermal Cycler. Each sample was analyzed in triplicate
and an equal amount of cDNA was plated for each sample. Values
were chosen in the linear range of amplification and the compara-
tive Ct method was used to assess differences in gene expression
between samples. b-actin was used as an internal control for
normalization.
2.10. Subcellular fractionation
Subcellular fractionation of adult mouse brain was conducted as
follows. The hippocampus was dissected and homogenized to 10%
(wt/vol) in ice cold 0.32 M sucrose buffer (0.32 M sucrose, 20 mM
HEPES (pH 7.4), 5 mM EDTA, and protease inhibitor cocktail
(Sigma) using a tissue lyser (Qiagen). The homogenate was pro-
cessed by sequential centrifugation to collect different fractions
(P2, LS1, LP1, LS2, LP2, SM) as described previously (Suh et al.,
2010). For each fraction, 20 lg of total protein were used for Wes-
tern blotting and the following primary antibodies: mouse mono-
clonal anti-KIBRA, mouse monoclonal anti-PSD-95 (7E3-1B8,
Sigma), and mouse monoclonal anti-mouse GluN1 (54.1,
Millipore).
2.11. Neuronal cultures
Primary hippocampal neurons were isolated from embryonic
day 18 (E18) control or transgenic mice and rat pups hippocampi.
Mouse hippocampal neurons were cultured on 12-well dishes as
previously described (Kaech & Banker, 2006). KIBRA transgene
expression was induced by adding dox (1 lM) to the culture med-
ium for 48 h. To knockdown KIBRA in neurons, we used commer-
cially available lentiviral particles containing several expression
constructs each harboring a target-specific shRNA (sc-146464-V,
Santa Cruz Biotechnology). Lentiviral particles (5  106 infectious
units/ml) containing a construct encoding a scrambled shRNA
sequence were used as control (sc-108080, Santa Cruz Biotechnol-
ogy). Neuronal infections were performed at DIV12-18 by using
2 ll of lentivirus per well on a 12-well dish. Rat hippocampal
neurons were cultured onto poly-L-lysine coated coverslips in
Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
Neurobasal growth medium supplemented with 2% B27, 2 mM
Glutamax and 5% FBS.
Mouse neurons in culture were stained using mouse mono-
clonal anti-KIBRA, rabbit polyclonal anti-GluA1 (ABN241, Milli-
pore) and mouse monoclonal anti-PSD-95 (7E3-1B8, Sigma)
primary antibodies. Images were acquired with a Zeiss LSM 410
confocal laser-scanning microscope equipped with a 63 oil-
immersion objective (N.A. = 1.32) and laser excitation at 488 nm
(FITC) and 570 nm (TRITC). Each image was a z series projection
of 16–17 stacks taken at 0.27 lm depth intervals.
2.12. Colocalization and co-immunoprecipitation assays
Green monkey CV1 and HEK293T cells were cultured as
described previously (Duning et al., 2008; Kremerskothen et al.,
2003). CV1 and HEK293T cells were transfected with native
pQCXIP-mCherry, pQCXIP-mCherry-KIBRA and pcDNA6-Flag-
KIBRA. Modified pEGFP-C1 and V37/V180-3-Flag plasmids were
used to express the full-length human KIBRA sequence (Duning
et al., 2008; Kremerskothen et al., 2003) and the rat GluA1
C-Terminus (aa 821-907) fused to EGFP (pEGFP-C1-KIBRA and
pEGFP-C1-GluA1 C-term) and to a Flag tag (V37/V180-3-Flag-
KIBRA and V37/V180-3-Flag-GluA1 C-term). Rat hippocampal
neurons were co-transfected with pEGFP-C1-GluA1 C-term or pSIN
REP5-GFP-GluA2 and pQCXIP-mCherry or pQCXIP-mCherry-KIBRA
at DIV 14-15. Live-imaging assays were performed at 488 nm and
561 nm excitation to detect GFP and mCherry 48 h post-
transfection. Transient transfection was performed using Lipofec-
tamine 2000 (Invitrogen, Germany) according to the manufac-
turer’s instructions. For immunocytochemical staining, CV1 cells
grown on coverslips were fixed 24 h post-transfection in 4%
paraformaldehyde (PFA) in PBS at room temperature for 20 min.
Samples were washed with PBS and incubated for 10 min in
50 mM NH4Cl in PBS to quench reactive amino groups. Staining
was performed using mouse monoclonal JL8 antibody directed
against EGFP (Invitrogen). After washing in PBS, coverslips were
rinsed in water and cells were mounted in Moviol. Images were
obtained using a Leitz Observer Z1 fluorescence microscope. For
co-IP assays, lysates from co-transfected HEK293T cells were pre-
pared by scraping cells into IP buffer (1% Triton-X 100, 20 mM
Tris-HCl (pH 7.5), 25 mM NaCl, 50 mM NaF, 15 mM Na4P2O7, and
1.5 mM EDTA) containing protease inhibitor (Complete; Roche,
Mannheim, Germany). Lysates were centrifuged at 10,000g for
30 min at 4 °C. For precipitation of EGFP-tagged KIBRA and GluA1
C-term, GFP-Trap-A (Chromotec) was used according to the manu-
facturer’s instructions. For precipitation of Flag-tagged KIBRA and
GluA1 C-term, supernatants were incubated with anti-FLAG affin-
ity beads (Sigma-Aldrich) overnight at 4 °C on a rocking platform.
For endogenous IP, rat brains were washed and homogenized in
buffer (4 mM HEPES/KOH, pH 7.4; 320 mM sucrose) and cen-
trifuged at 800g for 10 min at 4 °C. Supernatant was centrifuged
at 9,000g for 15 min at 4 °C and resulted pellet was resuspended
in homogenization buffer and centrifuged at 11,000g for 15 min
at 4 °C. The resulting pellet (P2) was solubilized in 0.5% Tri-
ton/50 mM Tris/HCl, pH 9.0 and centrifuged 10 min at 100,000g.
800 lg of each P2 supernatant was used for each immunoprecipi-
tation. Extracts were pre-cleared for 2 h with 40 ll of protein A/G
sepharose beads (Pierce) at 4 °C. Rabbit KIBRA antibodies or rabbit
IgG control were coupled 90 min at 4 °C to 40 ll protein A/G
sepharose beads. Pre-cleared P2 fraction was added to protein
A/G sepharose beads and incubated overnight at 4 °C on an over-
head rotator. For elution of bound proteins, washed beads were
resuspended in sample buffer and boiled for 5 min at 95 °C.
Samples were subsequently subjected to SDS-PAGE and Western
blot analysis as described earlier (Duning et al., 2008). 3Flag
was used as negative control samples.
 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx
2.13. Recycling and internalization assays
AMPAR recycling was measured in primary hippocampal neu-
rons as previously described (Suh et al., 2010), with some modifi-
cations. Briefly, dissociated neurons in culture (DIV17-23) were
incubated on coverslips in conditioned medium for 15 min at
20–25 °C with rabbit polyclonal anti-GluA1 primary antibody
(ABN241, Millipore) directed to the N-terminus to label surface
AMPARs. Neurons were then incubated in conditioned medium
for 30 min at 37 °C to allow internalization of the receptors. Non-
internalized surface bound antibody was removed by stripping in
0.5 M NaCl and 0.2 M acetic acid for 2 min at 4 °C. Stripping was
performed directly after primary antibody incubation as control.
Neurons were then incubated in conditioned medium for 1 h at
37 °C to allow the recycling of receptors back to the plasma mem-
brane. Neurons were fixed in 4% PFA in 0.1 M PBS for 10 min at
4 °C, washed (in PBS 0.1 M, 3% BSA), and incubated for 45 min at
room temperature (RT) with an excess of Cy2-conjugated sec-
ondary antibody (in PBS 0.1 M, 3% BSA) to label surface-recycled
receptors. Internalized receptors that failed to recycle back to the
plasma membrane were subsequently labeled by permeabilization
with 0.1% Triton X-100 (Sigma) in PBS 0.1 M for 30 min at RT and
by incubation with Cy5-conjugated rabbit secondary antibody (in
PBS 0.1 M, 3% BSA) for 45 min at RT. To control for the specificity
of internalized receptors staining, some neurons did not undergo
permeabilization. Endocytosis of GluA1-AMPARs was evaluated
by simultaneous staining surface and internalized receptors. The
assay was performed as described for the recycling of GluA1-
AMPARs, except that neurons were incubated for 15 min at 37 °C
to allow AMPARs internalization and immediately fixed before
incubation with the secondary antibody. Imaging was performed
with a Zeiss LSM 410 confocal laser-scanning microscope equipped
with a 40 oil-immersion objective (N.A. = 1.25) and laser excita-
tion at 488 nm (Cy2) and 633 nm (Cy5). Each image was a z series
projection of 10 stacks taken at 1 lm depth intervals and using
identical acquisition parameters. The amount of recycling and
endocytosis was quantified using Imaris 7 software (Bitplane).
Data for recycled receptors are presented as a recycling index,
which is the ratio of the surface (recycled) fraction and the total
(internalized and recycled) fraction. Data for internalized receptors
are presented as an internalization index, which is the ratio of the
internalized fraction and the total (surface and internalized)
fraction.
2.14. Live-cell imaging
Hippocampal neurons from E18 rat pups were plated onto
poly-L-lysine coated coverslips in Neurobasal growth medium sup-
plemented with 2% B27, 2 mM Glutamax, 50 U/mL penicillin,
50 lg/mL streptomycin and 5% FBS. Neurons were transfected at
DIV 14-17 using lipofectamine 2000 (Invitrogen) and SEP-GluA1
live-imaging assays were performed 48 h post-transfection as
described previously (Ashby et al., 2004). Briefly, coverslips con-
taining neurons were assembled onto a closed perfusion chamber
and continuously perfused with recording buffer (125 mM NaCl,
25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgCl2,
25 mM glucose, 2 mM CaCl2, 1 lM TTX, pH 7.4), After 3 min of
baseline recording (F0), neurons were perfused with recording
buffer supplemented with 200 lM glycine for 3 min before the
perfusion was switched back to recording buffer for the remainder
of the recording session. All imaging experiments were performed
at 37 °C using a Leica SP5 confocal microscope. The pHluorin
fluorescence was imaged at 488 nm excitation and collected
through a 505–550 nm filter, while the mCherry signal was imaged
at 561 nm excitation and 575–615 nm emission. Neurons were
Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
5
imaged through a 63 oil objective (N.A. = 1.10–1.30) and
collected at a rate of 1 image per min. Images were analyzed using
ImageJ software (NIH) by calculating the normalized change in
average pHluorin over mCherry fluorescence intensities from a
somatic and dendritic shaft area defined manually to compensate
for x-y movements of the recorded neurons. The fluorescence
intensity change is expressed as DF/Fo and the maximal amplitude
of fluorescence change (DFmax/Fo) represents the extent of GluA1
exocytosis.
2.15. Morphological analysis
Immunostaining with a mouse monoclonal anti-MAP2 primary
antibody (AP20, Novus Biologicals) was used on mouse primary
neurons. Golgi staining was performed on mouse brain slices using
FD Rapid GolgiStain Kit (FD NeuroTechnologies) and according to
the manufacturer’s instructions. Reconstruction and morphological
analyses were performed with Imaris 7 software (Bitplane).
Fluorescent images were taken with a Zeiss LSM 410 confocal
laser-scanning microscope equipped with a 63 oil objective
(N.A. = 1.32) and laser excitation at 488 nm. Each image was a z
series projection of 16–17 stacks taken at 0.27 lm depth intervals.
Golgi images were acquired on an Axiophot microscope (Zeiss). We
also used unstained brain slices of Thy1-YFP (Yellow Fluorescent
Protein) mice crossed with KIBRA transgenic mice (F4) to analyze
the morphology of YFP expressing neurons in the CA1 area of the
hippocampus. Transgenic males heterozygous for YFP (control
mice) or for both YFP and KIBRA (OE mice) were fed with
doxycycline-containing food for 10 days prior sacrifice and histo-
logical analysis.
2.16. Statistical analysis
Data are presented as mean normalized to baseline or con-
trol ± SEM. Student’s t-test and analysis of variance (ANOVA) were
used to compare non-normalized data. Statistical significance was
set at ⁄p 6 0.05, ⁄⁄p 6 0.01 and ⁄⁄⁄p 6 0.001.
3. Results
3.1. KIBRA overexpression in hippocampal neurons in vivo
We studied the role of KIBRA in memory formation and hip-
pocampal plasticity by selectively modulating its level of expres-
sion in mouse forebrain neurons in vitro and in vivo. KIBRA was
conditionally overexpressed selectively in forebrain neurons by
generating transgenic mice carrying a full-length KIBRA gene
under the control of a tetracycline-responsive bidirectional pro-
moter (BiTetO) fused to a green fluorescent protein (acGFP) repor-
ter. These animals were crossed with mice expressing rtTA2 under
the control of a CaMKIIa promoter (CaMKIIa-rtTA2) (Michalon
et al., 2005) (Fig. 1A). In double transgenic mice (heterozygous
for both transgenes), treatment with doxycycline (dox) induced
KIBRA mRNA overexpression in neurons in forebrain structures
including the prefrontal and somatosensory cortex, and the hip-
pocampus (Fig. 1B). Quantitative real-time PCR and Western blot
analysis using a selective antibody against KIBRA (Supplementary
Fig. 1A) showed that KIBRA (Kremerskothen et al., 2003;
Papassotiropoulos et al., 2006) (Fig. 1C) was expressed in the trans-
genic mouse hippocampus (Supplementary Fig. 1B), with a 2-fold
increase in KIBRA protein (Fig. 1D). The increase in KIBRA protein
was detected in neurons, particularly in CA1 pyramidal neurons
as shown by immunostaining (Fig. 1E and F).
6 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx

Fig. 1. Conditional KIBRA overexpression (OE) in the adult mouse forebrain. (A) Schematic representation of the rtTA2 system used for inducible and neuron-specific
expression of KIBRA in the mouse brain. Doxycycline (Dox) binds to rtTA2, which is expressed in forebrain neurons due to the CaMKIIa promoter, and leads to rtTA2-mediated
activation of the tetracycline-responsive BiTetO promoter, and KIBRA and acGFP expression in forebrain neurons. (B) RT-PCR analyses of acGFP expression reflecting KIBRA
expression in prefrontal cortex (PfCx), cortex (Cx), and hippocampus (Hi) in transgenic (OE + dox) and control (Control) littermates treated with dox, and in transgenic mice
not treated with dox (OE-dox). No transgene expression was detected in the absence of dox. -RT = no reverse transcription, H2O = water control. (C) Schematic representation
of full-length KIBRA protein including from the N- (N-term) to C-terminal (C-term) region two WW domains for binding to proline-rich sequences, a putative nuclear
localization signal (NLS), a calcium-dependent binding domain (C2), a glutamic acid-rich (Glu-rich) domain, two phosphorylation sites on Ser residues (p-Ser sites), and a PDZ
binding motif. (D) Immunoblot analyses of KIBRA protein in two independent samples from transgenic (OE) and control mice treated with dox (left panel). b-actin was used as
loading control. Quantification relative to control (right panel; KIBRA protein level: control = 1 ± 0.1 (n = 4) versus OE = 2.6 ± 0.7 (n = 3), t(5) = 2.67, ⁄p < 0.05. Data are
expressed as fold change (mean ± SEM). (E) Immunohistochemical analyses of KIBRA expression (green) in the hippocampus in transgenic (OE, upper panel) and control
(control, middle panel) mice. No fluorescence was observed in the presence of a KIBRA antibody blocking peptide (bottom panel), indicating no unspecific staining. Scale bar:
200 lm. (F) KIBRA (green) co-immunostaining with the neuronal marker NeuN (red), and DAPI (blue) in hippocampus area CA1 in transgenic (OE, left) and control (Control,
right) mice. Scale bar: 50 lm and 10 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. KIBRA overexpression impairs spatial memory
Since KIBRA has been implicated in human memory, we exam-
ined the impact of its overexpression on memory in the transgenic
animals using a water maze (Morris, 1984; Vorhees & Williams,
2006). On a non-spatial version of the task, both control and trans-
genic animals learned the platform position similarly well, and had
comparable swimming distance and latency to reach the platform
(Supplementary Fig. 2A). In contrast, on a spatial version of the
task, while learning was normal in both groups (Fig. 2A), spatial
memory 24 h after the end of training was impaired in the trans-
genic mice. Transgenic mice spent significantly less time in the tar-
get quadrant (Fig. 2B, left) and had fewer platform crossings
(Fig. 2B, right), suggesting impaired spatial memory. The memory
impairment was persistent and could still be detected based on
time spent in the target quadrant after 28 days (Fig. 2C, left). Both
control and transgenic mice showed few platform crossings
(Fig. 2C, right), indicative of a gradual decline in memory strength.
No difference in performance between control and KIBRA
transgenic mice was however observed on the open field
(Supplementary Fig. 2B), or in contextual or tone fear memory
(Supplementary Fig. 2C).
3.3. KIBRA acts as a positive regulator of synaptic potentiation in CA1
neurons
We next examined the effect of KIBRA overexpression on synap-
tic plasticity in the hippocampus after one- or three-train of high
frequency stimulation (HFS) of Schaffer collaterals. LTP was signif-
icantly increased in hippocampal area CA1 after one-train HFS in
slices from the transgenic mice compared to controls, but it was
lower after three-train HFS (Fig. 3A and B). In the slices from trans-
genic mice, the overall level of potentiation after one or three-train
HFS was similar, suggesting that LTP is at its maximum level in
transgenic slices already after only one tetanus. Indeed, when a
second tetanus was delivered 15 min after the first tetanus, LTP

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx 7

Fig. 2. KIBRA overexpression in forebrain neurons impairs spatial memory. (A) Path length (left panel) and escape latency (right panel) to find a hidden platform in a water
maze were similar in control (n = 9) and transgenic mice (OE, n = 8). Comparable decrease in path length (control: F(5,20) = 5.3, ⁄⁄p < 0.01; OE: F(5,40) = 3.0, ⁄p < 0.05; left
panel) and escape latency (control: F(5,20) = 5.0, ⁄⁄p < 0.01; OE: F(5,40) = 3.3, ⁄p < 0.05; right panel) over the 6 days of acquisition (4 trials per day) in control and transgenic
mice. (B) Performance on a spatial memory test 24 h after the last acquisition trial. Percentage of total time spent in each quadrant (left panel); chance is at 25%. Time spent in
the target quadrant was significantly above chance in control and transgenic mice (control: t9 = 3.4, ⁄p < 0.05; OE: t8 = 4.4, ⁄⁄p < 0.01) but, transgenic mice spent significantly
less time in the target quadrant (control: 57.8 ± 5.4%; OE: 34.1 ± 2.1%; F(1,11) = 8.5, ⁄⁄p < 0.01), and had fewer crossings of the platform location (control: 4.2 ± 0.4; OE:
2.1 ± 0.5; F(1,12) = 6.5, ⁄⁄p < 0.01; right panel) than control mice. (C) Performance on a spatial memory test 28 days after the last acquisition trial. Transgenic mice spent
significantly less time in the target quadrant than control mice (control: 60.4 ± 5.3%; OE: 35.2 ± 2.2%; F(1,11) = 12.9, ⁄⁄p < 0.01; right panel), with no differences in crossings of
the platform location (control: 1.7 ± 0.8; OE: 1.9 ± 0.3; right panel). Chance is at 25%. Target, left, opposite, right indicate the quadrant position relative to the platform’s last
position. Data are mean ± SEM.

was not further increased in the transgenic slices unlike in controls
(Supplementary Fig. 3A and B). The effect on LTP observed in trans-
genic slices resulted from KIBRA overexpression and not from any
effect of transgene integration since LTP was unaffected in double
transgenic mice not treated with dox (Supplementary Fig. 3C). Fur-
ther, LTP change upon KIBRA overexpression was not due to any
gross alteration in basal synaptic transmission since field excita-
tory postsynaptic potentials (f-EPSPs) in response to increasing
stimulation intensity were similar in transgenic and control slices
(Fig. 3B, right). To confirm the involvement of KIBRA in LTP, we
next tested the effect of KIBRA knockdown. For this, a lentivirus
expressing a KIBRA shRNA was injected in the CA1 pyramidal cell
layer in organotypic hippocampal slices (Koshibu et al., 2009),
and fEPSPs were recorded five days later. KIBRA shRNA signifi-
cantly decreased the expression of full-length KIBRA mRNA
(Fig. 3C). This led to an impairment of one-train LTP in CA1 neurons
(Fig. 3D), an effect that was not seen in neurons infected with a
scrambled control shRNA. These data reveal that KIBRA
overexpression and knockdown have an opposite effect on LTP
and confirm the involvement of KIBRA in this form of synaptic
plasticity. Next, we examined whether KIBRA is also involved in
LTD. Prolonged low frequency stimulation (LFS: 15 min at 1 Hz)
at Schaffer collaterals induced LTD in control slices, but not in
transgenic slices in which it rather led to a robust LTP in the CA1
area (Fig. 3E). This LTP slowly increased and reached an amplitude
comparable to that induced by one-train HFS about 35 min after
stimulation. Together, the data show that KIBRA overexpression
shifts hippocampal plasticity in favor of LTP and prevents LTD
(Fig. 3F), pointing to a function of KIBRA as positive regulator of
synaptic potentiation. The saturating effect of KIBRA overexpres-
sion on LTP and the impairing effect on LTD may explain the mem-
ory impairment, as inappropriate synaptic strengthening in the
hippocampus is likely detrimental to memory processes. It is also
consistent with the hypothesis that hippocampal LTD is needed
for memory formation (Kemp & Manahan-Vaughan, 2007, 2008;
Manahan-Vaughan & Braunewell, 1999; Migaud et al., 1998;

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
8 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx

Fig. 3. KIBRA overexpression increases hippocampal CA1 LTP while KIBRA knockdown blocks LTP. (A) f-EPSP slope is increased in transgenic slices (OE) compared to controls
following one-train of 100 Hz stimulation (1  100 Hz HFS) (f-EPSP slope averaged from min 20 to 45: control = 119.1 ± 6.9% (n = 6) versus OE = 152.1 ± 11.2% (n = 7), t(11)
=2.40, ⁄p < 0.05). (B) LTP induced by three-train HFS (3  100 Hz HFS) is higher in control slices than in transgenic slices (f-EPSP slope averaged from min 20 to 45:
control = 206.3 ± 14.5% (n = 9) versus OE = 162.1 ± 12.3% (n = 7), t(14)=2.24, ⁄p < 0.05). Stimulation was delivered at time zero. Inset: input-output curve with f-EPSPs over
increasing stimulation intensity showing comparable basal synaptic transmission in CA1 neurons in transgenic slices overexpressing KIBRA (OE, n = 5) and control slices
(n = 5). Data are expressed as mean f-EPSP slope. (C) Quantitative real-time PCR analyses of KIBRA full-length mRNA expression in hippocampal slice cultures injected in area
CA1 with a lentivirus expressing a KIBRA shRNA for knockdown (KD) or a lentivirus expressing a scrambled shRNA (control). Five days after infection, full-length mRNA was
significantly decreased in slices injected with the KIBRA shRNA lentivirus (control = 1.00 ± 0.08 (n = 3) versus KD = 0.71 ± 0.05 (n = 4), t(5) = 3.25, ⁄p < 0.05). b-actin was used
as internal control. Data are expressed as fold change (mean ± SEM). (D) No LTP was observed in slices with KIBRA knockdown while control slices have normal LTP following
one-train of 100 Hz stimulation (1  100 Hz HFS) delivered at time zero (f-EPSP slope averaged from min 20 to 30: control = 146.3 ± 8.6% (n = 3) versus KD = 90.2 ± 6.6%
(n = 4), t(5) = 5.28, ⁄⁄p < 0.01). Horizontal bars indicate the time window for calculation of mean f-EPSPs in control and KIBRA knockdown slices before (1) and after (2) LTP
induction, as shown in representative traces (top insets). (E) Low frequency stimulation (15 min at 1 Hz, delivered at time zero) results in LTD in control slices, but induces LTP
in transgenic slices (f-EPSP slope averaged from min 20 to 45: control = 76.9 ± 4.3% (n = 4) versus OE = 151.2 ± 13.1% (n = 4), t(6) = 5.389, ⁄⁄p < 0.01). (F) Frequency response
curve in control and transgenic slices. 0.033 Hz corresponds to the stimulation frequency used to record basal synaptic transmission. (G) Whole-cell two-pathway recording
of CA1 pyramidal neurons in acute slices from control and KIBRA OE mice. EPSCs were evoked at membrane potential by presynaptic stimulation and LTP was induced by
postsynaptic depolarization (delivered at time zero). LTP is increased in the stimulated pathway in transgenic neurons (OE LTP-path, n = 9) compared to controls (control
LTP-path, n = 6). No LTP was induced in the non-stimulated pathway (control and OE Ctrl-path). (H) Analysis of mean EPSCs amplitude in control and KIBRA OE CA1 neurons
at baseline (averaged from min 10 to 0), 10 min after LTP induction (averaged from min 0 to 10; stimulated pathway (LTP-path): control = 147 ± 16% (n = 6) versus
OE = 229 ± 14% (n = 9), t(13) = 3.80, ⁄⁄p < 0.01; non-stimulated pathway (Ctrl-path): control = 99 ± 8% (n = 6) versus OE = 90 ± 6% (n = 9), t(13) = 0.92, n.s.), and over the last
20 min (averaged from min 20 to 40 after pairing; stimulated pathway: control = 123 ± 12% (n = 6) versus OE = 183 ± 13% (n = 9), t(13) = 3.20, ⁄⁄p < 0.01; non-stimulated
pathway: control = 98 ± 9% (n = 6) versus OE = 72 ± 4% (n = 9), t(13) = 2.76, n.s.). Baseline (100%) is represented by a dashed line. Data are expressed as mean f-EPSP slope and
mean EPSC amplitude of five-binned consecutive sweeps normalized to baseline (100%) ± SEM.

Moser, Krobert, Moser, & Morris, 1998; Nakao, Ikegaya, Yamada,
Nishiyama, & Matsuki, 2002; Willshaw & Buckingham, 1990).
3.4. KIBRA is specifically involved in activity- and GluA1-dependent
synaptic potentiation in CA1 neurons
The expression and maintenance of LTP in the hippocampus lar-
gely depend on postsynaptic AMPARs, and require the insertion of
new AMPARs in the postsynaptic membrane (Hayashi et al., 2000;
Makino & Malinow, 2009; Takahashi, Svoboda, & Malinow, 2003).
Since KIBRA is present in synaptic terminals (Schneider et al.,
2010), we tested whether it can modulate a form of
GluA1-dependent LTP in CA1 pyramidal neurons using whole-cell
patch-clamp recording. AMPAR-dependent excitatory post-
synaptic currents (EPSCs) were measured following a paired stim-
ulation using a protocol known to induce GluA1-dependent LTP
(Frey, Sprengel, & Nevian, 2009). This stimulation induced a robust
LTP that was specific to the stimulated pathway in control neurons.
In transgenic neurons, this form of LTP was significantly increased
(Fig. 3G and H). Again, this effect was not due to a change in basal

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx 9

synaptic transmission since current-voltage relationship of
AMPAR-mediated EPSCs at different holding potential was compa-
rable in control and transgenic neurons (Fig. 4A and B). A rectifica-
tion index calculated from the ratio of EPSCs amplitude at 60 and
+40 mV holding potential was also similar in control and trans-
genic slices (data not shown). Further, the effect was not linked
to a change in NMDAR currents since the AMPAR/NMDAR ratio
was comparable in control and transgenic cells (Fig. 4C). AMPAR-
mediated spontaneous activity was also unaffected in transgenic
neurons as seen by comparable spontaneous AMPARs EPSC (sEPSC)
amplitude, sEPSC frequency, and sEPSC decay time course in CA1
neurons in transgenic and control slices (Fig. 4D and E). The mea-
surements were specific to AMPAR EPSCs, since the AMPAR chan-
nel blocker CNQX abolished spontaneous responses (Fig. 4E).
Moreover, bath application of AMPA evoked a comparable
response in control and transgenic slices (Fig. 4F). These results
demonstrate that KIBRA is involved in GluA1-dependent synaptic
plasticity, but does not alter AMPAR subunit composition at
synapses or the overall AMPAR content at the cell surface in resting
conditions (Meng, Zhang, & Jia, 2003).
3.5. KIBRA colocalizes and interacts with GluA1-AMPARs in
hippocampal neurons
To further explore the relationship between KIBRA and GluA1-
containing AMPARs (GluA1-AMPARs), we next examined the dis-
tribution of KIBRA in hippocampal neurons and its localization
with respect to AMPARs. Various subcellular fractions were pre-
pared from adult mouse hippocampus, and assayed for KIBRA by
Western blotting. KIBRA was enriched in synaptic fractions, specif-
ically in synaptic membranes that also contained the postsynaptic
components PSD-95 and GluN1 (Fig. 5A), consistent with the pres-
ence of KIBRA in PSDs (Johannsen et al., 2008). At a subcellular
level, immunostaining showed that KIBRA colocalizes with GluA1
clusters on dendrites, but is also present in somato-dendritic com-
partments in cultured neurons (Supplementary Fig. 4A and B). It
also colocalizes with PSD-95 in these structures, confirming its
presence in the PSD (Supplementary Fig. 4C and D). KIBRA is also
distributed in the nucleus of hippocampal neurons, consistent with
its putative nuclear localization sequence and nuclear function
(Rayala et al., 2006). Next, the link between KIBRA and GluA1

Fig. 4. KIBRA overexpression does not affect AMPAR spontaneous activity in CA1 neurons. (A) Mean amplitude of AMPAR EPSCs recorded at different membrane holding
potential (70 mV to +40 mV) in patched CA1 pyramidal neurons in acute hippocampal slices from control (n = 7) and transgenic mice (OE, n = 4) using the same stimulation
position and intensity as during LTP recording. The amplitude of the evoked AMPAR EPSCs component was calculated over the first 20 ms and averaged for each potential. (B)
Representative traces in control and transgenic slices recorded at a holding potential of +40 and 70 mV. (C) AMPAR/NMDAR ratio calculated as the mean AMPAR EPSC
amplitude at 70 mV compared to the mean NMDAR EPSC amplitude at +40 mV (AMPAR/NMDAR ratio: control = 2.1 ± 0.3 (n = 7) versus OE = 2.3 ± 0.6 (n = 4), t(9) = 0.34, n.
s.). (D) Quantitative histograms showing AMPAR sEPSC amplitude (control = 21.2 ± 2.1 pA (n = 6) versus OE = 19.4 ± 1.4 pA (n = 8), t(12) = 0.74, n.s.), frequency
(control = 0.5 ± 0.2 Hz (n = 6) versus OE = 0.4 ± 0.1 Hz (n = 8), t(12) = 0.48, n.s.), and decay time (control = 6.8 ± 0.9 ms (n = 6) versus OE = 5.5 ± 1.6 ms (n = 8), t(12) = 0.64,
n.s.) over 15 min starting 10 min after cell clamping. (E) Representative traces of AMPAR sEPSCs at 70 mV holding potential in CA1 neurons from control and transgenic (OE)
slices. AMPAR sEPSCs were blocked by bath perfusion of the AMPAR blocker 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 3 lM). (F) KIBRA OE (n = 10) does not change the
response to bath application of 500 nM AMPA when compared to control (n = 8). Data are expressed as mean ± SEM.

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
10 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx

Fig. 5. KIBRA is enriched at synapses and interacts with GluA1. (A) Subcellular fractionation of mouse hippocampal extracts showing that KIBRA is present in the synaptic
membrane (SM) fraction but not in other fractions except in low amount in crude synaptosomes (P2). The postsynaptic components PSD-95 and GluN1 are abundant in SM.
Input, crude hippocampal fraction; P2, crude synaptosomal fraction; LS1, synaptic vesicle and cytosolic supernatant; LP1, synaptosomal membrane fraction; LS2,
synaptosomal-cytosolic fraction; LP2, crude synaptic vesicle-enriched fraction; SM, synaptic membrane fraction. (B) and (C) CV1 cells and hippocampal neurons
co-transfected with constructs encoding the intracellular GluA1 C-terminal domain fused to EGFP (EGFP-GluA1 C-term) and mCherry as control (upper panels) or mCherry-
KIBRA (lower panels), respectively. EGFP-GluA1 C-term and mCherry are diffusely distributed in transfected cells, while expression of mCherry-KIBRA leads to the formation
of cytoplasmic aggregates containing EGFP-GluA1 C-term in CV1 cells and to somato-dendritic EGFP-GluA1 C-term redistribution with mCherry-KIBRA in hippocampal
neurons. Scale bar: 20 lm. (D) Colocalization of EGFP-GluA1 C-term and mCherry-KIBRA on dendrites of hippocampal neurons in culture (DIV17-23). Images were generated
by confocal immunofluorescence microscopy with a 63 oil-immersion objective. Scale bar: 5 lm. (E) Immunoprecipitation (IP) assays with KIBRA and GluA1 C-term.
HEK293 cells were co-transfected with constructs encoding EGFP-KIBRA and Flag-GluA1 C-term (left panel) or with Flag-tagged KIBRA and EGFP-GluA1 (right panel). Flag
only was used as negative control. Lysates from transfected cells were incubated with immobilized anti-GFP and anti-Flag antibody and immunoprecipitates were isolated by
centrifugation. Western blot (WB) analysis with anti-GFP and anti-Flag antibodies. (F) Immunoprecipitation (IP) assay on rat P2 brain fractions with rabbit anti-KIBRA
antibodies and normal rabbit IgG. Western blot analysis with anti-KIBRA and anti-GluA1 antibodies.

was examined by co-expression assays. A KIBRA variant fused to
the mCherry reporter (mCherry-KIBRA) was co-expressed with
the GluA1 cytoplasmic C-terminus domain (aa 821–907) fused to
EGFP (EGFP-GluA1 C-term) in CV1 cells and hippocampal neurons.
24 h after transfection, KIBRA and GluA1 colocalized in these two
cell types (Fig. 5B and C). In CV1 cells, KIBRA and GluA1 formed
large clusters, while in the absence of KIBRA, GluA1 was diffusely
distributed in the cytoplasm (Fig. 5B). When alone, KIBRA itself
remained concentrated in punctates, probably due to its oligomer-
ization activity after prolonged expression (Johannsen et al., 2008).
Likewise in hippocampal neurons, KIBRA colocalized with GluA1
clusters, and both proteins were found in soma and dendrites
including in dendritic spines (Fig. 5C and D). Moreover, co-
immunoprecipitation assays using recombinant EGFP- and Flag-
tagged KIBRA and EGFP-GluA1 C-term showed that KIBRA and
GluA1 are present in the same protein complex (Fig. 5E). These
results were confirmed in vivo on brain fractions (Fig. 5F). Overall,
these results strongly suggest that KIBRA preferentially colocalizes
with GluA1 in hippocampal neurons.
3.6. KIBRA regulates constitutive and activity-dependent GluA1
recycling in hippocampal neurons
In resting conditions in neurons, GluA1-containing AMPARs
constitutively traffic to the membrane, while during LTP, new
AMPARs are recruited by exocytosis and lateral diffusion, and
incorporate into synapses (Hanley, 2008; Makino & Malinow,
2009). Trafficking is controlled in part by the interaction of the
C-terminus cytoplasmic tail of GluA1 with postsynaptic scaffolding
proteins (Bredt & Nicoll, 2003; Collingridge, Isaac, & Wang, 2004;
Groc & Choquet, 2006; Hayashi et al., 2000; Malinow & Malenka,
2002). Since KIBRA is associated with GluA1, we examined
whether it can modulate the constitutive recycling of GluA1-
containing AMPARs in basal conditions. We monitored GluA1 traf-
ficking in primary hippocampal neurons in which KIBRA was either
knockdown or overexpressed. For knockdown, cultured neurons
were infected with a lentivirus expressing a KIBRA shRNA, and
for overexpression, neurons were prepared from KIBRA transgenic
mice and cultured. Quantitative immunostaining confirmed that
KIBRA expression was significantly decreased by the shRNA and
increased in transgenic neurons under these experimental condi-
tions (Supplementary Fig. 5). We then measured the constitutive
recycling of GluA1-AMPARs in the soma and proximal dendrites
of the cultured neurons using a GluA1 (N-terminus)-specific anti-
body for detection of both internalized and surface receptors
(Bhattacharyya, Biou, Xu, Schluter, & Malenka, 2009; Suh et al.,
2010). The signal for internalized GluA1 was significantly increased
by KIBRA knockdown in the soma and dendrites of infected neu-
rons, suggesting an accumulation of intracellular GluA1
(Fig. 6A and B). This effect was likely due to an impaired GluA1
recycling, since GluA1 recycling index was decreased by KIBRA
knockdown (Fig. 6B). In contrast, KIBRA overexpression increased

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx 11

Fig. 6. KIBRA regulates the constitutive and activity-dependent recycling of GluA1-AMPARs. (A) Differential staining of surface (recycled, top panels) and internalized
(bottom panels) GluA1-AMPARs in mouse primary hippocampal neurons infected with a scrambled shRNA lentivirus (Control) or a KIBRA shRNA lentivirus for knockdown
(KD), and in transgenic neurons overexpressing KIBRA (OE, 48 h of dox treatment). Images of representative neurons show that internalized GluA1 staining is increased by
KIBRA KD while surface GluA1 staining is increased by KIBRA OE in soma and dendrites. Neurons stripped immediately after incubation with GluA1 primary antibody
(Stripping 00 ) and neurons not permeabilized prior to secondary antibody incubation (Non perm.) were used as controls. Images were generated by confocal
immunofluorescence microscopy with a 40 objective. Scale bar: 60 lm. (B) Quantitative analyses of GluA1 recycling. A GluA1 recycling index was calculated as the
percentage of recycled receptors compared to total number of receptors (recycled and internalized) for the soma (Control = 1.00 ± 0.02 (n = 13); KD = 0.90 ± 0.03 (n = 13), t
(24) = 2.77, ⁄p < 0.05; OE = 1.12 ± 0.01 (n = 10), t(21) = 4.89, ⁄⁄⁄p < 0.001) and dendrites (Control = 1.00 ± 0.02 (n = 13); KD = 0.86 ± 0.03 (n = 10), t(21) = 4.03, ⁄⁄p < 0.01;
OE = 1.11 ± 0.01 (n = 11), t(22) = 4.65, ⁄⁄⁄p < 0.001). Quantitative histograms are mean ± SEM. (C)–(E) Cultured hippocampal neurons (DIV14-17) were co-transfected with
SEP-GluA1 and mCherry (control, n = 8) or SEP-GluA1 and mCherry-KIBRA (OE, n = 8)) constructs to analyze activity-dependent increase in GluA1 surface expression by
KIBRA. Surface SEP-GluA1 fluorescence intensity was measured 48 h post-transfection by live-cell confocal microscopy upon neuronal stimulation with 200 lM glycine for
3 min. C, Representative images of control and KIBRA overexpressing (OE) neurons with SEP and mCherry fluorescence visualization at low (top panels) and high (bottom
panels) magnification to observe individual dendrites. Images were generated by live confocal fluorescence microscopy with a 63 oil-immersion objective and converted to
a monochrome signal. Scale bar: 20 lm. (D) Time course of SEP-GluA1 fluorescence change (DF/F0) in dendrites and soma (left and right panels). Horizontal bars indicate the
time window of glycine perfusion delivered at 3 min. (E) Quantitative histogram showing the averaged maximum amplitude of SEP-GluA1 fluorescence change (DFmax/F0) in
response to glycine in dendrites (control = 1.11 ± 0.02 (n = 8) versus OE = 1.22 ± 0.04 (n = 8), t(14)=2.46, ⁄p < 0.05) and soma (control = 1.11 ± 0.09 (n = 3) versus
OE = 1.33 ± 0.12 (n = 8), t(9) = 1.002, n.s.). Data are mean ± SEM.

GluA1-AMPARs exocytosis and return to the membrane in soma
and dendrites (Fig. 6A and B). No fluorescence was detected when
surface GluA1 antibody was stripped before recycling, or only sur-
face signal in non-permeabilized neurons indicating no unspecific
staining. Further, there was no change in GluA1-AMPARs recycling
with a scrambled shRNA, suggesting no unspecific effect.
We next examined the effect of KIBRA overexpression on
activity-dependent GluA1 recycling in cultured hippocampal
neurons. Chemical LTP (cLTP) was induced by perfusion of glycine
for 3 min and GluA1 surface expression was monitored by live
confocal imaging using an adapted pH-sensitive GFP (super ecliptic
pHluorin) fused to GluA1 (SEP-GluA1) (Ashby et al., 2004; Lin &
Huganir, 2007) (Fig. 6C–E). Surface expression of SEP-GluA1 was
rapidly increased on soma and dendrites in control neurons upon
glycine perfusion, before reaching a plateau. However,
somato-dendritic SEP-GluA1 recycling rate and surface number
were further increased in response to glycine in neurons
overexpressing KIBRA when compared to control neurons
(Fig. 6D and E). These results demonstrate that KIBRA is involved
in activity-dependent GluA1-AMPAR surface expression, and are

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
12 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx

consistent with the effect of KIBRA overexpression and knockdown
observed on LTP.
3.7. KIBRA overexpression promotes neuronal growth in vitro and in
the adult mouse hippocampus in vivo
GluA1-AMPAR recycling has been implicated in neuronal
growth and dendritic branching complexity (Haas, Li, & Cline,
2006; Prithviraj et al., 2008; Zhou et al., 2008). Therefore, we
examined whether KIBRA influences these processes. Primary hip-
pocampal neurons in which KIBRA was knocked-down or overex-
pressed were stained with an antibody directed against the
dendritic marker protein MAP2 and the morphology of dendrites
was analyzed. KIBRA knockdown led to a significant decrease in
the total dendritic volume, in the mean number of branch points,
and in the mean number of tertiary dendrites, as well as in the
length of primary dendrites (Fig. 7A). In contrast, KIBRA overex-
pression significantly increased the dendritic volume, the number
of branch points, and the number of tertiary dendrites, although
it had no effect on primary dendritic length. These effects were
not only observed in cultured neurons in vitro, but were also pre-
sent in the brain of adult transgenic mice overexpressing KIBRA.
In these mice, dendritic volume, mean number of branch points,
and mean number of secondary dendrites were significantly
increased in the hippocampus only a few days after induction of
KIBRA transgene by doxycycline (Fig. 7B and C). These results
strongly suggest a critical role for KIBRA in dendritic organization
in the adult hippocampus.
4. Discussion
Here, we provide novel evidence that KIBRA, a protein associ-
ated with memory performance in human (Papassotiropoulos
et al., 2006; Schneider et al., 2010; Wersching et al., 2011) is a crit-

Fig. 7. Dendritic growth and arborization are regulated by KIBRA. (A) Representative images with reconstructions of the dendritic arborization and morphometric analyses of
cultured hippocampal neurons infected with a scrambled shRNA lentivirus (Control) or with a KIBRA shRNA lentivirus (KD), and of neurons from transgenic mice
overexpressing KIBRA (OE). To reveal neuronal morphology, neurons were fixed and stained with an anti-MAP2 primary antibody and a Cy2-conjugated secondary antibody
5 days after lentiviral infection, and 48 h after the induction of KIBRA expression with dox in OE neurons. The following parameters were analyzed: total dendritic volume
(R dendrite volume), number of branch points, number of primary (1°), secondary (2°) and tertiary (3°) dendrites, primary dendrite length (1° dendrite length). While control
neurons have a complex dendritic arborization, neurons with KIBRA KD show a decrease in dendritic volume (KD = 1996.7 ± 55.5 lm3 (n = 9) versus con-
trol = 2901.5 ± 239.1 lm3 (n = 15), t(22) = 2.88, ⁄⁄p < 0.01), in the mean number of branch points (KD = 22.1 ± 1.8 (n = 10) versus control = 39.7 ± 2.6 (n = 23), t(31) = 4.14,
⁄⁄⁄
p < 0.001), in the mean number of tertiary dendrites (KD = 1.2 ± 0.3 (n = 9) versus control = 5.5 ± 0.8 (n = 15), t(22) = 3.57, ⁄⁄p < 0.01), and in primary dendrite length
(KD = 131.4 ± 2.4 lm (n = 6) versus control = 175.3 ± 4.9 lm (n = 15), t(19) = 5.44, ⁄⁄⁄p < 0.001). In contrast, neurons OE KIBRA have a more complex arborization with an
increased dendritic volume (OE = 3677.1 ± 242.2 lm3 (n = 9) versus control = 2901.5 ± 239.1 lm3 (n = 15), t(22) = 2.14, ⁄p < 0.05), more branch points (OE = 50.4 ± 1.6 (n = 11)
versus control = 39.7 ± 2.6 (n = 23), t(32) = 2.65, ⁄p < 0.05), and more tertiary dendrites (OE = 9.1 ± 1.1 (n = 9) versus control = 5.5 ± 0.8 (n = 15), t(22) = 2.51, ⁄p < 0.05). KIBRA
KD or OE did not significantly affect the mean number of primary (KD = 4.4 ± 0.2 (n = 9) or OE = 4.6 ± 0.5 (n = 9) versus control = 3.1 ± 0.5 (n = 15), t(22) = 1.93 or t(22) = 1.90,
n.s.) or secondary dendrites (KD = 9.6 ± 0.9 (n = 9) or OE = 16.8 ± 2.8 (n = 9) versus control = 11.6 ± 2.2 (n = 15), t(22) = 0.68 or t(22) = 1.44, n.s.). Scale bar: 60 lm. (B) Golgi
staining and morphometric analyses of CA1 hippocampal neurons in adult control and KIBRA OE mice treated with dox. Top images show whole neurons in control and KIBRA
OE mice. Neurons overexpressing KIBRA have increased dendrite volume (OE = 280.3 ± 59.0% (n = 4) versus control = 100.0 ± 3.8% (n = 3), t(5) = 2.58, ⁄p < 0.05), more branch
points (OE = 48.8 ± 2.5 (n = 4) versus control = 35.7 ± 4.9 (n = 3), t(5) = 2.59, ⁄p < 0.05), and more secondary dendrites (OE = 30.5 ± 3.0 (n = 4) versus control = 18.7 ± 2.7 (n = 3),
t(5) = 2.81, ⁄p < 0.05). KIBRA OE did not significantly affect the number of primary dendrites (OE = 4.3 ± 0.3 (n = 4) versus control = 4.2 ± 0.6 (n = 3), t(5) = 0.10, n.s.). Scale bar:
30 lm. Morphometric analyses were performed with Imaris software. Data are mean ± SEM. (C) Images of yellow fluorescent protein (YFP)-containing neurons (green) from
hippocampus CA1 area in Thy1-YFP and KIBRA double transgenic mice (OE) compared to Thy1-YFP transgenic mice (control). Scale bar: 30 lm. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx
ical regulator of synaptic plasticity and memory that controls
AMPARs. We show that the selective overexpression of KIBRA in
forebrain neurons in adult mice does not affect learning but
impairs spatial long-term memory. We show further that KIBRA
overexpression increases hippocampal LTP, in particular a GluA1-
dependent form of LTP, but prevents LTD in area CA1, and results
in a shift of synaptic plasticity in favor of potentiation. KIBRA
knockdown in contrast, abolishes LTP. These effects are associated
with a modulation of the constitutive recycling of GluA1-AMPARs
that is increased by KIBRA overexpression and decreased by KIBRA
knockdown, and with an activity-dependent recruitment of GluA1
at the surface. They also correlate with an increase and decrease in
dendritic growth and arborization respectively, both in vitro and
in vivo. Overall, the present data establish KIBRA as a novel regula-
tor of GluA1 essential for bidirectional synaptic plasticity in the
hippocampus and for long-term memory.
We show that KIBRA co-localizes with AMPARs in hippocampal
neurons both in vitro and in vivo, and that both proteins co-
immunoprecipitate. In dendrites, KIBRA preferentially colocalizes
with GluA1 and favors the constitutive and activity-dependent
recycling of GluA1-containing AMPARs in hippocampal neurons.
In resting conditions, this does not affect basal synaptic transmis-
sion or the activity of AMPARs, since more GluA1 is not expected to
be recruited and stabilized at synapses. However upon neuronal
activity, it leads to an increase in GluA1 surface receptors that
likely leads to the enhanced LTP and impaired LTD observed after
KIBRA overexpression. This effect may be mediated by an increase
in recycling and receptor delivery at perisynaptic sites followed by
lateral diffusion to synapses. More surface GluA1 due to KIBRA
would lead to synaptic strengthening and favor synaptic potentia-
tion over depression, which likely induces LTP saturation and pre-
vent the induction of LTD. This is consistent with the demonstrated
role of AMPAR recycling and activity-dependent surface expression
in LTP in CA1 pyramidal neurons (Park et al., 2004; Petrini et al.,
2009; Yang, Wang, Frerking, & Zhou, 2008), that specifically impli-
cates calcium-permeable GluA1-containing AMPARs (Makino &
Malinow, 2009; Plant et al., 2006). It is also consistent with the
impairing effect of GluA1 knockout on CA1 hippocampal LTP
(Mack et al., 2001; Zamanillo et al., 1999), but the lack of effect
of GluA2 or GluA3 knockout (Meng et al., 2003). In addition, LTP
saturation identified after 3-train HFS and repeated 1-train HFS
in transgenic mice could be indicative of deficits in synaptic scaling
reflecting a lack of homeostatic compensation (Vitureira & Goda,
2013). In this view, KIBRA overexpression may increase synaptic
density of GluA1-containing AMPA receptors at activated synapses
upon LTP induction but may also lead to recruitment and stabiliza-
tion of GluA1-containing AMPA receptors at a larger number of
synapses including synapses whose strengths are normally weak-
ened by homeostatic mechanisms.
We show that KIBRA overexpression in transgenic mice impairs
spatial long-term memory on the hidden platform Morris water
maze task, but does not affect spatial learning. In KIBRA transgenic
mice, spatial memory deficits are associated with enhanced
hippocampal CA1 LTP and impaired LTD, together with increased
surface GluA1 receptors in CA1 pyramidal neurons. This is
consistent with spatial memory deficits in mice with mutations
in GluA1 phosphorylation sites critical for LTP and LTD expression
(Lee et al., 2003). In particular, GluA1 phosphomutant mice essen-
tially show a lack of LTD and a reduced LTP in the CA1 region of the
hippocampus associated with a deficit in spatial memory but not
spatial learning on the Morris water maze task. These findings
together with our results establish that activity-dependent regula-
tion of GluA1-containing AMPA receptor function at synapses is
critical for spatial memory. However, these findings contrast with
normal performance on the spatial memory water maze task in
Please cite this article in press as: Heitz, F. D., et al. The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult
brain. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.07.028
13
GluA1 knockout mice (Zamanillo et al., 1999; Reisel et al., 2002)
that may be caused by compensatory homeostatic mechanisms.
Since LTD impairment is associated with spatial memory deficit
in both GluA1 phosphomutant and KIBRA overexpressing mice,
these data also confirm the essential role of hippocampal CA1
LTD in long-term spatial memory (Ge PNAS 2010).
The exact mechanisms by which KIBRA influences AMPAR
trafficking are not known but likely involve some of its known
neuronal binding partners. This may include partners implicated
in AMPAR regulation such as dynein light chain 1, a cytoplasmic
protein required for the transport of AMPARs to dendrites
(Kapitein et al., 2010; Rayala et al., 2006), the exocyst complex
proteins Sec3/5/8 involved in synaptic insertion of AMPARs
(Gerges, Backos, Rupasinghe, Spaller, & Esteban, 2006; Rosse
et al., 2009), or the early endosome-to-endocytic recycling
compartment protein sorting nexin 4 (SNX4) involved in protein
recycling (Traer et al., 2007). It should be noted that, since KIBRA
also co-localizes with GluA2 to some extent, and a large number
of AMPARs are heteromeric and contain GluA1 and GluA2 (Shi,
Hayashi, Esteban, & Malinow, 2001), KIBRA can also regulate GluA2
as reported in a recent study (Makuch et al., 2011).
Further to synaptic plasticity, KIBRA overexpression and knock-
down are also shown to alter the morphology and dendritic orga-
nization of hippocampal neurons. They oppositely modulate the
length, number and volume of dendrites, and the number of branch
points. Such bidirectional effect of KIBRA is striking and points to a
remarkable property of KIBRA to actively contribute to dendritic
remodeling, in particular in the adult brain. This suggests that
KIBRA is likely an important player in the structural dynamics of
neuronal networks in adulthood, and may be implicated in
structural changes induced by activity-dependent experiences or
environmental enrichment, but also after stress and neurodegener-
ation (Holtmaat & Svoboda, 2009; Tavosanis, 2012). Notably, the
extent of the effect of KIBRA overexpression and knockdown on
different parameters of neuronal morphology varies. For instance,
the number of tertiary dendrites is more affected than the number
of primary dendrites maybe because tertiary dendrites have a dif-
ferent requirement for KIBRA and their growth and establishment
may be easier to modulate than primary dendrites. Finally, the
effect of KIBRA on neuronal morphology is likely linked to GluA1,
since GluA1 has been demonstrated to be implicated in dendrite
growth (Haas et al., 2006; Prithviraj et al., 2008; Zhou et al., 2008).
Finally, the present data are clinically relevant since KIBRA has
been associated with memory performance in human, and a SNP
(rs17070145) has been linked to an increased risk for late-
onset Alzheimer’s disease (Rodriguez-Rodriguez et al., 2009;
Corneveaux et al., 2010). This link may also be related to the obser-
vation that both AMPAR trafficking and LTP are impaired in Alzhei-
mer’s patients (Hsieh et al., 2006; Rui, Gu, Yu, Hartzell, & Zheng,
2010). It is also supported by our observation that KIBRA plays a
critical role in dendritic arborization and neuronal morphology,
and may therefore contribute to altered neuronal structures and
integrity in neurodegeneration, Alzheimer’s disease and other cog-
nitive pathologies. The present data may ultimately provide a
novel therapeutic target for the development of potential treat-
ment of disorders associated with impaired plasticity and cognitive
defects.
Author contributions
F.D.H. and I.M.M. conceived the project. F.D.H. designed and
conducted the experiments, and prepared the manuscript with I.
M.M. All authors technically contributed to the results. All the
authors approved the final version of the manuscript.
14 F.D. Heitz et al. / Neurobiology of Learning and Memory xxx (2016) xxx–xxx
Acknowledgements
We are grateful to Joachim Kremerskothen for generously pro-
viding KIBRA vectors and antibody, for support with the IPs and for
reading an initial version of the manuscript, Aniña Pescatore,
Osvaldo Mirante, Rreze Gecaj-Mehmeti and Nina Meyer for techni-
cal help, Dr. Holger Russig for advice on behavioral testing, Dr. Cris-
tina Fernandez for conceptual contribution, Philipp Schätzle, Stella
Kramer and Giovanna Bosshard for providing mouse and rat neu-
ronal cultures We acknowledge staff members of the Center for
Microscopy and Image Analysis of the University of Zürich (ZMB).
This work was supported by the University of Zürich, the Swiss
Federal Institute of Technology, the Human Frontier Science Pro-
gram, the Swiss National Foundation, The National Center for Com-
petence in Research ‘‘Neural Plasticity and Repair”, the Slack-Gyr
and Bitterlin foundations, and a DFG grant PA 483/14-2 to H.P.
The authors declare no conflict of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.nlm.2016.07.028.
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