The Journal of Physiological Sciences (2019) 69:453–463
https://doi.org/10.1007/s12576-018-00654-5

ORIGINAL PAPER

Overexpression of neuronal ­K+–Cl− co‑transporter enhances dendritic
spine plasticity and motor learning
Kayo Nakamura1,2 · Andrew John Moorhouse3 · Dennis Lawrence Cheung3 · Kei Eto1 · Ikuko Takeda1 ·Paul Wiers Rozenbroek3 · Junichi Nabekura1,2Received: 18 November 2018 / Accepted: 17 December 2018 / Published online: 13 February 2019
© The Physiological Society of Japan and Springer Japan KK, part of Springer Nature 2019

Abstract
The neuronal ­K+–Cl− cotransporter KCC2 maintains a low intracellular ­Cl− concentration and facilitates hyperpolarizing­GABAA receptor responses. KCC2 also plays a separate role in stabilizing and enhancing dendritic spines in the developing
nervous system. Using a conditional transgenic mouse strategy, we examined whether overexpression of KCC2 enhances
dendritic spines in the adult nervous system and characterized the effects on spine dynamics in the motor cortex in vivo
during rotarod training. Mice overexpressing KCC2 showed significantly increased spine density in the apical dendrites oflayer V pyramidal neurons, measured in vivo using two-photon imaging. During modest accelerated rotarod training, miceoverexpressing KCC2 displayed enhanced spine formation rates, greater balancing skill at higher rotarod speeds and a fasterrate of learning in this ability. Our results demonstrate that KCC2 enhances spine density and dynamics in the adult nervous
system and suggest that KCC2 may play a role in experience-dependent synaptic plasticity.
Keywords Two-photon microscopy · In vivo · Motor learning · KCC2 · Synaptic plasticity

Introduction
The neuron-selective isoform of the ­K+–Cl− cotransporter
KCC2 plays an important role in regulating the intracellular
chloride concentration. In most adult neurons, KCC2 simultaneously exports ­K+ and ­Cl− and thereby maintains a low
intracellular ­[Cl−] that facilitates subsequent gamma-amino
butyric acid receptor ­(GABAARs)-mediated ­Cl− influx,
hyperpolarization and neuronal inhibition [1–3]. Indeed,
GABAergic inhibition can be diminished when KCC2
is absent or has reduced function, such as occurs during
neuronal development and/or after injury. Given this rolein influencing GABA function, there has been extensive
investigation into the contributions that KCC2 may make to* Junichi Nabekura
nabekura@nips.ac.jp
1

Division of Homeostatic Development, Departmentof Fundamental Neuroscience, National Institutes
for Physiological Sciences, Okazaki 444‑8585, Japan2

Department of Physiological Sciences, Sokendai,
Hayama 240‑0193, Japan3

Department of Physiology, School of Medical Sciences,
University of New South Wales, Sydney 2052, Australia

neuronal circuit development, the pathogenesis of different
diseases and the mechanisms that regulate KCC2 expression
and transport function (reviewed in Kaila et al. [3], Watanabe et al. [4] and Kahle et al. [5]).
However, KCC2 has also been reported to have additional morphogenic effects on neuronal circuits independent of its ­Cl− transport function. In neuronal cultures from
KCC2 knockout mice, dendritic spines exhibited a more
immature filopodia-like phenotype, and the number of
immunohistochemically and functionally identified excitatory synapses was markedly decreased [6]. Remarkably,
this immature dendritic spine phenotype was rescued byoverexpression of C
­ l− transport-deficient KCC2 mutant
constructs [6]. Furthermore, in utero overexpression of
KCC2, or a C­ l− transport-defective mutant KCC2, resulted
in an increase in the density of mature dendritic spines inthe somatosensory cortex (layer II/III) that was sustained
at least until 90 days after birth [7]. Pertinently, KCC2
appears to form a complex with the actin-associated proteins 4.1 N and βPIX and can interact with the kinase andcofilin signaling pathways associated with dendritic spine
development and stabilization [6, 8, 9]. Thus, it would
appear that KCC2 supports the development of excitatory
synapses by enhancing spinogenesis and/or maturation.

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Such a role is consistent with the upregulation of KCC2
expression in development coinciding with the start ofspinogenesis and may (at least partly) explain the presenceof KCC2 on dendritic spines at glutamatergic synapses[4, 10–12].
Maintenance of dendritic spines in the adult nervous
system is also highly dynamic, underpinned by numerous
molecular mechanisms, and facilitates the functional andstructural plasticity required for memory and behavioral
adaptations in response to training [13–16]. Specifically,in vivo experiments have demonstrated a strong correlation between the rate of newly formed dendritic spines in
the motor cortex and the extent and rate at which micecan improve specific motor behaviors during learning[17, 18]. In addition, long-term potentiation (LTP) of
synaptic transmission is associated with the appearanceof new dendritic spines and an increase in the volume ofdendritic spine heads, while long-term depression (LTD)is correlated with dendritic spine shrinkage and removal(reviewed in Kasai et al. [16] and Bosch and Hayashi[19]). Together this suggests an activity-dependent modulation of dendritic spine appearance, growth and stability
during learning. Recent evidence suggests that KCC2 candirectly contribute to aspects of dendritic spine plasticity.
Suppression of KCC2 expression in juvenile rats and incultured neurons prevented the induction of LTP and theassociated increases in dendritic spine volume and AMPA
receptor insertion [9], pointing to a possible link between
KCC2 and learning-induced synaptic plasticity in adultneuronal circuits.
Therefore, to address whether altered KCC2 modulates
dendritic spine and synaptic plasticity in adult animals,
we have utilized a transgenic mouse wherein KCC2 canbe conditionally overexpressed by altering dietary doxycycline. We overexpressed KCC2 and quantified the effectsof this on dendritic spine density. Furthermore, we examined how overexpressed KCC2 affects dendritic spine plasticity during motor learning and the functional implications of this. We hypothesized that increasing KCC2 levelsin the adult brain would increase the density of dendritic
spines, as seen in developing brains. We also proposedthat increasing KCC2 would enhance the dendritic spine
plasticity associated with motor learning. Using in vivo
two-photon imaging of dendrites and spines in apicaldendrites of layer V motor cortex pyramidal neurons andGolgi-Cox staining of hippocampal and layer II/III somatosensory cortex pyramidal neurons in fixed tissues, weshow that overexpression of KCC2 does indeed increase
dendritic spine density. Using time-lapse imaging, we thendemonstrate that KCC2 overexpression increases dendritic
spine formation in the motor cortex during rotarod motor
learning and increases the extent and rate at which rotarod
performance is enhanced during training.

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Materials and methods
All animal experiments were carried out according to theguidelines defined by the National Institutes of Natural Sciences (NIPS) and approved by the Okazaki Institutional
Animal Care and Use Committee or by the UNSW Sydney
Animal Care and Ethics Committee.

Animals
We used a conditional transgenic mouse in which overexpression of KCC2 can be induced by cessation of dietary
supplementation with doxycycline [20]. KCC2 expression
was driven by the tetracycline transactivator (tTA) binding to the tetracycline operator construct (tet-O), and thisinteraction is prevented by doxycycline [21]. The tet-O
construct was introduced upstream of the KCC2 gene, and
KCC2 overexpression was restricted to excitatory neuronsof the forebrain by using the CaMKIIα promoter to drivetTA expression [22]. Mice were housed in standard cageswith a reversed light/dark cycle (light on 6:00 a.m., lightoff 6:00 p.m.) and fed with doxycycline (Dox, 100 mg/kg) laced chow. Adult (8–12 weeks old) male mice wereused for experiments, and feed was exchanged to Dox-freestandard chow prior to experiments as indicated (typically
2–3 weeks).

Accelerating rotarod experiments
One week prior to behavioral experiments, mice were identified, weighed and health-checked, before being acclimatized
to the behavioral rooms for at least 30 min prior to testing. The accelerating rotarod test used a rotarod with fivelanes, as previously described in Kakegawa et al. [23] andRothwell et al. [24]. Mice were placed into the rotarod, andthe rotation speed was increased from 4 to 40 rotations per
minute (r.p.m) over 5 min. The time at which a mouse fell
from the rotarod was recorded. Six trials were performed
over a period of 90 min, and this was repeated daily over 5
successive days.
Surgery and virus injection for in vivo imaging
To facilitate in vivo imaging, we made cranial windows
over the M1 motor cortex using the open skull approachas described in Kim et al. [25] and Wake et al. [26]. Surgery was performed over 2 days, 2–3 weeks before imaging
and rotarod experiments. On the 1st surgery day, a metal
head-plate, used to secure the mouse head under the microscope objective lens, was mounted with dental cement onto
exposed skull under anesthesia (ketamine:xyalizine mix,

The Journal of Physiological Sciences (2019) 69:453–463

0.13: 0.01 mg/g, i.p.). On the 2nd surgery day, the skull
over the M1 motor cortex was removed under anesthesia(isoflurane, 1.5% vol/vol in air), leaving a 2.0–3.0-mmdiameter window centered at 1.5 mm lateral to the bregma
and corresponding to the mouse’s forepaw. Subsequently,
adeno-associated virus (AAV) containing an enhanced green
fluorescent protein (eGFP) coupled to a CaMKIIα promoter(AAV2-CaMK2-eGFP; University of Pennsylvania vectorcore) was injected into layer V of the motor cortex (250 nl
over 5 min). After injection, the cortical surface was washedwith artificial cerebrospinal fluid for > 20 min before theopen window was sealed using double-cover glass and 2–5%
agarose. The cover glass was then secured to the adjacentskull using dental cement and cyanoacrylate glue.

Two‑photon imaging of dendrites and spines in vivo
Mice were anesthetized with isoflurane (~ 1.5% v/vol inair) and secured via their head-plate under the microscope
objective lens. The motor cortex was initially imaged at
low resolution at a depth of 500–600 μm to identify eGFPexpressing layer V pyramidal neurons. The apical dendritesof these neurons were traced toward the brain surface andhigher resolution imaging of dendrites and spines performed
within 100 μm from the cortical surface. On average, 2–5
apical dendrites were imaged in each mouse.

Dendritic spine analysis in vivo
Dendrite and spine structures were analyzed as described in
Kim et al. [25] and Kim and Nabekura [27] using ImageJ(http://rsbwe​b.nih.gov/ij/). Briefly, we manually identified
any dendritic protrusion and classified all these protrusions as spines regardless of length or shape. Thin filopodia
without clear spine heads were excluded. Spine formationand elimination rates were determined as the percentagesof spines in a single dendrite that appeared or disappeared,
respectively, between two successive imaging sessions andwere expressed relative to the total spine number in the former session. Spine turnover was defined as the average number of formed and eliminated spines.

Golgi‑Cox staining and in vitro spine analysis
Golgi-Cox staining was performed using a method modified from Levine et al. [28]. Mice were deeply anesthetized(sodium pentobarbital, 33 mg/kg, i.p.) and cardiac perfused with phosphate-buffered saline (PBS) followed by
4% paraformaldehyde. Brains were then dissected out andsectioned at 100 µm thickness in PBS using a vibratome.
These sections were then sandwiched between two microscope slides and immersed in Golgi-Cox fixative (1:1:1% w/
vol ­HgCl2:K2CrO4:K2Cr2O7) for 14 days and 1% K
­ 2Cr2O7455

for a subsequent 24 h in a light-free environment. Following
this, the sections were removed from the microscope slide
sandwich and successively exposed to 28% vol/vol ­NH3 for
20 min and 15% Kodak Polymax T Fixer for 8 min. After air
drying for 1 week, the sections were dehydrated through anincreasing alcohol series, incubated for 5 min in limonene
and mounted on glass microscope slides with EntellanNew (Merck). Spine density was manually counted for
30–100 μm sections of the first or second dendritic branch
off the apical dendrites.

Immunostaining
Mice were deeply anesthetized (ketamine:xyalizine mix,
0.13: 0.01 mg/g, i.p.) and cardiac perfused with phosphatebuffered saline (PBS) followed by 4% paraformaldehyde.
Brains were dissected out and stored in 4% paraformaldehyde at 4 °C overnight, which was replaced with 30% w/
vol sucrose the next day. Brains were then snap frozen withliquid nitrogen and sections cut at 35 μm thickness using
a microtome and stored at 4 °C in PBS. Immunostaining
was conducted using primary antibodies anti-NeuN (Mouse,
Monoclonal, Millipore, 1:500 dilution) and anti-VP16,
amino acids 413–490 (Rabbit, Polyclonal, Abcam, 1:100
dilution) and secondary antibodies Alexa Fluor 564 (AntiMouse, Life Technologies, 1:2000 dilution) and Alexa Fluor
488 (Anti-Rabbit, Life Technologies, 1:2000 dilution).

Western blotting
Mice were deeply anesthetized with ketamine (0.13 mg/g,
i.p.) and xylazine (0.01 mg/g, i.p.) and cardiac perfused with
PBS. After most of the blood was cleared, brains were dissected out and sectioned at 300 μm thickness in carbogenated artificial cerebrospinal fluid (aCSF) using a vibratome.
Motor cortex was isolated from these sections and homogenized in cold lysis buffer (0.5 M Tris-HCl, 150 mM NaCl,
0.1% Triton x-100, 10% sodium dexycholate, pH 6.8) withprotease inhibitor (11697498001, Roche). Cell lysates were
centrifuged (for 12,000 rpm for 10 min at 4 °C), with thesupernatants collected as samples and protein concentration
determined using the Pierce BCA assay kit (Pierce, Rockford, IL). The samples were then diluted in SDS sample
buffer before being heated to 100 °C for 5 min. Sample proteins were then separated by gel electrophoresis (7% SDSPAGE gel, 0.04 A, 100 min) and transferred to a nitrocellulose membrane. This was then incubated with anti-KCC2
primary antibody (rabbit, 1:1000, 07-432, Millipore) andanti-βactin (mouse, 1:1000, 030M4788, Sigma–Aldrich) andsecondary antibody (anti-rabbit immunoglobulin; 1:5000,
HAF008, R&D systems, and anti-mouse immunoglobulins;
1:1000, HAF007, R&D systems). Protein bands were analyzed for protein concentration using ImageJ.

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Data analysis and statistics
Data are presented as mean ± SEM. Comparisons between
control and KCC2 upregulated mice used an unpaired t test
or a one- or two-way ANOVA, followed by a post hoc Tukey’s or Bonferroni’s test as indicated. Significant differenceswere defined by p < 0.05.

Results
Overexpression of KCC2 in cortex
To overexpress KCC2, we utilized a conditional transgenic mouse that incorporates the tetracycline operator
construct (tet-O) upstream of the KCC2 gene. Overexpression of KCC2 was driven by the expression of thetetracycline trans-activator (tTA) protein and restricted toexcitatory neurons of the forebrain (cortex, hippocampus,
amygdala) by coupling to the CaMKIIα promoter [22, 29].
In mice that expressed both tet-O and tTA, overexpression of KCC2 was prevented by dietary supplementationof doxycycline (Dox) and triggered by cessation of Dox
supplementation. Details of this transgenic mouse havebeen recently reported as a preprint in Goulton et al. [20],which demonstrates that Dox cessation increases KCC2mRNA and protein expression throughout the cortex,
amygdala and hippocampus. Overexpression of KCC2
does not induce obvious changes in any fundamental
physiologic parameters. For example, in 7–11-week-old
male mice without KCC2 overexpression, body weightwas 25.1 ± 1.4 g (n = 13), while in age-matched micewith KCC2 overexpression, body weight was 27.0 ± 1.0 g(n = 11), with both values similar to the average weight of
11-week-old male C57Bl6 mice supplied by the JacksonLaboratory (26.7 ± 1.7 g). Furthermore, increasing KCC2
by withdrawing Dox for 1 week has no significant effecton basal locomotor activity or anxiety as measured in open
field and elevated plus maze tests, respectively (see Goulton et al. [20]). In the current study, we first confirmed theoverexpression of KCC2 protein in the motor cortex 7 days
after cessation of Dox supplementation, using Western blot experiments (Fig. 1a). The expression of KCC2
(relative to β-actin intensity) was significantly increased
compared with control mice, from 40.4 ± 9.3% (n = 3mice) to 87.8 ± 9.8% (n = 3), representing an approximate
doubling of protein expression (unpaired t test, p = 0.025;
Fig. 1b). The control or wild-type (WT) mice used in thisstudy were from the same colony and with the same diet,which lacked both of the two gene constructs required toinduce KCC2 overexpression (tet-O, tTA). To confirm that
KCC2 overexpression was reliable across the majority

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Fig. 1  Conditional overexpression of KCC2 in motor cortex in transgenic mice. a Representative Western blots for KCC2 immunoreactivity in tissue from the motor cortex obtained from three WT (left)
and KCC2-overexpressing (right) mice. Samples were obtained frommice 7 days after withdrawal of doxycycline (Dox) from the diet to
induce overexpression in KCC2 mice. b Quantification of the intensity of KCC2 immunoreactivity, expressed relative to β-actin. The
relative expression of KCC2 was significantly greater in KCC2-overexpressing mice compared with WT mice, with the absolute KCC2/β-actin ratios being 40.4 ± 9.3% for WT (n = 3) and 87.8 ± 9.8% for
KCC2 mice (n = 3; unpaired t test,*p < 0.05). This corresponded to a
117% increase in KCC2 protein expression levels. c Representative
immunohistochemical images of a brain slice from a KCC2-overexpressing transgenic mouse showing the primary motor cortex (left)
stained positive for NeuN (red, neuronal marker) and VP16 (green,marker for the tetO-KCC2 construct). Most larger layer V pyramidal neurons were double-labeled (yellow). Bar; 500 μm (left), 10 μm(upper right) and 50 μm (bottom right)of excitatory pyramidal neurons in the motor cortex, we
immunostained neurons using a label against the virion
protein 16 (VP16), a component of the tTA construct [21],with neurons identified by NeuN expression. As shown in
Fig. 1c, the majority of excitatory pyramidal neurons inlayer V of the M1 motor cortex co-expressed VP16. (e.g.,
Fig. 1c). In four KCC2-overexpressing mice, 62 ± 3.4% oflayer II/III and 69 ± 3.3% of layer V neurons that expressed
NeuN also expressed VP16. Given that about 15% of these
NeuN-positive cells should be inhibitory neurons [30], we
estimate that withdrawal of doxycycline would overexpress
KCC2 in about 75% of all excitatory neurons. Such mosaicism for tTA/tet-O-driven constructs in the absence of Dox
has been previously characterized, with between 34 and
72% of CA1 pyramidal neurons expressing tet-O-driven
reporter proteins in different transgenic lines [31].

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Overexpression of KCC2 increases spine density
and the formation of new spines during motor
learning
We initially conducted preliminary experiments in GolgiCox-stained brain slices from adult (≈ 6 months) mice toexamine whether overexpression of KCC2 affected spine
density. For these experiments, mice (and mothers) wereraised in the absence of Dox, and thus KCC2 overexpression would have occurred from 1 to 2 weeks after birth,paralleling CaMKIIα expression [32]. Dendritic spines
were manually counted from the primary branch of apicaldendrites. As shown in Fig. 2a, b, the density of dendritic
spines in hippocampal neurons was significantly increased(unpaired t test, p = 0.003) by ≈ 50%. Dendritic spine counts
in the somatosensory cortex were more variable, and no significant differences were detected (WT: 0.70 ± 0.11 spines/µm, 9 dendrites, 4 mice; KCC2: 0. 82 ± 0.08 spines/µm, 14
dendrites, 5 mice; not shown).
To evaluate whether increasing KCC2 in the adult brain
could also increase dendritic spine density, we used in vivo
two-photon imaging. Using this approach, we could also
examine whether spine density changed over time and during motor learning (see below). As shown in Fig. 2c, d, basal
spine density (pre-training levels) in KCC2-overexpressing
mice was significantly increased (≈ 40%) compared with
WT mice. Under pre-training control conditions, WT micehad 0.23 ± 0.01 spines/µm (28 dendrites, 11 animals), while
KCC2-overexpressing mice had 0.32 ± 0.02 spines/µm (34dendrites, 16 animals; unpaired t test, p = 0.0007). There wasmore variability in spine counts for KCC2-overexpressing
mice (Fig. 2c), perhaps reflecting that KCC2 was not overexpressed in every pyramidal neuron (see above). Nevertheless, our data confirm that KCC2 overexpression can
increase dendritic spine density and notably confirm that
this capability is still retained in the adult brain. We did not
examine whether the increase in spine density was reversible, although returning Dox to the diet for 3 weeks returns
KCC2 levels back to control values [20].
Subjecting mice to repeated trials on an acceleratingrotarod enables them to increase the time/speed before theyfall off, and this motor learning has been associated with anincreased formation rate of new dendritic spines specificallyin layer V pyramidal neurons within the forelimb-associatedregion of the M1 motor cortex [17]. Previous studies haveshown that motor learning and other forms of plasticity may
be associated with modest increases in dendritic spine density (approximately 5–10% in Yang et al. [18] and Xu et al.
[17]) or may be associated with no overall change depending on the balance of formation and elimination [15]. Under
our conditions, total dendritic spine density was relatively
stable throughout 5 successive days of repeated imaging of
the same dendrite, with mice undergoing rotarod training

Fig. 2  Overexpression of KCC2 increases spine density. a Representative CA1 pyramidal neuron dendrites in Golgi-stained hippocampal slices from WT and KCC2-overexpressing mice. Scalebars, 5 μm. b The density of spines in CA1 hippocampal pyramidal
neurons was significantly increased in KCC2-overexpressing mice.
Spine density in the KCC2 mice was 1.06 ± 0.10 spines/μm (15dendrites from 5 mice), while the spine density in the control group
was 0.69 ± 0.07 (12 dendrites from 4 mice), corresponding to a 54%
increase (**p < 0.01, unpaired t test). c Representative images of single dendrites in WT and KCC2-overexpressing mice using in vivo
two-photon imaging of dendrites and spines in apical dendrites oflayer V pyramidal neurons of the motor cortex. Scale bars, 5 μm. d
In KCC2-overexpressing mice (34 dendrites, 16 animals), there wasa significant increase in spine density compared with WT mice (18dendrites, 11 animals ***p < 0.001, unpaired t test). Graphs show
individual data with mean ± SEM)

during the latter 3 days (see below, Fig. 3a). Over the 5
imaging days, dendritic spine density was consistently
higher in KCC2-overexpressing mice (by 30–40%) acrosseach of these days (Fig. 3b–d). When images of dendrites
and their spines were compared between 2 consecutive days,
a significant increase in the proportion of newly appeared
dendritic spines (dendritic spine formation rate) was seenin mice overexpressing KCC2 following a single day ofrotarod training (Fig. 3b, c, e). Between the 2 pre-training
control days (day − 1 to day 0), the dendritic spine formation
rate in KCC2 mice was 6.94 ± 1.11%, whereas after 1 dayof training the dendritic spine formation rate increased to13.49 ± 1.58% (day 0 to day 1, 23 dendrites, 7 animals; oneway ANOVA, Tukey’s test, p = 0.036, Fig. 3e). The corresponding rates of new dendritic spine formation pre- and

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The Journal of Physiological Sciences (2019) 69:453–463◂Fig. 3  Increasing KCC2 induces a sustained increase in spine density

and a more rapid rate of spine formation during rotarod learning. a
Schematic diagram of experimental protocol. Imaging and rotarodtraining began 3 weeks after withdrawing Dox from the diet. The
same dendrites were imaged over 5 consecutive days. From days 1
to 3, rotarod training was performed in the morning prior to imaging
later that day. b, c Representative single dendrites of pyramid neurons
from WT (b) and KCC2-overexpressing (c) mice during repeatedin vivo imaging before and during rotarod training. Red arrowheads
indicate spines that newly appeared compared with the previous day’s
image (newly formed spines), while blue arrowheads indicate thosethat disappeared (eliminated spines). Scale bars in each image in b
and c, 5 μm. d Spine density measured at each imaging day was consistently higher in KCC2-overexpressing mice compared with that in
WT mice. e The relative number of new spines that formed during
the 1st day of rotarod learning was significantly greater in KCC2overexpressing mice, but not in WT mice. However, subsequent newspine formation rates were not different. The graph plots the percentage of spines in a dendrite that was not present at the previous day’s
imaging (7 KCC2 over-expressing mice, 23 dendrites; 7 WT mice,
18 dendrites; one-way ANOVA followed by Tukey’s test, 0 day vs.
1 day KCC2 mice, *p < 0.05). f The proportion of spines that disappeared (spine elimination) across the rotarod training period was not
significantly different between WT and KCC2-overexpressing mice.
g The spine turnover rate (numerical addition of formation and elimination) was significantly increased following 1 day of rotarod training for KCC2-overexpressing mice (one-way ANOVA, Tukey’s test,**p < 0.01), but not for WT mice—nor was it significantly different
for WT or KCC2 mice at 2 and 3 days

post-1 day training in WT mice was 5.28 ± 1.52% and12.00 ± 1.57% (18 dendrites, 7 animals; one-way ANOVA,
Tukey’s test, not significantly different, Fig. 3e). In both WT
and KCC2 mice, the proportion of dendritic spines that disappeared across consecutive imaging days (dendritic spine
elimination rate) was relatively constant across the 5 days ofimaging (between 5 and 10%) and not significantly different between WT and KCC2 mice (Fig. 3f). Corresponding
with the increased dendritic spine formation in KCC2 mice,the dendritic spine turnover rate (an average of eliminationand formation) seen after 1 day of training was also significantly increased in KCC2 mice (11.45 ± 1.13; one-way
ANOVA, Tukey’s test, p = 0.0097; Fig. 3g), but not in WT
mice (6.76 ± 0.98%).

KCC2‑overexpressing mice show a more rapid
acquisition of improved motor performance
Given the correlations between motor learning and dendritic spine formation rates previously reported, we examined and quantified the performance of mice during therotarod task. We used the same mouse cohort as subjected
to 5 days of imaging, but also included mice in which reliable imaging across the entire protocol was unsuccessful.
A series of six rotarod trials was performed over 5 consecutive days, which included imaging on days 1–3 in Fig. 3aand 2 additional training days. Performance was measured
by quantifying the time it took for mice to be no longer

459

able to hold on to the rotarod as its speed was gradually
increased in a step-wise fashion. As shown in Fig. 4a, both
WT and KCC2-overexpressing mice showed similar motor
performance levels in the first rotarod session, and bothcohorts improved in their motor performance during the
5 days of training. Nevertheless, the motor performance
curves across the 5 days of training were significantly different between KCC2-overexpressing mice and WT mice
(Fig. 4a; n = 15 WT mice with 6 trials per day, n = 15
KCC2 mice with 6 trials per day, p = 0.00095, two-way
ANOVA with Bonferroni’s test). Furthermore, the pooled
motor performance from days 4 and 5 of training was significantly better in KCC2-overexpressing mice comparedwith WT mice (Fig. 4a; n = 15 WT mice, 15 KCC2 mice;p = 0.0267, unpaired t test). Motor performance across trials for each day was also averaged and compared with
the initial motor performance level at day 1 (Fig. 4b). In
WT mice, a significant improvement in performance (i.e.,
motor learning) was first seen at day 4 of training, whereas
for KCC2-overexpressing mice, a significant improvementin performance was first seen at day 2 of training. Thus, asignificant improvement in motor performance occurredearlier in KCC2-overexpressing mice compared with WT
mice, which suggests an enhanced learning capacity.

Stability of newly formed and existing dendritic
spinesMost newly formed dendritic spines are labile and are
eliminated over the subsequent weeks. However, a proportion of dendritic spines that is newly formed during learning of a specific motor task, such as accelerating rotarodtraining or forelimb reaching, persists for a longer time,suggesting they help encode the storage of that learning
information [17, 18]. In addition, training can enhance theelimination of existing dendritic spines [18]. Hence, we
next examined the stability of existing and newly formed
dendritic spines. Although the absolute and relative numbers of new dendritic spines seen in KCC2-overexpressing
mice were greater than in WT mice (Fig. 2), these newdendritic spines were similarly labile, and both WT and
KCC2 mice had similar rates of elimination of dendritic
spines that existed prior to the rotarod training (Fig. 5a).
We then examined the stability of dendritic spines that
newly formed over the first 2 days of training (Fig. 5b). In
both WT and KCC2-overexpressing mice, approximately
20% of the new dendritic spines seen after 1 or 2 daysof rotarod training had disappeared by about 24 h later(Fig. 5c). Hence, under our conditions, we could not detect
a population of more stable dendritic spines associatedwith the enhanced learning in KCC2-overexpressing mice.

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Fig. 4  Performance in rotarod training trials over 5 days of training for KCC2 mice and WT mice. Following withdrawal of Dox for
2–3 weeks, mice underwent 6 trials of accelerating rotarod training
per day over 5 successive days (WT mice: black; n = 15; KCC2-overexpressing mice: red; n = 15). a Rotarod performance was measured
as the time before falling off as the rotarod was accelerated from 4
to 40 rpm over 300 s in each trial. The improvement of performance
during the successive 5-day training sessions was significantly greaterin KCC2 mice compared with WT mice (***p < 0.001, 2-way
ANOVA). b Comparison of the increase in rotarod performanceacross each day of training in WT (left, black) and KCC2-overexpressing mice (right, red). The performance in each of the six trials
was averaged for each day and the averaged performance comparedwith that on day 1 to evaluate the time course of the motor learning.
For WT mice a significant improvement was seen by day 4 (n = 15,*p < 0.05, one-way ANOVA, Bonferroni’s test), while for KCC2overexpressing mice, a significant increase in performance was seen
earlier, on the 2nd day of training (n = 15, *p < 0.05, ***p < 0.001,
one-way ANOVA, Bonferroni’s test). Data are shown as mean ± SEM

Discussion
Using a conditional transgenic mouse where KCC2 is overexpressed in excitatory pyramidal neurons by ceasing dietary doxycycline supplementation, we have shown that: (1)increasing KCC2 in the brain after the major developmental
period of synaptogenesis can increase dendritic spine density, (2) increasing KCC2 enhances the capacity of motor
learning to form new dendritic spines, and (3) increasing
KCC2 increases the extent and rate of performance increase

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Fig. 5  Stability of newly formed and pre-existing spines. a Survival
rates of spines that existed prior to the rotarod training in both WT
and KCC2 mice indicate a similar rate of spine elimination. b Schematic of the protocol used to define the stability of newly formed
spines. Spines that appeared on the 1st day of training (closed circles, relative to 1 day pre-training) were either present (closed circle)
or eliminated by the next day (open circle). Similarly, new spines
observed after day 2 of rotarod training (closed squares, relative today 1) were either present (closed square) or eliminated (open square)by the next day. c The relative elimination rates of newly formed
spines indicate that about 80% of the new spines survive the next day
with no difference between WT and KCC2-overexpressing mice

during training. Hence, our data strongly support the growing appreciation of the role of KCC2 in dendritic spine
development, maturation and/or maintenance and extend
this to a putative role in enhancing synaptic plasticity andperformance during learning in vivo.
In utero transfection of KCC2 into the cortex results in apermanent increase in dendritic spine density of between 50
and 100% when measured at different postnatal times and indifferent regions of layer II/III neurons. For example, in apical and basal dendrites from mice at 3 months of age, spine
density was approximately 90% higher [7]. This increase indendritic spines was supported by an approximately 70%
increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs; at postnatal day 30–34; [7]). Conversely, neurons cultured from KCC2 knockout mice showed
an approximate halving of synapses identified by both glutamate transporter (VGLUT) staining and mEPSC frequency

The Journal of Physiological Sciences (2019) 69:453–463[6]. It has been debated whether these striking effects of
KCC2 on synapse numbers may be at least partly due toan impact on developmental spinogenesis. Knocking down
KCC2 in more mature hippocampal cultures has not been
associated with significant changes in dendritic spine density
or mEPSC frequency, although knocking out KCC2 in developing neuronal cultures did alter dendritic spine volume,
AMPA receptor aggregation and quantal size [12]. Our present results show that overexpressing KCC2 in the postnatal
brain in vivo leads to a significant increase in dendritic spine
density in the hippocampus and motor cortex (but not somatosensory cortex). However, the extent of this increase in
the motor cortex was 40–50% less than that achieved when
KCC2 was manipulated prior to spinogenesis in development [7, 33]. While two reports have shown that shRNAinduced knockdown of KCC2 decreases hippocampal dendritic spine density [8], a more recent report demonstrated
that overexpression of KCC2 decreases dendritic spine
density in CA1 neurons, both in vivo and in slice cultures.
In the same study, dendritic spine density was increased inpyramidal neurons of the cortex after KCC2 overexpression,indicating regional differences [34]. The effects of KCC2 on
dendritic spine density may depend on the extent of effectsof KCC2 on spinogenesis and neuronal excitability, which
may further relate to regional and age-dependent differencesin relative membrane levels of KCC2 and on other neuroactive molecules such as brain-derived neurotrophic factor[34]. It will be important to also determine if adult KCC2
expression is associated with an increased dendritic spine
head diameter and the number of functional excitatory synapses, as has been observed with in utero electroporation of
KCC2 [7]. In the present study, while we did not specificallycompare the morphology of dendritic spines or the numberof functional synapses, the elimination rates of the dendritic
spines in KCC2-overexpressing and WT mice over the 3-day
imaging period were similar, which suggests that the additional dendritic spines in KCC2 mice could be similar tomature dendritic spines in WT mice.
Experience-dependent changes in dendritic spine number,size and spatial arrangements are important components ofthe structural synaptic plasticity that accompanies learningand memory formation [35, 36]. In the adult brain, most dendritic spines are generally very stable with a small proportion(≈ 5%) being continuously formed and eliminated. Learning can increase this new dendritic spine formation rate andpromote elimination of pre-existing dendritic spines. Motor
training on an accelerated rotarod, for example, causes anapproximate 5% increase in dendritic spine formation in theforelimb area of the motor cortex in adult mice [18]. Similar increases in dendritic spine formation rates are seen inother motor tasks, such as learning to reach for a pellet [17,37]. The extent of the increase in formation rate can dependon many factors including age, specific cortical layer and461

dendritic branch as well as sleep and glucocorticoid status[37–39]. Some of the newly formed dendritic spines persist,with the degree of dendritic spine stability correlated withthe degree of learning [17]. In our current study, we useda more modest rotarod training regime and did not detectsignificant increases in dendritic spine formation rates in
WT mice, nor did we detect correlations between the extent
of performance increments and new dendritic spine formation or stability, as has been previously reported in Yanget al. [18] and Liston et al. [38]. Our rotarod accelerated toa maximum of 40 rpm, and we trained with six trials persession (for 5 days), whereas Yang et al. [18], for example, accelerated to 100 rpm and trained with 20 trials persession (for 2 days). Despite this modest training protocol,
we still observed a significant increase in dendritic spine
formation rate in mice overexpressing KCC2. Hence, as
well as increasing basal spine levels, KCC2 overexpression
may facilitate the capacity for motor training to form new
dendritic spines. Related to this, KCC2 overexpression was
also associated with subtle yet significant increases in therate and extent of performance increase resulting from therotarod training. We speculate that the significant increasein performance seen at day 2 in the KCC2-overexpressing
mice may be related to the increase in dendritic spine formation seen following training on day 1, enabling at least partof the structural component of synapses to be more readily
available to encode the learning associated with subsequent
training. Indeed, daily significant increments in motor performance improvement and dendritic spine formation were
only seen on all days of the training protocol in KCC2overexpressing mice. This assumes that the locus of therotarod learning resides in the motor cortex, although otherregions related to motor activity, e.g., thalamus and cerebellum, may be affected by KCC2 and also be involved in the
motor performance changes. Clearly KCC2 overexpression
will also affect C­ l− homeostasis, GABAergic inhibition andneuronal excitability, and we are as yet unable to distinguishthe aspects of enhanced behavioral learning that are solely
attributable to the dendritic spine effects. Transport-deficient
KCC2 mutations may be able to probe this, as describedin Li et al. [6] and Fiumelli et al. [7], but see Awad et al.
[34]. Nevertheless, our tantalizing results raise the possibility that KCC2 may be involved in synaptic plasticity in theadult nervous system and should encourage further studies
to more closely examine this hypothesis.
Acknowledgements We thank Ms. Tatsuko Oba for the support of animal maintenance and preparation and Dr. Miho Watanabe at Hamamatsu Medical University for a useful suggestion for the analysis of
KCC2 expression and staining.
Author contributions KN, DLC, PWR, KE and IT conducted experiments; KN, AJM and JN wrote the paper; AJM and JN conceived thestudy. All authors approved the final version of the manuscript.

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462
Funding This study was supported by JSPS KAKENHI grant no.
JP17H01530 and JP25253017 to JN. The funders had no role in the
study design, data collection and analysis, decision to publish or preparation of the manuscript.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.
Ethical approval This article does not contain any studies with humanparticipants. All animal experiments were approved by the Okazaki
Institutional Animal Care and Use Committee or by the UNSW Sydney
Animal Care and Ethics Committee.

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