1HXURVFLHQFH/HWWHUV  ²Contents lists available at ScienceDirect

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Research article

Calcineurin knockout mice show a selective loss of small spines
a,b

a,c,d

a

a,b

Hitoshi Okazaki , Akiko Hayashi-Takagi, Akira Nagaoka , Makiko Negishi , Hasan Ucar⁎
Sho Yagishitaa,b, Kazuhiko Ishiia,b, Taro Toyoizumie, Kevin Foxf, Haruo Kasaia,b,

7
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,

a

Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, Bunkyo-ku, Tokyo, Japanc
Laboratory of Medical Neuroscience, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi-city, Gunma, Japand
PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japane
RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, JapanfSchool of Bioscience, Cardiﬀ University, Cardiﬀ, UKb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Dendritic spines
Synaptic plasticity
Schizophrenia
Calcineurin

Calcineurin is required for long-term depression and activity-dependent spine shrinkage, and calcineurin mutations have been identiﬁed in patients with schizophrenia. Moreover, mice with conditional knockout of calcineurin B (CNB-KO) exhibit behavioral abnormalities suggestive of schizophrenia. Changes in the dendritic
spines of these mice, however, have not been investigated. We therefore examined the dendritic spines of CNBKO mice, and observed a signiﬁcant reduction in small spines and an increase in large spines in the prefrontal
and visual cortices. The eﬀect of CNB-KO on the spine sizes was relatively moderate, possibly due to the presenceof spontaneous ﬂuctuations (dynamics) in the dendritic spines themselves. Thus, CNB-KO mice showed a spine
phenotype similar to those recently reported in patients with schizophrenia.

1. Introduction
Calcineurin is the only serine/threonine phosphatase controlled by
Ca2+/calmodulin [1], and is involved in various neuronal functions[2], including long-term depression (LTD) [3–5], spine shrinkage [6],and axonal guidance [7]. Calcineurin is a heterodimer comprising acatalytic subunit, calcineurin A (CNA), and a regulatory subunit, calcineurin B (CNB) [2]. CNB1 is an isoform of CNB and the only regulatory subunit expressed in the brain. In contrast, several diﬀerent
isoforms of CNA are expressed in the brain [8,9], with the PPP3CCencoded γ isoform showing single-nucleotide polymorphisms in patients with schizophrenia [10–12]. CNB knockout (CNB-KO) mice exhibit certain behavioral abnormalities typically observed in schizophrenia [13], including impairments in hippocampus-dependentworking memory and episodic-like memory, while contextual memory,
cued fear conditioning, and spatial reference memory are all preserved[13,14].
To gain insight into the possible relationship between schizophrenic-like behavior and calcineurin dysfunction, it is valuable tostudy the eﬀect of CNB-KO on spine structure in vivo—especially since
CNB-KO should abolish activity-dependent decreases in spine size [6].
However, it is important to note that, in addition to activity-dependent

spine enlargement and shrinkage, activity-independent slow changes inspine size occur [15–21]. Such intrinsic ﬂuctuations (dynamics) havebeen detected in the presence of inhibitors for NMDAR, as well as
voltage-dependent Ca2+ and Na+ channels, and are known to reﬂect
cell metabolism [15,22]. A recent study revealed that such dynamics
also occur in vivo, and are exacerbated in autism spectrum disorders[22], further emphasizing the need to take into account activity-independent spine dynamics when assessing the inﬂuence of activitydependent changes on spine structures.
We examined visual area V1 and frontal area 2 (Fr2), which is therodent analogue of the dorsolateral prefrontal cortex [23], to evaluate
possible diﬀerences in the distribution of spine sizes between wild-type(WT) and CNB-KO mice. We found signiﬁcant diﬀerences in the distribution of spine sizes in both cortical areas. These diﬀerences, however, were relatively small, consistent with the presence of intrinsic
spine dynamics.
2. Materials and methods
2.1. CNB-KO mice
Forebrain-speciﬁc CNB knockout mice, which harbor yellow⁎
Corresponding author at: Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo,
Japan.
E-mail address: hkasai@m.u-tokyo.ac.jp (H. Kasai).

https://doi.org/10.1016/j.neulet.2018.02.006
Received 22 December 2017; Received in revised form 26 January 2018; Accepted 3 February 2018
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ﬂuorescent protein (YFP H-line in [24]) in a small subset of neurons,
were generated by mating a male mouse heterozygous for ﬂoxed CNB(CNBﬂox/wild in [13]) and homozygous for the YFP allele (YFP+/+) with afemale CNBﬂox/wild mouse carrying the α-calcium–calmodulin-dependent
kinase II (αCaMKII)-Cre transgene [αCaMKII-Cre (+/−)]. This pairing led
to the generation of CNBﬂox/ﬂox, αCaMKII-Cre (+/−), YFP(+/−) mice for
the CNB-KO group and CNBwild/wild, αCaMKII-Cre (+/−), YFP (+/−) forthe littermate control (WT) group. The background strain used to generate the mutation was C57BL/6J. Mice were housed in a room with a
12-h light-dark cycle (lights on at 9:00 a.m.), with access to food andwater ad libitum. Tail DNA was collected to identify the genotype of eachanimal by using polymerase chain reaction. All procedures were approved by the Animal Experiment Committee of the University of Tokyo.
Procedures were carried out in accordance with the University of Tokyo
Animal Care and Use Guidelines. All surgeries were performed under
isoﬂurane anesthesia, and all eﬀorts were made to minimize suﬀering.
2.2. Slice preparation
Mice were anesthetized with isoﬂurane, and transcardially perfusedwith 4% paraformaldehyde (PFA; pH 7.4); thereafter, their brains weredissected and post-ﬁxed in 4% PFA for 12 h at room temperature. Coronal
sections (150-μm) were obtained using a vibratome (VT1000, Leica
Biosystems, Germany), and brain slices were subjected to free-ﬂoating
immunoﬂuorescence staining with a slight modiﬁcation to the method
described in a previous study [25]. In brief, the slices were permeabilizedwith perm/blocking buﬀer (2.5% normal goat serum [v/v] in phosphatebuﬀered saline [PBS] with 0.1% Triton X-100 [v/v]), followed by incubation for 48 h at 4 °C with the primary antibodies against anti-green
ﬂuorescent protein (anti-GFP, D153-3, MBL, Japan). After rinsing with
PBS (8 times, 5 min each), the slices were stained with the Alexa 488 goatanti-Rat IgG and mounted on slides.
2.3. Image analysis

Fig. 1. Distributions of spine areas in WT and CNB-KO groups.
(A) Fluorescence images of representative dendrites in the Fr2 and V1 areas of P26–32
mice. Scale bar, 1 μm.
(B, C) Distribution densities of spine areas in (B) Fr2 of P26–32 WT [574 spines, 14
dendrites, 3 mice] versus KO [709 spines, 20 dendrites, 3 mice] mice; (C), V1 of P26–32
WT [281 spines, 9 dendrites, 3 mice] versus KO [372 spines, 10 dendrites, 3 mice] mice.
Vertical dashed lines indicate 0.3 μm2, and Fisher’s exact tests were performed for the two
populations separated by the dashed lines. n.s., not signiﬁcant; **p < 0.01.
(D, E) Cumulative distributions of spine areas in Fr2 (D) and V1 (E). The ﬁndings ofKolmogorov-Smirnov tests are presented in D and E. Statistical signiﬁcances are also
indicated. Error bars indicate the standard errors of the mean.

Measurements of spine head cross-sectional areas and densities were
performed using National Institutes of Health ImageJ software. Before
quantifying the spine area, we binarized each imaged dendrite using thefollowing threshold: mean value plus 4 times the standard deviation of
the background intensity around the dendrite.
2.4. Statistical analysis
Distributions of spine areas were compared using theKolmogorov–Smirnov test. Two-way analyses of variance (ANOVAs),
with genotype (i.e., CNB and wild-type) and cortical areas (i.e., Fr2 andV1) as between-subjects factors, were performed to assess diﬀerences inspine areas and densities. ANOVAs were performed using type II sum ofsquares (SS) to adjust for unequal sample sizes, along with post hoc
Bonferroni multiple comparison tests. We calculated eta squared η2,which represents the coeﬃcient of determination for ANOVA, as

which is within the critical period for V1. In each case, spine head sizes
were measured in the focal plane yielding the largest area in each case.
Using this approach, we observed signiﬁcant changes in the distributionof spine head sizes in both cortical areas of CNB-KO mice compared tocontrol mice (Fig. 1B–E).
To assess whether small spines (< 0.3 μm2) were less frequent in thecortices of CNB-KO mice than in those of WT mice, we used Fisher’s
exact test for WT and CNB-KO mice (Fig. 1B and C). We adopted
0.3 μm2 as the threshold for the smallest spines because morphologicalplasticity has been shown to decrease by at least 0.3 μm2 (∼0.1 μm3 interms of volume) when assuming that spine heads are spherical[26,27]. We found that small spines were signiﬁcantly less frequent,and large spines more frequent, in CNB-KOs than in WTs for both Fr2
and V1 cortices.
Previous studies have reported that mean spine head area varies bycortical region and that, in the mouse, spine heads are largest in the
motor cortex [28]. Similarly, in this study, we found that mean spine
head area varied between Fr2 and V1 in WT mice (Fig. 2), with spines
in Fr2 being signiﬁcantly larger than those in V1 (Fig. 2). On the other
hand, mean spine areas in CNB-KO mice were larger in both Fr2 and V1
than in WT mice (Fig. 2). A two-way ANOVA showed a signiﬁcant eﬀect

η2 = SSbetween/ SStotal

where SSbetween and SStotal represent the SS between groups and theoverall SS, respectively. Within the WT and CNB-KO groups, spines
were categorized into small (< 0.3 μm2) and large sizes using Fisher’s
exact test.
3. Results
A confocal microscope was used to image spines in ﬁxed slice preparations of YFP-expressing H-line mice mated with CNB-KO mice or
their WT litter-mates. We investigated spines on the ﬁrst branches ofapical dendrites of layer 5 pyramidal neurons, where they appeared inlayer 2/3 (located 200–300 μm away from the soma), in two neocorticalareas (Fr2 and V1). Mice used in this study were aged P26–32 (Fig. 1),1HXURVFLHQFH/HWWHUV  ²H. Okazaki et al.

Acknowledgements
We thank T. Miyakawa for providing the CNB-KO mice and for thehelpful discussions. We also thank Ogasawara, H. Ohno, Tajiri, M. and
M. Nakamura for their technical assistance. This work was supported by
Grants-in-Aid for Scientiﬁc Research (S) (No. 26221001 to HK), (B) (No.
26293260 to AH-T), Young Scientists (B)(15K18333 to SY) and
Scientiﬁc Research on Innovative Areas (16H06396 to SY) from JSPS,
and CREST (JPMJCR1652 to HK) from JST, and the SICP, Brain/MIND,
Strategic Research Program for Brain Sciences projects (SRPBS,
17dm0107120h0002) from AMED (to HK), and World PremierInternational Research Center Initiative (WPI) from MEXT (to HK). KF
was supported by an MRC/JST International collaborative award (MR/
M501670/1). TT was supported by Brain/MINDS from AMED and
RIKEN Brain Science Institute.

Fig. 2. Mean spines areas in WT and CNB-KO groups.
Mean spine areas were signiﬁcantly diﬀerent according to genotypes and cortical areas, as
determined by two-way analyses of variance. All spine area p values were adjusted by the
Bonferroni method for multiple comparisons; ***p < 0.001. Error bars indicate thestandard errors of the mean.

Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.neulet.2018.02.006.

of genotype across the two cortical areas (15–18%; ANOVA:
p < 0.001, η2 = 0.014) but no signiﬁcant interaction between genotype and cortical area (p > 0.05). Interestingly, the diﬀerences between WTs and CNB-KOs did not appear to be larger than the diﬀerence
between the two cortical areas in WT or CNB-KO mice (21–24%;
ANOVA: p < 0.001, η2 = 0.021). Moreover, overall, the diﬀerence inspine head size due to CNB knockout was rather small.
Since spine density is often aﬀected in patients with schizophrenia[29–31], we also examined the number of spines in WT and CNB-KO
mice. However, no signiﬁcant diﬀerence in spine density was foundbetween phenotypes for Fr2 nor V1; spine density (spine number/μm)
in Fr2 was 1.45 ± 0.08 (mean ± standard error, n = 16) and1.63 ± 0.12 (n = 8) for WT and KO mice, respectively (p = 0.22),
while the spine density in V1 was 1.25 ± 0.07 (n = 14) and1.60 ± 0.18 (n = 6) for WT and KO mice, respectively (p = 0.06).

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4. Discussion
Consistent with our hypothesis, we found a reduction in small spines
in CNB-KO mice, suggesting that the prevention of spine shrinkage inthese animals allows small spines to be readily enlarged. Previous
studies have shown that CNB-KO mice exhibit schizophrenia-like behaviors, such as weak spatial working memory and normal spatial reference memory [14]. The formation of working memory requires rapid
synaptic changes and likely involves small spines which undergo more
pronounced enlargement [15,26,27,32,33]. In CNB-KO mice, working
memory deﬁcits may reﬂect a reduction in the number of small spinesthat are available for potentiation and enlargement. Recently, a systematic postmortem study of human patients with schizophrenia reported a speciﬁc loss of small spines in the neocortices [31,34]—consistent with our current ﬁndings in CNB-KO mice.
The diﬀerences between CNB-KO and WT mice observed in thecurrent study were relatively moderate (Fig. 1F). In contrast, activitydependent spine shrinkage is typically severe [6], and predicts themarked enlargement of spines. This discrepancy may be explained byintrinsic dynamics in spine volume which, unlike activity-dependent
plasticity, occur continuously over long periods of time and are themajor factor determining spine size distributions [15,35]. Due to these
intrinsic dynamics, large spines can shrink back to small spines, and
CNB-KO may only have a more moderate inﬂuence on spine size distribution than the diﬀerences between cortical regions.
In conclusion, our current ﬁndings of a moderate reduction in small
spine densities in CNB-KO mice are akin to observations made in patients with schizophrenia. Further, we hypothesize that CNB-KO-induced reductions were moderate because of the presence of spontaneous dynamics in dendritic spine sizes.
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