Pcdhβs affects synchronous activity in the hippocampus
Hirotaka Asai1,2, Noriaki Ohkawa1,2,3, Yoshito Saitoh1,2,3, Khaled Ghandour1,2,3, Hirofumi Nishizono2,4, Mina Matsuo4, Teruyoshi Hirayama7,8, Shin-ichi Muramatsu5, 6,97
1,2
Ryosuke Kaneko , Takeshi Yagi , and Kaoru Inokuchi
1. Department of Biochemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. 2. CREST, JST, University of Toyama, Toyama, Japan. 3. PRESTO, JST, Univ. of Toyama, Toyama, Japan. 4. Division of AnimalExperimental Laboratory, Life Science Research Center, Univ. of Toyama, Toyama, Japan. 5. Division of Neurology, Department of Medicine, Jichi Medical Univ., Tochigi, Japan. 6. Center for Gene and Cell Therapy, The Institute of Medical Science, The
Univ. of Tokyo, Tokyo, Japan. 7. KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka Univ., Osaka, Japan. 8. Department Anatomy and Develop Neurobiology, Tokushima Univ., Tokushima, Japan.
9. Bioresource center, Gunma Univ. Graduate School of Medicine, Maebashi, Japan

Summary

Pcdhb-deletion decreased repetitive activity during context exploration
ABackground: Clustered protocadherins (Pcdhs), a large subgroup of adhesion
molecules, are important for neural morphology such as axonal projection
and dendrite spread. However, little is known how Pcdhs affect neuralactivity.
Methods: Observing neural activity in Pcdhβ-deletion mice with in vivo Ca2+imaging, we demonstrate that Pcdhβs affect neural activity in thehippocampus during active state rather than resting state.
Results: Pcdhβ-deletion reduced repetitive synchronous activity during novel
context exploration and increased large size cell ensembles, which wereextracted with non-negative matrix factorization analysis based onsynchronous activity. Majority of the large ensembles was rarely activated.
Furthermore, larger portion of cells in Pcdhβ-deletion mice were shared
between ensembles than wild type mice, which may impair the discriminationof context in contextual fear conditioning task. These phenotypes may result
from the involvement of Pcdhβs in adjustment of neural activity throughensemble formation in the hippocampus without affecting cellular activity.
Conclusion: Together, Pcdhβs modulate neural ensemble activity in thehippocampus.

Pcdhb-deletion did not affect cellular level activity

Bbc100100200200300

300

Cell#Cell#a400

Figure 4. The cellular activity. (a, b) Representative
images of raster plot of all recorded cells (a: Wt, b:Δꞵ). (c) Cumulative curve of proportion of cells
against the number of active events during whole
recoding session (pre 10 min + sq 10 min). (d, e)
Proportion of cells on the number of Ca2+ events ineach session. (f) The ratio of the number of Ca2+
events in sq to pre session. n = 5 mice for Wt, n = 4mice for Δꞵ, Mean ± S.E.M. No significant
difference was observed in all figures (c:Kolmogorov-Smirnov test, d-f: Bo f rro iʼmultiple comparison test).

400
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500

600

600

b98

b52
700
2

68 10 12Time (min)141618d202

468 10 12Time (min)14161820e

f2+ event
2+Ratioof ofCaCa(sq/pre)
Ratio
event
(sq/pre)0.8

Proportion of Cells

Figure 1. Deletion of cPcdhbs reduced repetitive activity. (A, B) Representative image of correlation matrix (A: Wt, B: Δϐ). (C) Summation ofcorrelation coefficient in session by session. n = 5 mice for Wt, n = 4 mice for Δϐ. Mean±S.E.M. **p < 0.01, (Adjusted p-value of Bo f rro iʼmultiple comparison test).

4

Pcdhb-deletion increased large ensembles and cells shared between ensembles
A0.6
0.4
0.2
0.0

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2

3

4

567Group of event ratio

140
Ensemble ID

1120

Pcdhb-deletion decreased the discrimination of contextual fear conditioning

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Introduction

200300400 500
Cell ID

600700BPcdhs are thought to be important for forming diverse neural networks

160

Diverse expression patternEnsemble ID

140

Homophilic interaction

120
1008060

Figure 5. The contextual fear discrimination. (A) Schematic diagrams of experiment. (B) Freezing level in test session. (C) Discrimination index intest session. n = 19-22 mice in each group. Mean±S.E.M. *p < 0.05 (Unpaired t-test).

40
20100

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Cell ID

600700Figure 2. Deletion of cPcdhbs increased large ensembles. (A, B) Representative image of binarized basis matrix. (C, D) Normalized number ofensembles in each ensemble size. n = 5 mice for Wt, n = 4 mice for Δϐ, Mean±S.E.M. **p < 0.01 (Adjusted p-value of Bo f rro iʼ multiple
comparison test). (E, F) Ratio of cells contributing singular (open bar) and plural (pattern or solid bar) ensembles. ****p < 0.0001 (Chi-square test)
Ref: Yagi, Front. Mol. Neurosci., 2012

Ref: Thu et al., Cell, 2014

Large ensembles in Pcdhb-deletion mice showed rare activity

Large Ensemble

Olfactory Bulb

Hayashi and Takeichi, J Cell Sci., 2015

Figure 3. The activity oflarge ensembles were notso frequently. The numberof activity event of extra
large (XL, 20 or more cells;top right), large (L, 15-19cells; top middle), medium(M, 10-14 cells; tope left),small (S, 5-9 cells; bottomright), several cells (SCs, 2-4cells; bottm middle), single
cell (SC; bottom left) incontext session. n = 5 micefor Wt, n = 4 mice for Δϐ.
Mean±S.E.M. *p < 0.05,**p < 0.01, (Adjusted pvalue
ofBo f rro iʼmultiple comparison test).

Projection
Serotonergic projection

Mountoufaris et al., Science, 2017

Katori et al., J Neuro Sci., 2009

Question
How do Pcdhs affect neural activity in the hippocampus ?

Experiments
Overview of analysis
Preprocessing

Correlation(nVista)
CaMKII

GCaMP7

III.Imaging
Home cage (pre)context (sq)*Based on Ghandour et al., Nat. Commun., 2019

i. Cell identification (HOTARU algorithm†)
Detected cells

Activity

Ensemble activity(Fig. 2-4)

ii. Correlation matrix analysis(Occurrence)‡Non-negative Matrix Factorization

Raw data matrix

Raw data Matrix (t × C)
Cells (C)

Reduction

Clustered protocadherinβs do not affect the activity on thecellular level, but they are involved in population/ensemble
activity and forming ensembles, which are sets of cells
activated synchronously.

Fig. 5

Contextual discrimination
ImpairSimilarIncrease the sharing cells between ensembles may impair
contextual fear discrimination.

➢ Schematic diagrams of expected function

Clustered protocadherinβs
make ensembles diverse toavoid confusing information

Ca2+ trace

Basis matrix

Cells (C)t₁

1. Raw data
matrix was binned
every 200 msec.

t₂

𝑫 ≈ 𝑩𝑪𝑬 ≡ ෍(𝑫𝒊𝒋 − ෍ 𝑩𝒊𝒌 𝑪𝒌𝒋 )𝟐
𝒊𝒋𝒌NMF

Occurrence matrix
Ensemble (En)

Cells (C)Time (t)NMF

Ensemble (En)

II. Setting microscopy

Increase

Cellular activity

Conclusion
Clustered protocadherinβs tune ensemble activity in the hippocampus
and context discrimination

Cellular activity

Time (t)I. GCaMP induction by AAV

Cell sharing

Repetitive activity(Basis)Time (t)dF data

Cell identification
Ref: Hasegawa et al., Front. Mol. Neurosci., 2016Fig. 2

Fig. 4

Cellular components

Repetitive activity(Fig. 1)

Correlation Matrix

2. Correlation
coefficients werecalculated
between all timebinned framest₂Time (t)Raw data

Increase
Rare activity

Fig. 1ii. Extraction of ensemble by NMF‡

IV.Analysis

Mutant mice

➢ Summary of Results & Discussion
Fig. 2, 3

Pcdhs affect neuronal morphology
Dendritic spread

Discussion & Conclusion

Correlation coefficeint
between t₁ and t₂†Takekawa et al., bioRxiv, 2017
t₁

Time (t)No significant difference wasobserved in the normalized numberof ensembles between genotypes
in both sessions

Acknowledgements
We thank Dr. Hiroyuki Okuno (Kagoshima Univ.) for advising cFos immunohistochemistry; Dr. TomokiFukai, Dr. Chi Chung Alan Fung (OIST), and Dr. Takashi Takekawa (Kogakuin Univ.) for the analysis ofCa2+ imaging; Dr. Tsuyoshi Koide and Dr. Akira Tanave (NIG) for modifying tracking software
DuoMouse; Dr. Emi Murayama, Dr. Reiko Okubo-Suzuki and Dr. Masanori Nomoto (Inokuchi Lab.) for
Ca2+ imaging experiments.

We have no COI (Conflict of Interest) with regard to this presentation.