Fig. 2A). This raised the possibility of mul-
tiple subpopulations of calcium events. Indeed,
visual inspection suggested four qualitatively
distinct types of activation patterns in type
1PTNs(singlebranch,subtree,hemi-tree,whole
tree; Fig. 1, H and I; Fig. 2B; and fig. S2). To
perform quantitative analysis of these differ-
ent event types, we subdivided the events into
four clusters according to either their average
amplitudes over all ROIs (see materials and
methods) or the number of activated ROIs
during each event (spatial activation extent)
using the k-means algorithm (average percent-
age of events in cluster 1 = 66%, cluster 2 =17%, cluster 3 = 10%, cluster 4 = 8% of the
events;Fig.1H,Fig.2B,andfigs.S2and
S3). With the calcium activity vectors and
the structural distance matrix at hand, we
constructed a pairwise Pearson correlation
activity matrix arranged according to the
structural distance matrix for each of the268 15 APRIL 2022¥VOL 376 ISSUE 6590 science.orgSCIENCE
Fig. 1. M1 contains two popu-
lations of thick-tufted layer-5
PTNs with distinct morpholo-
gies and functional proper-
ties.(A) (Top) Behavioral setup
for hand reach task. (Bottom)
An ethogram and histogram
annotating the behavior of
an expert mouse. (B) (Top)
Behavioral setup of head-fixed
mice running on a treadmill.
(Bottom) Example of velocity
(cm/s) over time (s). (Cand
D) Z-stack of a single PTN acquired
in vivo and its three-dimensional
anatomical reconstruction.
(E) Single-imaging plane of a
GCaMP6s-positive layer 5 PTNÕs
tuft dendrites. ROIs are marked
in yellow. (F) Schematics of
the tuft morphology; dots rep-
resent ROIs. Same neuron in (C)
to (F). (G) (Top) Hierarchical
clustering of thick-tufted layer
5 PTNs based on five morpho-
logical parameters. (Bottom)
Three example apical morpholo-
gies from each PTN type. Basal
and oblique dendrites are not
shown. Red numbers indicate
the corresponding neurons in
the clustering. (H) Calcium
events (DF/Fheat map) in single
trials from the same three type 1
(left) and type 2 (right) PTNs
shown in (G), arranged by the
tree structure as indicated by
the dendrogram. (I) Illustration
of structure-activity correlations.
Schematic representation of a
layer 5 PTN with structural
distance matrix presented as a
heat map of the shortest path
distance between each two ROIs
(left) and illustrated activity
traces for ROIs (right upper
panel, dashed rectangles mark
events belonging to different
event clusters) and activity
matrix showing Pearson correla-
tion between ROI pairs
presented for all events and for
each event cluster (right lower
panel). (J) (Left) Normalized calcium events histogram from 21 neurons for type 1 (black) and type 2 (gray) PTNs. (Right) Event distribution color coded by event
cluster type for individual sessions in multiple neurons.
031
21
11
80
40(^0) -2 -1 041 2 3
Time (sec)
(^) la
irT#
Event count
Time (sec)-4 0 8
Tone+Rotation 0.2 sec
Lift
SupinationGrabAt mouth
Time (sec)
C
Soma
D
EF
50 μm
150 μm from pia
800 μm
pia
L1
L2/3
L5
Hierarchical clustering
G
1 243 5678910 11 12 13 14 15 1617 18 19 20 1234567 8 9 10 1 1 12 13 14 15 16171819 20 21 2223
Type 1 Type 2
Type 1 Type 2 2425
Examples type 1 PTNs
5 10 16 17 19 22
H Examples type 2 PTNs
Trial #
123456
Neuron 16
-0.3
4
-0.3
4
F/F F/F
1234567
Neuron 5
Trial #
1234
Neuron 10
567
Trial #
1234
Neuron 22
Trial #
1234567
Neuron 17
Trial #
1234
Neuron 19
Trial #
I J
Left
Right
2 nd bifurcation
Structural distance
matrix
(^11)
4
4
8
8
0
(^1000) (μm)
12 34 56 78
Cluster 1 Cluster 2 Cluster 3 Cluster 4
Activity matrix
1
0
All events
Shortest path pairwise
structural distance
Activity vectors
Pearson correlation pairwise activity distance^1
0
1
4
5
8
(^4) Time
F/F
1 1 2 2 3 3
Events distribution
Events cluster distribution
Cluster 4
Cluster 3
Cluster 2
Cluster 1
0
50
100
150
200
250
300
350
400
450
Events coun
t
Session #
0 1020304050 60
F/F events histogram
F/F (%)
Events count
0
200
400
600
800
1000
1200
1600
0 0.2 0.4 0.6 0.8 1
Type 2
Type 1
1400
16 x
A B
16
0 0.20.40.60.8 1 1.21.41.61.8 2 2.22.4
6
4
2
0
)c
es/
mc(
yti
col
eV
x
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