Nature - USA (2020-10-15)

Nature | Vol 586 | 15 October 2020 | 421

absence of strong neuronal activation, microglial motility is character-
ized by the constant extension and retraction of microglia protrusions,
which has been defined as microglial baseline or surveillance activity^9 –^12.
The activation of neurons resulted in a decrease in microglial base-
line motility indicated by reduced protrusion velocity (Fig. 3h) while

enhancing microglial targeted motility (directional branch extension

towards target sites^3 ,^9 ,^10 ,^27 ), leading to increased microglia–neuron

proximity (Fig. 3h). Following neuronal activation, an increased num-

ber of microglia protrusions were recruited to the synaptic boutons

of active thalamocortical projection neurons in an ATP-dependent

Relative

uorescence units

h

D1 agonist +

A 1 R agonist

D1 agonist

i D1 agonist

DARPP32

pThr75

0

0.5

1.0

1.5

2.0

2.5

32 kDa

32 kDa

DARPP32pThr34

DARPP32

DARPP32

32 kDa pThr75

100 kDa GLUR1pSer845

GLUR1

Normalized to GLUR1

Il34

fl/fl

Drd1a

Cre/+

Il34

fl/fl

100 kDa

GLUR1

pSer845

0

1

2

3

4

5

Normalized to DARPP32

DARPP32

pThr34

P2RY12 antagonist

D1 agonist + A 1 R agonist

Adenosine

400

0

100

200

300

D1 + A 1 R

agonist

P2RY12 antagonist

D1

agonist

P = 0.016

P = 0.004

P = 0.0055

P = 0.02

P = 0.0019

9/10

P = 0.0001

l m

0

20

40

60

80

(^100) P = 0.01
3/ 13 9/ 11
Mice with stage IV–V
seizures (%)
Mice with stage IV–V
seizures (%)
Mice with stage IV–V
seizures (%)
Mice with stage IV–V
seizures (%)
Mice with stage IV–V
seizures (%)
Mice with stage IV–V
seizures (%)
Mice with stage IV–V
seizures (%)
0
20
40
60
80
100
0/10 0/10 7/10
D1 agonist(3 mg kg
–1)
g
j
A^1 R antagonist
D1 agonist (3 mg kg
–1) +
A^1 R anta
gonis
t
Low D1 agonist + k
A 1 R antagonist
D1 agonist + P2RY12 antagonist
Padj = 0.0023
Padj = 0.0023
Contro
l
P2ry12
–/–
f D1 agonist
Adora1
fl/fl
Adora1
fl/fl
Drd1a
Cre/+
D1 agonist
Brain-impermeable
P2RY12 antagonist
Active Inactive
P2RYBrain- 12 permeableantagonist
Padj = 0.0075
Padj = 0.0012
PKA D1R
A 1 R
f, g, l P2RY12 ablation/
antagonism
DARPP32
pThr34
GLUR1
pSer845
h, i CD39
ablation
j, k A 1 R ablation/
antagonism
i, l, m A 1 R agonism
D1 neuron
Microglia
e
Microglia
ablation
D1
agonism
d
0
20
40
60
80
100
2/12 5/6
P = 0.02
0
20
40
60
80
100
13/16^13 /1^6 2/13 1/7 0
20
40
60
80
100
1/11 9/11 0/5 1/9
D1 agonist +
A 1 R agonist
Cd39
fl/fl
Cx3cr1
CreErt2/+
Contro
l
2/11 8/9 0/11 0/9
e, m
a
b
e
±CD39 inhibitor
±CD73 inhibitor
Neonatal CD11b+ microglia

• 100 μM ATP
  HPLC analysis
  of supernatant ATP
  ATP + CD39i
  0
  20
  40
  60
  80
  100
  120 P = 0.018
  P = 0.0013
  0
  10
  20
  30
  40
  (^50) P = 0.001P = 0.0006
  0
  1
  2
  3
  P = 0.04
  P = 0.02
  ATP + CD73i
  ATP
  ATP + CD39iATP + CD73i
  ATP
  ATP + CD39iATP + CD73i
  ATP AMP Adenosine
  Concentration (
  μM)
  0
  253
  507
  760
  1,014
  –10^2103104105 –10^210310410501 –10^203104
  –10^2
  102
  103
  104
  105
  –10^2
  102
  103
  104
  105
  74% 13%
  7% 7%
  93% 0%
  CD73highCD39–
  CD73–CD39+
  CD73lowCD39+
  CD39–A700
  CD73–PE CD73–PE CD73–PE
  Cell count
  CD39–A700
  ControlMicroglia-
  decient
  0
  200
  400
  600
  (^800) P = 0.037
  c Adenosine
  Microdialysis
  (striatum)
  Mass
  spectrometry
  Il34
  fl/fl
  Drd1a
  Cre/+
  Il34
  fl/fl
  Wild type CD73-decient
  Concentration (
  μM)
  Concentration (
  μM)
  Il34
  fl/fl
  Drd1a
  Cre/+
  Il34
  fl/fl
  Il34
  fl/fl
  Drd1a
  Il34fl/fl Cre/+ Il34fl/fl
  Drd1a
  Cre/+
  Il34
  fl/fl
  Il34
  fl/fl
  Drd1a
  Cre/+
  Il34
  fl/fl
  0
  20
  40
  60
  80
  100
  0
  20
  40
  60
  80
  100
  Cd39
  fl/fl
  Cx3cr1
  Control CreErt2/+
  Cd39
  fl/fl
  Cx3cr1
  Control CreErt2/+
  0/10
  P = 1.0
  P = 0.98
  P = 0.75
  Concentration (nM)
  Fig. 4 | See next page for caption.