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Gamma control of cognitive maturation Bitzenhofer et al.
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Activity-dependent maturation of prefrontal gamma 8
oscillations sculpts cognitive performance 9
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Sebastian H. Bitzenhofer1,*,§, Jastyn A. Pöpplau1, Mattia Chini1, Annette 11
Marquardt1 & Ileana L. Hanganu-Opatz1,* 12
1 Developmental Neurophysiology, Institute of Neuroanatomy, University Medical Center 13
Hamburg-Eppendorf, Hamburg, Germany 14 §Current address: Center for Neural Circuits and Behavior, Department of 15
Neurosciences, University of California, San Diego, La Jolla, CA, USA. 16
17
* Corresponding author: Ileana L. Hanganu-Opatz 18
hangop@zmnh.uni-hamburg.de 19
Falkenried 94, 20251 Hamburg, Germany 20
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Sebastian Bitzenhofer 22
sebbitz@zmnh.uni-hamburg.de 23
Falkenried 94, 20251 Hamburg, Germany 24
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4 figures 26
Words: Abstract 70, Main 1883, 39 references 27
Supplementary Material: Methods, Supplementary Fig. 1-4, Supplementary Tab. 1 28
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Running title 30
Gamma control of cognitive maturation 31
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One sentence summary 33
Fast oscillatory activity in layer 2/3 sculpts the maturation of prefrontal cortex and 34
cognitive performance. 35
36
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Gamma control of cognitive maturation Bitzenhofer et al.
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Abstract 37
Gamma oscillations are the neural network attribute of cognitive processing. They 38
emerge early in life, yet their contribution to cortical circuit formation is unknown. We 39
show that layer 2/3 pyramidal neurons entrain mouse prefrontal cortex in fast oscillations 40
with increasing frequency across development. Chronic boosting of fast oscillations at 41
neonatal age reversibly alters neuronal morphology, but cause permanent circuity 42
dysfunction and impairs recognition memory and social interactions later in life. 43
44
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Gamma control of cognitive maturation Bitzenhofer et al.
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Main 45
Synchronization of neuronal activity in fast rhythms is a commonly observed feature in 46
the cerebral cortex 1. Although its contribution to cognitive performance is still a matter of 47
debate, high frequency oscillatory activity facilitates the communication between 48
neuronal ensembles and is thought to shape information processing in cortical networks 49
2-4. Oscillatory activity at frequencies within gamma band has been proposed to emerge 50
from reciprocal interactions of excitatory and inhibitory neurons 1. Fast inhibitory 51
feedback via soma-targeting parvalbumin (PV)-expressing inhibitory interneurons leads 52
to fast gamma (30-80 Hz) 5,6, whereas dendrite-targeting somatostatin (SST)-expressing 53
inhibitory interneurons contribute to beta/low gamma (20-40 Hz) activity 6,7. 54
Interneuronal dysfunction and ensuing abnormal gamma activity in the medial prefrontal 55
cortex (mPFC) has been related to impaired cognitive flexibility 8. Moreover, pyramidal 56
neurons critically contribute to the generation of fast oscillatory rhythms, beyond solely 57
providing the excitatory drive. As recently shown, their feed-forward excitation 58
determines oscillatory dynamics 9. 59
Despite substantial knowledge linking fast oscillations in the mature cortex and 60
cognitive abilities 8,10,11, their role during development is still largely unknown. Here we 61
examined the emergence of gamma activity in the mouse mPFC across development 62
and assessed the role of fast oscillations for the maturation of prefrontal networks and 63
cognitive abilities. Shifting the level of early oscillatory activity to a transient excessive 64
boosting of fast frequency events, we provide evidence that gamma entrainment of 65
neonatal circuits is critical for the prefrontal function and behavioral performance of 66
adults. 67
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Gamma control of cognitive maturation Bitzenhofer et al.
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Extracellular recordings in the mPFC of postnatal day (P) 5-40 mice revealed that 68
fast frequency oscillations occur spontaneously in awake and urethane-anesthetized 69
mice starting around P8 (Fig. 1a,b and S1a-d). While anesthesia reduced the oscillatory 70
power during development across a broad range of frequencies (Fig. S1e-g), it did not 71
affect the spectral composition of oscillations 12. In both states, fast frequency 72
oscillations increased in power and frequency with age, reaching stable values around 73
P25 with an adult-like gamma frequency peak at ~50 Hz (Fig. 1c,d). Using a recently 74
established protocol for optogenetic manipulation in neonatal mice 13,14, we interrogated 75
the neuronal substrate of fast oscillatory activity across development. To this end, we 76
transfected ~25% of pyramidal neurons in layers 2/3 (L2/3) by in utero electroporation 77
(IUE) with the light-sensitive channelrhodopsin 2 derivate E123T T159C (ChR2(ET/TC)) 78
and stimulated them with ramp light pulses. This stimulation type activates the network 79
without imposing a specific rhythm, thus enabling neurons to fire at their preferred 80
frequencies. We focused on neurons from L2/3 because our previous investigations 81
identified them as drivers of fast oscillatory rhythms at neonatal age 13. This layer-82
specificity seems to be a developmental feature of mPFC, since recent work in adult 83
sensory cortices has shown that gamma rhythms can be driven by pyramidal neurons 84
from all layers 15,16. We found that light-driven fast oscillatory activity increased in power 85
and frequency across development, similarly to what we observed for spontaneous 86
oscillations (Fig. 1e-k and S1h-l). This increase co-occurred with the maturation of PV-87
expressing interneurons (Fig. S2a,b) and the maturation of inhibitory synapses on 88
excitatory neurons 17,18. Therefore, a developmental transition from SST-dominated to 89
PV-dominated feedback inhibition might underlie the gamma frequency increase across 90
development. Alternatively, increased gamma frequency with age might result from 91
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Gamma control of cognitive maturation Bitzenhofer et al.
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alterations in the biophysical properties underlying excitability and maximal firing rate of 92
cortical pyramidal neurons 19. Indeed, we found that the firing rate and amplitude of 93
single unit activity (SUA) increased with age, whereas its half width decreased (Fig. S2c-94
e). To disentangle the role of interneurons from that of pyramidal neurons for the 95
generation of fast oscillations across development, we transfected L2/3 pyramidal 96
neurons with ChR2(ET/TC) by IUE in transgenic mice expressing archaerhodopsin3 97
(ArchT) in all interneurons. Silencing of interneurons led to a broadband increase of 98
activity in fast frequencies (>12 Hz) that was most pronounced in neonatal mice (Fig. 99
S2f-i). However, silencing of interneurons did not affect gamma activity driven by light 100
stimulation of L2/3 neurons, demonstrating a critical role of pyramidal neurons for the 101
generation of gamma rhythmicity. 102
The presence of neuronal ensembles capable of generating fast oscillatory 103
rhythms during early development raises the question of whether this pattern of activity 104
solely reflects or directly contributes to the maturation of cortical circuits and their 105
functions. Abundant literature documented the role of coordinated electrical activity for 106
dendrite formation, synaptic pruning and apoptosis 20-22. To elucidate the role of 107
neonatal fast oscillatory activity for prefrontal network maturation, we established here 108
an optogenetic protocol for chronic early stimulation (ES) of L2/3 pyramidal neurons at 109
the age when fast oscillations start to occur in the mPFC (Fig. 2a) 23. ES mice were 110
stimulated daily from P7-11 with blue light ramps (473 nm), whereas control animals 111
were similarly stimulated, but with yellow light ramps (594 nm) that do not activate 112
ChR2(ET/TC). Both intra- and transcranial ramp light stimulation acutely boosted 113
network oscillations in beta-low gamma frequency range in neonatal mice (Fig. 2b). 114
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Gamma control of cognitive maturation Bitzenhofer et al.
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First, we assessed the effects of ES on the morphology of L2/3 pyramidal 115
neurons. Immediately after stimulation, light-induced boosting of fast oscillatory activity 116
led to an exhaustive and premature growth of dendrites of L2/3 pyramidal neurons (Fig. 117
2c,d). At P11-12, stimulated neurons reached a dendritic length (3166 ± 632 µm) 118
significantly higher than of age-matched controls (2029 ± 244 µm, p=0.007) but 119
comparable to that of control animals at P23-25 (3356 ± 356 µm). These effects were 120
transient, such that the dendritic complexity and length of L2/3 pyramidal neurons was 121
largely unchanged in P23-25 (ES: 2906.5 ± 860.5 µm, p=0.631) and P38-40 (control: 122
4202 ± 625.5 µm; ES: 3746. 5± 630.5 µm, p=0.162) ES mice. Surprisingly, ES caused a 123
transient reduction of GABA-positive neurons (Fig. 2e). The survival of interneurons has 124
been shown to depend on the level of excitatory input during early postnatal 125
development 20,24. Therefore, the reduction of interneuron density might be explained by 126
the fact that optogenetic stimulation and subsequent augmented firing of transfected 127
neurons (~25% of total pyramidal neurons) reduces the overall level of activity, due to 128
surround suppression 7,15. 129
Next, we monitored the ES effects on functional network maturation. Extracellular 130
recordings from the mPFC of P11-12, P23-25 and P38-40 control and ES mice were 131
performed simultaneously with light stimulation of ChR2(ET/TC)-transfected L2/3 132
pyramidal neurons. Acutely stimulated activity was similar in control and ES mice at 133
P11-12, whereas at P23-25 the induced oscillatory peak slightly decreased, yet not at 134
significant level. At P38-40, the amplitude of driven oscillations in gamma band was 135
significantly reduced in both anesthetized (modulation index, P38-40: control: 0.72 ± 136
0.16; ES: 0.19 ± 0.24, p=0.025) (Fig. 3a-c) and awake (modulation index, P38-40: 137
control: 0.72 ± 0.06; ES: 0.50 ± 0.17, p=0.043) (Fig. S3f,g) ES mice when compared to 138
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Gamma control of cognitive maturation Bitzenhofer et al.
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controls. Bilateral recordings from all prefrontal layers in ES mice revealed that interlayer 139
as well as interhemispheric gamma frequency coherence was reduced at P38-40 when 140
compared to controls (Fig. 3d-i). In contrast, spontaneous activity of mPFC did not differ 141
between controls and ES mice across development (Fig. S3a-e), indicating that 142
perturbation of fast neonatal rhythms might solely disrupt the ability of local circuits to 143
respond to activation (i.e. under physiological conditions: stimulus, task). Of note, the 144
reduction of L2/3 driven gamma activity in ES mice occurred towards the end of juvenile 145
development, when dendritic morphology and interneuron number had largely recovered 146
(Fig. 2d,e). 147
Gamma activity is thought to affect information processing in adult mPFC and, 148
ultimately, behavior 8,10,11. Weaker gamma entrainment of prefrontal circuits after 149
interfering with fast oscillatory activity through ES at neonatal age might come along with 150
compromised cognitive abilities. We used a battery of tests to examine mPFC-151
dependent behavior of ES and control mice at P16-36. To avoid confounding effects, we 152
ascertained that ES mice have normal somatic and reflex development (Fig. S4a). Their 153
open field behavior was also normal (Fig. S4b). However, ES mice failed to distinguish 154
between objects in mPFC-dependent novel object and recency recognition tasks, but not 155
in a hippocampus-dependent object location recognition task (Fig. 4a,b and S4c). 156
Moreover, they showed abnormal social interactions as mirrored by reduced interaction 157
time with the mother (Fig. 4c). Spatial working memory was also impaired as revealed 158
by significant deficits of ES mice in 8-arm maze test (Fig. 4d). In contrast, spatial 159
alteration and tail suspension were not affected (Fig. S4d,e). Combined analysis of 160
behavioral performance throughout the different tests with support vector classification 161
and 5-fold cross-validation yielded a correct classification of control and ES mice in 77% 162
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Gamma control of cognitive maturation Bitzenhofer et al.
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in the training and 78% in the test data set (Fig. 4e). Thus, the cognitive deficits at 163
adulthood are reliable markers of functional imbalance within neonatal prefrontal 164
circuitry. 165
Gamma oscillations are highly relevant for adult cortical function 3,4,25 and 166
dysfunction 8,26, yet their development is still poorly understood. The results of the 167
present study (i) provide insights into the age-dependent mechanisms of early fast 168
oscillations and (ii) demonstrate their relevance for the functional maturation of prefrontal 169
networks as well as cognitive and social abilities. Throughout development, L2/3 170
pyramidal neurons have been identified as key players for the generation of fast 171
oscillations in the mPFC. Their activation led to activity patterns with similar features as 172
spontaneously-generated gamma oscillations. With age, the fast rhythms become more 173
prominent. The frequency and amplitude increase results from the maturation of 174
electrical properties of pyramidal neurons but also from the progressive embedding of 175
PV-expressing fast-spiking interneurons into circuits initially controlled by SST-176
expressing interneurons 27,28. 177
Do fast oscillations during development simply mirror neural maturation or do they 178
contribute to circuit refinement? This key question has been previously approached for 179
sensory systems with defined cortical topography. Neonatal fast oscillations have been 180
proposed to facilitate the formation of topographic units through input replay in 181
somatosensory thalamocortical circuits 29. In visual cortex, gamma activity depends on 182
visual experience and has been proposed as an indicator of cortical maturity 30. Being 183
not directly driven by corresponding environmental stimuli or sensory periphery, limbic 184
circuits follow distinct developmental rules. Emergence of oscillation-coupled prefrontal 185
ensembles at a developmental stage when the mice are blind, deaf and lack whisking 186
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Gamma control of cognitive maturation Bitzenhofer et al.
9
and motor abilities, is driven by hippocampal theta oscillations 23,31. They result, on their 187
turn, from activation of lateral entorhinal cortex under the control of olfactory stimuli 32,33. 188
Excitatory hippocampal projections targeting L5/6 neurons promote interlaminar 189
interactions within mPFC centered on coordinated activation of L2/3 pyramidal neurons. 190
Boosting this neonatal process through chronic light stimulation of L2/3 pyramidal 191
neurons seems to push the system out of an optimum level of activity and synchrony. 192
The direct consequences are transient structural changes (exuberant dendritic 193
arborization, decreased density of interneurons) that are compensated before adulthood. 194
However, the function of prefrontal circuits appears permanently compromised after ES. 195
The weaker gamma entrainment of adult prefrontal circuits contributes to poorer 196
performance in behavioral tasks that require mPFC, such as novel and recency 197
recognition, working memory as well as social interaction. These results uncover the role 198
of neonatal oscillations for the maturation of limbic circuit function and cognitive abilities. 199
The mechanisms described here might explain cognitive difficulties of preterm 200
born humans that experience excessive sensory stimulation in neonatal intensive care 201
unit (NICU) at a comparable stage of brain development (2nd-3rd gestational trimester) 202
34,35. These stressful stimuli might trigger premature gamma entrainment, perturbing the 203
activity-dependent maturation of cortical networks (Moiseev et al., 2015). Frontal regions 204
have been reported to be particularly vulnerable to NICU conditions 36 and 205
correspondingly, preterm children highly prone to frontally-confined impairment, such as 206
memory and attention deficits (Taylor and Clark, 2016). Thus, our findings lend support 207
to the concept that fast network oscillations have a central role for neurodevelopmental 208
disorders 37,38. 209
210
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Acknowledgments 300
We thank P. Putthoff, A. Dahlmann, and K. Titze for excellent technical assistance. This 301
work was funded by grants from the European Research Council (ERC-2015-CoG 302
681577 to I.L.H.-O.) and the German Research Foundation (Ha 4466/10-1, Ha4466/11-303
1, Ha4466/12-1, SPP 1665, SFB 936 B5 to I.L.H.-O.). 304
I.L. H.-O. is founding member of FENS Kavli Network of Excellence. 305
306
Author contributions: S.H.B. and I.L.H.-O. designed the experiments, S.H.B., J.A.P 307
and A.M. carried out the experiments, S.H.B., J.A.P. and M.C. analyzed the data, 308
S.H.B., I.L.H.-O., M.C and J.A.P interpreted the data and wrote the manuscript. All 309
authors discussed and commented on the manuscript. 310
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Gamma
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Gamma control of cognitive maturation Bitzenhofer et al.
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stimulations (473 nm, 3 s) of L2/3 pyramidal neurons in mPFC at different ages (left) 324
accompanied by the corresponding modulation index (MI) of power spectra normalized 325
to pre-stimulus (right). (g) Color-coded normalized (during-to-before stimulation) MI of 326
power spectra averaged over age for P5-40 mice (n=80). (h) Scatter plot displaying the 327
age-dependence of stimulus induced peak frequencies across development for 328
anesthetized (n=80) and awake mice (n=20, 35 recordings). Marker size displays peak 329
strength. (i-k) Same as F-H for control ramp light stimulations (594 nm, 3s). See 330
Supplementary Tab. 1 for detailed information on statistics. Average data is presented 331
as median ± MAD. 332
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333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
Gamma
Fig. 2.
mPFC.
behavio
(right)
togethe
Exampl
control
dendriti
the som
(conditi
group;
(e) Left
mice at
positive
a control of
Structura
(a) Schem
oral assess
ramp light
er with the
le pictures
(left) and
ic intersect
ma center
on p=2.2e
ES 18 cell
t, GABA an
t P11-12,
e and GAB
f cognitive
l alteration
matic timeli
sment. (b)
t stimulatio
e correspon
and avera
ES mice
tions avera
for contro
-16) and P
ls of 3 mic
nd CaMKII
P23-25 an
BA-positive
maturation
ns in mPF
ne of mani
Example o
ons of L2
nding MI o
age heat m
(right) at
aged for L2
ol and ES
P38-40 (co
ce / age gr
immunost
nd P38-40
e neuron d
n
FC as resu
ipulations a
of LFPs driv
2/3 pyramid
of power s
maps of IUE
P11-12,
2/3 pyramid
S mice at
ndition p=2
roup). Thin
tainings of
0. Right, vi
density in
ult of ES o
as well as
ven by intr
dal neuron
spectra (bo
E-transfecte
P23-25 an
dal neuron
P11-12 (c
2.4e-5) (co
lines corr
prefrontal
iolin plots
prefrontal
B
of L2/3 pyr
morpholog
racranial (le
ns in mPF
ottom) for
ed L2/3 py
nd P38-40
s within a
condition p
ontrol, 18 c
respond to
neurons f
of RFP-tra
L2/3 of co
Bitzenhofer
ramidal ne
gical, functi
eft) and tra
FC (top) d
a P11 m
yramidal ne
0. (d) Line
250 μm ra
p=2.2e-16)
cells of 3 m
individual
rom contro
ansfected,
ontrol and
r et al.
15
eurons in
onal, and
anscranial
displayed
ouse. (c)
eurons for
e plots of
dius from
), P23-25
mice / age
neurons.
ol and ES
CaMKII-
ES mice
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Gamma control of cognitive maturation Bitzenhofer et al.
16
(right). *P<0.05, **P<0.01 and ***P<0.001. See Supplementary Tab. 1 for detailed 348
information on statistics. Average data is presented as median ± MAD. 349
.CC-BY-NC 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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350
351
352
353
354
355
356
357
358
359
360
361
362
363
Gamma
Fig. 3.
(a) Top
ES mic
corresp
right, sa
mice (c
(d) Sca
of imag
control
Scatter
imagina
control
Same a
a control of
Prefronta
left, MI of
ce at P1
ponding MI
ame as top
control n=1
atter plots d
ginary cohe
and ES m
plots disp
ary cohere
and ES m
as (d,e) for
f cognitive
l dysfunct
power spe
1-12 (con
power pe
p for contro
0, ES n=1
displaying
erence bet
mice for s
playing the
ence betwe
mice for st
r P23-25 m
maturation
tion as res
ectra of ligh
trol n=10,
ak strength
ol stimulatio
1). (c) Sam
the peak s
tween ligh
stimulation
peak stren
een light-dr
timulation
mice. (h,i) S
n
sult of ES
ht-driven ac
, ES n=1
h as a func
on (ramp, 5
me as (a) f
strength as
t-driven L2
(473 nm)
ngth as a f
riven L2/3
(473 nm)
Same as (d
S of L2/3 p
ctivity (ram
0). Top r
ction of pe
594 nm, 3s
for P38-40
s a function
2/3 and ips
and cont
function of
and contr
and contro
d,e) for P3
B
pyramidal
mp, 473 nm
right, scat
ak frequen
s). (b) Sam
mice (con
n of peak fr
silateral L5
trol stimula
f peak freq
ralateral L2
ol stimulat
38-40 mice
Bitzenhofer
neurons i
m, 3s) for co
tter plot d
ncy. Bottom
me as (a) fo
ntrol n=9, E
requency f
5/6 in the
ation (594
quency for
2/3 in the
tion (594 n
. *P<0.05,
r et al.
17
n mPFC.
ontrol and
displaying
m left and
or P23-25
ES n=12).
for the MI
mPFC of
nm). (e)
the MI of
mPFC of
nm). (f,g)
**P<0.01
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Gamma control of cognitive maturation Bitzenhofer et al.
18
and ***P<0.001. See Supplementary Tab. 1 for detailed information on statistics. 364
Average data is presented as median ± MAD. 365
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The copyright holder for this preprint (whichthis version posted February 22, 2019. ; https://doi.org/10.1101/558957doi: bioRxiv preprint
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
Gamma
Fig. 4.
pyrami
Bottom
control
Bottom
control
Bottom
(n=19)
memory
in an 8
matrix (
machin
combin
conditio
informa
a control of
Poorer co
idal neuro
, discrimin
(n=28) an
, discrimin
(n=28) an
, discrimina
and ES m
y (conditio
8-arm radia
(top) and d
e classifica
ed behav
on. *P<0.0
ation on sta
f cognitive
ognitive a
ons in mP
nation ratio
nd ES mic
ation ratio
nd ES mic
ation ratio
mice (n=2
n p=0.001)
al maze w
decision sp
ation used
ioral perfo
5, **P<0.0
atistics. Ave
maturation
nd social
PFC. (a) T
o of interac
ce (n=30).
of interact
ce (n=30).
of interacti
1). (d) Sp
) performa
ith 4 baite
pace (botto
d to predic
ormance.
01 and ***P
erage data
n
performa
Top, schem
ction time
(b) Top,
tion time w
(c) Top, s
ion time wi
patial work
nce for P2
ed arms ov
om) as well
t if an anim
Fill color
P<0.001. S
a is present
ance of mi
matic of n
with a no
schematic
with a less-
schematic
ith a mothe
king (condi
3-36 contr
ver 14 con
l as single
mal belong
of single
See Suppl
ted as med
B
ice experi
novel objec
ovel-to-fam
of recenc
-to-more re
of matern
er-to-empty
ition p=0.0
rol (n=10) a
nsecutive d
data point
gs to contr
data poin
ementary
dian ± MAD
Bitzenhofer
encing ES
ct recognit
miliar object
cy recognit
ecent objec
nal interact
y bin for P2
007) and r
and ES mic
days. (e) C
ts for suppo
rol or ES b
nts represe
Tab. 1 for
D.
r et al.
19
S of L2/3
tion task.
t for P17
tion task.
ct for P22
tion task.
21 control
reference
ce (n=12)
Confusion
ort vector
based on
ents true
r detailed
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Gamma control of cognitive maturation Bitzenhofer et al.
20
List of supplementary materials 383
384
Methods 385
386
Fig. S1 (related to Fig. 1): Spontaneous and L2/3-driven activity in awake and 387
anesthetized mice across development 388
389
Fig. S2 (related to Fig. 1): Neurochemical profile and firing patterns in PFC across 390
development. 391
392
Fig. S3 (related to Fig. 3): ES effects on spontaneous and L2/3 pyramidal neuron-393
driven activity in anesthetized and awake head-fixed mice across development 394
395
Fig. S4 (related to Fig. 4): Developmental milestones and behavioral performance after 396
ES of L2/3 pyramidal neurons in the mPFC 397
398
Supplementary Tab. 1: Statistics summary 399
400
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Gamma control of cognitive maturation Bitzenhofer et al.
1
Supplementary materials for: 1
2
3
Activity-dependent maturation of prefrontal gamma 4
oscillations sculpts cognitive performance 5
Sebastian H. Bitzenhofer1,*, Jastyn A. Pöpplau1, Mattia Chini1, Annette Marquardt1, 6
Ileana L. Hanganu-Opatz1,* 7
1 Developmental Neurophysiology, Institute of Neuroanatomy, University Medical Center 8
Hamburg-Eppendorf, Hamburg, Germany 9
10
11
* Corresponding authors: Ileana L. Hanganu-Opatz 12
hangop@zmnh.uni-hamburg.de 13
Falkenried 94, 20251 Hamburg, Germany 14
15
Sebastian H. Bitzenhofer 16
sebastian.bitzenhofer@zmnh.uni-hamburg.de 17
Falkenried 94, 20251 Hamburg, Germany 18
19
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Gamma control of cognitive maturation Bitzenhofer et al.
2
List of supplementary materials 20
21
Methods 22
23
Supplementary Fig.1 (related to Fig. 1): Spontaneous and L2/3-driven activity in 24
awake and anesthetized mice across development 25
26
Supplementary Fig.2 (related to Fig. 1): Neurochemical identity and firing patterns in 27
PFC across development. 28
29
Supplementary Fig.3 (related to Fig. 3): ES effects on spontaneous and L2/3 30
pyramidal neuron-driven activity in anesthetized and awake head-fixed mice across 31
development 32
33
Supplementary Fig.4 (related to Fig. 4): Developmental milestones and behavioral 34
performance after ES of L2/3 pyramidal neurons in the mPFC 35
36
Supplementary Tab. 1: Statistics summary 37
38
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Gamma control of cognitive maturation Bitzenhofer et al.
3
Methods 39
Animals 40
All experiments were performed in compliance with the German laws and the guidelines 41
of the European Community for the use of animals in research and were approved by 42
the local ethical committee (G132/12, G17/015, N18/015). Experiments were carried out 43
on C57Bl/6J mice of both sexes. To achieve interneuron-specific expression of 44
archaerhodopsin-3 (ArchT), Gad2 driver line (Gad2-IRES-Cre knock-in, The Jackson 45
Laboratory, ME, USA) was crossed with Ai40 reporter line (Ai40(RCL-ArchT/EGFP)-D, 46
The Jackson Laboratory, ME, USA). Timed-pregnant mice from the animal facility of the 47
University Medical Center Hamburg-Eppendorf were housed individually at a 12 h 48
light/12 h dark cycle and were given access to water and food ad libitum. The day of 49
vaginal plug detection was considered E0.5, the day of birth was considered P0. 50
In utero electroporation 51
Pregnant mice received additional wet food on a daily basis, supplemented with 2-4 52
drops Metacam (0.5 mg/ml, Boehringer-Ingelheim, Germany) one day before until two 53
days after in utero electroporation. At E15.5, pregnant mice were injected 54
subcutaneously with buprenorphine (0.05 mg/kg body weight) 30 min before surgery. 55
Surgery was performed under isoflurane anesthesia (induction 5%, maintenance 3.5%) 56
on a heating blanket. Eyes were covered with eye ointment and pain reflexes and 57
breathing were monitored to assess anesthesia depth. Uterine horns were exposed and 58
moistened with warm sterile PBS. 0.75-1.25 µl of opsin- and fluorophore-encoding 59
plasmid (pAAV-CAG-ChR2(E123T/T159C)-2A-tDimer2, 1.25 µg/µl) purified with 60
NucleoBond (Macherey-Nagel, Germany) in sterile PBS with 0.1% fast green dye was 61
injected in the right lateral ventricle of each embryo using pulled borosilicate glass 62
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Gamma control of cognitive maturation Bitzenhofer et al.
4
capillaries. Electroporation tweezer paddles of 5 mm diameter were oriented at a rough 63
20° leftward angle from the midline of the head and a rough 10° downward angle from 64
the anterior to posterior axis to transfect precursor cells of medial prefrontal L2/3 65
pyramidal neurons with 5 electroporation pulses (35 V, 50 ms, 950 ms interval, CU21EX, 66
BEX, Japan). Uterine horns were placed back into the abdominal cavity. Abdominal 67
cavity was filled with warm sterile PBS and abdominal muscles and skin were sutured 68
with absorbable and non-absorbable suture thread, respectively. After recovery from 69
anesthesia, mice were returned to their home cage, placed half on a heating blanket for 70
two days after surgery. Fluorophore expression was assessed at P2 in the pups with a 71
portable fluorescence flashlight (Nightsea, MA, USA) through the intact skin and skull 72
and confirmed in brain slices post mortem. 73
Early stimulation (ES) 74
A stimulation window was implanted at P7 for chronic transcranial optogenetic 75
stimulation in mice transfected by in utero electroporation. Mice were placed on a 76
heating blanket and anesthetized with isoflurane (5% induction, 2% maintenance). 77
Breathing and pain reflexes were monitored to assess anesthesia depth. The skin above 78
the skull was cut along the midline (3 mm) at the level of the mPFC and gently spread 79
with a forceps, before covering the incision with transparent tissue adhesive (Surgibond, 80
SMI, Belgium). Mice were returned to the dam in the home cage after recovery from 81
anesthesia. From P7-11 mice were stimulated daily under isoflurane anesthesia (5% 82
induction, 2% maintenance) with ramp stimulations of linearly increasing light power 83
(473 nm wavelength, 3 s duration, 7 s interval, 180 repetitions, 30 min total duration). 84
Light stimulation was performed using an Arduino uno (Arduino, Italy) controlled laser 85
system (Omicron, Austria) coupled to a 200 µm diameter light fiber (Thorlabs, NJ, USA) 86
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Gamma control of cognitive maturation Bitzenhofer et al.
5
positioned directly above the tissue adhesive window. Light power attenuation was set to 87
reach 10 mW in the brain, adjusted for measured light attenuation by the tissue adhesive 88
(~30%) and by the immature skull (~25%). Control animals were treated identical, but 89
stimulated with light of 594 nm wavelength that does not activate the expressed opsin 90
ChR2(ET/TC). 91
Optogenetics and electrophysiology in vivo 92
Acute recordings. Multi-site extracellular recordings of local field potential (LFP) and 93
multi-unit activity (MUA) were performed unilaterally or bilaterally in the mPFC of non-94
anesthetized or anesthetized P5-40 mice. Pups were on a heating blanket during the 95
entire procedure. Under isoflurane anesthesia (induction: 5%; maintenance: 2.5%), a 96
craniotomy was performed above the mPFC (0.5 mm anterior to bregma, 0.1-0.5 mm 97
lateral to the midline). Neck muscles were cut and 0.5% bupivacaine / 1% lidocaine was 98
locally applied to cutting edges. Pups were head-fixed into a stereotaxic apparatus using 99
two plastic bars mounted on the nasal and occipital bones with dental cement. Multi-site 100
electrodes (NeuroNexus, MI, USA) were inserted into the mPFC (four-shank, A4x4 101
recording sites, 100 µm spacing, 125 µm shank distance, 1.8-2.0 mm deep). A silver 102
wire was inserted into the cerebellum and served as ground and reference. Pups were 103
allowed to recover for 30 min prior to recordings. For anesthetized recordings, urethane 104
(1 mg/g body weight) was injected intraperitoneally prior to the surgery. Extracellular 105
signals were band-pass filtered (0.1-9,000 Hz) and digitized (32 kHz) with a multichannel 106
extracellular amplifier (Digital Lynx SX; Neuralynx, Bozeman, MO, USA). Electrode 107
position was confirmed in brain slices post mortem. 108
Chronic recordings. Multisite extracellular recordings were performed in the mPFC of 109
P23-25 and P38-40 mice. The adapter for head fixation was implanted at least 5 days 110
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Gamma control of cognitive maturation Bitzenhofer et al.
6
before recordings. Under isoflurane anesthesia (5% induction, 2.5% maintenance), a 111
metal head-post (Luigs and Neumann, Germany) was attached to the skull with dental 112
cement and a craniotomy was performed above the mPFC (0.5-2.0 mm anterior to 113
bregma, 0.1-0.5 mm right to the midline) and protected by a customized synthetic 114
window. A silver wire was implanted between skull and brain tissue above the 115
cerebellum and served as ground and reference. 0.5% bupivacaine / 1% lidocaine was 116
locally applied to cutting edges. After recovery from anesthesia, mice were returned to 117
their home cage. After recovery from the surgery, mice were accustomed to head-118
fixation and trained to run on a custom-made spinning disc. For recordings, craniotomies 119
were uncovered and multi-site electrodes (NeuroNexus, MI, USA) were inserted into the 120
mPFC (one-shank, A1x16 recording sites, 100 µm spacing, 2.0 mm deep). Extracellular 121
signals were band-pass filtered (0.1-9000 Hz) and digitized (32 kHz) with a multichannel 122
extracellular amplifier (Digital Lynx SX; Neuralynx, Bozeman, MO, USA). Electrode 123
position was confirmed in brain slices post mortem. 124
Optogenetic stimulation. Pulsed (light on-off) and ramp (linearly increasing light power) 125
light stimulation was performed using an Arduino uno (Arduino, Italy) controlled laser 126
system (473 nm / 594 nm wavelength, Omicron, Austria) coupled to a 50 µm (4 shank 127
electrodes) or 105 µm (1 shank electrodes) diameter light fiber (Thorlabs, NJ, USA) 128
glued to the multisite electrodes, ending 200 µm above the top recording site. 129
Histology 130
P5-40 mice were anesthetized with 10% ketamine (aniMedica, Germanry) / 2% xylazine 131
(WDT, Germany) in 0.9% NaCl (10 µg/g body weight, intraperitoneal) and transcardially 132
perfused with 4% paraformaldehyde (Histofix, Carl Roth, Germany). Brains were 133
removed and postfixed in 4% paraformaldehyde for 24 h. Brains were sectioned 134
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Gamma control of cognitive maturation Bitzenhofer et al.
7
coronally with a vibratom at 50 µm for immunohistochemistry or 100 µm for examination 135
of dendritic complexity. 136
Immunohistochemistry. Free-floating slices were permeabilized and blocked with PBS 137
containing 0.8% Triton X-100 (Sigma-Aldrich, MO, USA), 5% normal bovine serum 138
(Jackson Immuno Research, PA, USA) and 0.05% sodium azide. Slices were incubated 139
over night with primary antibody rabbit-anti-GABA (1:1000, #A2052, Sigma-Aldrich, 140
MMO, USA), rabbit-anti-Ca2+/calmodulin-dependent protein kinase II (1:200, #PA5-141
38239, Thermo Fisher, MA, USA; 1:500, #ab52476, Abcam, UK), rabbit-anti-142
parvalbumin (1:500, #ab11427, Abcam, UK) or rabbit-anti-somatostatin (1:250, 143
#sc13099, Santa Cruz, CA, USA), followed by 2 h incubation with secondary antibody 144
goat-anti-rabbit Alexa Fluor 488 (1:500, #A11008, Invitrogen-Thermo Fisher, MA, USA) 145
or goat-anti-rat Alexa Fluor 488 (1:750, #A11006, Invitrogen-Thermo Fisher, MA, USA). 146
Sections were transferred to glass slides and covered with Fluoromount (Sigma-Aldrich, 147
MO, USA). 148
Cell quantification. Images of immunofluorescence in the right mPFC as well as IUE-149
induced tDimer2 expression were acquired with a confocal microscope (DM IRBE, 150
Leica, Germany) using a 10x objective (numerical aperture 0.3). tDimer2-positive and 151
immunopositive cells were automatically quantified with custom-written algorithms in 152
ImageJ environment. The region of interest (ROI) was manually defined over L2/3 of the 153
mPFC. Image contrast was enhanced before applying a median filter. Local background 154
was subtracted to reduce background noise and images were binarized and segmented 155
using the watershed function. Counting was done after detecting the neurons with the 156
extended maxima function of the MorphoLibJ plugin. 157
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Dendritic complexity. Image stacks of tDimer2-positive neurons were acquired with a 158
confocal microscope (LSN700, Zeiss, Germany) using a 40x objective. Stacks of 6 159
neurons per animal were acquired as 2048x2048 pixel images (voxel size 156*156*500 160
nm). Dendritic complexity was quantified by Sholl analysis in ImageJ environment. 161
Images were binarized using auto threshold function and the dendrites were traced 162
using the semi-automatic simple neurite tracer plugin. The geometric center was 163
identified and the traced dendritic tree was analyzed with the Sholl analysis plugin. 164
Behavior 165
Mice were handled and adapted to the room of investigation daily starting two days prior 166
to behavioral examination. Arenas and objects were cleaned with 0.1% acetic acid 167
before each trial. Animals were tracked automatically (Video Mot2, TSE Systems GmbH, 168
Germany). 169
Developmental milestones. Somatic and reflex development was examined from P2-20 170
at a 3-day interval. Weight, body length and tail length were measured. Grasping reflex 171
was assessed by touching front paws with a toothpick. Vibrissa placing was measured 172
as head movement in response to gently touching the vibrissa with a toothpick. Auditory 173
startle was assessed in response to finger snapping. The days of pinnae detachment 174
and eye opening were monitored. Surface righting was measured as time to turn around 175
after being positioned on the back (max 30 s). Cliff avoidance was measured as time 176
until withdrawing after being positioned with forepaws and snout over an elevated edge 177
(max 30 s). Bar holding was measured as time hanging on an toothpick grasped with the 178
forepaws (max 10 s). 179
Open field. Each mouse was positioned in the center of a circular arena individually (34 180
cm in diameter) at P16 for 10 min. Behavior was quantified by measuring: discrimination 181
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Gamma control of cognitive maturation Bitzenhofer et al.
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index of time spent in the center and the border of the arena ((time in surround - time in 182
center) / (time in surround + time in center)), grooming time, average velocity and 183
number of rearing, wall rearing and jumping. 184
Object recognition. Novel object recognition (NOR, P17), object location recognition 185
(OLR, P18) and recency recognition (RR, P21) were performed in the same arena as 186
the open field examination. Mouse center, tail and snout position were tracked 187
automatically. Object interaction was defined as the snout being within <1 cm distance 188
from an object. For NOR, each mouse explored two identical objects for 10 min during 189
the sample phase. After a delay period of 5 min in a break box, the mouse was placed 190
back in the arena for the test phase where one of the objects was replaced by a novel 191
object. Behavior was quantified as discrimination index of time spent interacting with the 192
novel and familiar object ((time novel object - time familiar object) / (time novel object + 193
time familiar object)). OLR was performed similarly, but one object was relocated for the 194
test phase instead of being exchanged. For RR, each mouse explored two identical 195
objects during the first sample phase for 10 min, followed by a delay phase of 30 min, 196
and a second sample phase of 10 min with two novel identical objects. After a second 197
break of 5 min, time interacting with an object of the first sample phase (old) and an 198
object from the second sample phase (recent) was assessed during the test phase for 2 199
min. Behavior was quantified as discrimination index of time spent interacting with the 200
novel and familiar object ((time old object – time recent object) / (time old object + time 201
recent object)). 202
Maternal interaction. Maternal interaction was performed at P21 in the same arena as 203
the open field examination with two plastic containers, one empty and one containing the 204
dam of the mouse pup examined. Small holes in the containers allowed the pup and the 205
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Gamma control of cognitive maturation Bitzenhofer et al.
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mother to interact. Behavior was quantified as discrimination index of time spent 206
interacting with the empty container and the container containing the mother ((time 207
mother container – time empty container) / (time mother container + time empty 208
container)). 209
Spontaneous alteration. At P15, 18 and 21, each mouse was positioned on the central 210
arm of an elevated t-maze. After 1 min, the mouse had access to one of the other arms. 211
The mouse was placed back in the start arm for 1 min, before a second run. Behavior 212
was quantified as alteration or no alteration between the two arms in the first and second 213
run. 214
Tail suspension. Mice were fixed with their tail on a bar 30 cm above ground for 5 min at 215
P21. Behavior was quantified as time spent inactive, passively hanging. 216
Spatial working memory. Mice were positioned in the center of an elevated 8-arm radial 217
maze daily from P23-36. Arms were without walls and 4 arms contained a food pellet at 218
the distal end (baited). On the first day, mice were allowed to examine the maze for 20 219
min or until all arms were visited. During the following days, mice were allowed to 220
examine the maze until all baited arms were visited, but for max 20 min and arm entries 221
were assessed. Visit of a non-baited arm was considered as reference memory error, 222
repeated visit of the same arm in one trial as working memory error. 223
Data analysis 224
In vivo data were analyzed with custom-written algorithms in Matlab environment. Data 225
were band-pass filtered (500-9000 Hz for spike analysis or 1-100 Hz for LFP) using a 226
third-order Butterworth filter forward and backward to preserve phase information before 227
down-sampling to analyze LFP. 228
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Gamma control of cognitive maturation Bitzenhofer et al.
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Power spectral density. For power spectral density analysis, 2 s-long windows of LFP 229
signal were concatenated and the power was calculated using Welch’s method with non-230
overlapping windows. Spectra were multiplied with squared frequency. 231
Imaginary coherence. The imaginary part of complex coherence, which is insensitive to 232
volume conduction, was calculated by taking the absolute value of the imaginary 233
component of the normalized cross-spectrum. 234
Modulation index. For optogenetic stimulations, modulation index was calculated as 235
(value stimulation - value pre stimulation) / (value stimulation + value pre stimulation). 236
Peak frequency and strength. Peak frequency and peak strength were calculated for the 237
most prominent peak in the spectrum defined by the product of peak amplitude, peak 238
half width and peak prominence. 239
Single unit analysis. Spikes were detected and sorted with Ultra Mega Sort 2000 240
software in Matlab. 241
Support vector classification. Model training and performance evaluation were carried 242
out using the scikit-learn toolbox in Python. The set was iteratively (n=500) divided using 243
5-fold cross-validation in a training (4/5) and a test (1/5) set. The value of the model 244
regularization parameter “C” was tuned on the training set, which was further split using 245
3-fold cross-validation. Model prediction was assessed on the test set. Performance was 246
stable across a wide range of regularization parameter values. To plot the classifier 247
decision space, we used t-sne to reduce the feature space to two dimensions, while 248
preserving the hyper-dimensional structure of the data. The decision space was then 249
approximated by imposing a Voronoi tessellation on the 2d plot, using k-nearest 250
regression on the t-sne coordinates of the predicted classes of the mice. 251
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Gamma control of cognitive maturation Bitzenhofer et al.
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Statistics. Statistical analyses were performed in the Matlab environment or in R 252
Statistical Software (Foundation for Statistical Computing, Austria). Data are presented 253
as median ± median absolute deviation (MAD). Data were tested for significant 254
differences (*P<0.05, **P<0.01 and ***P<0.001) using non-parametric Wilcoxon rank 255
sum test for unpaired and Wilcoxon signed rank test for paired data or Kruskal-Wallis 256
test with Bonferroni corrected post hoc analysis. Nested data were analyzed with linear 257
mixed-effect models considering within animal variance with Turkey multi comparison 258
correction for post hoc analysis. More information about statistic results are provided in 259
Supplementary Tab. 1. 260
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Supplementary figures 261
262
Supplementary Fig.1 (related to Fig. 1). Spontaneous and L2/3-driven activity in 263
awake and anesthetized mice across development. (a) Setup for recordings from 264
awake head-fixed mice on a spinning disc with one shank 1x16 electrode in L2/3. (b) 265
Example LFPs from mPFC of non-anesthetized mice at different ages. (c) Setup for 266
recordings from anesthetized head-fixed mice with four shank 4x4 electrode. (d) 267
Example LFPs from mPFC of anesthetized mice at different ages. (e) Left, average 268
power spectra of prefrontal activity for awake (n=6, 6 recordings) and anesthetized 269
(n=10) P11-12 mice. Right, scatter plot displaying corresponding peak strength as a 270
function of peak frequency. (f) Same as (e) for P23-25 mice (awake n=5, 13 recordings; 271
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anesthetized n=10). (g) Same as (e) for P38-40 mice (awake n=5, 12 recordings; 272
anesthetized n=9). (h) Example prefrontal LFPs driven by ramp light stimulation of L2/3 273
pyramidal neurons in non-anesthetized mice at different ages. (i) Example prefrontal 274
LFPs driven by ramp light stimulation of L2/3 pyramidal neurons in anesthetized mice at 275
different ages. (j) Scatter plots displaying peak strength as a function of peak frequency 276
for the MI of power spectra during-to-before stimulation (ramp, 473 nm or 594 nm) in the 277
mPFC of awake (n=6, 6 recordings) and anesthetized (n=10) P11-12 mice. (k) Same as 278
(j) for P23-25 mice (awake n=5, 13 recordings; anesthetized n=10). (l) Same as (j) for 279
P38-40 mice (awake n=5, 12 recordings; anesthetized n=9). *P<0.05, **P<0.01 and 280
***P<0.001. See Supplementary Tab. 1 for detailed information on statistics. Average 281
data is presented as median ± MAD. 282
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283
Supplementary Fig.2 (related to Fig. 1). Neurochemical identity and firing patterns 284
in PFC across development. (a) Left, examples of SST immunostaining of prefrontal 285
neurons from P6, P15, P26, and P34 mice. Right, scatter plot displaying the density of 286
SST-immunopositive neurons in the mPFC of P5-40 mice (n=39). (b) Same for PV-287
immunopositive neurons (n=38). (c) Scatter plot displaying prefrontal SUA firing rate for 288
P5-40 mice (806 units of 71 mice). (d) Same for SUA amplitude. (e) Same for unit half 289
width. (f) Example of EGFP-positive (arrow) and RFP-positive cells showing no overlap 290
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in expression. (g) Top, MI of power spectra during-to-before stimulation (ramp, 473 nm) 291
of L2/3 pyramidal neurons (ramp, 473 nm), inhibition of interneurons (square pulse, 594 292
nm) or a combination of both in the mPFC of P11-12 mice (n=5). Bottom, scatter plot 293
displaying corresponding peak strength as a function of peak frequency. (h) Same as (g) 294
for P23-25 mice (n=6). (i) Same as (g) for P38-40 mice (n=6). *P<0.05, **P<0.01 and 295
***P<0.001. See Supplementary Tab. 1 for detailed information on statistics. Average 296
data is presented as median ± MAD. 297
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298
Supplementary Fig.3 (related to Fig. 3). ES effects on spontaneous and L2/3 299
pyramidal neuron-driven activity in anesthetized and awake head-fixed mice 300
across development. (a) Left, average power spectra of spontaneous mPFC activity for 301
anesthetized head-fixed P11-12 control (n=10) and ES mice (n=10). Right, scatter plot 302
displaying corresponding peak strength as a function of peak frequency. (b) Same as (a) 303
for P23-25 mice (control n=10, ES n=11). (c) Same as (a) for P38-40 mice (control n=9, 304
ES n=12). (d) Same as (b) for awake head-fixed control (n=6, 13 recordings) and ES 305
mice (n=5, 14 recordings) on a spinning disc. (e) Same as (c) for awake head-fixed 306
control (n=5, 12 recordings) and ES mice (n=5, 12 recordings) on a spinning disc. (f) 307
Top left, MI of power spectra during-to-before stimulation (ramp, 473 nm) of L2/3 308
pyramidal neurons in the mPFC of P23-25 awake control (n=6, 13 recordings) and ES 309
mice (n=5, 14 recordings). Top right, scatter plot displaying corresponding peak strength 310
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Gamma control of cognitive maturation Bitzenhofer et al.
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as a function of peak frequency. Bottom, same as top for control stimulation (ramp, 594 311
nm) (g) Same as (f) for P38-40 mice (control n=5, 12 recordings; ES n=5, 12 312
recordings). *P<0.05, **P<0.01 and ***P<0.001. See Supplementary Tab. 1 for detailed 313
information on statistics. Average data is presented as median ± MAD. 314
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315
Supplementary Fig.4 (related to Fig. 4). Developmental milestones and behavioral 316
performance after ES of L2/3 pyramidal neurons in the mPFC. (a) Age dependence 317
of developmental milestones for control (n=11) and ES mice (n=11). (b) Left, schematic 318
of open field task (top) and discrimination ratio of time spent in border-to-center in an 319
open field (bottom). Right, behavioral quantification during exploration averaged for P16 320
control (n=28) and ES mice (n=30). (c) Top, schematic of object location recognition 321
task. Bottom, discrimination ratio of interaction time with an object in a novel-to-familiar 322
location averaged for P18 control (n=28) and ES mice (n=30). (d) Spontaneous 323
alteration in t-maze test for control (n=19) and ES mice (n=21) at P15, 18 and 21. (e) 324
Tail suspension test for P21 control (n=19) and ES mice (n=21). *P<0.05, **P<0.01 and 325
***P<0.001. See Supplementary Tab. 1 for detailed information on statistics. Average 326
data is presented as median ± MAD. 327
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