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NEUROSCIENCE
Chemogenetic stimulation of tonic locus coeruleus
activity strengthens the default mode network
Esteban A. Oyarzabal1,2,3,4†, Li-Ming Hsu1,2,3†, Manasmita Das1,2,3†,
Tzu-Hao Harry Chao1,2,3, Jingheng Zhou5, Sheng Song1,2,3, Weiting Zhang1,2,3,
Kathleen G. Smith6, Natale R. Sciolino6, Irina Y. Evsyukova6, Hong Yuan2, Sung-Ho Lee1,2,3,
Guohong Cui5, Patricia Jensen6, Yen-Yu Ian Shih1,2,3*
The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this
study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral
fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)–releasing noradrenergic
neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cere-
bral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the
frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation
induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate
cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the
frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional con-
nectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retro-
splenial and hippocampal regions to the association cortices of the DMN.
INTRODUCTION
Functional magnetic resonance imaging (fMRI) has been widely
used to demonstrate the presence of spatiotemporally consistent in-
trinsic functional brain networks during resting state. The default
mode network (DMN), composed of the prefrontal, orbitofrontal,
prelimbic, cingulate, retrosplenial, posterior parietal, and temporal
association cortices as well as the dorsal hippocampus, is among the
most robust intrinsic networks because of its highly synchronized
activity in the absence of cognitive tasks or saliency (1). The DMN
is vulnerable in several neurological and neuropsychiatric disorders
(2), is functionally associated with a wide range of behaviors (3),
integrates interoceptive and exteroceptive information from multiple
brain networks (4), and maintains the brain in a semivigilant state
(5). To make causal interpretations of behaviorally relevant DMN
changes and design network-based interventions for disorders that
afflict DMN activity, identifying the modulatory mechanisms con-
trolling the DMN is of paramount importance.
The locus coeruleus (LC), a small nucleus within the pons, is a
potential DMN modulator (6,7). A large portion of the neuro-
modulator norepinephrine (NE) originates from the LC and is re-
leased in the brain regions that are considered DMN nodes (8,9).
Accumulating evidence suggests that LC-NE may be essential for
DMN modulation because (i) NE receptors are prominently expresse d
in DMN-related brain structures (9); (ii) LC-NE can bidirectionally
modulate attention reorientation in a dose-dependent manner (8);
(iii) LC-NE neuron degeneration and DMN disruption are coincidently
found in depression (10), traumatic brain injury (11), Parkinson’s
disease (12), Alzheimer’s disease (13), and aging (14); and (iv)
pharmacological treatment of pathological LC-NE levels reduces
attentional lapses (15) and restores DMN integrity in attention defi-
cit hyperactivity disorder (ADHD) patients (16). Despite these findings ,
the modulatory association between LC-NE and the DMN remains
circumstantial because pharmacological interventions using NE-
related agents inherently result in nonselective binding on dopaminergic,
cholinergic, and serotonergic receptors (7,17,18). Furthermore,
systemic administration of these agents indiscriminately targets all
NE-producing neurons in the brain and sympathetic nervous system,
making it difficult to determine the role of LC-NE in modulating the
DMN. Although selective manipulation of LC-NE while imaging
the DMN is currently impossible in humans due to technical and
ethical constraints (7,19), such studies are feasible in rodent models
because structural and functional homologs of the human DMN have
been identified in mice (20–28).
In this study, we used an established data-driven approach to
identify DMN modules (29) and an intersectional chemogenetic
strategy to selectively and reproducibly induce tonic LC-NE activity
in mice (30,31). To reveal potential confounders that could affect
our interpretations of LC-NE influence on the DMN, we measured
changes in several fMRI metrics, neuronal calcium activity, and glu-
cose uptake across different spatial and temporal scales. Through
modeling the signal dynamics, we revealed the circuit mechanism
by which LC-NE activation modulates the DMN. Our findings should
pave the way toward a better understanding of how large-scale
brain networks are mediated by a specific neuromodulatory system.
RESULTS
To selectively and reproducibly activate LC-NE neurons, we used
an intersectional chemogenetic approach in which the excitatory G
protein (heterotrimeric GTP-binding protein)–coupled receptor
hM3Dq, fused to mCherry, is expressed in 99.6% of the anatomically
1Center for Animal MRI, University of North Carolina, Chapel Hill, NC, USA. 2Biomedical
Research Imaging Center, University of North Carolina, Chapel Hill, NC, USA.
3Department of Neurology, University of North Carolina, Chapel Hill, NC, USA.
4Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC, USA. 5In
Vivo Neurobiology Group, Neurobiology Laboratory, NIEHS/NIH, Research Triangle
Park, NC, USA. 6Developmental Neurobiology Group, Neurobiology Laboratory,
NIEHS/NIH, Research Triangle Park, NC, USA.
*Corresponding author. Email: shihy@unc.edu
†These authors contributed equally to this work.
Copyright © 2022
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defined LC located within the central gray and a small portion of the
dorsal subcoeruleus immediately adjacent to and continuous with the
LC (LC-NE/hM3Dq) (Fig.1) (31). This population of NE neurons
robustly innervates canonical DMN regions including cingulate 1
(Cg1) and retrosplenial (RSC) cortices (fig. S1) (32).
To functionally delineate the DMN and study selective NE mod-
ulatory effects, we performed invivo cerebral blood volume (CBV)–
weighted fMRI scans on LC-NE/hM3Dq mice and littermate controls
(n=9 and 12, respectively) under light isoflurane (~1%) anesthesia
using a previously described isotropic echo planar imaging (EPI)
protocol (33). Although medetomidine with low-dose isoflurane is
considered the preferred sedative for rodent fMRI (34), we avoided
its usage because it suppresses NE release (35). We collected a
10-min resting-state baseline scan before administering clozapine-
n- oxide (CNO; 1 mg/kg, intraperitoneally) (Fig.2A). CNO was
selected because it was previously used for behavior studies of the
same mouse line (30,31). This protocol has been shown to activate
LC-NE neurons at tonic frequency and to suppress locomotion in
LC-NE/hM3Dq mice (30,31). Subsequent comparisons were made
against littermate controls to account for off-target effects of CNO
and/or the back-metabolized clozapine (36). We spatially warped each
imaging dataset into the Allen Mouse Common Coordinate Framework
(fig. S2), functionally parcellated the baseline fMRI data from all
subjects (n = 21) by performing a 100-component independent
component analysis (ICA) (Fig.2, Band C), and verified their re-
producibility (fig. S3). We identified 17 DMN-related independent
components (ICs) (fig. S4A) according to previous rodent DMN
studies (22,24,25,28,29,37). The areas showing significant tempo-
ral correlation associated with the 17 identified DMN ICs were re-
constructed using dual regression (DR), and a one-sample two-sided
t test was performed to generate the group-level maps representing
the connectivity of these ICs. These maps showed high spatial simi-
larity with an RSC seed-based connectivity map commonly used to
depict DMN (fig. S4, B to D). Louvain community modularity anal-
ysis clustered the 17 DMN ICs into three distinct modules (Q=0.10,
P<0.01; Fig. 2D): a Frontal module composed of prelimbic/
infralimbic (PrL/IL), lateral orbital (LO), Cg1, and anterior cingu-
late 2 (aCg2) cortices; an RSC-HIPP module composed of the dorsal
hippocampus (HIPP) and posterior Cg2 (pCg2), medial parietal
association (MPtA), retrosplenial granular (RSG), and retrosplenial
dysgranual/visual (RSD/Vis) cortices; and an Association module
composed of posterior parietal (PPtC) and auditory (Aud) cortices.
No significant difference among the connectivity of DMN ICs was
found in the pre-CNO baseline data between LC-NE/hM3Dq and
control groups (PFDR-corrected>0.05; fig. S4E).
Using the Frontal, RSC-HIPP, and Association module regions
of interest (one-sample two-sided t test, P<0.0001) and striatum as
a reference region due to sparse innervation from LC-NE neurons,
we examined how NE release from LC modulates fMRI-derived CBV,
regional homogeneity (ReHo), and amplitude of low-frequency
fluctuation (ALFF) changes. We compared the 10-min pre-CNO
baseline fMRI data against data acquired between 20- and 30-min
post-CNO. We selected a later phase of the CNO response to avoid
the transition time period that has been shown to have greater
intrasubject variability and weaker behavioral effects in designer
receptor exclusively activated by designer drugs induced activity
kinetics as observed by fMRI (6,38–42) and behavior studies
(30,31). CNO-evoked LC-NE activation significantly decreased CBV
from pre-CNO baseline to post-CNO in all DMN modules in LC-NE/
hM3Dq. These changes are significant (two-sided two-sample t test,
PFDR-corrected<0.005) compared to control mice (Fig.3A). LC-NE
activation induced robust CBV changes in the striatum despite sparse
innervation from LC-NE neurons (6), possibly because of NE-induced
vasoconstriction at watershed arteries upstream of the striatum (43).
While CBV changes appeared less specific, ReHo (Fig.3B) and ALFF
(Fig.3C) changes were more localized, and the increase of these
signals contradicted the intuitive interpretation of CBV, suggesting
a possible increase of synchronous, low-frequency activity in the
Frontal and RSC-HIPP DMN modules by LC-NE. Specifically,
LC-NE activation enhanced ALFF changes within 0.01- and 0.05-Hz
band (unpaired t test, PFDR-corrected<0.05; fig. S5A).
To validate these fMRI findings, we used spectral fiber photometry
(Fig.4A) in LC-NE/hM3Dq (n =5) and control mice (n =4). We
virally expressed a genetically engineered NE2.1 sensor (44) and a
red-shifted jRGECO1a calcium activity sensor (45) under the pan-
neuronal human Synapsin-1 (hSyn) promoter in the Cg1 of the
Frontal DMN module (Fig.4B), where the effects of LC-NE activa-
tion were most robust, and intravenously administered a CY5-
conjugated dextran far-red fluorescent dye to measure CBV (fig. S6).
Collectively, this allowed us to simultaneously detect changes in syn-
aptic NE release, neuronal activity–mediated calcium influx, and
CBV in Cg1 (Fig.4C). After CNO administration, we observed sig-
nificant increases in NE release (unpaired t test, PFDR-corrected<0.05;
Fig. 1. Intersectional chemogenetic strategy to selectively activate LC-NE neu-
rons. (A) Schematic illustration of the intersectional genetic strategy. (B) A sagittal
schematic diagram of the hindbrain compressed along the mediolateral axis illus-
trates the approximate position of NE neurons. Recombination of the RC::FL-hM3Dq
allele by the noradrenergic-specific driver DbhFlpo and En1cre results in expression
of the excitatory G protein–coupled receptor hM3Dq fused to mCherry in LC-NE
neurons (magenta cells in schematic). Expression of DbhFlp by all remaining NE
neurons results in expression of green fluorescent protein (GFP) (green neurons in
schematic). (C) Immunofluorescent labeling of sections from the adult brainstem
of LC-NE/hM3Dq mice reveals hM3Dq-mCherry–expressing NE neurons in the LC
(magenta) and GFP-expressing NE neurons (green) in the SubC, A5, C2/A2, and C1/
A1 nuclei. Scale bars, 50 m.
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Fig. 4D), decreases in CBV (unpaired t test, PFDR-corrected <0.05;
Fig.4E), and increases in the neuronal activity (unpaired t test,
PFDR-corrected<0.05; Fig.4F) of LC-NE/hM3Dq mice compared to
controls. Notably, the number of calcium spikes also increased signifi-
cantly following CNO administration (unpaired t test, PFDR-corrected<
0.05; Fig.4G), a hallmark of NE that tunes the signal-to-noise ratio
of downstream neuronal firing (46). These findings corroborate well
with our fMRI results (Fig.3) and indicate that driving LC-NE re-
lease can concurrently induce regional vasoconstriction while enhancing
neuronal excitability in Cg1. Given that fMRI does not directly
measure neuronal activity, these results highlight the importance to
cautiously interpret fMRI-derived DMN results when NE is involved,
as inferring neuronal activity by direct fMRI signal changes in this
case may be erroneous.
We also examined the effect of NE on brain glucose uptake in a sub-
set of LC-NE/hM3Dq (n=5) and control (n=5) mice using an
established 18F-fluorodeoxyglucose (FDG) positron emission tomog-
raphy (PET) protocol (47). We found that CNO-induced LC-NE acti-
vation significantly decreased glucose uptake in all three DMN
modules (paired t test, PFDR-corrected<0.001), but not in control subjects
(Fig.5 and fig. S7). Although there are mixed findings on how NE alters
glucose uptake (48–53), many studies using pharmacology to enhance
NE release have also shown glucose uptake suppression (48–53) but
are often difficult to disambiguate. One possible mechanism
governing this effect is a shift in metabolic pathway by astrocytes to
preferentially use glycogen reserves (51,52). Together, these find-
ings suggest that additional caution needs to be considered for FDG
PET data interpretation when triggering NE release.
To access how selective NE release modulates DMN connectivity
and network properties, we examined functional connectivity (FC)
changes within and between Frontal, RSC-HIPP, and Association
DMN modules in LC-NE/hM3Dq (n=9) and control mice (n=12).
We found that CNO significantly enhanced FC within Frontal
(t=2.89, PFDR-corrected< 0.05) and between Frontal and Association
modules (t=2.63, PFDR-corrected<0.05) of the DMN in LC-NE/hM3Dq,
but not in control mice (Fig.6, Aand B). Notably, the high intra-
modular connectivity characterized by within-module degree (WD),
which quantifies the level of node connectivity within a module, indi-
cated that Cg1 and RSC serve as provincial hubs for the Frontal and
RSC-HIPP modules of the DMN, respectively (fig. S8, A and B). The
high intermodular connectivity characterized by the partition coef-
ficient (PC), which estimates the level of interaction with nodes of
other modules, indicated that the aCg2 of the Frontal module serves
as a connector hub throughout the entire DMN and may control the
FC between Frontal and other modules (fig. S8, A and B). Given
the putative causal control of the anterior insular (AI) cortex on the
Fig. 2. Experimental design and identification of mouse DMN modules. (A) CBV fMRI experimental design includes a 10-min baseline scan before CNO administration
and 30 min of scans after CNO. i.p., intraperitoneally. (B) One hundred ICs were derived from group ICA of baseline scans among all subjects. Specific IC masks were de-
termined by a winner-take-all strategy by comparing mean z values from all ICs on a voxel basis and then color-coded. DMN-related ICs were then identified according to
rodent DMN topology in the literature. (C) Correlation among the 100 ICs was plotted with a threshold Fischer |z| > 0.3. DMN ICs were labeled in red. (D) Modularity anal-
ysis of DMN ICs showing that the mouse DMN is composed of Frontal (yellow), RSC-HIPP (pink), and Association modules (blue).
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Frontal module of the DMN (26,54), we also examined the effects of
LC-NE activation on their FC changes and found that CNO-evoked
activation significantly enhanced anticorrelation between AI and
DMN frontal module (fig. S8, C and D).
To further unravel the causal influences on FC in DMN modules by
LC-NE activation, we conducted a dynamic causal modeling (DCM)
analysis and found that LC-NE activation significantly reduced effec-
tive connectivity (EC) from RSC-HIPP to the Association module
(paired two-sided t test, PFDR-corrected<0.05; Fig.7A), whereas no
change was detected in the littermate controls (fig. S9A). Coactivation
pattern (CAP) (55) analyses may support these findings, as the CAP
representing distinct RSC-HIPP and Association states was also sup-
pressed following LC-NE activation (fig. S9, B to D). Together with
the robust NE modulatory effects in the Frontal module, these findings
prompted us to conduct a mediation analysis to determine the origin
of the FC changes in the Frontal module. A moderation analysis model
was constructed using the structural equation modeling method in
AMOS 17.0. We first demonstrated that the reduction in EC observed
from RSC-HIPP to the Association module causally manipulates the
FC changes within the Frontal module (coefficient=−0.53±0.13,
P< 0.001; Fig.7B). Then, when we incorporated the FC increases
between the Frontal and Association modules as a mediator, we ob-
served a full mediation effect of the reduced EC to the Frontal module
FC changes, while the direct relationship between the reduced EC
from RSC-HIPP to the Association module and the increased FC
within the Frontal module became insignificant (coefficient= −0.26±
0.13, no significance). The Sobel test further indicated the significant
mediation effect (Sobel z=−2.02, P<0.05; Fig.7B). In contrast, we
did not observe the reduced EC from the RSC-HIPP to Association
module being mediated by the FC increases between Frontal and Asso-
ciation modules (fig. S10A). Collectively, these findings indicate that
LC-NE activation modulates the DMN by (i) strengthening FC within
the Frontal module, (ii) strengthening FC between Frontal and Asso-
ciation modules, and (iii) reducing RSC-HIPP control over the Asso-
ciation module, which causally alters Frontal module FC, with the FC
between Frontal and Association modules serving as a key mediator.
Fig. 3. CBV, ReHo, and ALFF changes in DMN modules following activation of LC-NE neurons. (A) CBV decreased (post-CNO, baseline) significantly in LC-NE/hM3Dq
following LC-NE activation across all DMN modules compared to controls. LC-NE activation significantly increased (B) ReHo change in the Frontal DMN module and
(C) ALFF changes in Frontal and RSC-HIPP modules compared to controls. *PFDR-corrected < 0.005; horizontal dotted lines represent means, and error bars represent ±SD. The
brain maps indicate the significant difference of CBV, ReHo, and ALFF changes between LC-NE/hM3Dq and controls (P3dClustSim-corrected < 0.05).
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Fig. 4. Triple-spectral fiber photometry measuring NE release, CBV, and calcium-weighted neuronal activity in Cg1. (A) Experimental setup of the fiber photometry
system with 488-, 561-, and 640-nm lasers simultaneously used to detect NE release (NE2.1), neuronal calcium activity (jRGECO1a), and CBV (CY5-dextran dye) changes,
respectively. i.v., intravenously. (B) Cg1 neurons were confirmed to be transfected to express NE2.1 (green) and jRGECO1a (red) sensors. (C) Spectral profiles of NE neuronal
activity and CBV sensor emissions used to resolve signals via an established spectral unmined approach. (D to F) Respective effects of CNO on NE release, CBV, and neu-
ronal activity in Cg1 from representative subjects and group level bar graphs. Subjects were continuously recorded for 40 min with a dose of CNO (1 mg/kg) administered
via an intraperitoneal catheter at 10 min (t = 0) after scan onset. (G) CNO-induced LC-NE activation altered postsynaptic calcium spiking patterns, resulting in a significant
increase in spike counts. Red dots represent the Ca2+ spikes (z score > 1.96). *P < 0.05 and **P < 0.01; horizontal lines represent means, and error bars represent ±SD.
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DISCUSSION
The mouse DMN is well documented in the literature (20–25,28),
and its structural foundation has been recently established by a piv-
otal study by Whitesell etal. (25). Most studies analyzing the mouse
DMN using ICA or seed-based connectivity analyses report consist-
ent DMN architectures that are homologous to the human DMN. A
multicenter study compiled by Grandjean etal. (22) analyzed several
resting-state mouse fMRI datasets acquired under various condi-
tions (e.g., magnetic field strengths, coils, imaging parameters, and
anesthesia protocols) and generated a group ICA atlas delineating
mouse brain connectivity. The results pulled three DMN modules
that include prefrontal, cingulate/retrosplenial, and temporal asso-
ciation areas. Similar to those findings, our data analysis revealed
three modules including Frontal, RSC-HIPP, and Association modules,
where the Frontal and RSC-HIPP modules included prefrontal and
posterior cingulate components, respectively (Fig.2C). Note that
the involvement of the hippocampus in the mouse DMN remains
disputed because of the lack of direct anatomical projections
(22,24,25). This study includes hippocampus as part of the DMN
because we followed established studies identifying the DMN con-
stituents across rodents (29,37) and humans (4). In addition, sever-
al unbiased hierarchical clustering analyses (24,56), including our
own (29,33), functionally classified the hippocampus as part of the
DMN and lend further support to the validity of the DMN regions
used for the analysis in this study. Aside from hippocampus, note
that thalamus has been recently shown to alter large-scale cortical
rhythms and affects fMRI-derived functional connectivity in the
cortex, including several putative DMN regions (57). Given the robust
Fig. 5. Glucose uptake changes in DMN modules following activation of LC-NE neurons. (A) Activation of LC-NE neurons significantly decreased FDG uptake com-
pared to saline-treated sham and littermate controls receiving CNO. (B) LC-NE activation significantly reduced standardized uptake values (SUVs) in all DMN regions. SUV
changes were derived from each subject that underwent two scans (vehicle and CNO administration) following co-registration. Comparisons were made against litter-
mate controls. *P < 0.001 and **P < 0.0001; horizontal dotted line represents the mean, and error bars represent ±SD.
Fig. 6. FC changes among DMN modules following activation of LC-NE neurons. (A) Within and between DMN module FC changes. (B) Network-based sta-
tisti cal analysis showing significant differences in FC among edges in LC-NE/hM3Dq > Control. *PFDR-corrected < 0.05; horizontal dotted lines represent means, and error
bars represent ±SD.
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LC innervations into thalamus (32), it is crucial for future studies to
examine network interactions among LC, thalamus, and DMN.
Several research groups have pioneered chemogenetic fMRI
(6,38–42) and PET (47) approaches to selectively map the influence
of neurotransmitters on brain networks in rodents. Our experimen-
tal design using an intersectional chemogenetic mouse line presented
a unique opportunity to selectively investigate functional DMN
modulation. It is our hope that through dissemination of raw data,
this work, together with a seminal study by Zerbi etal. (6), will help
set the foundation for future studies examining other network sys-
tems manipulated by LC-NE.
We demonstrated that chemogenetic activation of LC-NE neu-
rons significantly reduced CBV and glucose uptake among all three
DMN modules compared to littermate controls (Figs.3and5).
While CBV measures may suffer from systemic effects from LC-NE
activation on sympathetic outputs (58), our study used an array of
multimodal techniques to help interpret the influence of LC-NE ac-
tivation on DMN. We found that directional CBV changes are not
a proper metric to explain network activity or connectivity changes.
Future studies should incorporate central and peripheral NE levels
as confounders of hemodynamic-based brain mapping techniques.
Both fMRI and fiber photometry corroborated the findings of
CBV reduction in the Frontal DMN module following activation of
LC-NE neurons. Concurrently, we observed robust increases in
synchronous low-frequency activity as measured by ReHo, ALFF,
and photometry-derived calcium activity in our experimental con-
dition (Fig.4,FandG). The most straightforward interpretation of
the fMRI data based on well-documented neurovascular coupling
rules (59) does not apply, likely because of the potent vasoconstric-
tive properties of LC-NE (60). It is not surprising that the neuro-
modulatory effect induced by NE release appears inconsistent in the
literature owing to various brain states and basal firing rate examined,
as well as the distinct experimental approaches used to promote/
benchmark NE release (6,61–63). The literature also shows mixed
findings regarding the effects of NE on both cerebral hemodynamics
and metabolism that are difficult to disambiguate because common
pharmacological agents used to induce NE release suffer from dif-
ferential actions on NE receptor subtypes and nonselective binding
(64) that can either increase (65) or decrease perfusion (60,66) and
increase (48,53) or decrease glucose metabolism (49,50). One possible
mechanism governing the observed changes in glucose metabolism
following LC-NE stimulation could also be attributed to a dose-
dependent metabolic shift in astrocytes to preferentially use glyco-
gen reserves (51,52). Unlike many studies that use pharmacological
manipulations of NE release, our chemogenetic approach coupled
with multimodal measurement of NE, neuronal activity, and CBV
revealed an effect of NE in increasing synchronized low-frequency
activity, strengthening neuronal firing, and decreasing CBV. This has
substantial implications when using fMRI to interpret DMN neuro-
nal activity, as “deactivation” of raw fMRI signal caused by LC-NE
activation may not necessarily represent reduced DMN neuronal act ivity.
Recent animal and human fMRI studies support the role of NE
in brain network reorganization (6,62,63,67). NE release has been
shown to promote topological integration within the network (68,69).
This aligns well with our findings showing enhanced ReHo and FC
within the Frontal DMN module following CNO-evoked LC-NE
activation. In addition, our ALFF results show that LC-NE activa-
tion enhanced low-frequency power of the Frontal DMN that was
restricted to a frequency band ranging from 0.01 to 0.05 Hz—a
range associated with robust changes in human DMN (70). Alter-
ations in the power within this subfrequency band of ALFF also
plays a vital role in attention reorientation during visual-motor
attentional tasks (70), which has also been linked to changes in cor-
tical NE levels (71).
Fig. 7. DCM and mediation analysis among DMN modules. (A) DCM analysis among DMN modules found that RSC-HIPP reduced its causal modulation to the Association
module upon LC-NE activation (PFDR-corrected < 0.05; horizontal dotted lines represent means, and error bars represent ±SD). (B) Mediation analysis was performed with
direct effect (a, b, and c) and with a mediator (c′). A Sobel test was also performed (red dotted line) to evaluate the significance of mediation effect (Sobel z value = −2.02,
P < 0.05). *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., no significance.
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LC-NE enhanced FC within the Frontal module and between
Frontal and Association modules. In agreement with our finding, a
seminal study virally transfecting hM3Dq via the Dbh promotor
into the LC found significant FC enhancement in the Cg1, Cg2, and
RSC following chemogenetic stimulation (6). Abnormally height-
ened FC in Frontal DMN regions has been associated with anxiety
and depression (72). Anxiety-like behavioral phenotypes such as
reduced locomotor activity in a novel environment and anhedonia
have also been found following chemogenetic-evoked LC-NE acti-
vation in awake mice (30). Such a behavior response is similar to
optogenetic-induced LC-NE neuronal activation at tonic frequencies
(73). The intersectional chemogenetic strategy used in the current
study has been shown to result in approximately 2Hz of tonic firing
(31), comparable to that in (74). Unlike phasic LC bursting that in-
duces arousal by desynchronizing cortical electroencephalography
(EEG) states (75), the low tonic LC firing induced by CNO as seen
in our experimental condition has been shown to strengthen theta
and suppress delta power (74) and does not trigger sufficient arousal
activity under anesthesia (76). Together with our data showing
tonically elevated NE release (Fig.4D) and strengthened DMN con-
nectivity (Fig.6), these results suggest that tonic firing of LC-NE
neurons shifts the brain toward a DMN-dominated state and there-
fore facilitates DMN-associated behaviors (77).
As we did not observe any FC decreases in the DMN and only
identified strengthening FC within and between modules, our re-
sults support the functional integration theory of NE proposed by
Shine (68), complemented by the results of Zerbi etal. (6), and
suggest that LC-NE–induced functional integration could occur at
a rather focal, subnetwork level within the DMN. In addition, we
found that FC strengthened anticorrelated coupling between the
Frontal DMN module and AI, a key node of the salience network
(SN) that may causally suppress the DMN (fig. S8, C and D) (54).
This also indicates that integration between the DMN and SN is
greater following LC-NE activation under our experimental condi-
tion. This finding may suggest a putative role of tonic LC-NE activity
in improving efficient cognitive control and reducing behavioral
variability by fostering SN-DMN anticorrelation (78). In addition,
our findings point to the possibility that tonic and phasic outputs
from LC-NE neurons may preferentially drive DMN and SN, re-
spectively. Furthermore, the enhanced anticorrelation between net-
works also supports the plausible mechanism by which NE-targeting
pharmacological agents like atomoxetine, clonidine, and guanfacine
are effective in treating ADHD patients because they strengthen the
FC within the Frontal DMN module and result in improved atten-
tion (79).
Our DCM and mediation analyses show that enhanced Frontal
module connectivity in the DMN was causally manipulated by re-
ducing RSC-HIPP control of the Association module, with the con-
nectivity between the Association and Frontal modules serving as a
key mediator. These findings reveal a new understanding of how
LC-NE activation controls the signaling cascades within DMN modules
and achieves its control of the Frontal cortical regions, which are
among the most well-studied projection targets of LC-NE because
of their importance in shaping multiple behaviors (80). LC-NE neu-
ronal loss and the subsequent depletion of cortical NE levels are
widely considered to be among the first sites of neurodegeneration
in Parkinson’s and Alzheimer’s diseases (81), resulting in behavioral
pathologies linked to alterations in DMN such as delayed attention
shifting (82), enhanced mind-wandering (83), and reduced cognitive
and emotional processing of sensory information (82,83). Optogenetic-
induced LC-NE activation at lower tonic frequencies into prefrontal
and orbitofrontal cortices enhances stimulus and goal-directed
attention with decreased impulsivity (84). Conversely, the suppres-
sion of LC-NE activation exacerbates distractibility and impulsivity
(84), similar to that observed in Parkinson’s and Alzheimer’s dis-
ease patients (83). Together, alterations within Frontal and between
Frontal and RSC-HIPP DMN modules have potential to serve as
early biomarkers for pathophysiological changes in LC-NE neurons.
Our circuit-level findings could also pave the way toward novel tar-
gets to causally control the Frontal DMN via the RSC-HIPP module
when LC neurons have degenerated such that the behavioral traits
relevant to the Frontal DMN may be restored when endogenous NE
is pathologically diminished.
MATERIALS AND METHODS
Animals
All animal procedures were performed in strict compliance with
ethical regulations for animal research and approved by the Institu-
tional Animal Care and Use Committee of the University of North
Carolina at Chapel Hill. En1cre (85), DbhFlpo (32), and RC::FL-hM3Dq
(31) mouse colonies are maintained on a C57BL/6J background.
Male and female triple-transgenic animals were generated at the
National Institute of Environmental Health Sciences by crossing
En1cre mice to double-transgenic DbhFlpo;RC::FL-hM3Dq mice. Single-
and double-transgenic littermates served as controls essential for rigor
in chemogenetic studies (86) because off-target binding of CNO or
clozapine through reverse metabolism of CNO may occur (36). All
animals were maintained on a 12-hour/12-hour light-dark cycle
with access to food and water ad libitum.
CBV-fMRI acquisition
For fMRI studies, LC-NE (n=9) and control mice (n =12) were
initially anesthetized using 2 to 3% isoflurane and maintained un-
der light anesthesia (1% isoflurane) while preserving physiological
homeostasis. All MRI experiments were performed on a Bruker
BioSpec 9.4-T, 30-cm bore system (Bruker BioSpin Corp., Billerica,
MA) with ParaVision 6.0.1 on an AVANCE II console. An RRI BFG
150/90 gradient insert (Resonance Research Inc., Billerica, MA) paired
with a Copley C700 gradient amplifier (Copley Controls Corp., Canton,
MA) was used for all experiments. A 72-mm-volume coil was used
as the transmitter, and a quadrature mouse brain coil was used as
the receiver (Bruker BioSpin Corp., Billerica, MA). Magnetic field
homogeneity was optimized first by global shimming, followed by
local second-order shims using a MAPSHIM protocol. All CBV-fMRI
data were acquired using a two-dimensional (2D) multislice, single-
shot, gradient-echo EPI sequence: TR (repetition time)=3000 ms,
TE (echo time)=7.9 ms, bandwidth=250 kHz, flip angle=70°, FOV
(field of view)=19.2mm × 19.2 mm, matrix size=64 × 64, slice
number=26, slice thickness=0.3 mm, resulting in an isotropic
voxel size of 0.3mm3. Subjects were continuously recorded for
40min with a dose of CNO (1 mg/kg) administered via an intraper-
itoneal catheter 10min after scan onset. CBV-weighted fMRI was
achieved by a bolus dose of an in-house–developed iron oxide
nanoparticle (30mg of Fe/kg, intravenously) (87). Rectal body tem-
peratures were continuously maintained at 37°±0.5°C with a tem-
perature controller (Oakton Temp9500, Cole-Parmer, Vernon Hills,
IL, USA) coupled to a circulating water bath (Haake S13, Thermo
Oyarzabal et al., Sci. Adv. 8, eabm9898 (2022) 29 April 2022
SCIENCE ADVANCES | RESEARCH ARTICLE
9 of 12
Fisher Scientific, Waltham, MA, USA) that heats the MRI mouse
cradle. Respiration was monitored through a pneumatic pillow
(Respiration/EEG Monitor, SA Instruments, Stony Brook, NY, USA)
and maintained between 90 and 110 breaths/min through fine ad-
justments in the inhaled isoflurane concentration.
Physiological parameters were monitored during fMRI. The only
change observed following CNO administration was an increase in
respiratory rate, which rapidly recovered within a few seconds and
did not follow the sustained CBV changes measured by fMRI and
photometry. Moreover, these changes were observed in both LC-NE/
hM3Dq and control groups, indicating that it is likely because of the
well-known injection volume stress.
fMRI data analysis
Preprocessing of images
All fMRI data were corrected for slice timing and motion using Anal-
ysis of Functional NeuroImages. Brain data were isolated using a
U-Net deep-learning skull stripping tool and spatially normalized
to our EPI template using Advanced Normalization Tools. In addi-
tion, despiking, ICA denoising, and nuisance variable regression of
the six motion parameters estimated from motion correction and the
cerebrospinal fluid signal extracted using a mask of the major brain
ventricles were performed. Datasets were then smoothed using a
Gaussian kernel with full width at half maximum (FWHM) at 0.6 mm,
detrended, and temporally filtered by applying a high-pass filter
between >0.01Hz. Datasets underwent quality control by measur-
ing frame-wise displacement (FD), temporal signal-to-noise ratio
(tSNR), and DVARS (temporal derivative of the root mean square
variance over voxels). A detailed description of the preprocessing
pipeline (56) can be found in Supplementary Methods.
ICA and modularity analyses
MRI data were decomposed into 100 functional components using
baseline data from all subjects via a group-level ICA (FSL MELODIC).
Functional modules of the identified 17 DMN components were
parcellated using the Louvain community detection algorithm.
Within- and between-module connectivity was then defined as the
average of FC across node pairs within or between the identified DMN
modules. The ICA and modularity analysis method are detailed in
Supplementary Methods.
DCM and mediation analysis
We specified a DCM model with full connectivity consisting of
three modules from DMN to estimate pairwise EC among the DMN
modules and constructed a directed and weighted graph (representing
an EC network) for each subject. We applied serial multiple media-
tion analysis model in AMOS 17.0 (SPSS Inc., Chicago, IL, USA) to
uncover underlying functional pathways within DMN. Specifically,
we first estimated the direct relationships between dependent vari-
able (EC from RSC-HIPP module to Association module) and inde-
pendent variable (FC within Frontal module). Then, in a mediation
model, the FC between Association and Frontal module was added
as a mediator. In this context, full mediation occurs when the rela-
tionship between the independent variable and the dependent variable
is no longer significant with the inclusion of a mediator variable.
Detailed data analysis is further described in Supplementary Methods.
FDG PET procedure
Mice were fasted 12hours before undergoing 18F-FDG PET scans to
reduce variability in blood glucose levels that could alter 18F-FDG
uptake (47). Static PET scans were collected on the same cohort of
animal over two scan sessions using LC-NE/hM3Dq (n=5) and
control (n=6) mice to represent sham-treated baseline or CNO-
treated condition. Mice were briefly anesthetized under 1 to 3% iso-
flurane and injected with either a saline + dimethyl sulfoxide
(DMSO) vehicle or CNO dissolved in DMSO (1 mg/kg, intraperito-
neally) and subsequently received an intravenous injection of
∼0.2 mCi of 18F-FDG after 5min. Mice were recovered in their home
cages for a 45-min uptake period. Mice were subsequently anesthe-
tized with isoflurane (2%) and underwent a 10-min computed to-
mography (CT) and 20-min PET scan on a small-animal PET/CT
scanner (Argus-2R, Sedecal, Madrid, Spain). PET data were recon-
structed using the 2D ordered subset expectation maximization
(OSEM) algorithm expressed in standardized uptake values (SUVs)
and normalized using arm muscle uptake of 18F-FDG using PMOD
(PMOD Technologies LLC, Zurich, Switzerland). Data were repre-
sented as % changes in SUV between vehicle and CNO scans.
Fiber photometry procedure
Surgical adeno-associated virus (AAV) microinjection and
fiber implantation
LC-NE (n=5) and control (n =5) mice were microinjected
with 0.3 l of AAV5-hSyn-NE2.1 (h-N01, WZ Biosciences) and
0.5 l of AAV9-hSyn-jRGECO1a (100854, Addgene) to the left Cg1
(Anterior- Posterior=2.2 mm, Medial-Lateral=0.2 mm, Dorsal-
Ventral =−1.6 mm). NE2.1 (a green fluorescent NE sensor) and
jRGECO1a (a red-shifted intracellular calcium sensor) were used
for determining the NE release and neuronal activity, respectively.
An optic fiber was implanted 0.3mm above the injection site
and imbedded to the skull using cement (C&B Metabond,
S380, Parkell).
Fiber photometry recording
All recordings began at least 4 weeks after surgery. CY5-conjugated
dextran fluorescent dye (20 mg/kg; R-FN-006, RuixiBio) was injected
intravenously for CBV measurements. Animals were prepared and
maintained under the same conditions as fMRI experiments. A spectral
fiber photometry system capable of recording NE2.1, jRGECO1a,
and CY5 signals was simultaneously recorded during the experiments.
Detailed methods can be found in Supplementary Methods.
Immunohistology procedure
Mice were deeply anesthetized with sodium pentobarbital and tran-
scardially perfused with 0.1M phosphate-buffered saline (PBS)
followed by 4% paraformaldehyde (PFA). Brains were postfixed
overnight by immersion in 4% PFA at 4°C. Following a rinse in
PBS, brains were cryoprotected in 30% sucrose in PBS and embedded
in Tissue Freezing Medium. Forty-micrometer free-floating coronal
cryosections were collected in PBS and processed for immunohisto-
chemistry according to previously published protocol (32). Briefly,
free-floating sections were blocked in 5% normal goat serum in PBS
with 0.1% Triton X-100 for 1hour before incubating in primary
antibody overnight at 4°C. All NE cell bodies were labeled with rabbit
anti-tyrosine hydroxylase (TH) antibody (AB152, Millipore). hM3Dq-
mCherry-expressing NE neurons were labeled with rat anti-mCherry
primary antibody (EST202, Kerafast) and green fluorescent protein
(GFP)–expressing NE neurons were labeled with chicken anti-GFP
primary antibody (AB13970, Abcam). Sections were washed three
times in PBS and incubated for 2 hours in Alexa Fluor 648 anti-rabbit,
Alexa Fluor 568 anti-rat, and Alexa Fluor 488 anti-chicken secondary
antibodies (Invitrogen). Sections were mounted onto glass slides,
Oyarzabal et al., Sci. Adv. 8, eabm9898 (2022) 29 April 2022
SCIENCE ADVANCES | RESEARCH ARTICLE
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coverslipped with Vectashield hard-set mounting medium with
4′,6-diamidino-2-phenylindole (DAPI) (H-1500, Vector Labs), and
imaged on a Zeiss LSM 880 inverted confocal microscope.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at https://science.org/doi/10.1126/
sciadv.abm9898
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Acknowledgments: We thank the members of the UNC Center for Animal MRI and F. Crews,
Z. McElligott, and B. Roth for inputs. We thank the UNC Small Animal Imaging Core Facility
staff J. Frank and J. Merrill for assistance in PET data acquisition. Funding: This research was
supported by the Extramural Research Programs of U.S. National Institutes of Health, NINDS
(R01NS091236), NIMH (R01MH126518, R01MH111429, and RF1MH117053), NIAAA
(P60AA011605 and U01AA020023), and NICHD (P50HD103573) to Y.-Y.I.S. and the Intramural
Research Program of the U.S. National Institutes of Health, National Institute of Environmental
Health Sciences (ZIA-ES102805 to P.J. and 1ZIAES103310 to G.C.). Author contributions:
E.A.O., M.D., and Y.-Y.I.S. designed the study. E.A.O. and M.D. collected the imaging data. E.A.O.,
L.-M.H., and S.-H.L. analyzed the imaging data. J.Z. and G.C. performed the viral injections and
fiber implantation. E.A.O., T.-H.H.C., and W.Z. collected and analyzed the fiber photometry
data. K.G.S. processed and K.G.S. and P.J. analyzed the histology data. N.R.S., I.Y.E., and P.J.
developed and shared the transgenic mice. E.A.O., L.-M.H., P.J., and Y.-Y.I.S. wrote the
manuscript with input from all authors. Competing interests: The authors declare that they
have no competing interests. Data and materials availability: All data needed to evaluate
the conclusions in the paper are present in the paper and/or the Supplementary Materials. All
MRI, PET, and photometry data from this study are openly available on Mendeley Data
(https://doi.org/10.17632/hxch8htz84.2).
Submitted 22 November 2021
Accepted 15 March 2022
Published 29 April 2022
10.1126/sciadv.abm9898
