2024 11 03 621722v1 Full

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Neuron-astrocyte Coupling in Lateral Habenula Mediates Depressive-like
Behaviors
Qianqian Xin1,2,5, Junying Wang1,2,5, Jinkun Zheng2, Yi Tan1,2,3, Xiaoning Jia2, Zheyi Ni2,
Jiesi Feng4, Zhaofa Wu4, Yulong Li4, Xiaoming Li2, Huan Ma2, Hailan Hu1,2,3,6,*
1
Department of Neurobiology, Affiliated Mental Health Center & Hangzhou Seventh People's
Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
2
Nanhu Brain–Computer Interface Institute, Liangzhu Laboratory, MOE Frontier Science Center for
Brain Science and Brain–Machine Integration, State Key Laboratory of Brain–Machine Intelligence,
New Cornerstone Science Laboratory, Zhejiang University, Hangzhou, China.

3
Department of Psychiatry and International Institutes of Medicine, The Fourth Affiliated Hospital,
Zhejiang University School of Medicine, Yiwu, China.

4
State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing
100871, China
5
These authors contributed equally
6
Lead contact
*Correspondence:
[email protected]
Tel: +86-(0)571-8898-1720
Fax: +86-(0)571-8820-8920
1
bioRxiv preprint doi: https://doi.org/10.1101/2024.11.03.621722; this version posted November 3, 2024. The copyright holder for this preprint
Summary
The lateral habenular (LHb) neurons and astrocytes have been strongly implicated in
depression etiology but it was not clear how the two dynamically interact during
depression onset. Here, using multi-brain-region calcium photometry recording in
freely-moving mice, we discover that stress induces a unique, bimodal neuronal
response and a most rapid astrocytic response in the LHb. LHb astrocytic calcium
requires the α1A-adrenergic receptor, and depends on a recurrent neural network
between the LHb and locus coeruleus (LC). Through the gliotransmitter glutamate and
ATP/Adenosine, LHb astrocytes mediate the second-wave activation of local LHb
neurons as well as release of norepinephrine (NE). Activation or inhibition LHb
astrocytic calcium signaling facilitates or prevents stress-induced depressive-like
behaviors respectively. These results identify a stress-induced positive feedback loop in
the LHb-LC axis, with astrocytes being a critical signaling relay. The identification of
this prominent neuron-glia interaction may shed light on stress management and
depression prevention.
Keywords: lateral habenula, locus coeruleus, astrocytes, norepinephrine, stress,
depression
2
Introduction
Stress is a prominent risk factor for the development of depressive illness.1 Modern
perspectives on the etiology of major depressive disorder (MDD) suggest that the molecular
expression, cellular activities, and synaptic connections of specific brain regions are altered in
response to external stressful stimuli.2-4 Recently, the lateral habenula (LHb), known as the
brain’s ‘anti-reward center’ has been strongly implicated in the pathophysiology of major
depression.5-7 LHb regulates essentially all neuromodulatory systems, including the
serotonergic, dopaminergic, and noradrenergic systems.7-9 LHb neurons are activated by
stress10-13 and are hyperactive under the depressive state.14-19 Although neurons have been the
primary focus for depression etiology, recent studies have begun to implicate malfunctions of
LHb astrocytes, including increased expression of Kir4.1 for potassium buffering20 and
reduced expression of GLT1 for glutamate uptake.21,22 However, it has remained elusive how
neurons and astrocytes in the LHb coordinate their activity in response to stress, and how
their dynamic interaction contributes to depression etiology.
The synergy between neurons and astrocytes is increasingly recognized as crucial in brain
physiology, animal behavior, and disease.23-28 Astrocytes communicate with neurons through
chemically based dialogue.29,30 Astrocytes possess dense fine processes and express various
types of G protein-coupled receptors (GPCRs), bestowing them to sense neuronal and
synaptic activities with intracellular calcium transients.31-35 Astrocytic calcium levels can be
elevated by neuromodulators, such as acetylcholine (ACh),36-38 dopamine,32,39 serotonin40 or
norepinephrine (NE).41-43 In turn, astrocytes can regulate neuronal functions at diverse
3
timescales through the uptake of extracellular ions and neurotransmitters20,44-46 or release of
gliotransmitters.27,30,47-52 Gliotransmitters, including glutamate, gamma-aminobutyric acid
(GABA), D-serine and ATP/Adenosine, can lead to synchronized neuronal excitation,29,53-55
enhanced temporal fidelity of firing,56 or fine-tuned synaptic transmission.32,39,57-61 However,
in the context of stress response and depression, the bi-directional signaling of how neurons
and astrocytes communicate with each other has remained much less studied.
In this study, we set out to apply fiber photometry in freely-moving mice to record the
calcium activities of neurons and astrocytes in response to stress in multiple brain regions.
We found that stress induced a most rapid astrocytic response and a unique, biphasic neuronal
response in the LHb. Using cell-type-specific manipulations in combination with fiber
photometry and two-photon imaging recordings, we identified a bidirectional
neuron-astrocyte interaction in the LHb-LC circuit during stress response, and pinpointed a
critical role of LHb astrocytes in the onset of depression.
Results
Foot-shock-stress induces most rapid calcium response in LHb astrocytes
We performed in vivo multi-site fiber photometry to record astrocytic calcium activities
during foot-shock stress (FS, duration: 1 s; intensity: 1 mA; Figure 1A). We recorded a total
of 6 brain regions known to be responsive to stress, including the medial prefrontal cortex
(mPFC), hippocampus (HPC), lateral habenula (LHb), lateral hypothalamus (LH), striatum
(STR) and basolateral amygdala (BLA). Adeno-associated viral (AAV) 2/5 expression of the
4
glia-specific GfaABC1D promoter62 driving the genetically encoded calcium indicator
GCaMP6f63 allowed the recording of calcium signals specifically in astrocytes (Figure 1B).
Among the GCaMP6f positive cells, 91.9% were S100β (astrocytic marker) positive and 0.3%
were NeuN (neuronal marker) positive (Figure 1C and S1A), suggesting a highly specific
expression. Three brain regions were simultaneously recorded per animal and their calcium
responses were aligned to the onset of FS to allow the comparison of signals across different
brain regions (Figure 1D and 1E). While all six recorded regions showed increased astrocytic
calcium activities in response to FS (Figure 1E), signals in the LHb peaked significantly
earlier than those of other five regions (mean latency to peak 1.68 ± 0.08 s in LHb, 2.22 ±
0.12 s in LH, 3.17 ± 0.24 s in PFC, 3.37 ± 0.22 s in HPC, 3.45 ± 0.13 s in BLA, 4.98 ± 0.89 s
in STR; LHb versus PFC, HPC, BLA, STR, p < 0.0001; LHb versus LH, p = 0.001;
Mann-Whitney test; Figure 1D, 1E and 1G). The peak amplitude of FS-evoked LHb
astrocytic calcium signals was also larger than those of other five regions (LHb versus LH,
PFC, p = 0.01; LHb versus HPC, p = 0.005; LHb versus BLA, STR, p = 0.0002;
Mann-Whitney test; Figure 1H). By analyzing the kinetics of calcium signals in mice with
varying levels of peak fluorescence in the LHb, we found that the time to peak did not depend
on the peak amplitude (Figure S1B), suggesting that the kinetics of calcium signals is not
affected by signal intensity, but rather reflects intrinsic regional properties. Thus, among the
multiple regions examined, the LHb astrocytes showed the most rapid response to FS,
suggesting that it may play a unique role in processing stress-related information.
Foot-shock-stress induces biphasic calcium activities in LHb neurons
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We next expressed GCaMP6s under the CaMKIIα promoter in the above six brain regions
and performed calcium photometry recording in neurons (Figure 2A and S2A-S2E).
Consistent with previous reports that LHb neurons encode aversive signals,10,12 LHb neuronal
calcium signals were strongly activated in response to FS stress (Figure 2B, 2C, S3A and
S3B). Interestingly, the FS-evoked calcium signals in LHb neurons first peaked at 0.9 ± 0.03
s (Figure 2D), and was followed by a second phase of activation with the inflection point at
2.54 ± 0.16 s (Figure 2C, 2D and S3C). The mean half-decay time of the first fast and the
second slower phase was 0.83 ± 0.05 s and 22.4 ± 1.77 s respectively (Figure S3D). Although
the second-phase signal has a smaller peak, due to its prolonged decay time, its area under the
curve (AUC) is 3.46 ± 0.78 folds of that of the first phase (Figure 2D and S3E), suggesting
that even more calcium entered during the second phase. Such FS-evoked bimodal neuronal
calcium activation was not observed in the other five brain regions examined (Figure S2).
To better understand the relationship between stress-activated neuronal and astrocytic
calcium activities in the LHb, we simultaneously recorded their activities by expressing the
red calcium sensor jRGECO1a64 under the neuron-specific human synapsin (hSyn) promoter
and green GCaMP6f under the GfaABC1D promoter (Figure 2E). Consistent with results
from their separate recordings in Figure 1D-H and 2A-D, dual-color fiber photometry
recording revealed that FS triggered sequential activation of LHb neurons and astrocytes,
with the neuronal signals peaking at 0.64 ± 0.04 s and astrocytic signals peaking at 1.64 ±
0.05 s (Figure 2F-2H). It is also of interest to note that LHb astrocytic signals peaked between
the first and second phase of LHb neuronal signals (Figure 2F-2H), suggesting a possibility
6
that LHb astrocytes may be regulated by the first-phase neuronal activation, and contribute to
the second-phase neuronal activity.
Neuron-to-astrocyte crosstalk in the LHb during FS
In order to characterize the mechanism of LHb neuron-astrocyte interaction in stress
processing, we first explored the potential crosstalk from neurons to astrocytes (Figure 3). We
used AAV virus to express ChrimsonR65 in LHb neurons and GCaMP6f in LHb astrocytes.
Subsequently, we recorded calcium activities in astrocytes following photostimulation of
LHb neurons both in vivo and in vitro (Figure 3A and 3D). For the in vivo experiments, we
tracked LHb astrocytic calcium activities using fiber photometry while optogenetically
activating LHb neurons in freely moving mice (Figure 3A). For the in vitro experiments, we
monitored LHb astrocytic calcium activities using two-photon imaging while optogenetically
activating the LHb neurons in brain slices (Figure 3D). Surprisingly, while activation of LHb
neurons robustly activated LHb astrocytic calcium signals in a frequency-dependent manner
in vivo (Figure 3B and 3C), it failed to do so in vitro (Figure 3E and 3F). Even under
stimulation frequency as high as 70 Hz, still no evoked astrocytic calcium signal was detected
in brain slices (Figure 3F). We also coexpressed ChrimsonR and GCaMP6s in LHb neurons,
and found that the same optogenetic stimulation protocol was able to strongly activate LHb
neurons in brain slices (Figure S4A-S4D). These results suggest that first, activation of LHb
neurons can lead to activation of LHb astrocytes in vivo; second, the lack of neuron-activated
astrocytic calcium signals in vitro was not due to inefficient activation of LHb neurons, but
more likely a lack of mediator of the neuron-astrocyte crosstalk in the brain slice setup.
7
To search for the mediator of the neuron-astrocyte crosstalk in the LHb, we first looked for
the astrocytic receptor(s) involved in this process in vivo. We measured astrocytic calcium
photometry signals in response to FS while intraperitoneally (i.p.) injecting animals with
blood-brain-barrier (BBB)-permeable inhibitors or antagonists of receptors for different
neurotransmitters or neuromodulators (Figure 3G). We tested receptors for three classes of
neurotransmitter/modulators, which were previously implicated in astrocytic Ca2+ signaling:
the metabotropic glutamate receptors (mGluR),31,66 acetylcholine receptors (AChR) 36,67,68
and norepinephrine receptors (NER).42,69,70 While antagonists for mGluR5, mGluR2/3,
muscarinic AChR (mAChR) or ionotropic AChR (nAChR), namely MPEP, LY341495,
scopolamine and mecamylamine respectively, induced no change (p = 0.87 for MPEP and p =
0.52 for LY341495, p = 0.67 for scopolamine and p = 0.71 for mecamylamine,
Mann-Whitney test), antagonists for the NE receptors caused strong impacts on FS-evoked
LHb astrocytic Ca2+ (Figure 3H, S5A and S5B). In particular, α1-adrenergic receptors (α1-AR)
antagonist prazosin largely suppressed FS-evoked LHb astrocytic calcium signals (p = 0.0001,
Mann-Whitney test; Figure 3H and S5A). On the other hand, atipamezole, the antagonist of
α2-adrenergic receptors (α2-AR), which are mostly expressed on presynaptic terminals
serving autoinhibitory function,71 increased FS-evoked LHb astrocytic calcium signals (p =
0.0006, Mann-Whitney test; Figure 3H and S5B), possibly due to the release of autoinhibition.
Among the α1-adrenergic receptors, there are three subtypes: α1A-, α1B- and α1D-adrenergic
receptors (AR). By using their respective antagonists, we further delineated the primary
contributor of FS-evoked LHb astrocytic calcium signals to be the α1A-AR (Figure 3I).
8
To further confirm that NE can activate LHb astrocytes through the α1-adrenergic receptors,
we performed calcium imaging of astrocytes on LHb brain slices (Figure S6). Bath
application of either NE or the α1-adrenergic receptor agonist phenylephrine (PE) increased
astrocytic calcium signals in the presence of tetrodotoxin (TTX, which blocks all neuronal
action potentials) (Figure S6A-S6E). The NE-elicited LHb astrocytic calcium signals were
blocked by the α1-adrenergic receptor antagonist prazosin (Figure S6F and S6G). These data
demonstrated that NE can activate astrocytic calcium signals through its action on astrocytic
α1-adrenergic receptors in the LHb independent of local neural activity.
To detect whether NE is indeed released during FS in the LHb, we virally expressed in the
LHb genetically encoded G-protein-coupled receptor activation-based NE (GRABNE) sensor,
NE2h (Figure 3J).72 Interestingly, in response to FS, NE signals exhibited both rapid phasic
activity and slower ramping activity (Figure 3K and S7A-S7C). The rapid phasic increases in
NE signals were time-locked to the delivery of FS, and similar as LHb neuronal Ca2+ signals,
displayed bimodal distribution (Figure 3K and 3L). Over the course of 6 FSs, the LHb-NE
signals kept ramping up and remained elevated for the duration of the 8 min session in the
shock chamber (Figure 3K, S7A and S7C). Furthermore, alignment of LHb-neuron calcium,
LHb-NE sensor and LHb-astrocyte calcium signals to the FS onset revealed a sequential
activation of these three signals (Figure 3M and 3N).
The above data suggests that NE may be the potential link mediating the neuron to astrocyte
9
crosstalk in the LHb. To further substantiate this hypothesis, we next explored the anatomical
and functional connections between the LHb and LC, the hub for noradrenergic neurons and
potential source of NE.73-77 First to look at the connection from the LHb to the LC, we
injected AAV2/9 virus expressing hSyn promoter-driven ChrimsonR in the LHb, and AAV2/9
virus expressing Cre-dependent GCaMP6s into the LC of TH-Cre transgenic mice for
NE-neuron-specific expression (Figure 3O and S8A). In these mice, optogenetic activation of
LHb terminals in the LC evoked calcium response of LC-NE neurons (Figure 3P and S8B),
suggesting that there is functional input from LHb neurons into the LC to activate NE
neurons. Next, for the LC to LHb connection, we examined the samples from experiments in
Figure 3O, and detected GCaMP signals from the TH+ LC neurons in the LHb (Figure S8A),
verifying that anatomically there is a direct afferent input from the LC-NE neurons to the
LHb region. Furthermore, optogenetic activation of LC-NE-neuron terminals in the LHb
evoked calcium response of LHb astrocytes (Figure 3Q, 3R, S8C and S8D), which was
blocked by α1-adrenergic receptor antagonist prazosin (p = 0.009, Mann-Whitney test; Figure
3S). Prazosin also abolished LHb astrocytic calcium signals evoked by activation of LHb
neurons in vivo (p = 0.0095, Mann-Whitney test; Figure S8E-S8H). Furthermore, optogenetic
inhibition of LHb neurons or chemogenetic inhibition of LC-NE neurons significantly
decreased the calcium responses of LHb astrocytes evoked by FS stress (Figure S8I-S8P).
Collectively, this set of data delineates a pathway, in which stress activates LHb neurons, in
turn recruiting LC-NE neurons, which through the release of NE, stimulates the astrocytic
calcium in the LHb (Figure 3T).
10
Astrocyte-to-neuron crosstalk in the LHb
We next explored the signaling pathway mediating the potential crosstalk from LHb
astrocytes to neurons. We first determined the effect of LHb astrocytic activation on LHb
neuronal activities in freely moving mice (Figure 4A-4E). To mimic the activation of
Gq-coupled α1-adrenergic receptor signaling,78 we expressed the Gq-coupled hM3Dq under
the GfaABC1D promoter for chemogenetic activation of LHb astrocytes (Figure 4A). Upon
injection of the ligand of hM3Dq clozapine, in vivo fiber photometry recording of the
CaMKIIα-GCaMP6s-expressing LHb neurons revealed significantly elevated,
dose-dependent, bulk calcium activity (Figure 4B and 4C). There was also an increase in the
immunohistochemical signals of c-Fos, a cellular marker of cell activation, in LHb neurons
(Figure 4D-4E).
In LHb brain slices, calcium imaging recording also revealed successful activation of
astrocytes and neurons upon chemogenetic activation of astrocytes (Figure S9A-S9F). In
particular, 55% of the recorded neurons (148 of 267) showed significantly increased calcium
signals upon CNO application (Figure S9F). Among these, 38% were silent, 55% and 7%
showed irregular and regular baseline activity respectively before the CNO application
(Figure S9F). To achieve astrocytic activation in a more transient and temporally controlled
manner, we then tried optogenetic activation by expressing chicken opsin 5 (cOpn5), a
light-sensitive Gq-coupled GPCR that can trigger a blue-light-induced elevation in
intracellular calcium,79 in the LHb astrocytes (Figure 4F). Light stimulation (1 s, 473 nm)
significantly evoked calcium activities in LHb astrocytes (Figure 4G and 4H). And the signals
11
decayed much faster than the CNO-evoked ones (7.84 ± 2.25 s vs. 62.23 ± 23.92 s, Figure 4H
and S9C), closer to the FS-evoked physiological condition (Figure 1G). Using this approach,
we found that upon optogenetic activation of LHb astrocytes, 48% (103 of 216) recorded
LHb neurons were excited (Figure 4I, 4J and S10A-S10B). Among these, 47% were silent, 39%
and 14% showed irregular and regular baseline activity respectively before the stimulation
(Figure 4J). Overall, both chemogenetic and optogenetic activation of astrocyte-Gq pathway
significantly increased the activities of LHb neurons.
While the optogenetic activation of astrocyte-Gq may be less efficient than the chemogenetic
method, it caused a more physiological change (in terms of signal duration) that allowed us to
identify the gliotransmitter(s) mediating the LHb astrocyte-neuron crosstalk. Calcium signals
in astrocytes are known to facilitate the release of various gliotransmitters, including
glutamate,80-82 GABA83,84, ATP or its immediate degradation product adenosine.61,85,86
Therefore we measured LHb neuronal calcium activities triggered by optogenetic stimulation
of LHb astrocytes in brain slices in the presence of antagonists for these gliotransmitter
receptors (Figure 4K). While antagonists for the GABAA (picrotoxin) and adenosine A1 (A1R,
DPCPX) receptors did not show any effect (Figure 4K), those blocking adenosine A2A (A2AR,
SCH58261, Figure 4K and S10D) or ionotropic glutamate receptors (iGluR, APV+NBQX,
Figure 4K and S10E) significantly suppressed astrocyte-stimulated neuronal calcium
responses (34.8% reduction and p = 0.0002 for SCH58261; 32% reduction and p = 0.0008 for
APV+NBQX; paired t test, Figure 4K). 52% and 44% of astrocyte-excited neurons exhibited
significantly reduced calcium activities in response to adenosine A2AR and iGluR blockade
12
respectively (Figure S10D and S10E). On the other hand, blockade of ATP receptors (ATP-R,
PPADS + suramin) had a significant but much smaller effect on astrocyte-stimulated neuronal
calcium responses (10% reduction and p = 0.012; paired t test, Figure 4K). Using
genetically-encoded sensors of adenosine (ADO1.0m)87 or glutamate (iGluSnFR),88 we found
that activation of astrocyte-Gq pathway (CNO, 1 μM, with TTX to exclude indirect release
from neuronal activation) significantly increased the level of adenosine (Figure 4L) and
glutamate (Figure 4M). These results suggest that activation of LHb astrocytes can lead to
activation of LHb neurons through the release of gliotransmitters ATP/Adenosine and
glutamate.
LHb astrocytes account for the second-phase of LHb neural activity and NE release
We next investigated the functional impact of LHb astrocytic activities on the FS-evoked
LHb neuronal activity in vivo (Figure 5). We employed GfaABC1D promoter to drive the
expression of either a short hairpin RNA (shRNA) targeting intracellular IP3R2 for its
knockdown (Figure 5A), or a 122-residue inhibitory peptide from β-adrenergic receptor
kinase1 (iβARK, Figure 5K) to specifically block astrocytic calcium signaling. These
approaches have been developed and validated to attenuate Gq-GPCR-mediated calcium
signaling in astrocytes.89,90 Expression of these two viral constructs in the LHb significantly
reduced FS-evoked calcium in LHb astrocytes by 68% and 51% respectively (p = 0.0002 for
IP3R2-shRNA group, unpaired t test; p = 0.008 for iβARK group, Mann-Whitney test; Figure
S11A-S11F). To examine the contribution of LHb astrocytes to neuronal calcium activities
during FS stress, we then injected either one of these two viruses into the bilateral LHb and
13
expressed GCaMP6s under CaMKIIα promoter in the unilateral LHb, and installed optic
fibers above the LHb for in vivo photometry recording (Figure 5B, 5L, S11G and S11H).
Notably, the second-phase LHb neuronal activity evoked by FS was largely eliminated by
expression of IP3R2-shRNA (Figure 5C and 5D) or iβARK (Figure 5M and 5N) in LHb
astrocytes. For the first-phase FS-evoked neuronal calcium signals, the time to peak, peak
amplitude and AUC from 0 to 2.5s (AUC1) were not changed (Figure 5E-5G and 5O-5Q).
However, for the second-phase FS-evoked neuronal calcium signals, the AUC from 2.5 to 40s
(AUC2) was greatly reduced in mice expression IP3R2-shRNA compared with control (p =
0.005, Mann-Whitney test; Figure 5H and 5I). Consequently, the total AUC from the two
phases was reduced by 55.7 % (p = 0.005, Mann-Whitney test; Figure 5J). Similar effects
were observed in mice infected with iβARK (Figure 5R-5T). These results suggest that LHb
astrocytic calcium activity are required for the second-phase LHb neuronal activity in
response to stress.
Given the identification of the stress-induced positive feedback interactions between LHb and
LC-NE neurons (Figure 3P and 3R) and between LHb neurons and astrocytes (Figure 3A-3C
and 4A-4C), we next sought to examine whether LHb astrocytes can regulate NE release in
the LHb. Following FS, both neuronal calcium and NE-sensor signals in the LHb showed a
biphasic pattern, and the second peak of NE signals followed the second peak of neuronal
calcium signals, both following the peak of astrocytic signals (Figure 3M and 3N). Such
temporal pattern, combined with results in Figure 5A-T, suggest that the reactivation of LHb
neurons by local astrocytes may lead to subsequent reactivation of LC-NE neuron and NE
14
release. To test this possibility, we simultaneously expressed IP3R2-shRNA and NE sensor in
the LHb and performed photometry recording during FS stress. Expression of IP3R2-shRNA
significantly reduced the second-phase NE signals (Figure 5U and 5V). While the AUC of
first-phase FS-evoked NE signals showed no difference, the AUC of second-phase FS-evoked
NE signals was largely reduced in mice expressing IP3R2-shRNA compared with control (p
= 0.004, Mann-Whitney test; Figure 5V). Additionally, the ramp-up of NE signals over
multiple FSs was also inhibited (p = 0.026, Mann-Whitney test; Figure 5U and 5W). These
results suggest that LHb astrocytes do not affect the first-phase, but strongly contribute to the
second-phase NE release in response to stress.
Activation of LHb astrocytes facilitates depression-like behaviors
We next explored the functional consequence of LHb-astrocytic activation on behavioral
response to stress (Figure 6). We first tried to activate LHb astrocytes, by expressing
GfaABC1D-hM3Dq in the LHb (Figure 6D), in a subthreshold stress protocol (1 mA, 1 s, 6◊
FS delivered within 6 min; Figure 6A). This protocol by itself did not induce depression-like
phenotypes (Figure 6B). However, when LHb astrocytes were chemogenetically activated
(i.p., DCZ, deschloroclozapine, 0.5 mg kg-1) before the first FS, mice developed
depression-like phenotypes after this subthreshold protocol (Figure 6C-6E). Compared with
the mCherry-expressing group, mice expressing hM3Dq in LHb-astrocytes were more
immobile in the forced swim test (FST), which models behavioral despair, and showed less
preference for sucrose water in the sucrose preference test (SPT), which models anhedonia
(Figures 6E).
15
Inhibition of LHb astrocytes prevents stress-induced depression-like behaviors
We next tested whether LHb-astrocytic activity is necessary for stress-induced depression.
We tried to inactivate LHb astrocytes by expressing the IP3R-shRNA under the GfaABC1D
promoter (Figure 6I). Mice were then exposed to a series of inescapable and unpredictable FS
stress (1 mA, 1 s, 20◊FS delivered within 20 min; Figure 6F), which was shown to induce
depression-like behaviors (Figure 6G).16,91 Compared with mice expressing the control
shRNA, inhibition of LHb astrocytes with IP3R2-shRNA caused a pronounced reduction in
depressive-like phenotypes (Figure 6H-6J), reducing immobility in the FST (p = 0.0003,
unpaired t-test; Figure 6J) and increasing sucrose preference in the SPT (p = 0.0049,
Mann-Whitney test; Figure 6J).
Discussion
Among the six stress-related brain regions examined (mPFC, HPC, LHb, LH, STR and BLA),
we found that LHb astrocytes and neurons exhibit the most rapid and unique, biphasic
calcium pattern respectively in response to stress. Following FS, there is a sequential
activation of neurons, release of NE from the LC, followed by activation of astrocytes in the
LHb (Figure 3M and 3N). Manipulation experiments revealed a functional bi-directional
signaling between LHb neurons and astrocytes: stress first induces LHb neuronal activation,
which triggers astrocytic calcium signals through LC- and α1A-AR-dependent noradrenergic
signaling (Figure 3); in tandem, through gliotransmitters glutamate and ATP/Adenosine, LHb
16
astrocytes prompt the second-phase of both local LHb neural activity and NE release (Figure
4 and 5). Through such a positive reinforcing loop, LHb-astrocytes facilitate stress-induced
depressive-like behaviors (Figure 7).
Functional implication and possible mechanisms underlying stress-induced biphasic
calcium activities in LHb neurons
An important question in neurophysiology is how neurons maintain persistent activity in
response to transient sensory stimuli. Previously several elegant mechanisms have been
demonstrated, e.g. in the hypothalamus in maintaining a persistent internal emotional state92
or in the cortex in mediating working memory,93,94 involving slow-acting neuromodulators or
recurrent neural network.92-95 Here our study reveals a new mechanistic foundation for a
persistent neural dynamic pattern, in the context of FS-stress, driven by astrocyte signaling.
In particular, we observed that LHb neuronal calcium signals exhibit a biphasic pattern,
incorporating time-locked, fast first-phase transient and heavy-tailed, slow second-phase
signals (Figure 2C and S3A-S3D). Importantly, the second-phase calcium signals, despite
their smaller amplitude, lasted for many tens of seconds and had an AUC that was 3.46 ± 0.78
times that of the first phase (Figure 2C, 2D, S3D and S3E). Therefore, even more calcium
enters into neurons during the second phase, which may activate calcium-dependent genes.
These sets of genes are crucial for driving transcriptional programs, leading to persistent
remodeling of neuronal network.96,97
In the other five brain regions examined, we did not observe such biphasic neural calcium
17
activation in response to FS-stress (Figure S2A-S2E), suggesting that the above mechanism is
region-specific. Such unique dynamics in the LHb may be accounted for by several potential
mechanisms. First, the intrinsic properties of LHb neurons may be distinct. LHb neurons
contain calcium-permeable AMPA receptors (CP-AMPARs)15,17,98 and pacemaker channels,
such as hyperpolarization-activated cyclic nucleotide-gated (HCN) and T-type calcium
channels.18,99 Through the glutamate-CP-AMPARs and adenosine-A2-receptors-coupled
HCN channels,86,100 gliotransmitters released from LHb astrocytes may contribute to the
second-phase neural calcium activation. Second, different types of gliotransmitters and
different dynamics (e.g., onset and duration) of astrocytic activities in different brain regions
may also contribute to this distinct neuronal response.
Functional implication and possible mechanisms underlying stress-induced unique
activities in LHb astrocytes
Compared with the five other brain regions recorded, astrocytic calcium dynamic in the LHb
showed the earliest onset in response to FS-stress (Figure 1D-1G). This unique property may
also be explained by several potential mechanisms. One possibility is that the strength of
astrocytic coupling can potentially influence the speed of calcium propagation.
Astrocytes form interconnected networks via gap junctions, enabling locally-coupled calcium
activity among neighboring astrocytes.35,47,101,102 Heterogeneity of astrocytic gap-junction
coupling in the central nervous system (CNS) may yield diverse calcium dynamics.
Alternatively, different neuromodulator receptor signaling in astrocytes of different regions
may account for varying time course and magnitude of calcium signals.103 For instance, in the
18
context of FS-stress, astrocytes in auditory cortex are regulated by ACh,67 whereas in the LHb,
they are regulated by NE (Figure 3H). Another possibility is that the sensitivity of astrocytes
to NE may be differentially regulated by local neuronal activities. Supporting this, a previous
study in visual cortex demonstrated that local neuronal activity serves as gain control to
modulate the response of astrocytes to NE.42 On the other hand, our study demonstrates that
local neural activity itself is not sufficient (Figure 3D-3F), and the long-range LHb-LC loop
is required, to drive astrocytic calcium in the LHb (Figure 3G-3T).
The function of the stress induced LHb astrocytic activity may go beyond gene regulation in
the local neuron-astrocyte network. Through the second-wave activation of LC-NE neurons,
which have brain-wide projections,104,105 local LHb astrocytes may impact on global neural
activity.
Phasic and tonic NE release
Monoamine release is often subdivided into phasic (rapid kinetics) and tonic (slow kinetics)
modes.106-109 Recent studies using biosensors have revealed both slowly ramping and phasic
signals of monoamines in behaving mice, such as striatal dopamine release during
reward-directed virtual reality and basal forebrain NE release under FS stress.110,111 During
virtual reality experiments, tonic and phasic dopamine signals convey information of speed
and changes in teleportation, respectively.110 These findings provide key insights into
monoamine coding, suggesting that different modes of monoamine release may support
different brain function.106,112
19
Here we found that NE release in the LHb during FS stress exhibited rapid phasic as well as
slowly ramping tonic signals (Figure 3K-3L, S7A and S7C). The phasic NE release appears to
be critical in activating LHb astrocytic calcium signals. Our recording showed that large
increases in LHb astrocytic calcium activity were time-locked to the phasic NE signals
(Figure 3M). Interestingly, although toward later shocks, the absolute amplitude of tonic NE
signals was also high, as high as the amplitude of the early phasic signals, astrocytic calcium
was still not induced (Figure S7). This suggests that to activate
α1-adrenergic-receptor-dependent calcium signaling in LHb astrocytes, what matters is not
only the amount but also the pattern of NE release. Different ligand-binding kinetics (e.g., the
duration of dwell-time) can bias GPCR signaling toward the G-protein- biased or
β-arrestin-biased pathways.113,114 In comparison to acute, sharp increase during phasic NE
release, tonic stead-state increase of NE occurs over minutes time scale, which may induce
bias towards β-arrestin-recruitment and receptor desensitization.113 It will be interesting to
clarify the spatiotemporal organization and functional significance of these distinct NE signal
patterns in the future.
As to the tonic NE signal, it displays the intriguing “ ramp up ” pattern over repeated
footshocks (Figure 3K). We found that LHb astrocytes not only contribute to the
second-phase phasic NE activity, but are also responsible for this ramp-up signal (Figure 5U
and 5W). It will be very interesting to explore whether this increased basal NE activity may
represent a change relevant for depression pathology, and through which mechanism
20
astrocytes contribute to this change.
Astrocyte function in behavioral regulation
Astrocytes were long considered passive bystanders of the brain. However, they are now
emerging as important orchestrators of brain function and behaviors.115,116 Recent studies
have highlighted the essential nature of a cohesive neuron-astrocyte program in influencing
behavioral outcomes. Their coordinated activities span various processes including sensory
information processing,56,117 brain metabolism,118-121 sleep regulation,122 memory
consolidation,123,124 dominance behavior,60 and behavioral-state switching.60,69,125-127 These
interactions are implicated in animal models of psychiatric disorders,128,129 including
hyperactivity with attention deficit,130 obsessive-compulsive-like behavior,55
schizophrenia,131 drug addiction,132 and depression.20,40,133 Adding to this general theme, our
results demonstrate that LHb astrocytes play an essential role in stress-induced
depression-like behaviors. Furthermore, we reveal a dynamic interaction and information
flow between neurons and astrocytes in this process. By dissecting the cellular and signaling
pathways involved, we outline a stress-induced recurrent network defined by LHb neurons,
LC-NE neurons and LHb astrocytes, highlighting LHb astrocytes as a critical signaling relay
in this positive feedback loop. Through the broadcast by the LHb-LC neural circuit, local
LHb astrocytes can then regulate global brain activity. Collectively, our findings provide a
framework for understanding mechanisms of neuron-astrocyte interaction underlying
stress-induced depression, with significant implications for the LHb-LC loop as a central hub
in psychiatric disorders.
21
Limitations of the study
One of the limitations is that the biphasic calcium activity of LHb neurons was derived from an
analysis of bulk signals. Without recording at single-cell solution, we could not differentiate
whether the two-phase activity involves the same or different subpopulations of LHb neurons.
Miniscope/endoscope imaging in the LHb, which is technically highly challenging, should be
explored in the future. Another limitation is that we did not provide experimental data to
explain why LHb astrocytes respond most rapidly to stress. We have discussed multiple
candidate mechanisms above, and further investigation is required to elucidate the underlying
processes.
Acknowledgments
We thank Yingzhuo Gou, Yang Xiong, Min Chen, Huafeng Zhang, Shiqi Wang and colleagues
from Hu lab for assistance with experiments; Minmin Luo for providing the
GfaABC1D-cOpn5-mCherry virus; Xiaowei Chen and Kuan Zhang for gifting the
GfaABC1D-lck-GCaMP6f virus; Qin Han and Hangjun Wu at the Center of Cryo-Electron
Microscopy for technical assistance with two-photon imaging; and Hui Li and Xiaoke Xie for
advice on two-photon imaging. Some graphic components were created with BioRender. This
work was supported by the STI2030-Major Projects (2021ZD0203000 and 2021ZD0203001),
the National Natural Science Foundation of China (32130042, 31830032 and 82288101), the
New Cornerstone Science Foundation to H.H., Key R&D Program of Zhejiang
(2024SSYS0016), the Fundamental Research Funds for the Central Universities
22
(2023ZFJH01-01 and 2024ZFJH01-01), the Non-profit Central Research Institute Fund of
Chinese Academy of Medical Sciences (2023-PT310-01), and the Project for Hangzhou
Medical Disciplines of Excellence, and Key Project for Hangzhou Medical Disciplines to
H.H..
Author Contributions
H.H., Q.X., and J.W. designed the study. Q.X., J.W., and, J.Z. conducted the behavioral
pharmacology experiments and the related behavioral analysis. Q.X., and, J.W. performed the
in vivo fiber photometry recording. J.W., and, Q.X. performed the in vitro two-photon
imaging. Q.X. performed immunohistochemistry experiments with the assistance of J.Z., Y.T.,
X.J. Z.N. assisted J.W., and, Q.X. in analyzing two-photon imaging data. Y.L., J.F., and, Z.W.
shared NE sensor and adenosine sensor. X.L., and, H.M. contributed to experimental design
and discussions. H.H. supervised the project and wrote the manuscript with the assistance of
Q.X. and J.W.
Figure legends
Figure 1. Foot-shock-stress Induces Most Rapid Calcium Response in LHb Astrocytes
(A) Schematic illustrating viral construct for astrocyte-specific expression of GCaMP and multi-site fiber
photometry setup for recording astrocytic calcium activities during FS.
(B) Illustration of viral expression (stained with antibodies against GFP and Hoechst) and optic fiber
placement (indicated by the yellow dotted line) in 6 central nervous system (CNS) regions, the
boundary of which is outlined by the white dotted line. Green, GCaMP6f; blue, Hoechst. Scale bar, 200
μm.
(C) GCaMP expression is highly specific in astrocytes. Top: representative image of LHb brain slices
stained with antibodies against GFP (which indicates GCaMP positive cells, green) and S100β
(astrocytic marker, red). Scale bar, 100 μm. Bottom: bar graph showing percentage of GCaMP-positive
cells that are S100β-positive (n = 7 slices from 3 mice).
(D) Representative traces (left) and heatmap (right) of delta F/F ratio of simultaneously recorded
23
astrocytic calcium signals in LHb, LH and PFC over three continuous FSs. Scale bars, 50% delta F/F,
5s (left).
(E) Alignment of FS-onset plots of calcium signal changes of simultaneously recorded multi-region
astrocytes during FS.
(F) Illustration of calculation of rise and decay time.
(G) Average time to peak (left), rise time (middle) and decay time (right) of astrocytic calcium signals
evoked by FS in 6 brain regions. Each circle represents one mouse.
(H) Average peak amplitudes of astrocytic calcium signals evoked by FS in 6 brain regions. Each circle
represents one mouse.
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. Data are represented as mean
± SEM.
Figure 2. Foot-shock-stress Induces Biphasic Calcium Activities in LHb Neurons

(A) Illustration of viral construct for neuron-specific expression of GCaMP, viral expression and optic
fiber placement (indicated by the yellow dotted line) in the LHb, the boundary of which is outlined by
the white dotted line. Green, GCaMP6s; blue, Hoechst. Scale bars, 100 μm.
(B) Heatmap of delta F/F ratio of calcium signals from LHb neurons (n = 47 trials from 8 mice) aligned
to onset of FS. Color bars on the left indicate different mice.
(C) Average delta F/F ratio of calcium signals from LHb neurons aligned to onset of FS. Solid lines
indicate mean and shaded areas indicate SEM.
(D) Average time to peak (left), average time to inflection (middle) of LHb neuronal calcium signals
from FS onset, and ratio of area under curve (AUC) of FS-evoked signals after and before the inflection
point (right). AUC1: AUC of neuronal calcium signal in 0-inflection from FS onset. AUC2: AUC of
neuronal calcium signal in inflection-10%peak. Each circle represents one mouse.
(E) Illustration of dual-color neuronal expression of jRGECO1a and astrocytic expression of GCaMP6f
and optic fiber placement (indicated by the yellow dotted line) in LHb. Scale bars, 100 μm.
(F) Representative traces of simultaneously recorded neuronal and astrocytic calcium signals in LHb
over three continuous FSs. Scale bars, 20% delta F/F, 10s.
(G) Delta F/F ratio of calcium signals from LHb neurons and LHb astrocytes aligned to the onset of FS.
Solid lines indicate mean and shaded areas indicate SEM.
(H) Average time to peak of LHb neuronal (N-Peak, left) and astrocytic (A-Peak, middle) calcium
signals and average time to inflection of LHb neuronal calcium signals from FS onset (N-inflection,
right) in dual-color fiber photometry system. Each circle represents one mouse.
Figure 3. Neuron-to-Astrocyte Crosstalk in LHb during FS

(A) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons and fiber
photometry recording of nearby GCaMP6f-expressing astrocytes in LHb in vivo.
(D) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons and two-photon
imaging of nearby GCaMP6f-expressing astrocytes in LHb slices in vitro.
(B, E) Astrocytic calcium responses to LHb neuronal ChrimsonR activation (1s, 40 Hz) in vivo (B) and
in LHb slices (E).
(C, F) Mean astrocytic calcium responses to varying stimulation frequencies in vivo (C) and in LHb
slices (F).
(G) Schematic of in vivo fiber photometry recording of LHb astrocytes in response to FS after i.p.
24
injection of various receptor blockers.

(H) Bar graph showing effects of MPEP (mGluR5 antagonist), LY341495 (mGluR2/3 antagonist),
Scopolamine (mAChR antagonist), Mecamylamine (nAChR antagonist), Prazosin (α1-AR antagonist),
Propranolol (β-AR antagonist) and Atipamezole (α2-AR antagonist) on FS-evoked calcium signals in
LHb astrocytes. Each circle represents one mouse.
(I) Bar graph showing effects of a1-AR subtype-selective antagonists, including Silodosin (α1A-AR
antagonist), L-765314 (α1B-AR antagonist), BMY 7378 (α1D-AR antagonist) on FS-evoked calcium
signals in LHb astrocytes. Each circle represents one mouse.
(J-L) In vivo fiber photometry recording of NE sensor signals in LHb during FS. Viral expression (J,
stained with antibodies against GFP and Hoechst) after injection of AAV2/9-hSyn-NE2h and optic fiber
placement (indicated by the yellow dotted line) in LHb, the boundary of which is outlined by the white
dotted line. Scale bar, 100 μm. Example trace (K) of LHb NE sensor signals during FS (black) and
homecage (HC, grey). Scale bars, 5% delta F/F, 50s. Plots (L, left) of delta F/F ratio of NE sensor
signals aligned to the onset of FS. Solid lines indicate mean and shaded areas indicate SEM. Heatmap
(L, right) of delta F/F ratio of NE sensor signals in the LHb aligned to the onset of FS (n = 46 trials from
5 mice). Color bars on the left indicate different mice.
(M) Plots of delta F/F ratio of calcium signals from LHb neurons, LHb astrocytes, and LHb-NE-sensor
signals aligned to onset of FS stress. Solid lines indicate mean and shaded areas indicate SEM. Data
from 2E-H and 3L.
(N) Average time to peak of LHb neuronal calcium signals, LHb-NE-sensor signals and LHb astrocytic
calcium signals from FS onset (left) and average time to inflection of LHb neuronal calcium signals and
LHb-NE-sensor signals (right). Data from 2E-H and 3L.
(O) Schematic illustrating optogenetic activation of LHb-LC terminals and fiber photometry recording of
LC NE neurons in TH-Cre mice.
(P) Calcium response of LC NE neurons to optogenetic activation of LHb-LC terminals (1s, 40 Hz).
Plots of delta F/F ratio of calcium signal aligned to laser onset. Red line represents light-on. Solid lines
(Q) Schematic illustrating optogenetic activation of LCTH-LHb terminals and fiber photometry recording
of LHb astrocytes in TH-Cre mice.
(R) Calcium response of LHb astrocytes to optogenetic activation of LCTH-LHb terminals (1s, 40Hz).
Plots of averaged delta F/F ratio of astrocytic calcium signal induced by optogenetic activation of
LCTH-LHb terminals after i.p. injection of saline (black) or Prazosin (green, α1-AR antagonist) aligned
to laser onset. Red line represents light-on. Solid lines indicate mean and shaded areas indicate SEM.
(S) Bar graph showing effects of Prazosin (α1-AR antagonist) on astrocytic calcium signals induced by
optogenetic activation of LCTH-LHb terminals. Each circle represents one mouse.
(T) Working model summarizing the neuron-to-astrocyte crosstalk in the LHb during FS. Stress
activates LHb neurons, which recruit LC NE neurons to elevate LHb astrocytic calcium via NE-α1A
signaling.
± SEM.
Figure 4. Astrocyte-to-Neuron Crosstalk in the LHb

(A) Representative image showing viral expression of GCaMP6s in LHb neurons and hM3Dq in LHb
astrocytes. Scale bar, 100 μm.
25
(B-C) In vivo fiber photometry recording of LHb neuronal calcium in response to LHb astrocytic hM3Dq
activation. Representative raw trace (B) and bar chart (C) showing mean AUC of LHb neuronal calcium
signals following an intraperitoneal injection of saline or clozapine. Each circle represents one mouse.
(D) Representative images of c-Fos IHC signals following unilateral astrocytic hM3Dq activation in LHb.
Note c-Fos signals are only present in injected side. Green, c-Fos; blue, Hoechst. Scale bar, 40 μm.
(E) Quantification of total c-Fos+ cells in LHb.
(F) Representative image of LHb brain slices with viral expression of cOpn5 in astrocytes. Scale bar,
40 μm.
(G) Schematic (left) and representative two-photon image (right) illustrating optogenetic activation of
cOpn5-expressing astrocytes while recording two-photon images of LHb astrocytic calcium responses.
Scale bar: 50 μm.
(H) Plots of delta F/F ratio of calcium signals in LHb astrocytes aligned to laser onset. Solid lines
(I) Schematic (left) and representative two-photon image (right) illustrating optogenetic activation of
cOpn5-expressing astrocytes while recording two-photon images of LHb neuronal calcium responses.
Scale bar: 50 μm.
(J) Pie chart illustrating percent abundance of excited neurons by astrocytic cOpn5 activation (left, n =
4 slices from 4 mice). Colored outer circle indicates percentage of three types (according to baseline
activity) among astrocytic-hM3Dq-excited neurons. Representative raw calcium traces from the three
types of astrocytic cOpn5-excited neurons (right). Blue line represents light-on.
(K) Bar graph showing effects of ACSF (control), picrotoxin (GABAA receptor antagonist), PPADS and
suramin (ATP-R antagonists, ATP receptor antagonists), DPCPX (ADO A1R antagonists, adenosine A1
receptor antagonist), SCH58261 (ADO A2AR antagonist, adenosine A2A receptor antagonist) and APV
and NBQX (iGlu receptor antagonists) on astrocytic cOpn5-evoked neuronal calcium signals in LHb
slices. Each circle represents one neuron.
(L, M) Illustration of AAVs used for expressing hM3Dq and ADO1.0m (L) or iGluSnFR (M) in LHb
astrocytes (left top), fluorescence images (left bottom) and traces (right) illustrating astrocyte-induced
adenosine (L) or glutamate (M) release in LHb slice. Scale bar: 100 μm. (Experiments were performed
in TTX).
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Data are represented as mean
± SEM.
Figure 5. LHb Astrocytic Ca2+ Signaling Is Required for the Second-phase Neuronal Activities
and NE Release
(A) Left: illustration of blockade of astrocytic calcium signaling by IP3R2-shRNA. Right: viral
expression of IP3R2-shRNA in astrocytes. Scale bar, 100 μm.
(B) Schematic illustrating the viral expression of astrocyte-specific IP3R2-shRNA or Ctrl-mCherry in
bilateral LHb and neuron-GCaMP6s in unilateral LHb, and fiber photometry recording of neurons in
LHb.
(C, D) Plots of averaged delta F/F ratio (C) and heatmap (D) of LHb neuronal calcium signals aligned
to the FS onset in mice expressing Ctrl-mCherry (grey, n = 69 trials from 7 mice) or IP3R2-shRNA (red,
n = 50 trials from 5 mice) in LHb astrocytes.
(E-J) Quantification of Time to peak (E), Peak amplitude (F), AUC1 in 0-2.5s (G), AUC2 in 2.5-40s (H),
AUC ratio (I) and total AUC in 0-40s (J) of FS-evoked LHb neuronal calcium signals in mice expressing
26
Ctrl-mCherry or IP3R2-shRNA in LHb astrocytes. Each circle represents one mouse.

(K) Left: illustration of blockade of astrocytic calcium signaling by iβARK. Right: viral expression of
iβARK in astrocytes. Scale bar, 100 μm.
(L) Schematic illustrating the viral expression of astrocyte-specific iβARK or Ctrl-mCherry in bilateral
LHb and neuron-GCaMP6s in unilateral LHb, and fiber photometry recording of neurons in LHb.
(M, N) Plots of averaged delta F/F ratio (M) and heatmap (N) of LHb neuronal calcium signals aligned
to the FS onset in mice expressing Ctrl-mCherry (grey, n = 22 trials from 4 mice) or iβARK (red, n = 30
trials from 5 mice) in LHb astrocytes.
(O-T) Quantification of Time to peak (O), Peak amplitude (P), AUC1 in 0-2.5s (Q), AUC2 in 2.5-40s (R),
AUC ratio (S) and total AUC in 0-40s (T) of FS-evoked LHb neuronal calcium signals in mice
expressing Ctrl-mCherry or iβARK in LHb astrocytes. Each circle represents one mouse.
(U) Example trace (left) and plots of averaged delta F/F ratio (right) of LHb NE signals during FS in
mice expressing Ctrl-mCherry (grey, n = 42 trials from 7 mice) or IP3R2-shRNA (red, n = 42 trials from
7 mice) in LHb astrocytes.
(V, W) AUC1 in 0-inflection and AUC2 in inflection-40s of LHb NE signals aligned to FS onset (V) and
basal (tonic) delta F/F ratio of LHb-NE signals in response to FS (W) in mice expressing Ctrl-mCherry
(grey) or IP3R2-shRNA (red) in LHb astrocytes.
± SEM.
Figure 6. LHb Astrocytes Regulate Stress-induced Depression-like Behaviors
(A) Cartoon illustration of a subthreshold depression-induction protocol with 6◊FS.
(B) Subthreshold 6×-FS protocol did not induce depression-like behaviors in FST (n =8, 8 mice for
naive and FS group, respectively) and SPT (n =8, 8 mice for naive and FS group, respectively).
(C) Experimental paradigm for activating Gq pathway of LHb astrocytes during the subthreshold
depression-induction protocol.
(D) Illustration of viral expression of hM3Dq in LHb astrocytes. Scale bar, 100 μm.
(E) Depression-like behaviors in FST (n =13, 12, 10, 14 mice for Ctrl-mCherry/DCZ, hM3Dq/DCZ,
Ctrl-mCherry/saline and hM3Dq/Saline group, respectively) and SPT (n =10, 12, 10, 12 mice for
Ctrl-mCherry/DCZ, hM3Dq/DCZ, Ctrl-mCherry/saline and hM3Dq/Saline group, respectively) of mice
expressing Ctrl-mCherry (grey) or hM3Dq (blue) in LHb astrocytes.
(F) Cartoon illustration of a depression-induction protocol with 20◊FS.
(G) 20×-FS protocol induced depression-like behaviors in FST (n =16, 16 mice for naive and FS group,
respectively) and SPT (n =15, 17 mice for naive and FS group, respectively).
(H) Experimental paradigm for inhibiting calcium signaling of LHb astrocytes during the
(I) Illustration of viral expression of IP3R2-shRNA in LHb astrocytes. Scale bar, 100 μm
(J) Depression-like behaviors in FST (n =17, 17 mice for Ctrl-mCherry and IP3R2-shRNA group,
respectively) and SPT (n =15, 16 mice for Ctrl-mCherry and IP3R2-shRNA group, respectively) of mice
expressing Ctrl-mCherry (grey) or IP3R2-shRNA (red) in LHb astrocytes.
± SEM.
27
Figure 7. LHb neuron-astrocyte synergy in mediating depression-like behaviors

(A) A model summarizing the LHb neuron-astrocyte synergy in mediating depression-like behaviors.
Stress first activates LHb neurons, which in term recruit LC-NE neurons. NE release in LHb then
triggers astrocytic calcium activities through α1AAR-dependent signaling. In tandem, through
gliotransmitters glutamate (Glu) and ATP/Adenosine (Ado), LHb astrocytes prompt the second-phase
of both local LHb neural activities and NE release. Through such a positive reinforcing loop and
regulation of global gene expression, LHb astrocytes facilitate stress-induced depression-like
behaviors.
Supplementary figure legends

Figure S1. Foot-shock-stress Induces Most Rapid Calcium Response in LHb Astrocytes,
Related to Figure 1
(A) GfaABC1D-promoter-driven GCaMP is not expressed in neurons. Left and middle: representative
images of LHb brain slices stained with antibodies against GFP (indicating GCaMP-positive cells,
green) and NeuN (neuronal marker, red). Scale bar, 50 μm. Right: bar graph showing percentage of
GCaMP-positive cells that are NeuN-positive (n = 7 slices from 4 mice).
(B) Scatterplots showing the relationship between peak amplitude and time to peak of LHb astrocytic
calcium signals evoked by FS. Each circle represents one mouse.
± SEM.
Figure S2. Neuronal Calcium Response Evoked by Foot-shock-stress in Other Five Brain
Regions, Related to Figure 2
(A-E) Left: Illustration of viral expression and optic fiber placement (indicated by the yellow dotted line)
in BLA (A), mPFC (B), HPC (C), LH (D) and STR (E), the boundary of which is outlined by the white
dotted line. Green, GCaMP6s; blue, Hoechst. Scale bars, 100 μm. Middle: Average delta F/F ratio of
neuronal calcium signals from BLA (A, n = 3 mice), mPFC (B, n = 7 mice), HPC (C, n = 7 mice), LH (D,
n = 3 mice) and STR (E, n = 3 mice) aligned to the onset of FS. Solid lines indicate mean and shaded
areas indicate SEM. Right: representative traces of recorded neuronal calcium signals from the 5 CNS
regions during homecage (top) and continuous FSs (bottom).
Figure S3. Foot-shock-stress Induces Biphasic Calcium Activities in LHb Neurons, Related to
Figure 2
(A) Representative traces of recorded neuronal calcium signals in LHb over three continuous FSs.
Scale bars, 20% delta F/F, 10s.
(B) Alignment of FS-onset plots of average calcium signals of recorded neurons from individual animal
during FS (n = 8 mice). Each circle represents one mouse.
(C) Illustration of the segmented phases of FS-evoked LHb neuronal calcium signals.
(D) Average half-decay time in phase-1 and phase-2 of FS-evoked neuronal calcium signals. Each
circle represents one mouse.
(E) Average peak amplitude in phase-1 and phase-2 of FS-evoked neuronal calcium signals. Each
28
± SEM.
Figure S4. Optogenetic Activation of ChrimsonR-expressing Neurons Induces Neuronal

Calcium in LHb Slice, Related to Figure 3
(A) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons while recording
two-photon images of LHb neuronal calcium responses.
(B) Calcium fluorescence images of LHb neurons before (B, left) and after (B, right) optogenetic
activation of LHb neurons (1s, 40 Hz).
(C) Neuronal calcium responses to optogenetic activation of LHb neurons (1s, 40 Hz) in LHb slices.
(D) Mean neuronal calcium responses to varying stimulation frequencies in LHb slices.
Figure S5. Calcium Activities Evoked by FS in LHb Astrocytes Require Noradrenergic Signaling,
Related to Figure 3
(A-C) Plots of averaged delta F/F ratio (left) and heatmap (middle) of FS-evoked calcium signals in the
LHb astrocytes before and after i.p. injection of Prazosin (α1-AR antagonist, A), Atipamezole (α2-AR
antagonist, B) and Propranolol (β-AR antagonist, C) aligned to the FS onset. Bar graph showing
effects of NE receptor antagonists on FS-evoked calcium signals in LHb astrocytes (right). Each circle
± SEM.
Figure S6. NE Triggers Calcium Signals in LHb Astrocytes via α1-AR, Related to Figure 3
(A) Schematic illustrating in vitro calcium imaging of LHb astrocytes in slices.
(B) Calcium fluorescence images of LHb astrocytes showing calcium increase at different time points
after NE application (n = 8 slices from 8 mice). Scale bar: 50 μm. (Experiments were performed in
TTX).
(C) Plots of delta F/F ratio (left) and heatmap (right) of calcium signals in LHb astrocytes induced by
NE (n = 8 slices from 8 mice). Solid lines indicate mean and shaded areas indicate SEM.
(D, E) Calcium fluorescence images of LHb astrocytes before (D, left) and after (D, right) application of
phenylephrine (PE, α1-AR agonist). Traces (E) of LHb astrocytic calcium signals before and after PE
application (n = 6 slices from 4 mice). Scale bars, 20% delta F/F, 30s. (Experiments were performed in
TTX).
(F) Schematic illustrating viral injection of Cre-dependent GCaMP6s in LHb in Aldh1l1-Cre mice and in
vitro calcium imaging of LHb astrocytes in response to NE after application of NE receptor antagonist.
ROI detection using a grid array (see STAR Methods for details).
(G) Representative calcium fluorescence images (left) and bar graph (right) showing effects of
Prazosin (α1-AR antagonist), Propranolol (β-AR antagonist) and Atipamezole (α2-AR antagonist) on
LHb astrocytic calcium signals evoked by NE. Scale bar: 50 μm. Each circle represents one slice.
± SEM.
Figure S7. LHb NE Sensor and Astrocytic Calcium Signals Induced by FS, Related to Figure 3
(A) Example trace of LHb-NE-sensor signals over multiple FSs. Scale bars, 5% delta F/F, 50s.
29
(B) AUC of basal (tonic) signal of LHb NE sensor in homecage (grey) and in response to FS (red).
Each circle represents one mouse.
(C) Plots showing basal (tonic) signal change of LHb NE sensor in response to FS.
(D) Example trace of LHb astrocytic calcium signals over multiple FSs. Scale bars, 5% delta F/F, 50s.
(E) AUC of basal signal of LHb astrocytic calcium in homecage (grey) and in response to FS (red).
Each circle represents one mouse.
(F) Plots showing basal signal change of LHb astrocytic calcium in response to FS.
± SEM.
Figure S8. Effects of manipulating LHb and LC-NE Neurons on LHb Astrocytic Calcium, Related
to Figure 3
(A, B) Effects of activating LHb-LC terminals on LC-NE neurons. (A) Representative images showing
viral expression of ChrimsonR in LHb neurons (left), GCaMP6s in LC-NE neurons (middle) and LC-NE
neuron terminals in LHb (right, stained with antibody against GFP) in TH-Cre mice. Scale bar, 100 μm.
(B) Calcium responses of LC-NE neurons to optogenetic activation of LHb-LC terminals (1s, 40 Hz) in
vivo.
(C, D) Effects of activating LCTH-LHb terminals on LHb astrocytes. (C) Representative images showing
viral expression of ChrimsonR in LC NE-neurons (left), LCTH-LHb terminals (middle, stained with
antibody against RFP) and GCaMP6f in LHb astrocytes (right) in TH-Cre mice. Scale bar, 100 μm. (D)
TH
Calcium responses of LHb astrocytes to optogenetic activation of LC -LHb terminals (1s, 40 Hz).
(E-H) Effects of Prazosin (α1-AR antagonist) on LHb astrocytic calcium signals evoked by LHb neuron
activation in vivo. (E) Schematic illustrating optogenetic activation of LHb neruons and fiber photometry
recording of LHb astrocytes. (F) Representative image showing viral expression of ChrimsonR in LHb
neurons and GCaMP6f in LHb astrocytes. Scale bar, 100 μm. (G) Plots of averaged delta F/F ratio of
LHb astrocytic calcium signals induced by LHb neurons before and after i.p. injection of Prazosin
(α1-AR antagonist) aligned to the laser onset. Solid lines indicate mean and shaded areas indicate
SEM. (H) Bar graph showing the effects of Prazosin (α1-AR antagonist) on astrocytic calcium signals
evoked by LHb neurons. Each circle represents one mouse.
(I-L) Effects of inhibiting LHb neurons on FS-evoked calcium signals in LHb astrocytes. (I) schematic
illustrating optogenetic inhibition of LHb neruon and fiber photometry recording of LHb astrocytes in
response to FS. (J) Representative image showing viral expression of eNpHR in LHb neurons and
GCaMP6f in LHb astrocytes. Scale bar, 100 μm. (O) Plots of averaged delta F/F ratio of FS-evoked
calcium signals in LHb astrocytes during light off and light on aligned to FS onset. Solid lines indicate
mean and shaded areas indicate SEM. (L) Bar graph showing effects of inhibiting LHb neurons on
FS-evoked calcium signals in LHb astrocytes. Each circle represents one mouse.
(M-P) Effects of inhibiting LC-NE neurons on FS-evoked calcium signals in LHb astrocytes. (M)
Schematic illustrating chemogenetic inhibition of LC-NE neurons and fiber photometry recording of
LHb astrocytes in response to FS. (N) Representative image showing viral expression of hM4Di in
LC-NE neurons. Scale bar, 100 μm. (K) Plots of averaged delta F/F ratio of FS-evoked calcium signals
in LHb astrocytes before and after i.p. injection of clozapine (Cloz) aligned to FS onset. Solid lines
indicate mean and shaded areas indicate SEM. (P) Bar graph showing effects of inhibiting LC
NE-neurons on FS-evoked calcium signals in LHb astrocytes. Each circle represents one mouse.
30
± SEM.
Figure S9. Chemogenetic Activation of Astrocytic Gq Pathway in LHb Slice, Related to Figure 4
(A, B) Schematic (A) and representative calcium fluorescence images (B) illustrating chemogenetic
activation of hM3Dq-expressing astrocytes while recording calcium fluorescence images of LHb
astrocytic calcium responses. Scale bar: 50 μm.
(C) Plots of delta F/F ratio (left) and heatmap (right) of calcium signals in LHb astrocytes before and
after CNO application (n = 3 mice). Solid lines indicate mean and shaded areas indicate SEM.
(D, E) Schematic (D) and representative two-photon image (E) illustrating chemogenetic activation of
hM3Dq-expressing astrocytes while recording two-photon image of LHb neuronal calcium responses.
Scale bar: 50 μm.
(F) Pie chart illustrating percent abundance of excited neurons by astrocytic hM3Dq activation (left, n =
3 slices from 2 mice). Colored outer circle indicates percentage of three types (according to baseline
activity) among astrocytic-hM3Dq-excited neurons. Representative raw calcium traces from the three
types of astrocytic hM3Dq-excited neurons (right).
Figure S10. Optogenetic Activation of Astrocytic Gq Pathway in LHb Slice, Related to Figure 4
(A) Schematic illustrating optogenetic activation of cOpn5-expressing astrocytes while recording
two-photon images of LHb neuronal calcium responses.
(B) Left: example spatial map of astrocytic cOpn5-excited and non-responsive neurons in the LHb.
Right: representative raw calcium traces from the three types of non-responsive neurons. Blue line
represents light-on.
(C-E) Traces of LHb neuronal calcium signals induced by astrocyte-cOpn5 before (left) and after
(middle) application of various receptor antagonists. Right: bar graph showing effects of ACSF (control,
C), SCH58261 (adenosine A2AR antagonist, D) and APV/NBQX (iGluR antagonists, E) on LHb
neuronal calcium signals induced by astrocyte-cOpn5. Each circle represents one neuron.
Figure S11. Inhibition of Astrocytic Calcium Signaling in LHb, Related to Figure 5

(A-C) Schematic (A) illustrating the viral expression of astrocyte-specific IP3R2-shRNA or Ctrl-mCherry
and GCaMP6f, and fiber photometry recording of astrocytes in LHb in response to FS. Representative
image (B) illustrating viral expression of IP3R2-shRNA and GCaMP6f in LHb astrocytes. Scale bar,
100 μm. Quantification of peak amplitude of FS-evoked LHb astrocytic calcium signals in mice
expressing Ctrl-shRNA (grey, n = 12 mice) or IP3R2-shRNA (red, n = 12 mice) in LHb astrocytes (C).
(D-F) Schematic (D) illustrating the viral expression of astrocyte-specific iβARK or Ctrl-mCherry and
GCaMP6f, and fiber photometry recording of astrocytes in LHb in response to FS. Representative
image (E) illustrating viral expression of iβARK and GCaMP6f in LHb astrocytes. Scale bar, 100 μm.
Quantification of peak amplitude of FS-evoked LHb astrocytic calcium signals in mice expressing
mCherry (grey, n = 7 mice) or iβARK (red, n = 6 mice) in LHb astrocytes (F).
(G) Schematic (left) illustrating the viral expression of astrocyte-specific IP3R2-shRNA or Ctrl-mCherry
and neuronal GCaMP6s, and fiber photometry recording of neurons in LHb in response to FS.
Representative image (right) illustrating viral expression of IP3R2-shRNA in LHb astrocytes and
GCaMP6s in LHb neurons. Scale bar, 100 μm.
(H) Schematic (left) illustrating the viral expression of astrocyte-specific iβARK or Ctrl-mCherry and
neuronal GCaMP6s, and fiber photometry recording of neurons in LHb in response to FS.
31
Representative image (right) illustrating viral expression of iβARK in LHb astrocytes and GCaMP6s in
LHb neurons. Scale bar, 100 μm.
± SEM.
STAR Methods
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit anti-c-fos Synaptic Systems Cat# 226008;
RRID:AB_2891278
Mouse anti-NeuN Millipore MAB337;
RRID:AB_2298772
Rabbit anti-S100β Dako Products Z0311;
RRID:AB_10013383
Chicken anti-GFP Aves Labs GFP-1010;
RRID:AB_2307313
32
Rabbit anti-GFP Invitrogen A11122;

RRID:AB_221569
Chicken anti-TH Aves Labs TYH-0020;
RRID:AB_10013440
Rabbit anti-TH Santa Cruz sc-14007;
Biotechnology RRID:AB_671397
Rabbit anti-RFP Rockland 600-401-379;
RRID:AB_2209751
Goat anti-rabbit Alexa Fluor 546 Invitrogen A11035;
RRID:AB_2534093
Goat anti-rabbit Alexa Fluor 488 Invitrogen A11034;
RRID:AB_2576217
Goat anti-mouse Alexa Fluor Cy5 Invitrogen A10524;
RRID:AB_10562712
Goat anti-chicken Alexa Fluor 488 Invitrogen A11039;
RRID:AB_2534096
Goat anti-mouse Alexa Fluor 488 Invitrogen A11029; RRID:AB_2534088
Goat anti- mouse Alexa Fluor 546 Invitrogen A11030; RRID:AB_2737024
Bacterial and virus strains
AAV2/9-CaMKIIa-GCaMP6s Addgene Cat# 107790-AAV9
AAV2/9-hSyn-jRGECO1a Addgene Cat# 100854-AAV9
AAV2/5-GfaABC1D-cyto-GCaMP6f Addgene Cat# 52925-AAV5
AAV2/5-GfaABC1D-lck-GCaMP6f Addgene Cat# 2924-AAV5
AAV2/9-CAG-Flex-GCaMP6s-WPRE-pA Taitool Bioscience Cat# S0354-9
(Shanghai)
AAV2/9-EF1a-DIO-Axon-GCaMP6s-WPRE-pA BrainVTA Cat# PT-1227
(Wuhan)
AAV2/9-hSyn-GRABNE2h-WPRE-SV40-pA Vigene Cat# YL003011-AAV9
(Shandong)
AAV2/5-GfaABC1D-SFiGluSnFR(A184S)-WPR BrainVTA Cat# PT-2450
E-SV40pA (Wuhan)
AAV2/5-GfaABC1D-Ado1.0m-WPRE-SV40pA BrainVTA Cat# PT-9019
(Wuhan)
AAV2/9-hSyn-eNpHR3.0-mCherry-WPRE-pA Taitool Bioscience Cat# S0463-9
(Shanghai)
AAV2/9-EF1a-DIO-hM4Di-EGFP-WPRE-pA BrainVTA Cat# PT-0987
(Wuhan)
AAV2/9-hSyn-mCherry-WPRE-pA Taitool Bioscience Cat# S0238-9
(Shanghai)
AAV2/5-GfaABC1D-hM3Dq-mCherry Taitool Bioscience Cat# S0483-5
(Shanghai)
AAV2/9-hSyn-ChrimsonR-tdTomato-WPRE-SV Taitool Bioscience Cat# S0459-9
40pA (Shanghai)
33
AAV2/9-hSyn-Flex-ChrimsonR-mCherry-WPRE Taitool Bioscience Cat# S0739-9

-pA (Shanghai)
AAV2/5-GfaABC1D-mCherry Sunbio Medical PAAVE3126
Biotechnology
(Shanghai)
AAV2/8-GfaABC1D-cOpn5-T2A-mCherry-WPR Luo Lab
E-pA (Chinese Institution for
Brain Research, Beijing)
AAV2/5-GfaABC1D-cOpn5-T2A-mCherry-WPR Taitool Bioscience AAV2/5-XT670
E-pA (Shanghai)
AAV2/5-GfaABC1D-iβARK-p2A-mCherry-WPR Sunbio Medical Cat# PSE5374
E-pA Biotechnology
(Shanghai)
AAV2/5-GfaABC1D-IP3R2-shRNA-mCherry-W Sunbio Medical PAAVE3465
PRE-pA Biotechnology
(Shanghai)
Chemicals, peptides, and recombinant proteins
Tetrodotoxin citrate alomone labs T-550
(TTX)
Norepinephrine (NE) Sigma HY-13715
Picrotoxin (PTX) Tocris 1128
Serotonin MCE HY-B1473A
γ-Aminobutyric acid Sigma A2129
Dopamine APExBIO B1482
Clozapine N-oxide Sigma C0832
Deschloroclozapine MCE HY-42110
Clozapine MCE HY-14539
PPADS tetrasodium MCE HY-101044
Suramin hexasodium salt APExBIO B6752
DPCPX MCE HY-100937
SCH582661 MCE HY-19533
APV Sigma A5282
NBQX MCE HY-15068
Prazosin hydrochloride MCE HY-B0193A
Atipamezole hydrochloride MCE HY-12380
Propranolol hydrochloride MCE HY-B0573
Scopolamine Tocris 114-49-8
Mecamylamine hydrochloride MCE HY-B1395
MPEP hydrochloride MCE HY-14609
LY341495 MCE HY-70059
Silodosin MCE HY-10122
34
L-765314 MCE HY-101385

BMY-7378 Tocris HY-100554
Experimental models: Organisms/strains
C57BL/6J mice Qizhen or Jihui N/A
Laboratory animal
Shanghai
Aldh1l1-Cre The Jackson Laboratory JAX. 023748
TH-Cre The Jackson Laboratory JAX. 008601
Software and algorithms
CamFibrePhotometry ThinkerTech, Nanjing http://www.thinkerbiotech.c
om/
InperStudioMultiColorEVAL15 Inper Ltd., China https://www.inper.com

InperStudio Inper Ltd., China https://www.inper.com
Inper Data Process Inper Ltd., China https://www.inper.com
MATLAB R2015b or R2019b MathWorks https://www.mathworks.com
/products/matlab.html
Prism GraphPad Software https://www.graphpad.com/
scientific-software/prism
BORIS Friard and Gamba http://www.boris.unito.it/

ImageJ National Institutes of https://imagej.nih.gov/ij/inde
Health x.html
FV31S-SW OLYMPUS https://www.olympus-lifesci

ence.com/en/downloads/de
tail-iframe/?0[downloads][id
]=847252002
HCImage Live HAMAMATSU Photonics https://hcimage.com/hcima
ge-overview/hcimage-live/
NIS-Elements AR4.5.00 OLYMPUS http://www.olympusconfocal
.com/products/fv1000/fv100
0software.html
Any-maze software Stoelting https://stoeltingco.com/Neur
oscience/ANY-maze
Electrical V1.0.4 SansBio, Jiangsu https://www.sansbio.com/
Other
473 nm, 589 nm and 635 nm Inper Ltd., China https://www.inper.com
laser LED
35
Fiber photometry system ThinkerTech, Nanjing or http://www.thinkerbiotech.c

Inper Ltd., China om/
https://www.inper.com
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals
Data for experiments were collected from male adult C57BL/6J strain mice (Qizhen or Jihui
Laboratory animal), Aldh1l1-Cre (The Jackson Laboratory, JAX. 023748) and TH-Cre (The
Jackson Laboratory, JAX. 008601) transgenetic mice, all over 8 weeks of age. Mice were
housed in groups of four or five randomly under standard conditions (12-hour light/dark
cycle) with food and water available ad libitum. Mice were habituated in the behavioral
rooms for 0.5-1h before all the behavioral experimentations. All animal-related experimental
procedures were under the guidelines of the Animal Care and Use Committee of the animal
facility at Zhejiang University.
METHOD DETAILS
Surgery and viral injection
Mice were deeply anaesthetized by 1% pentobarbital sodium (100mg/kg body weight) and
head-fixed in a stereotactic frame (RWD Instruments). Virus was injected into the target
brain regions (LHb: AP, - 1.72 mm from bregma; ML, ± 0.46 mm; DV, - 2.62 mm from the
brain surface; mPFC: AP, + 2.43 mm from bregma; ML, ± 0.4 mm; DV, - 1.3 mm from the
brain surface; LH: AP, - 0.90 mm from bregma; ML, ± 1.10 mm; DV, - 4.90 mm from the
brain surface; STR: AP, + 0.80 mm from bregma; ML, ± 2.0 mm; DV, - 2.20 mm from the
36
brain surface; BLA: AP, + 1.40 mm from bregma; ML, ±3.10 mm; DV, - 4.5 mm from the
brain surface; HPC: AP, -2.20 mm from bregma; ML, ± 1.35 mm; DV, - 1.20 mm from the
brain surface; LC: AP, -5.5 mm from bregma; ML, ± 0.9 mm; DV, - 3.40 mm from the brain
surface) using a pulled glass pipette connected to a pressure microinjector (Picospritzer III,
Parker). The injection needle remained in place for approximately 10 min before being
withdrawn to prevent fluid reflux. For optic fiber implantation, a 200-μm optic fiber was
placed 300 μm above the center of the viral injection site and cemented onto the skull with
dental acrylic. For bilateral inhibition of the LHb neurons or multi-regions photometry
recording, optic fiber was implanted to target brain regions (LHb: -1.72 mm AP, ±1.14 mm
ML, -2.35 mm DV, and angled at 15 from the vertical in the lateral direction; LH: -0.9 mm
AP, ±2.32 mm ML, -4.4 mm DV, and angled at 15 from the vertical in the lateral direction).
After surgery, mice were allowed to recover from anesthesia on a heat pad.
After all experiments were completed, mice were transcardially perfused under deep
anesthesia with 0.1 M phosphate buffered saline (PBS) followed 4% w/v paraformaldehyde
(PFA) to verify the sites of virus injection and fiber placement. Brains were postfixed in 4%
w/v PFA for 12 hours, followed by cryoprotection in a 30% w/v sucrose solution for 1~2
days. The dehydrated brains were then sectioned into 40 μm thick coronal slices using a
cryostat (Leica). The slices were counterstained with DAPI or Hoechst before imaging.
Fluorescent image acquisition was performed using an Olympus Fluoview FV1000 confocal
microscope. Only data from mice with correct virus injection site and optic location site were
used.
37
For calcium recording experiments, the following viruses were used:
AAV2/9-CaMKIIa-GCaMP6s (titre: 1.0 × 1013 vector genome (v. g.)/ml, dilution: 1: 8, 0.1μl
into LHb, BLA, 0.2μl into LH, mPFC, STR, HPC, Addgene), AAV2/9-hSyn-jRGECO1a
(titre: 1.0 × 1013 vector genome (v. g.)/ml, dilution: 1:3, 0.15μl into LHb, Addgene),
AAV2/5-GfaABC1D-cyto-GCaMP6f (titre: 7.0 × 1012 vector genome (v. g.)/ml, 0.25μl into
LHb, BLA, LH, mPFC, HPC, STR, Addgene), AAV2/5-GfaABC1D-lck-GCaMP6f (titre: 7.0
× 1012 vector genome (v. g.)/ml, 0.25μl into LHb, BLA, LH, mPFC, HPC, STR, Addgene),
AAV2/9-CAG-Flex-GCaMP6s-WPRE-pA (titre: 1.66 × 1013 vector genome (v. g.)/ml,
dilution: 1: 5, 0.2μl into LHb, Taitool Bioscience),
AAV2/9-EF1a-DIO-Axon-GCaMP6s-WPRE-pA (titre: 5.14 × 1012 vector genome (v. g.)/ml,
0.2μl into LC, BrainVTA).
For sensor recording experiments, the following viruses were used:
AAV2/9-hSyn-GRABNE2h-WPRE-SV40-pA (titre: 1.0 × 1013 vector genome (v. g.)/ml,
dilution: 1: 3, 0.2μl into LHb, Vigene), AAV2/5-GfaABC1D-SFiGluSnFR
(A184S)-WPRE-SV40pA (titre: 1.0 × 1013 vector genome (v. g.)/ml, 0.2μl into LHb,
BrainVTA), AAV2/5-GfaABC1D-ADO1.0m-WPRE-SV40pA (titre: 1.0 × 1013 vector
genome (v. g.)/ml, 0.2μl into LHb, BrainVTA).
For manipulation experiments of neurons, the following viruses were used:
AAV2/9-hSyn-ChrimsonR-tdTomato-WPRE-SV40pA (titre: 2.27 × 1013 vector genome (v.
g.)/ml, dilution: 1: 10, 0.1 μl into LHb, Taitool Bioscience),
38
AAV2/9-hSyn-eNpHR3.0-mCherry-WPRE-pA (titre: 1.76 × 1013 vector genome (v. g.)/ml,
dilution: 1: 8, 0.1 μl into LHb, Taitool Bioscience), AAV2/9-hSyn-mCherry-WPRE-pA (titre:
2.49 × 1013 vector genome (v. g.)/ml, dilution: 1: 5, 0.1 μl into LHb, Taitool Bioscience),
AAV2/9-hSyn-Flex-ChrimsonR-mCherry-WPRE-pA (titre: 1.30 × 1013 vector genome (v.
g.)/ml, dilution: 1: 5, 0.15 μl into LC, Taitool Bioscience),
AAV2/9-EF1a-DIO-hM4Di-EGFP-WPRE-pA (titre: 5.35 × 1012 vector genome (v. g.)/ml,
0.2μl per side bilateral into LC, BrainVTA).
For manipulation experiments of astrocytes, the following viruses were used: AAV2/5-
GfaABC1D-hM3Dq-mCherry (titre: 1.45 × 1013 vector genome (v. g.)/ml, dilution: 1: 5, 0.2μl
per side bilateral into LHb, plasmid from Addgene); AAV2/5- GfaABC1D-mCherry (titre:
2.08 × 1013 vector genome (v. g.)/ml, dilution: 1: 5, 0.2μl per side bilateral into LHb, Sunbio
Medical Biotechnology); AAV2/8-GfaABC1D-cOpn5-T2A-mCherry-WPRE-pA (0.2μl per
side bilateral into LHb, provided by M. Luo);
AAV2/5-GfaABC1D-cOpn5-T2A-mCherry-WPRE-pA (0.2μl per side bilateral into LHb,
Taitool Bioscience); AAV2/5-GfaABC1D-iβARK-p2A-mCherry-WPRE-pA (titre: 1.9 × 1013
vector genome (v. g.)/ml, 0.2μl per side bilateral into LHb, plasmid from Addgene);
AAV2/5-GfaABC1D-IP3R2-shRNA-mCherry-WPRE-pA (titre: 2.61 × 1013 vector genome
(v. g.)/ml, 0.2μl per side bilateral into LHb, Sunbio Medical Biotechnology).
In vivo fiber photometry recording
The calcium signals of multi-regions were simultaneously recorded using fiber photometry
39
systems (ThinkerTech, Nanjing). A beam of 488 nm excitation light was delivered, and
fluorescence signals were acquired at a sampling rate of 50 Hz. To minimize fluorescence
signal bleaching, the laser intensity was adjusted to a low level (40 μW) at the tip of optic
fiber.
The calcium signals of astrocytes and neurons were simultaneously recorded using dual-color
fiber photometry recording systems (ThinkerTech, Nanjing; Inper Ltd., China). System
delivered two excitation light sources, 470 nm and 580 nm, allowing simultaneous recording
of red and green indicators at a frequency of 40 or 25 frames per channel per second. The
light power was approximately 40 μW for the 470 nm wavelength and the 50 μW for the 580
nm wavelength at the tip of optic fiber.
During the recording of signals under the FS stress, the mice were placed into an FS chamber
(SansBio, Jiangsu) with metal grid floor and subjected to unpredictable FS (1s, 0.4-1mA). A
video camera was positioned above the chamber to track each mouse.
Optogenetic manipulations in fiber photometry recording
To synchronize optogenetic manipulation and photometry recording, we utilized fiber
photometry recording systems combined with photostimulation (Inper Ltd., China). In the
experiments involving the activation of LHb neurons, LHb-LC terminals and LCTH-LHb
terminals, we applied 1s red-light pulses (635 nm, 2-5 mW at fiber tip, 5 ms or 10 ms pulse
width). In the experiments involving inhibition of LHb neurons, we applied constant yellow
light (589 nm, 10 mW at fiber tip). When the light was delivered, a trigger simultaneously
40
sent a TTL pulse to the data recording systems, allowing verification of the exact time points
when the light was turned on.
Analysis of fiber photometry data
Data were analyzed using the codes (e. g. OpSignal, from Thinker Tech Nanjing Biotech Co.,
Ltd. and Inper Ltd., China) based on MATLAB. The fluorescence responses were indicated
by delta-F/F0 (calculated as (F-F0)/ F0). F0 represents the baseline average fluorescence
signals in a 2-second-long period prior to the onset of FS stress or light on. Delta-F/F0 are
presented as heatmaps and also as average plots with a shaded area indicating the SEM. For
the quantification of rise and decay of signals, rise time was defined as latency from 10%
peak signal timing to 80% peak signal timing. Decay time was defined as latency from peak
timing decay to 50% peak activity timing. The peak of signals during response period (10s
from the stimulus onset) was detected by finding a maximum response. For quantification of
inflection point, average fluorescence signals evoked by FS stress for each mouse were used
to define the point of decay slope change as the inflection point. For signal-phase analysis of
neuronal calcium, phase1 was defined as the period from the onset of FS timing to the
inflection point timing and phase2 was defined as the period from the inflection point timing
to 10% peak activity timing. For astrocyte-loss-of-function experiments, the phases of
neuronal calcium signals were defined as follows: phase1 was the period from the onset of FS
to 2.5s, and phase2 was from 2.5 s to 40 s (It is difficult to identify the inflection point of
neuronal calcium signals when inhibiting astrocytic calcium signaling). The quantification of
AUC1 and AUC2 of NE signals evoked by FS excluded ramp values.
41
Drugs application in vivo recording
The following drugs were administered intraperitoneally (i.p.). Concentrations were as
follows: Prazosin hydrochloride (4 mg/kg, MCE), Propranolol (40 mg/kg, MCE),
Atipamezole hydrochloride (0.5 mg/kg, MCE), Scopolamine (1 mg/kg, Tocris),
Mecamylamine hydrochloride (2.5 mg/kg, MCE), MPEP hydrochloride (20 mg/kg, MCE),
LY341495 (2.5 mg/kg, MCE), Silodosin (5 mg/kg, MCE), L-765314 (5 mg/kg, MCE) and
BMY-7378 (2.5 mg/kg, Tocris).
For inhibition of LC NE neurons, four weeks after injection of
AAV2/9-EF1a-DIO-hM4Di-EGFP-WPRE-pA into LC, clozapine (1mg/kg, MCE) or saline
was administered to TH-Cre mice by intraperitoneal injection.
For activation of LHb astrocytes, four weeks after injection of
AAV2/5-GfaABC1D-hM3Dq-mCherry or AAV2/5-GfaABC1D-mCherry and
AAV2/9-CaMKIIα-GCaMP6s into LHb, clozapine (0.25, 0.5, 1mg/kg, MCE) or saline was
administered to mice by intraperitoneal injection.
For experiments of inhibitors and antagonists administration, each recording session was
separated by 30-50 minutes to allow for drug effectiveness.
Acute brain slice preparation for imaging
42
Sagittal LHb slices were prepared from C57BL/6J or Aldh1l1-Cre mice with AAV virus
injection for in vitro calcium imaging. Briefly, mice were deeply anesthetized with
pentobarbital sodium and decapitated with sharp shears. The brains were sliced in ice-cold
modified artificial CSF (ACSF) (oxygenated with 95% O2 and 5% CO2) containing the
following (in mM): 220 sucrose, 2 KCl, 6 MgCl2, 0.2 CaCl2, 26 NaHCO3, 1.2 NaH2PO4, and
10 D-glucose. A vibratome (Leica2000) was used to cut 300 μm brain sections. The slices
were allowed to equilibrate for 30 min at 34-36 °C in normal ACSF containing (in mM): 125
NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, and 25 D-glucose with 1 mM
pyruvate added, continuously bubbled with 95% O2 and 5% CO2. Slices were allowed to
recover for at least 1h in the same buffer until use. All slices were used within 5-8 hours of
slicing.
In vitro imaging
Slice preparation was performed as described above. In neuronal calcium imaging and
sensors imaging experiments, images were captured on an Olympus two-photon microscope
(FVMPE-RS, Tokyo, Japan) equipped with a mode-locked Ti:Sapphire laser (MaiTai
DeepSee, Spectra-Physics, San Francisco, USA) set at 920 nm and a water-immersion
objective lens (25×, N.A. 1.05; Nikon, Tokyo, Japan). Regions with virus expression in the
LHb were identified and full-frame images were acquired at 0.92 Hz. In astrocytic calcium
imaging experiments, for optogenetic manipulation experiments, images were captured on an
Olympus two-photon microscope (FVMPE-RS, Tokyo, Japan). For other experiments,
images were captured on a digital CMOS camera (ORCA-Flash4.0 V3; HAMAMATSU and
43
FL 9BW; TUCSEN) with a water-immersion objective lens (40×, N.A. 0.85). Full-frame
images were acquired at 4 Hz. A constant flow of fresh buffer perfused the imaging chamber
at all times.
Optogenetic manipulations in two-photon imaging
In optogenetic manipulation experiments, an optical fiber connecting a photostimulation
system (Inper, China) was placed on the slices and delivered red light (635 nm, 5mW at fiber
tip, 5ms or 10ms pulse width) for neuronal ChrimsonR activation, or blue light (473 nm,
60µW at fiber tip, 1s constant) for astrocytic cOpn5 activation.
Drug applications in vitro
The following drugs were applied in the bath: Norepinephrine (MCE, HY-13715),
Phenylephrine (10 µM, Sigma), γ-Aminobutyric acid (300 µM, Sigma), Serotonin (20 µM,
MCE), Dopamine (10 µM, APExBIO), Clozapine N-oxide (CNO, Sigma), Picrotoxin (100
µM; Tocris), PPADS tetrasodium (50 µM; MCE), Suramin hexasodium salt (75 µM ;
APExBIO), DPCPX (300 nM; MCE), SCH582661 (100 nM; MCE), APV (50 µM; Sigma),
NBQX (10 µM; MCE), Prazosin hydrochloride (10 µM; MCE), Atipamezole hydrochloride
(1 µM; MCE), Propranolol hydrochloride (10 µM; MCE), Tetrodotoxin cltrate (TTX; 1 µM;
alomone labs).
For experiments of inhibitors and antagonists bath, each slice imaging session was separated
at least 10 min to permit drug penetration.
44
Analysis of imaging data
Calcium imaging analysis was conducted using FIJI (ImageJ) and MATLAB. Image XY drift
was corrected using MATLAB. For CMOS camera imaging data (excluding Figure S6F, G),
the entire field of view (FOV) was selected as the region of interest (ROI). In Figure S6F, G,
the FOV was uniformly divided into 128x128 segments and the segments with calcium
activities exceeding the threshold (delta-F/F0 > 10% in response to 5 µM NE) were selected
as ROIs. For two-photon imaging data of sensors and astrocytic calcium signals, regions
exhibiting fluorescence (including soma and process of astrocytes) during the experiment
were selected as ROIs. For two-photon imaging data of neuronal calcium signals, the
neuronal somatic regions were selected as ROIs. The fluorescence responses were indicated
by delta-F/F0 (calculated as (F-F0)/ F0). F0 represents the baseline average fluorescence
signals in a 10-second period prior to the onset of light on or a 60-second period prior to the
drug application. Delta-F/F0 were presented as heatmaps and also as average plots with a
shaded area indicating the SEM.
The recorded neurons show three modes of spontaneous calcium activity at resting conditions.
We calculated the standard deviation (SD) of calcium fluorescence value and the SD of
calcium-event intervals for each neuron during the resting conditions. Neurons were
classified as silent if they showed no calcium events during recording (SD of fluorescence
value < 30); Neurons were classified as regular if they showed periodic calcium events during
recording (SD of fluorescence value > 30 and SD of calcium-event interval < 10); Neurons
were classified as irregular if they showed irregular calcium events during recording (SD of
45
fluorescence value > 30 and SD of calcium-event interval > 10).
For the recording of LHb neurons during astrocytic-hM3Dq activation, we calculated the
mean fluorescence for each neuron during the control period (180-second period before CNO
application) and during the CNO-responsive period (180-second period during CNO
application). Neurons were identified as excited if their mean fluorescence during
CNO-responsive period was at least 2.5-fold above during the control period. For the
recording of LHb neurons during astrocyte-cOpn5 activation, photostimulations (473 nm, 60
µW at fiber tip, 1s constant) were performed 6-8 times. Neurons were identified as excited if
they exhibited high response fidelity (> 80%). To quantify the effect of inhibitors and
antagonists, neurons with stable responses (response fidelity > 80%, signal decay < 10%) to
cOPN5 in normal ACSF were included in the analysis. We separately calculated the mean
cOPN5-evoked calcium peak in normal ACSF (control) and in ACSF with inhibitors or
antagonists added. Neurons were identified as inhibited if their mean calcium peak in the
presence of inhibitors or antagonists decreased by at least 30% compared to the control.
Immunohistochemistry
In experiment of immunohistochemistry for c-Fos, four weeks after injection of
AAV2/5-GfaABC1D-hM3Dq-mCherry or AAV2/5-GfaABC1D-mCherry into LHb,
deschloroclozapine (0.5 mg/kg, MCE), clozapine (0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, MCE) or
saline was administered to mice by intraperitoneal injection. Two hours after administration,
mice were sacrificed for immunohistochemistry of c-Fos. In experiment of
46
immunohistochemistry for NeuN, S100β, GFP and RFP, mice were sacrificed, after all the
experiments were completed.
Mice were deeply anesthetized with 1% pentobarbital sodium and perfused transcardially
with 0.1 M phosphate buffer saline (PBS, pH = 7.4) followed by 4% paraformaldehyde in
PBS. Brains were removed and postfixed overnight and dehydrated in 30% sucrose in PBS.
Coronal brain sections (40 μm) were serially cut and divided for 6 interleaved sets.
The antibodies used were rabbit anti-c-Fos (1:2000, SYSY), mouse anti-NeuN (1:500,
Millipore), rabbit anti-S100β (1:1000, Dako Products), chicken anti-GFP (1:2000, Aves Labs),
rabbit anti-GFP (1:2000, Invitrogen), chicken anti-TH (1:1000, Aves Labs), rabbit anti-TH
(1:1000, Santa Cruz Biotechnology), rabbit anti-RFP (1:1000, Rockland); Alexa Fluor 546
goat anti-rabbit IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor Cy5 goat anti-mouse
IgG, Alexa Fluor 488 goat anti-chicken IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa
Fluor 546 goat anti-mouse IgG (all 1:1,000, Invitrogen).
Slices for checking the injection site were counterstained with Hoechst in the final incubation
step. Fluorescent image acquisition was performed with an Olympus Fluoview FV1000
confocal microscope.
Behavioral assays
Foot-shock stress (FS)
In FS-induced depression-like behaviors protocol, FS was performed as previously
47
described.16,91 Mice were placed into a standard FS chamber (SansBio, Jiangsu) with metal
grid floor and habituated to the new environment for 5 min. During a 20-min FS session,
mice were subjected to 20 unpredictable FS (1 mA, 1s) with an intershock interval of 45-75s.
In subthreshold FS protocol, mice were subjected to 6 unpredictable FS (1 mA, 1s) with an
intershock interval of 45-75s. For activation of LHb astrocytic Gq pathway,
Deschloroclozapine (0.5 mg/kg, MCE) was administered intraperitoneally (i.p.) 10 min
before FS.
Forced swim test (FST)
FST was performed as previously described.18,134 Mice were gently and individually placed in
a cylinder (12 cm diameter, 25 cm height) of water (23-24 °C) and swam for 6 min under
normal light (100-200 lux). Water depth (15 cm) was set to prevent mice from touching the
bottom with their tails or hind limbs. The entire test lasted for 6 min. A camera was set at the
side of the cylinder to record the behaviors. The immobile duration during the last 4-min test
were counted offline by an experienced observer blinded to the animal treatments. Immobility
was defined by animals remaining floating or motionless with only small and necessary
movements for keeping balance.
Sucrose preference test (SPT)
SPT was conducted as previously described.18,134 Animals were single housed and habituated
to two bottles of drinking water for 2 days, followed by two bottles of 2% sucrose for 2 days.
After habituation, the preference for any specific bottle was checked. Only mice without
48
basal preference (between 25-75%) were used. Mice with basal preference of one bottle on
the last day of habituation (below 25% or above 75%) were excluded. Mice were then water
deprived for 24 hours. In the test phase, mice exposed to one bottle of 2% sucrose and one
bottle of water for 2 hours in the dark phase. The positions of 2 bottles were switched after
1hour. The sucrose preference was calculated as the average of consumption of sucrose/ total
consumption of water and sucrose during the 2 hours.
Statistical analysis
All data are shown as mean ± SEM. Statistical analyses were done with Prism 6 (GraphPad)
or MATLAB. By pre-established criteria, values were excluded from analyses if virus
expression was poor or optic fiber location was out of the interested region. The data were
analyzed by Student’s test for Gaussian distributions, while Mann-Whitney test for
non-Gaussian distributions. Results were considered statistically significant when the P value
< 0.05. More details are provided in the Table S1.
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Figures
68
Figure 1. Foot-shock-stress Induces Most Rapid Calcium Response in LHb Astrocytes

(A) Schematic illustrating viral construct for astrocyte-specific expression of GCaMP and multi-site fiber
photometry setup for recording astrocytic calcium activities during FS.
(B) Illustration of viral expression (stained with antibodies against GFP and Hoechst) and optic fiber placement
(indicated by the yellow dotted line) in 6 central nervous system (CNS) regions, the boundary of which is outlined
by the white dotted line. Green, GCaMP6f; blue, Hoechst. Scale bar, 200 μm.
(C) GCaMP expression is highly specific in astrocytes. Top: representative image of LHb brain slices stained with
antibodies against GFP (which indicates GCaMP positive cells, green) and S100β (astrocytic marker, red). Scale
bar, 100 μm. Bottom: bar graph showing percentage of GCaMP-positive cells that are the S100β-positive (n = 7
slices from 3 mice).
(D) Representative traces (left) and heatmap (right) of delta F/F ratio of simultaneously recorded astrocytic
calcium signals in LHb, LH and PFC over three continuous FSs. Scale bars, 50% delta F/F, 5s (left).
(E) Alignment of FS-onset plots of calcium signal changes of simultaneously recorded multi-region astrocytes
69
during FS.
(F) Illustration of calculation of rise and decay time.
(G) Average time to peak (left), rise time (middle) and decay time (right) of astrocytic calcium signals evoked by FS
in 6 brain regions. Each circle represents one mouse.
(H) Average peak amplitudes of astrocytic calcium signals evoked by FS in 6 brain regions. Each circle represents
one mouse.
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. Data are represented as mean ± SEM.
70
Figure 2. Foot-shock-stress Induces Biphasic Calcium Activities in LHb Neurons

(A) Illustration of viral construct for neuron-specific expression of GCaMP, viral expression and optic fiber
placement (indicated by the yellow dotted line) in LHb, the boundary of which is outlined by the white dotted line.
Green, GCaMP6s; blue, Hoechst. Scale bar, 100 μm.
(B) Heatmap of delta F/F ratio of calcium signal from LHb neurons (n = 47 trials from 8 mice) aligned to onset of
FS. Color bars on the left indicate different mice.
(C) Average delta F/F ratio of calcium signal from LHb neurons aligned to onset of FS. Solid lines indicate mean
and shaded areas indicate SEM.
(D) Average time to peak (left), average time to inflection (middle) of LHb neuronal calcium signals from FS onset,
and ratio of area under curve (AUC) of FS-evoked signals after and before inflection point (right). AUC1: AUC of
neuronal calcium signal in 0-inflection from FS onset. AUC2: AUC of neuronal calcium signal in
inflection-10%peak. Each circle represents one mouse.
(E) Illustration of dual-color neuronal expression of jRGECO1a and astrocytic expression of GCaMP6f and optic
fiber placement (indicated by the yellow dotted line) in LHb. Scale bar, 100 μm.
(F) Representative traces of simultaneously recorded neuronal and astrocytic calcium signals in LHb over three
continuous FSs. Scale bars, 20% delta F/F, 10s.
(G) Delta F/F ratio of calcium signals from LHb neurons and LHb astrocytes aligned to the onset of FS. Solid lines
(H) Average time to peak of LHb neuronal (N-Peak, left) and astrocytic (A-Peak, middle) calcium signals and
average time to inflection of LHb neuronal calcium signals from FS onset (N-inflection, right) in dual-color fiber
photometry system. Each circle represents one mouse.
71
Figure 3. Neuron-to-Astrocyte Crosstalk in LHb during FS

(A) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons and fiber photometry recording
72
of nearby GCaMP6f-expressing astrocytes in LHb in vivo.

(D) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons and two-photon imaging of
nearby GCaMP6f-expressing astrocytes in LHb slices in vitro.
(B, E) Astrocytic calcium responses to LHb neuronal ChrimsonR activation (1s, 40 Hz) in vivo (B) and in LHb slices
(E).
(C, F) Mean astrocytic calcium responses to varying stimulation frequencies in vivo (C) and in LHb slices (F).
(G) Schematic of in vivo fiber photometry recording of LHb astrocytes in response to FS after i.p. injection of
various receptor blockers.
(H) Bar graph showing effects of MPEP (mGluR5 antagonist), LY341495 (mGluR2/3 antagonist), Scopolamine
(mAChR antagonist), Mecamylamine (nAChR antagonist), Prazosin (α1-AR antagonist), Propranolol (β-AR
antagonist) and Atipamezole (α2-AR antagonist) on FS-evoked calcium signals in LHb astrocytes. Each circle
(I) Bar graph showing effects of α1-AR subtype-selective antagonists, including Silodosin (α1A-AR antagonist),
L-765314 (α1B-AR antagonist) and BMY 7378 (α1D-AR antagonist) on FS-evoked calcium signals in LHb
astrocytes. Each circle represents one mouse.
(J-L) In vivo fiber photometry recording of NE sensor signals in LHb during FS. Viral expression (J, stained with
antibodies against GFP and Hoechst) after injection of AAV2/9-hSyn-NE2h and optic fiber placement (indicated by
the yellow dotted line) in LHb, the boundary of which is outlined by the white dotted line. Scale bar, 100 μm.
Example trace (K) of LHb NE sensor signals during FS (black) and homecage (HC, grey). Scale bars, 5% delta F/F,
50s. Plots (L, left) of delta F/F ratio of NE sensor signals aligned to the onset of FS. Solid lines indicate mean and
shaded areas indicate SEM. Heatmap (L, right) of delta F/F ratio of NE sensor signals in the LHb aligned to the
onset of FS (n = 46 trials from 5 mice). Color bars on the left indicate different mice.
(M) Plots of delta F/F ratio of calcium signals from LHb neurons, LHb astrocytes, and LHb-NE-sensor signals
aligned to onset of FS stress. Solid lines indicate mean and shaded areas indicate SEM. Data is from 2E-H and
3L.
(N) Average time to peak of LHb neuronal calcium signals, LHb-NE-sensor signals and LHb astrocytic calcium
signals from FS onset (left) and average time to inflection of LHb neuronal calcium signals and LHb-NE-sensor
signals (right). Data is from 2E-H and 3L.
(O) Schematic illustrating optogenetic activation of LHb-LC terminals and fiber photometry recording of LC NE
neurons in TH-Cre mice.
(P) Calcium response of LC NE neurons to optogenetic activation of LHb-LC terminals (1s, 40 Hz). Plots of delta
F/F ratio of calcium signal aligned to laser onset. Red line represents light-on. Solid lines indicate mean and
shaded areas indicate SEM.
TH
(Q) Schematic illustrating optogenetic activation of LC -LHb terminals and fiber photometry recording of LHb
astrocytes in TH-Cre mice.
(R) Calcium response of LHb astrocytes to optogenetic activation of LCTH-LHb terminals (1s, 40Hz). Plots of
TH
averaged delta F/F ratio of astrocytic calcium signal induced by optogenetic activation of LC -LHb terminals after
i.p. injection of saline (black) or Prazosin (green, α1-AR antagonist) aligned to laser onset. Red line represents
light-on. Solid lines indicate mean and shaded areas indicate SEM.
(S) Bar graph showing effects of Prazosin (α1-AR antagonist) on astrocytic calcium signals induced by
TH
optogenetic activation of LC -LHb terminals. Each circle represents one mouse.
(T) Working model summarizing the neuron-to-astrocyte crosstalk in the LHb during FS. Stress activates LHb
neurons, which recruit LC NE neurons to elevate LHb astrocytic calcium via NE-α1A-AR signaling.
73
Figure 4. Astrocyte-to-Neuron Crosstalk in the LHb

(A) Representative image showing viral expression of GCaMP6s in LHb neurons and hM3Dq in LHb astrocytes.
74
Scale bar, 100 μm.

(B-C) In vivo fiber photometry recording of LHb neuronal calcium in response to LHb astrocytic hM3Dq activation.
Representative raw traces (B) and bar chart (C) showing mean AUC of LHb neuronal calcium signals following an
intraperitoneal injection of saline or clozapine. Each circle represents one mouse.
(D) Representative images of c-Fos IHC signals following unilateral astrocytic hM3Dq activation in LHb . Note
c-Fos signals are only present in injected side. Green, c-Fos; blue, Hoechst. Scale bar, 40 μm.
(E) Quantification of total c-Fos + cells in the LHb.
(F) Representative image of LHb brain slices with viral expression of cOpn5 in astrocytes. Scale bar, 40 μm.
(G) Schematic (left) and representative two-photon image (right) illustrating optogenetic activation of
cOpn5-expressing astrocytes while recording two-photon image of LHb astrocytic calcium responses. Scale bar:
50 μm.
(H) Plots of delta F/F ratio of calcium signals in LHb astrocytes aligned to laser onset. Solid lines indicate mean
and shaded areas indicate SEM.
(I) Schematic (left) and representative two-photon image (right) illustrating optogenetic activation of
cOpn5-expressing astrocytes while recording two-photon images of LHb neuronal calcium responses. Scale bars:
50 μm.
(J) Pie chart illustrating percent abundance of excited neurons by astrocytic cOpn5 activation (left, n = 4 slices
from 4 mice). Colored outer circle indicates percentage of three types (according to baseline activity) among
astrocytic-hM3Dq-excited neurons. Representative raw calcium traces from the three types of astrocytic
cOpn5-excited neurons (right). Blue line represents light-on.
(K) Bar graph showing effects of ACSF (control), picrotoxin (GABAA receptor antagonist), PPADS and suramin
(ATP-R antagonists, ATP receptor antagonists), DPCPX (ADO A1R antagonists, adenosine A1 receptor
antagonist), SCH58261 (ADO A2AR antagonist, adenosine A2A receptor antagonist) and APV and NBQX (iGlu
receptor antagonists) on astrocytic cOpn5-evoked neuronal calcium signals in LHb slices. Each circle represents
one neuron.
(L, M) Illustration of AAVs used for expressing hM3Dq and ADO1.0m (L) or iGluSnFR (M) in LHb astrocytes (left
top), fluorescence images (left bottom) and traces (right) illustrating astrocyte-induced adenosine (L) or glutamate
(M) release in LHb slice. Scale bar: 100 μm. (Experiments were performed in TTX).
75
2+
Figure 5. LHb Astrocytic Ca Signaling Is Required for the Second-phase Neuronal Activities and NE
Release
(A) Left: illustration of blockade of astrocytic calcium signaling by IP3R2-shRNA. Right: viral expression of
IP3R2-shRNA in astrocytes. Scale bar, 100 μm.
(B) Schematic illustrating the viral expression of astrocyte-specific IP3R2-shRNA or Ctrl-mCherry in bilateral LHb
and neuron-GCaMP6s in unilateral LHb, and fiber photometry recording of neurons in LHb.
(C, D) Plots of averaged delta F/F ratio (C) and heatmap (D) of LHb neuronal calcium signals aligned to FS onset
in mice expressing Ctrl-mCherry (grey, n = 69 trials from 7 mice) or IP3R2-shRNA (red, n = 50 trials from 5 mice) in
LHb astrocytes.
76
(E-J) Quantification of Time to peak (E), Peak amplitude (F), AUC1 in 0-2.5s (G), AUC2 in 2.5-40s (H), AUC ratio (I)
and total AUC in 0-40s (J) of FS-evoked LHb neuronal calcium signals in mice expressing Ctrl-mCherry or
IP3R2-shRNA in LHb astrocytes. Each circle represents one mouse.
(K) Left: illustration of blockade of astrocytic calcium signaling by iβARK. Right: viral expression of iβARK in
astrocytes. Scale bar, 100 μm.
(L) Schematic illustrating the viral expression of astrocyte-specific iβARK or Ctrl-mCherry in bilateral LHb and
neuron-GCaMP6s in unilateral LHb, and fiber photometry recording of neurons in LHb.
(M, N) Plots of averaged delta F/F ratio (M) and heatmap (N) of LHb neuronal calcium signals aligned to FS onset
in mice expressing Ctrl-mCherry (grey, n = 22 trials from 4 mice) or iβARK (red, n = 30 trials from 5 mice) in LHb
astrocytes.
(O-T) Quantification of Time to peak (O), Peak amplitude (P), AUC1 in 0-2.5s (Q), AUC2 in 2.5-40s (R), AUC ratio
(S) and total AUC in 0-40s (T) of FS-evoked LHb neuronal calcium signals in mice expressing Ctrl-mCherry or
iβARK in LHb astrocytes. Each circle represents one mouse.
(U) Example trace (left) and plots of averaged delta F/F ratio (right) of LHb NE signals during FS in mice
expressing Ctrl-mCherry (grey, n = 42 trials from 7 mice) or IP3R2-shRNA (red, n = 42 trials from 7 mice) in LHb
astrocytes.
(V, W) AUC1 in 0-inflection and AUC2 in inflection-40s of LHb NE signals aligned to FS onset (V) and basal (tonic)
delta F/F ratio of LHb-NE signals in response to FS (W) in mice expressing Ctrl-mCherry (grey) or IP3R2-shRNA
(red) in LHb astrocytes.
77
Figure 6. LHb Astrocytes Regulate Stress-induced Depression-like Behaviors

(A) Cartoon illustration of a subthreshold depression-induction protocol with 6× FS.
(B) Subthreshold 6×-FS protocol did not induce depression-like behaviors in FST (n = 8, 8 mice for naive and FS
group, respectively) and SPT (n = 8, 8 mice for naive and FS group, respectively).
(C) Experimental paradigm for activating Gq pathway of LHb astrocytes during the subthreshold
(D) Illustration of viral expression of hM3Dq in LHb astrocytes. Scale bar, 100 μm.
(E) Depression-like behaviors in FST (n =13, 12, 10, 14 mice for Ctrl-mCherry/DCZ, hM3Dq/DCZ,
Ctrl-mCherry/saline and hM3Dq/Saline group, respectively) and SPT (n =10, 12, 10, 12 mice for
Ctrl-mCherry/DCZ, hM3Dq/DCZ, Ctrl-mCherry/saline and hM3Dq/Saline group, respectively) of mice expressing
Ctrl-mCherry (grey) or hM3Dq (blue) in LHb astrocytes.
(F) Cartoon illustration of a depression-induction protocol with 20× FS.
(G) 20×-FS protocol induced depression-like behaviors in FST (n = 16, 16 mice for naive and FS group,
respectively) and SPT (n = 15, 17 mice for naive and FS group, respectively).
(H) Experimental paradigm for inhibiting calcium signaling of LHb astrocytes during the depression-induction
protocol.
(I) Illustration of viral expression of IP3R2-shRNA in LHb astrocytes. Scale bar, 100 μm.
(J) Depression-like behaviors in FST (n =17, 17 mice for Ctrl-mCherry and IP3R2-shRNA group, respectively) and
78
SPT (n =15, 16 mice for Ctrl-mCherry and IP3R2-shRNA group, respectively) of mice expressing Ctrl-mCherry
(grey) or IP3R2-shRNA (red) in LHb astrocytes.
79
Figure 7. LHb neuron-astrocyte synergy in mediating depression-like behaviors

(A) A model summarizing the LHb neuron-astrocyte synergy in mediating depression-like behaviors. Stress first
activates LHb neurons, which in term recruit LC-NE neurons. NE release in LHb then triggers astrocytic calcium
activities through α1AAR-dependent signaling. In tandem, through gliotransmitters glutamate (Glu) and
ATP/Adenosine (Ado), LHb astrocytes prompt the second-phase of both local LHb neural activities and NE
release. Through such a positive reinforcing loop and regulation of global gene expression, LHb astrocytes
facilitate stress-induced depression-like behaviors.
80
Supplementary figures
Figure S1. Foot-shock-stress Induces Most Rapid Calcium Response in LHb Astrocytes, Related to Figure
1
(A) GfaABC1D-promoter-driven GCaMP is not expressed in neurons. Left and middle: representative images of
LHb brain slices stained with antibodies against GFP (indicating GCaMP-positive cells, green) and NeuN
(neuronal marker,red). Scale bar, 50 μm. Right: bar graph showing percentage of GCaMP-positive cells that are
NeuN-positive (n = 7 slices from 4 mice).
(B) Scatterplots showing the relationship between peak amplitude and time to peak of LHb astrocytic calcium
signals evoked by FS. Each circle represents one mouse.
81
Figure S2. Neuronal Calcium Response Evoked by Foot-shock-stress in Five Other Brain Regions,
Related to Figure 2
(A-E) Left: Illustration of viral expression and optic fiber placement (indicated by the yellow dotted line) in BLA (A),
mPFC (B), HPC (C), LH (D) and STR (E), the boundary of which is outlined by the white dotted line. Green,
GCaMP6s; blue, Hoechst. Scale bars, 100 μm. Middle: Average delta F/F ratio of neuronal calcium signals from
BLA (A, n = 3 mice), mPFC (B, n = 7 mice), HPC (C, n = 7 mice), LH (D, n = 3 mice) and STR (E, n = 3 mice)
aligned to the onset of FS. Solid lines indicate mean and shaded areas indicate SEM. Right: representative traces
of recorded neuronal calcium signals from the 5 CNS regions during homecage (top) and continuous FSs
(bottom).
82
Figure S3. Foot-shock-stress Induces Biphasic Calcium Activities in LHb Neurons, Related to Figure 2
(A) Representative traces of recorded neuronal calcium signals in LHb over three continuous FSs. Scale bars, 20%
delta F/F, 10s.
(B) Alignment of FS-onset plots of average calcium signal of recorded neurons from individual animal during FS (n
= 8 mice). Each circle represents one mouse.
(C) Illustration of segmented phases of FS-evoked LHb neuronal calcium signals.
(D) Average half-decay time in phase-1 and phase-2 of FS-evoked neuronal calcium signals. Each circle
(E) Average peak amplitude in phase-1 and phase-2 of FS-evoked neuronal calcium signals. Each circle
83
Figure S4. Optogenetic Activation of ChrimsonR-expressing Neurons Induces Neuronal Calcium in LHb
Slice, Related to Figure 3
(A) Schematic illustrating optogenetic activation of ChrimsonR-expressing neurons while recording two-photon
images of LHb neuronal calcium responses.
(B) Calcium fluorescence images of LHb neurons before (B, left) and after (B, right) optogenetic activation of LHb
neurons (1s, 40 Hz).
(C) Neuronal calcium responses to optogenetic activation of LHb neurons (1s, 40 Hz) in LHb slices.
(D) Mean neuronal calcium responses to varying stimulation frequencies in LHb slices.
84
Figure S5. Calcium Activities Evoked by FS in LHb Astrocytes Require Noradrenergic Signaling, Related
to Figure 3
(A-C) Plots of averaged delta F/F ratio (left) and heatmap (middle) of FS-evoked calcium signals in LHb astrocytes
before and after i.p. injection of Prazosin (α1-AR antagonist, A), Atipamezole (α2-AR antagonist, B) and
Propranolol (β-AR antagonist, C) aligned to FS onset. Bar graph showing effects of NE receptor antagonists on
FS-evoked calcium signals in LHb astrocytes (right). Each circle represents one mouse.
85
Figure S6. NE Triggers Calcium Signals in LHb Astrocytes via α1-AR, Related to Figure 3
(A) Schematic illustrating in vitro calcium imaging of LHb astrocytes in slices.
(B) Calcium fluorescence images of LHb astrocytes showing calcium increase at different time points after NE
application (n = 8 slices from 8 mice). Scale bar: 50 μm. (Experiments were performed in TTX).
(C) Plots of delta F/F ratio (left) and heatmap (right) of calcium signals in LHb astrocytes induced by NE (n = 8
slices from 8 mice). Solid lines indicate mean and shaded areas indicate SEM.
(D, E) Calcium fluorescence images of LHb astrocytes before (D, left) and after (D, right) application of
phenylephrine (PE, α1-AR agonist). Traces (E) of LHb astrocytic calcium signals before and after PE application
(n = 6 slices from 4 mice). Scale bars, 20% delta F/F, 30s. (Experiments were performed in TTX).
(F) Schematic illustrating viral injection of Cre-dependent GCaMP6s in LHb in Aldh1l1-Cre mice and in vitro
calcium imaging of LHb astrocytes in response to NE after application of NE receptor antagonist. ROI detection
using a grid array (see STAR Methods for details).
(G) Representative calcium fluorescence images (left) and bar graph (right) showing effects of Prazosin (α1-AR
antagonist), Propranolol (β-AR antagonist) and Atipamezole (α2-AR antagonist) on LHb astrocytic calcium signals
evoked by NE. Scale bar: 50 μm. Each circle represents one slice.
86
Figure S7. LHb NE Sensor and Astrocytic Calcium Signals Induced by FS, Related to Figure 3
(A) Example trace of LHb-NE-sensor signals over multiple FSs. Scale bars, 5% delta F/F, 50s.
(B) AUC of basal (tonic) signal of LHb NE sensor in homecage (HC, grey) and in response to FS (red). Each circle
(C) Plots showing basal (tonic) signal change of LHb NE sensor in response to FS.
(D) Example trace of LHb astrocytic calcium signals over multiple FSs. Scale bars, 5% delta F/F, 50s.
(E) AUC of basal signal of LHb astrocytic calcium in homecage (grey) and in response to FS (red). Each circle
(F) Plots showing basal signal change of LHb astrocytic calcium in response to FS.
87
Figure S8. Effects of manipulating LHb and LC-NE Neurons on LHb Astrocytic Calcium, Related to Figure
3
(A, B) Effects of activating LHb-LC terminals on LC-NE neurons. (A) Representative images showing viral
expression of ChrimsonR in LHb neurons (left), GCaMP6s in LC-NE neurons (middle) and LC-NE neuron
terminals in LHb (right, stained with antibody against GFP) in TH-Cre mice. Scale bar, 100 μm. (B) Calcium
responses of LC-NE neurons to optogenetic activation of LHb-LC terminals (1s, 40 Hz) in vivo.
(C, D) Effects of activating LCTH-LHb terminals on LHb astrocytes. (C) Representative images showing viral
88
expression of ChrimsonR in LC NE-neurons (left), LCTH-LHb terminals (middle, stained with antibody against RFP)
and GCaMP6f in LHb astrocytes (right) in TH-Cre mice. Scale bar, 100 μm. (D) Calcium responses of LHb
TH
astrocytes to optogenetic activation of LC -LHb terminals (1s, 40 Hz).
(E-H) Effects of Prazosin (α1-AR antagonist) on LHb astrocytic calcium signals evoked by LHb neuron activation
in vivo. (E) Schematic illustrating optogenetic activation of LHb neruons and fiber photometry recording of LHb
astrocytes. (F) Representative image showing viral expression of ChrimsonR in LHb neurons and GCaMP6f in
LHb astrocytes. Scale bar, 100 μm. (G) Plots of averaged delta F/F ratio of LHb astrocytic calcium signals induced
by LHb neurons before and after i.p. injection of Prazosin (α1-AR antagonist) aligned to the laser onset. Solid lines
indicate mean and shaded areas indicate SEM. (H) Bar graph showing the effects of Prazosin (α1-AR antagonist)
on astrocytic calcium signals evoked by LHb neurons. Each circle represents one mouse.
(I-L) Effects of inhibiting LHb neurons on FS-evoked calcium signals in LHb astrocytes. (I) schematic illustrating
optogenetic inhibition of LHb neruons and fiber photometry recording of LHb astrocytes in response to FS. (J)
Representative image showing viral expression of eNpHR in LHb neurons and GCaMP6f in LHb astrocytes. Scale
bar, 100 μm. (K) Plots of averaged delta F/F ratio of FS-evoked calcium signals in LHb astrocytes during light off
and light on aligned to FS onset. Solid lines indicate mean and shaded areas indicate SEM. (L) Bar graph showing
effects of inhibiting LHb neurons on FS-evoked calcium signals in LHb astrocytes. Each circle represents one
mouse.
(M-P) Effects of inhibiting LC-NE neurons on FS-evoked calcium signals in LHb astrocytes. (M) Schematic
illustrating chemogenetic inhibition of LC-NE neurons and fiber photometry recording of LHb astrocytes in
response to FS. (N) Representative image showing viral expression of hM4Di in LC-NE neurons. Scale bar, 100
μm. (O) Plots of averaged delta F/F ratio of FS-evoked calcium signals in LHb astrocytes before and after i.p.
injection of clozapine (Cloz) aligned to FS onset. Solid lines indicate mean and shaded areas indicate SEM. (P)
Bar graph showing effects of inhibiting LC NE-neurons on FS-evoked calcium signals in LHb astrocytes. Each
89
Figure S9. Chemogenetic Activation of Astrocytic Gq Pathway in LHb slice, Related to Figure 4
(A, B) Schematic (A) and representative calcium fluorescence images (B) illustrating chemogenetic activation of
hM3Dq-expressing astrocytes while recording calcium fluorescence images of LHb astrocytic calcium responses.
Scale bar: 50 μm.
(C) Plots of delta F/F ratio (left) and heatmap (right) of calcium signals in LHb astrocytes before and after CNO
application (n = 3 mice). Solid lines indicate mean and shaded areas indicate SEM.
(D, E) Schematic (D) and representative two-photon images (E) illustrating chemogenetic activation of
hM3Dq-expressing astrocytes while recording two-photon image of LHb neuronal calcium responses. Scale bar:
50 μm.
(F) Pie chart illustrating percent abundance of excited neurons by astrocytic hM3Dq activation (left, n = 3 slices
from 2 mice). Colored outer circle indicates percentage of three types (according to baseline activity) among
astrocytic-hM3Dq-excited neurons. Representative raw calcium traces from the three types of astrocytic
hM3Dq-excited neurons (right).
90
Figure S10. Optogenetic Activation of Astrocytic Gq Pathway in LHb Slice, Related to Figure 4
(A) Schematic illustrating optogenetic activation of cOpn5-expressing astrocytes while recording two-photon
images of LHb neuronal calcium responses.
(B) Left: example spatial map of astrocytic cOpn5-excited and non-responsive neurons in LHb. Right:
representative raw calcium traces from the three types of non-responsive neurons. Blue line represents light-on.
(C-E) Traces of LHb neuronal calcium signals induced by astrocyte-cOpn5 before (left) and after (middle)
application of various receptor antagonists. Right: bar graph showing effects of ACSF (control, C), SCH58261
(adenosine A2AR antagonist, D) and APV/NBQX (iGluR antagonists, E) on LHb neuronal calcium signals induced
by astrocyte-cOpn5. Each circle represents one neuron.
91
Figure S11. Inhibition of Astrocytic Calcium Signaling in LHb, Related to Figure 5

(A-C) Schematic (A) illustrating the viral expression of astrocyte-specific IP3R2-shRNA or Ctrl-mCherry and
GCaMP6f, and fiber photometry recording of astrocytes in LHb in response to FS. Representative image (B)
illustrating viral expression of IP3R2-shRNA and GCaMP6f in LHb astrocytes. Scale bar, 100 μm. Quantification of
peak amplitude of FS-evoked LHb astrocytic calcium signals in mice expressing Ctrl-mCherry (grey, n = 12 mice)
or IP3R2-shRNA (red, n = 12 mice) in LHb astrocytes (C).
(D-F) Schematic (D) illustrating the viral expression of astrocyte-specific iβARK or Ctrl-mCherry and GCaMP6f,
and fiber photometry recording of astrocytes in LHb in response to FS. Representative image (E) illustrating viral
expression of iβARK and GCaMP6f in LHb astrocytes. Scale bar, 100 μm. Quantification of peak amplitude of
FS-evoked LHb astrocytic calcium signals in mice expressing Ctrl-mCherry (grey, n = 7 mice) or iβARK (red, n = 6
mice) in LHb astrocytes (F).
(G) Schematic (left) illustrating the viral expression of astrocyte-specific IP3R2-shRNA or Ctrl-mCherry and
92
neuronal GCaMP6s, and fiber photometry recording of neurons in LHb in response to FS. Representative image
(right) illustrating viral expression of IP3R2-shRNA in LHb astrocytes and GCaMP6s in LHb neurons. Scale bar,
100 μm.
(H) Schematic (left) illustrating the viral expression of astrocyte-specific iβARK or Ctrl-mCherry and neuronal
GCaMP6s, and fiber photometry recording of neurons in LHb in response to FS. Representative image (right)
illustrating viral expression of iβARK in LHb astrocytes and GCaMP6s in LHb neurons. Scale bar, 100 μm.
93
Supplementary Table S1. Detailed statistical information

Figure Sample Sample Statistic method Statistic results
size
(Time to peak) LHb vs n = 14, 6 Mann-Whitney test p = 0.001
LH
(Time to peak) LHb vs n = 14, 6 Mann-Whitney test p < 0.0001
PFC
HPC
BLA
STR
(Rise time) LHb vs LH n = 14, 6 Mann-Whitney test p = 0.0015
(Rise time) LHb vs PFC n = 14, 6 Mann-Whitney test p = 0.0002
(Rise time) LHb vs HPC n = 14, 5 Mann-Whitney test p = 0.0003
(Rise time) LHb vs BLA n = 14, 6 Mann-Whitney test p < 0.0001

1G
(Rise time) LHb vs STR n = 14, 5 Mann-Whitney test p = 0.0002
(Decay time) LHb vs LH n = 14, 6 Mann-Whitney test p = 0.0004
(Decay time) LHb vs n = 14, 6 Mann-Whitney test p < 0.0001

PFC
(Decay time) LHb vs n = 14, 5 Mann-Whitney test p = 0.0002
HPC
(Decay time) LHb vs n = 14, 6 Mann-Whitney test p < 0.0001
BLA
(Decay time) LHb vs n = 14, 5 Mann-Whitney test p = 0.0072
STR
(Peak amplitude) LHb vs n = 14, 6 Mann-Whitney test p = 0.01
LH
PFC
1H (Peak amplitude) LHb vs n = 14, 5 Mann-Whitney test p = 0.005
HPC
BLA
94
STR
vehicle vs MPEP n = 7, 7 Mann-Whitney test p = 0.87
vehicle vs LY341495 n = 7, 7 Mann-Whitney test p = 0.52
vehicle vs scopolamine n = 7, 8 Mann-Whitney test p = 0.67
vehicle vs n = 7, 6 Mann-Whitney test p = 0.71
3H mecamylamine
vehicle vs prazosin n = 7, 10 Mann-Whitney test p = 0.0001
vehicle vs propranolol n = 7, 7 Mann-Whitney test p = 0.96

vehicle vs atipamezole n = 7, 7 Mann-Whitney test p = 0.0006
vehicle vs silodosin n = 6, 8 Mann-Whitney test p = 0.0007
3I vehicle vs L-765314 n = 6, 6 Mann-Whitney test p = 0.31
vehicle vs BMY 7378 n = 6, 7 Mann-Whitney test p = 0.02
3S (opto-stimulation Ca n = 4, 5 Mann-Whitney test p = 0.009
signal) vehicle vs
prazosin
(AUC of Ca signal) n = 8, 8 unpaired t test p > 0.99
hM3Dq/vehicle vs
mCherry/1.0 mg kg-1
Clozapine
(AUC of Ca signal) n = 8, 3 Mann-Whitney test p = 0.01
hm3Dq/vehicle vs
hM3Dq/0.25 mg kg-1
Clozapine
4C (AUC of Ca signal) n = 8, 10 unpaired t test p < 0.0001
hm3Dq/vehicle vs
hM3Dq/0.5 mg kg-1
Clozapine
(AUC of Ca signal) n = 8, 7 Mann-Whitney test p = 0.0003
hm3Dq/vehicle vs
hM3Dq/1.0 mg kg-1
Clozapine
(#cFos+/section) n = 5, 5 Mann-Whitney test p = 0.0079
hm3Dq-/0.5 mg kg-1
Clozapine vs
hM3Dq+/0.5 mg kg-1
Clozapine
4E (#cFos+/section) n = 5, 3 Mann-Whitney test p = 0.036
-1
hm3Dq+/0.5 mg kg
Clozapine vs hM3Dq+/
vehicle
(#cFos+/section) n = 3, 3 Mann-Whitney test p = 0.9
hm3Dq-/saline vs
95
hM3Dq+/saline
(opto-stimulation Ca n = 43 Paired t test p = 0.09
signal) before vs after
ACSF
(opto-stimulation Ca n = 11 Wilcoxon p = 0.32
signal) before vs after matched-pairs
picrotoxin signed rank test
PPADS+suramin
4K signal) before vs after
DPCPX
SCH58261
APV+NBQX
5E (Time to peak) n = 7, 5 Mann-Whitney test p = 0.41
Ctrl-mCherry vs
IP3R2-shRNA
5F (Peak dF/F) n = 7, 5 Mann-Whitney test p = 0.2
Ctrl-mCherry vs
IP3R2-shRNA
5G (Mean AUC1) n = 7, 5 Mann-Whitney test p = 0.2
Ctrl-mCherry vs
IP3R2-shRNA
5H (Mean AUC2) n = 7, 5 Mann-Whitney test p = 0.0051
Ctrl-mCherry vs
IP3R2-shRNA
5I (AUC2/AUC1) n = 7, 5 Mann-Whitney test p = 0.0025
Ctrl-mCherry vs
IP3R2-shRNA
5J (Total AUC) n = 7, 5 Mann-Whitney test p = 0.0051
Ctrl-mCherry vs
IP3R2-shRNA
5O (Time to peak) n = 4, 5 Mann-Whitney test p = 0.11
Ctrl-mCherry vs iβARK
5P (Peak dF/F) n = 4, 5 Mann-Whitney test p > 0.99
5Q (Mean AUC1) n = 4, 5 Mann-Whitney test p = 0.87
96
5R (Mean AUC2) n = 4, 5 Mann-Whitney test p = 0.016

5S (AUC2/AUC1) n = 4, 5 Mann-Whitney test p = 0.016
5T (Total AUC) n = 4, 5 Mann-Whitney test p = 0.016
5V (AUC1) Ctrl-mCherry vs n = 7, 7 Mann-Whitney test p = 0.31
IP3R2-shRNA
(AUC2) Ctrl-mCherry vs n = 7, 7 Mann-Whitney test p = 0.0041
IP3R2-shRNA
5W (Ramp) Ctrl-mCherry vs n = 6, 6 Mann-Whitney test p = 0.026
IP3R2-shRNA
(FST) Naïve vs shock n = 8, 8 Mann-Whitney test p = 0.43
6B (6◊)
(SPT) Naïve vs shock n = 8, 8 Mann-Whitney test p = 0.63
(6◊)
(FST) Ctrl-mCherry n = 13, 12 unpaired t test p = 0.011
/DCZ vs hM3Dq/DCZ
/saline vs hM3Dq/saline
(FST) hM3D/DCZ vs n = 12, 14 unpaired t test p = 0.0002
hM3Dq/saline
/DCZ vs Ctrl-mCherry
6E /saline
(SPT) Ctrl-mCherry n = 10, 12 unpaired t test p = 0.0038
/DCZ vs hM3Dq/DCZ
/saline vs hM3Dq/saline
(SPT) hM3D/DCZ vs n = 12, 12 unpaired t test p < 0.0001
hM3Dq/saline
/DCZ vs Ctrl-mCherry
/saline
(FST) Naïve vs shock n = 16, 16 unpaired t test p = 0.03
(20◊)
6G
(SPT) Naïve vs shock n = 15, 17 unpaired t test p = 0.0058
(20◊)
(FST) Ctrl-mCherry vs n = 17, 17 unpaired t test p = 0.0003
6J IP3R2-shRNA
(SPT) Ctrl-mCherry vs n = 15, 16 Mann-Whitney test p = 0.0049
IP3R2-shRNA
S1B Correlation of between n = 13 two tailed test p = 0.27
97
peak amplitude and time

to peak
S3D (Average half-decay n = 8, 8 unpaired t test p < 0.0001
time) Phase-1 vs Phase-2
S3E (Average peak n = 8, 8 unpaired t test p = 0.0003
amplitudes) Phase-1 vs
Phase-2
S5A (peak dF/F) Before vs n=8 paired t test p = 0.0002
after Prazosin
S5B (peak dF/F) Before vs n=7 Wilcoxon p = 0.015
after Atipamezole matched-pairs
signed rank test
S5C (peak dF/F) Before vs n=7 Wilcoxon p = 0.94
after Propranolol matched-pairs
signed rank test
S6G (NE-evoked astrocytic n = 4, 3, 4, 4 Mann-Whitney test ACSF vs Prazosin,
calcium signals) after p = 0.05; ACSF vs
ACSF, Prazosin, Propranolol, p =
Propranolol, 0.12; ACSF vs
Atipamezole Atipamezole, p =
0.2;
S7B (NE sensor) Homecage n=5 paired t test p = 0.0046
vs shock
(NE sensors basal n = 5, 5, 5, 5, 5, 5 paired t test S1 vs S2, p = 0.036;
change) S1, S2, S3, S4, S1 vs S3, p = 0.007;
S7C S5, S6 S1 vs S4, p =
0.0028; S1 vs S5, p
= 0.0016; S1 vs S6,
p = 0.0025
S7E (Astrocyte calcium) n=5 Wilcoxon p > 0.99
Homecage vs shock matched-pairs
signed rank test
S7F (Astrocyte calcium basal n = 5, 5, 5, 5, 5, Wilcoxon S1 vs S2, p = 0.63;
change) S1, S2, S3, S4, matched-pairs S1 vs S3, p = 0.44;
S5, S6 signed rank test S1 vs S4, p = 0.31;
S1 vs S5, p = 0.31
S8H Saline vs Prazosin n = 4, 6 Mann-Whitney test p = 0.0095
S8L LHb :: mcherry vs n = 5, 6 Mann-Whitney test p = 0.0043
LHb :: eNPHR
S8P Saline vs clozapine n = 4, 4 Mann-Whitney test p = 0.028
S11C LHb :: Ctrl-mCherry vs n = 12, 12 unpaired t test p = 0.0002
LHb :: IP3R-shrna
S11F LHb :: Ctrl-mCherry vs n = 7, 6 Mann-Whitney test p = 0.0082
98
LHb :: iβARK
99

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bioRxiv preprint doi: https://doi.org/10.1101/2024.11.03.621722; this version posted November 3, 2024.

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Neuron-astrocyte Coupling in Lateral Habenula Mediates Depressive-like

Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China

New Cornerstone Science Laboratory, Zhejiang University, Hangzhou, China.

Zhejiang University School of Medicine, Yiwu, China.

depression onset. Here, using multi-brain-region calcium photometry recording in

freely-moving mice, we discover that stress induces a unique, bimodal neuronal

requires the α1A-adrenergic receptor, and depends on a recurrent neural network

ATP/Adenosine, LHb astrocytes mediate the second-wave activation of local LHb

neurons as well as release of norepinephrine (NE). Activation or inhibition LHb

astrocytic calcium signaling facilitates or prevents stress-induced depressive-like

behaviors respectively. These results identify a stress-induced positive feedback loop in

Keywords: lateral habenula, locus coeruleus, astrocytes, norepinephrine, stress,

depression.5-7 LHb regulates essentially all neuromodulatory systems, including the

serotonergic, dopaminergic, and noradrenergic systems.7-9 LHb neurons are activated by

their dynamic interaction contributes to depression etiology.

types of G protein-coupled receptors (GPCRs), bestowing them to sense neuronal and

elevated by neuromodulators, such as acetylcholine (ACh),36-38 dopamine,32,39 serotonin40 or

norepinephrine (NE).41-43 In turn, astrocytes can regulate neuronal functions at diverse

timescales through the uptake of extracellular ions and neurotransmitters20,44-46 or release of

gliotransmitters.27,30,47-52 Gliotransmitters, including glutamate, gamma-aminobutyric acid

(GABA), D-serine and ATP/Adenosine, can lead to synchronized neuronal excitation,29,53-55

enhanced temporal fidelity of firing,56 or fine-tuned synaptic transmission.32,39,57-61 However,

response in the LHb. Using cell-type-specific manipulations in combination with fiber

photometry and two-photon imaging recordings, we identified a bidirectional

critical role of LHb astrocytes in the onset of depression.

Foot-shock-stress induces most rapid calcium response in LHb astrocytes

We performed in vivo multi-site fiber photometry to record astrocytic calcium activities

glia-specific GfaABC1D promoter62 driving the genetically encoded calcium indicator

suggesting that it may play a unique role in processing stress-related information.

Foot-shock-stress induces biphasic calcium activities in LHb neurons

and performed calcium photometry recording in neurons (Figure 2A and S2A-S2E).

To better understand the relationship between stress-activated neuronal and astrocytic

the second-phase neuronal activity.

Neuron-to-astrocyte crosstalk in the LHb during FS

In order to characterize the mechanism of LHb neuron-astrocyte interaction in stress

Subsequently, we recorded calcium activities in astrocytes following photostimulation of

neurons robustly activated LHb astrocytic calcium signals in a frequency-dependent manner

photometry signals in response to FS while intraperitoneally (i.p.) injecting animals with

blood-brain-barrier (BBB)-permeable inhibitors or antagonists of receptors for different

neurotransmitters or neuromodulators (Figure 3G). We tested receptors for three classes of

neurotransmitter/modulators, which were previously implicated in astrocytic Ca2+ signaling:

the metabotropic glutamate receptors (mGluR),31,66 acetylcholine receptors (AChR) 36,67,68

and norepinephrine receptors (NER).42,69,70 While antagonists for mGluR5, mGluR2/3,

muscarinic AChR (mAChR) or ionotropic AChR (nAChR), namely MPEP, LY341495,

α2-adrenergic receptors (α2-AR), which are mostly expressed on presynaptic terminals

serving autoinhibitory function,71 increased FS-evoked LHb astrocytic calcium signals (p =

application of either NE or the α1-adrenergic receptor agonist phenylephrine (PE) increased

α1-adrenergic receptors in the LHb independent of local neural activity.

LHb genetically encoded G-protein-coupled receptor activation-based NE (GRABNE) sensor,

activation of these three signals (Figure 3M and 3N).

NE-neuron-specific expression (Figure 3O and S8A). In these mice, optogenetic activation of

LHb region. Furthermore, optogenetic activation of LC-NE-neuron terminals in the LHb

blocked by α1-adrenergic receptor antagonist prazosin (p = 0.009, Mann-Whitney test; Figure

neurons in vivo (p = 0.0095, Mann-Whitney test; Figure S8E-S8H). Furthermore, optogenetic

inhibition of LHb neurons or chemogenetic inhibition of LC-NE neurons significantly

calcium in the LHb (Figure 3T).

Astrocyte-to-neuron crosstalk in the LHb