# Social Stress Increases Anxiety by GluA1-Dependent Synaptic Strengthening of Ventral Tegmental Area Inputs to the Basolateral Amygdala

Thomas Contesse, Joana Gomes-Ribeiro, Lea Royon, Hugo Fofo, Anaelle

Braine, Christelle Glangetas, Shiliang Zhang, M Flavia Barbano, Mariano

Soiza-Reilly, François Georges, et al.

► To cite this version:

Thomas Contesse, Joana Gomes-Ribeiro, Lea Royon, Hugo Fofo, Anaelle Braine, et al. Social Stress Increases Anxiety by GluA1-Dependent Synaptic Strengthening of Ventral Tegmental Area Inputs to the Basolateral Amygdala. Biological Psychiatry, In press, 10.1016/j.biopsych.2025.04.007. hal-05225418

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Archival Report

# Social Stress Increases Anxiety by GluA1-Dependent Synaptic Strengthening of Ventral Tegmental Area Inputs to the Basolateral Amygdala

Thomas Contesse, Joana Gomes-Ribeiro, Lea Royon, Hugo Fofo, Anaelle Braine, Christelle Glangetas, Shiliang Zhang, M. Flavia Barbano, Mariano Soiza-Reilly, François Georges, Jacques Barik, and Sebastian P. Fernandez

## ABSTRACT

**BACKGROUND:** Brain defensive mechanisms evolved to maintain low levels of state anxiety. However, risk factors such as stress exposure shift activity within defensive circuits, resulting in increased anxiety. The amygdala is a crucial node for maintaining adaptive anxiety levels, and amygdala hyperactivity can lead to pathological anxiety through mechanisms that are not well understood.

**METHODS:** We used chronic social defeat stress (CSDS) in mice. We combined anatomical tracing methods, patch-clamp recordings, and optogenetics to probe how synaptic inputs from the ventral tegmental area (VTA) to the basolateral amygdala (BLA) are affected by CSDS. We performed in vivo fiber photometry recordings to track inputs onto the BLA. Array tomography and electron microscopy were used to unravel the structural composition of VTA-BLA synapses.

**RESULTS:** We identified the VTA as a source of glutamatergic inputs to the BLA potentiated by stress. Inputs from the medial prefrontal cortex were not potentiated. BLA-projecting VTA glutamatergic neurons were activated by social stress, which increased their excitability and synaptic strength. In vivo potentiation of VTA glutamatergic inputs in the BLA was sufficient to increase anxiety. We showed that stress-induced synaptic strengthening was mediated by insertion of GluA1-containing AMPA receptors. Impeding GluA1 subunit trafficking in BLA neurons with VTA upstream inputs prevented stress-induced increase in synaptic firing and anxiety.

**CONCLUSIONS:** Potentiation of VTA inputs increased synaptic integration, thereby enhancing amygdala activity and promoting maladaptive anxiety. Understanding the impact of amygdala hyperactivity may lead to targeted therapies that restore circuit balance and offer new precision medicine approaches for anxiety disorders.

https://doi.org/10.1016/j.biopsych.2025.04.007

Anxiety is an adaptive response to threats, but when exaggerated, it negatively affects mental well-being (1,2). Anxiety disorders, the most prevalent psychiatric conditions, impose significant individual and societal burdens (3-5). Pathological anxiety is linked to heightened activity in fear circuits, particularly the amygdala, which evaluates emotional significance and the predictive value of stimuli (2,6,7). Disruptions in the balance of excitatory and inhibitory signals in amygdala principal neurons can lead to heightened emotional responses (8). Hyperactivity in the amygdala is central to anxiety disorders, with imaging showing increased reactivity in patients (9-13). However, the precise cellular mechanisms responsible for the increased activity of amygdalar principal neurons remain unclear. Understanding these mechanisms is crucial for both clinical insights and therapeutic advancements. Extensive research has shown that stress, a major contributor to anxiety, induces significant morphological changes in the amygdala,

such as increased dendritic branching and spine density in principal neurons (14-16). These findings highlight the potential role of glutamatergic inputs in driving amygdala hyperactivity and anxiety-related behaviors.

The amygdala receives excitatory inputs from multiple brain structures including cortical and subcortical regions (17,18). Excitatory inputs from the prefrontal cortex (PFC) region are important for fear processing (19-21). However, anatomical studies indicate the presence of noncortical excitatory inputs, notably glutamatergic neurons in modulatory regions (22-25). Recent research has linked a subpopulation of ventral tegmental area (VTA) VGluT2-expressing neurons to innate emotional regulation in response to threats (26,27). Neuroanatomical studies have revealed that glutamate neurons within the VTA extensively innervate the amygdala (28), although the precise impact of this innervation on amygdalar function remains to be fully elucidated.
In this study, we investigated the role of VTA glutamatergic (VTAGlu) neurons in amygdala hyperactivity following chronic stress. Using anatomical, electrophysiological, optogenetic, and fiber photometry techniques in a social defeat model, we found that stress strengthened VTA-amygdala connections, increasing synaptic firing in amygdala principal neurons. In contrast, medial PFC glutamatergic (mPFCGlu) inputs were unaffected. This synaptic potentiation was driven by increased GluA1-containing AMPA receptors (AMPARs) at VTAGlu-BLA synapses, ultimately promoting anxiety.

## METHODS AND MATERIALS

### Experimental Model and Subject Details

We used C57BL/6J, CD1, vGAT-CRE, and VGluT2-CRE mice (26). Transgenic mice were heterozygous and backcrossed on a C57BL/6J background. Mice were enriched and housed 4 to 5 mice per cage on a 12-hour light/dark cycle with lights on from 8 AM to 8 PM and with free access to food and water. All mice used in behavioral experiments were prehandled during daily 5-minute sessions for 1 week before each test to limit any stress induced by the manipulator. In addition, during these prehandling sessions, animals from in vivo optogenetic cohorts were habituated to being connected to laser cables for 20 minutes daily.

All behavioral tests and ex vivo and in vivo recordings were performed on adult mice that were at least 2 months old, and littermates were used as controls. All the experiments were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All procedures were in accordance with the recommendations of the European Commission (2010/63/EU) for care and use of laboratory animals and were approved by the French National Ethical Committee.

### Chronic Social Defeat

The chronic social defeat stress (CSDS) paradigm was performed as previously described (29). Wild-type mice, transgenic mice, and their respective control littermates were subjected to 10 consecutive days of social defeat by former CD1 breeder male mice. Each experimental mouse faced a new CD1 every day, and sessions of defeat were limited to a maximum of 5 minutes. Mice were then maintained in sensory, but not physical, contact with the dominant male through a semipermeable barrier. Undefeated mice (naïve) were not confronted with a dominant male but lived in similar housing conditions, separated by a semipermeable barrier. For social defeat of female mice, cotton swabs were used to apply urine of a male CD1 around the vaginal orifice, tail, and lower back of female mice, who were then placed in a resident CD1 home cage as previously described (30).

Details on behavioral testing are provided in the Supplement.

### Stereotaxic Virus Injections and Optical Fiber Implantations

Stereotaxic injections were performed using a stereotaxic frame (Kopf Instruments) under general anesthesia with xylazine and ketamine (10 mg/kg and 150 mg/kg, respectively).

The injection rate was set at 100 nL/min. Anatomical coordinates were adjusted from Paxinos and were as follows: VTA: anteroposterior (AP) -2.9 mm, mediolateral (ML) ±0.6 mm, dorsoventral (DV) -4.7 mm; BLA: AP -0.9 mm, ML ±3.22 mm, DV -4.6 mm. Detailed information concerning in vivo optogenetic manipulation and fiber photometry recordings is provided in the Supplement.

### Ex Vivo Patch-Clamp Recording

Mice were anesthetized (ketamine 150 mg/kg/xylazine 10 mg/kg) and transcardially perfused with artificial cerebrospinal fluid (aCSF) for slice preparation. For VTA recordings, horizontal 250-µM slices were obtained in bubbled ice-cold 95% O₂/5% CO₂ aCSF containing (in mM): KCl 2.5, NaH₂PO₄ 1.25, MgSO₄ 10, CaCl₂ 0.5, glucose 11, sucrose 234, and NaHCO₃ 26. Slices were then incubated in aCSF containing (in mM): NaCl 119, KCl 2.5, NaH₂PO₄ 1.25, MgSO₄ 1.3, CaCl₂ 2.5, NaHCO₃ 26, glucose 11 at 37 °C for 1 hour and then kept at room temperature. For amygdala recordings, coronal 250-µM sections were obtained using the same solutions. Detailed information concerning electrophysiology protocols used and single-cell polymerase chain reaction (PCR) is provided in the Supplement.

### Microscopy

Details concerning microscopy experiments for histology, c-Fos counting, array tomography quantification, and electron microscopy are provided in the Supplement.

### Data Analysis

Data were analyzed using Prism (GraphPad). The normality of the distribution was first tested using a Kolmogorov-Smirnov test. Depending on the number of groups and the results of the normality test, groups were then compared using a Student's *t* test, Mann-Whitney *U* test, or 2-way analyses of variance followed by Sidak's post hoc test. Data in the figures are presented as mean ± SEM. Statistical significance was conventionally established at \*p < .05, \*\*p < .01, and \*\*\*p < .001.

## RESULTS

### Chronic Social Stress Increases Anxiety and Synaptic Firing of BLA Principal Neurons

To study the mechanisms that result in increased anxiety, male and female mice were subjected to 10 days of CSD, followed by behavioral tests measuring anxiety and by patch-clamp recordings (Figure 1A). Stressed mice showed decreased exploration of open arms in the zero maze compared with controls (Figure 1B). In the open field, CSD mice ventured less to the center area, but the total distance traveled was similar for both groups (Figure 1C). These results showed that CSD promoted anxious behaviors in mice without altering locomotor function. After the behavioral tests, mice were used for patch-clamp recordings in acute brain slices (Figure 1D). Principal (glutamatergic) cells of the BLA participate in anxiety modulation (31). We identified this neuronal population by morphological as well as described electrophysiological properties (32,33). Patch-clamp recordings showed that CSD exposure did not alter passive membrane properties such as
Increased Anxiety Via VTA-BLA Synaptic Strengthening

resting membrane potential or membrane resistance or their
excitability in response to increasing current steps (Figure 1D).
However, CSD did significantly increase the frequency, but not
the amplitude, of spontaneous excitatory postsynaptic cur-
rents (sEPSCs) (Figure 1E), suggesting increased gluta-
matergic terminal release onto amygdala principal neurons. To
ascertain how synaptic inputs modulate output firing activity in
these cells, we applied a protocol of synaptic stimulation to
evoke action potentials. In this protocol, BLA neurons from
stressed mice showed a significantly higher probability of ac-
tion potential firing than BLA neurons from control mice
(Figure 1F). Importantly, both male and female mice subjected
to CSD showed similar changes in anxiety levels in the zero
maze and increased firing probability in patch-clamp re-
cordings compared with controls (Figure S1A). In turn, mice
subjected to a single defeat session did not show elevated
anxiety levels 24 hours post-acute defeat in either the zero
maze or the open field test (Figure S1A). In acute slices pre-
pared from these mice, BLA principal neurons did not show
significant changes in synaptic firing (Figure S1B). These re-
sults demonstrate that exposure to chronic stress increased
anxiety in mice, presumably by increasing glutamatergic
release and synaptic firing of BLA principal neurons.

VTAGlu Neurons Are Functionally Connected to BLA Principal Neurons

Recent studies have suggested that VTAGlu neurons modulate threat responses, which prompted us to explore their role in stress-induced anxiety via amygdala projections. We characterized VTAGlu neuron connectivity with amygdala nuclei anatomically and functionally. We expressed ChR2-mCherry and found that axonal projections were most abundant in the BLA and centromedial amygdala (Figure S2A). Ex vivo patch-clamp recordings with blue light stimulation of VTAGlu terminals showed the highest optostimulation responses and postsynaptic current amplitudes in the BLA (Figure S2B, C), identifying it as the primary target of VTAGlu projections.

Focusing on the BLA, we showed that in voltage-clamp recordings, short (5 ms) blue light pulses resulted in inward currents with an onset delay of 2.41 ± 0.17 ms (mean ± SEM), suggesting monosynaptic connectivity (34). These inward responses were blocked by application of the AMPAR antagonist DNQX, demonstrating that VTAGlu axons release glutamate and activate postsynaptic AMPARs (Figure 2A). Optogenetic activation of VTAGlu terminals was sufficient to elicit action potentials in BLA principal neurons (Figure 2A). We suctioned the cytoplasm of a subset of BLA-recorded neurons and conducted single-cell reverse transcriptase PCR for glutamatergic and GABAergic (gamma-aminobutyric acidergic) markers. Most recorded neurons (6 of 7) expressed VGluT1, consistent with principal glutamatergic neurons (Figure 2B). Activation of VTAGlu terminals within the BLA elicited a significant increase in the number of cFos+ cells, confirming a functional connection in vivo (Figure S3).

Next, we analyzed the characteristics of VTAGlu projections
in the BLA by confocal microscopy and immunoelectron mi-
croscopy. Taking advantage of CaMKII (calcium/calmodulin-
dependent protein kinase II)'s predominant expression in
VTAGlu neurons, we injected AAV-CaMKII-ChR2-mCherry into

the VTA and AAV-DIO-YFP into the BLA of VGluT1-Cre mice to
label glutamatergic populations in both regions (Figure 2C). A
subset of VTA dopamine neurons also expresses CaMKII (35);
however, we minimized this confounding factor by combining
axon labeling with detection of VGluT2 expression. Using
quadruple fluorescent immunolabeling, we found that mCherry
varicosities containing VGluT2 (derived from the VTAGlu neu-
rons) contacted VGluT1 (eYFP [enhanced yellow fluorescent
protein]-positive) cell bodies or dendrites in the BLA
(Figure 2D). Using electron microscopy, we confirmed that
mCherry-labeled varicosities corresponded to axon terminals.
Using triple immunoelectron microscopy, we found that
mCherry axon terminals containing VGluT2 from the VTA
established asymmetrical (putative excitatory) synapses on
VGluT1 (eYFP-positive) dendrites in the BLA (Figure 2E).
Altogether, this evidence demonstrates that VTAGlu neurons
are functionally connected to BLA principal neurons.

VTAGlu Neurons Are Activated by Social Stress

To determine whether VTAGlu neurons projecting to the BLA contribute to anxiety after CSD, we first tested their activation by social stress. Using a retrograde cre-dependent tracer in the BLA of VGluT2-Cre mice, we found BLA-projecting glutamatergic neurons primarily in the medial VTA (mVTA), with minimal coexpression of the dopaminergic marker tyrosine hydroxylase (Figure 3A). We also observed that most BLA-projecting neurons were located in the anterior VTA (Figure S4). Using the same viral strategy, cFos coimmunostaining revealed increased activation of BLA-projecting VTAGlu neurons by acute defeat (Figure 3B). Because cFos experiments only provided histological evidence, it remained unclear whether these neurons actively transmit social stress information. Thus, we conducted fiber photometry recordings to assess their response to defeat stress. We expressed the calcium indicator GCaMP8m in VTAGlu neurons, implanted fibers over the VTA, and exposed mice to a defeat session. Each aggressor attack triggered a transient Ca²⁺ increase in VTAGlu neurons (Figure 3C). Using whole-cell patch-clamp recordings from CSD and control mice, we observed that exposure to CSD produced a significant increase in excitability in VTAGlu→BLA neurons (Figure 3D). These findings suggest that VTAGlu neurons are activated by social stress and undergo maladaptive changes with chronic exposure, thereby heightening glutamatergic input to the BLA and promoting anxiety.

CSDS Specifically Potentiates VTA-BLA Synapses

BLA neurons receive different cortical and subcortical gluta-
matergic inputs (8). In particular, the mPFC is an upstream
regulator of amygdala activity involved in emotional regulation
(9,36). We expressed GCaMP8f in BLA neurons receiving in-
puts from the mPFC and conducted photometry recordings
over the BLA combined with stimulation of either the mVTA or
the mPFC (Figure 4A, B). Electrical stimulation of the mPFC in
burst-like or continuous patterns (10 or 20 Hz) produced strong
increases in Ca²⁺ activity in the BLA (Figure 4C). Interestingly
the same population of neurons also responded to electrical
stimulation in the medial VTA, where the vast majority of glu-
tamatergic neurons are located (Figure 4D). These results
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F

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**Figure 1.** CSD increases anxiety, glutamate release, and synaptic firing in BLA principal neurons. (A) Schematic experimental timeline of CSD protocol followed by behavioral and electrophysiology characterization. (B) Exposure to CSD decreases exploration time in open arms ($t_{57} = 6.506$) and open arm entries ($t_{57} = 5.920$) in the zero maze test compared with control mice ($n = 29$ control and $n = 30$ CSD, Student's t test, \*\*\*$p < .001$). (C) Exposure to CSD decreases exploration time in the center zone ($t_{35} = 5.637$) but not total distance traveled ($t_{35} = 1.707$) in the open field test compared with control mice ($n = 18$ control and $n = 19$ CSD, Student's t test, \*\*\*$p < .001$). (D) Patch-clamp recordings in acute brain slices containing the BLA. CSD exposure did not significantly affect RMP ($t_{27} = 0.4643$) or Rm ($t_{27} = 1.221$) of BLA principal neurons ($n = 14$ control and $n = 15$ CSD, Student's t test, ns). On the right, excitability curves to increasing current injection steps were not significantly different between control and CSD groups ($F_{9,195} = 0.6282$, $n = 14$ control and $n = 15$ CSD, 2-way repeated-measures ANOVA treatment $\times$ current, ns). Representative voltage traces for each condition are shown on the far right. (E) The frequency but not the amplitude of isolated excitatory postsynaptic currents onto BLA principal neurons is augmented after CSD compared with controls ($t_{amplitude} = 1.769$ and $t_{frequency} = 4.928$, df = 27, $n = 14$ control and $n = 15$ CSD, Student's t test, \*\*\*$p < .001$, ns). Each panel depicts the cumulative frequency distribution of total events and mean per condition (inset). Representative current traces per conditions are shown on the far right. (F) Synaptic stimulation to generate APs in BLA principal neurons. Three stimulation trains containing 5 pulses were delivered at 50 Hz. Exposure to CSD increases the probability of discharge compared with control conditions ($F_{4,120} = 10.36$, $n = 14$ control and CSD = 18, 2-way repeated-measures ANOVA followed by Sidak's post hoc test, \*\*\*$p < .001$). Representative voltage traces are depicted for each condition. All data are expressed as mean $\pm$ SEM. ANOVA, analysis of variance; AP, action potential; BLA, basolateral amygdala; CSD, chronic social defeat; ns, not significant; Rm, membrane resistance; RMP, resting membrane potential.

suggest that BLA neurons receive convergent excitatory inputs from the mPFC and the mVTA. To corroborate this hypothesis, we used a double opsin strategy to selectively activate

glutamatergic inputs from these 2 regions (Figure S5A). Patch-clamp recordings were conducted in BLA principal neurons, and light pulses of 470 nm or 590 nm were used to activate
Increased Anxiety Via VTA-BLA Synaptic Strengthening

<figure><img src="image_14.png" /><figcaption>**Figure 2.** VTAGlu neurons make functional synapses onto BLA principal neurons. (A) VGluT2-Cre mice were injected with AAV-DIO-ChR2-mCherry in the VTA. Patch-clamp recording of BLA principal neurons was combined with optogenetic stimulation of VTAGlu axon terminals. Representative traces of light-evoked currents and action potentials in the BLA. Optical EPSCs were blocked by the perfusion of the AMPA antagonist DNQX ($t_d = 5.325$, $n = 5$, Student's paired t test, $p < .01$). (B) In a subset of neurons, cytoplasm was aspirated in the pipette after electrophysiology recording was completed. This content was used to perform scPCR for the glutamatergic markers VGluT1, VGluT2, and the GABAergic markers GAD65 and GAD67. An intron genomic control is run parallel to verify that the nucleus was not aspired during cytoplasm extraction (GEN). (C) Viral injection of AAV2-CaMKII-ChR2-mCherry into the VTA and AAV2-DIO-eYFP into BLA of Vglut1-Cre mice to target BLA VGluT1 neurons. (D) Fluorescent micrographs showing VTA VGluT2 axon terminals (yellow arrows) coexpressing mCherry (red) and VGluT2 (white) making contacts on BLA VGluT1 somas or dendrites expressing eYFP (green, for BLA VGluT1 detection). High magnification images showing closer views of each channel and overlay for the yellow box in the low magnification image. (E) Electron micrograph showing an axon terminal (red outline) from VTA VGluT2 neurons coexpressing mCherry (scattered dark material) and VGluT2 (gold particles, red arrowhead) making an asymmetrical synapse (green arrow) with a BLA VGluT1 dendrite (green outline) expressing eYFP (gold particles, green arrowhead, for VGluT1 detection). All data are expressed as mean ± SEM. BLA, basolateral amygdala; EPSC, excitatory postsynaptic current; eYFP, enhanced yellow protein; GABAergic, gamma-aminobutyric acidergic; Glu, glutamatergic; scPCR, single-cell polymerase chain reaction; VTA, ventral tegmental area.</figcaption></figure>

channelrhodopsin-expressing fibers from the mPFC or Chrimson-expressing fibers from the VTA (Figure S5A). Most BLA neurons responded with an inward current to both light stimulations (12/16 recorded neurons), consistent with both inputs converging on the same neurons. Inward current responses from PFC inputs were generally greater in amplitude and were followed by a large outward phase, which was not seen in VTA inputs (Figure S5B).

We expressed channelrhodopsin in mPFCGlu or VTAGlu neurons and prepared brain slices from CSD or control mice (Figure 4E, F). Patch-clamp recordings were conducted in BLA principal neurons combined with blue light stimulation. We assessed the ratio of AMPAR- to NMDA receptor (NMDAR)-

mediated currents recorded at +40 mV using pharmacological blockers. CSD increased the AMPAR/NMDAR ratio compared with control at VTA-BLA but not mPFC-BLA synapses (Figure 4E, F). These findings reveal the VTA as a subcortical glutamatergic input to BLA neurons and showed that CSD results in increased glutamatergic drive from the VTA but not the mPFC.

CSD Enhances Trafficking of AMPAR Favoring Insertion of GluA1 Subunits and Synaptic Firing of BLA Neurons

Increases of AMPAR/NMDAR ratio have been attributed to insertion of AMPARs in the cell membrane, often accompanied
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**Figure 3.** VTAGlu→BLA neurons are activated by social stress. (A) VGluT2-Cre mice were injected with retrograde AAV-Flex-tdTomato in the BLA. Retrogradely labeled neurons were significantly higher in mVTA vs. IVTA parts ($t_{10} = 5.258, n = 5, \text{Student's t test, } **p < .01$). VTAGlu BLA-projecting neurons did not express the dopaminergic marker TH. (B) VGluT2-Cre mice were injected with a retrograde AAV-Flex-TdTomato in the BLA. Three weeks later, mice were subjected to an acute social defeat session (5 minutes) followed by 1 hour of sensory contact with the aggressor. An immunostaining against cFos (green) and TH (blue) was performed. The number of VGluT2 retrogradely labeled neurons (red) colabeled with cFos was significantly higher in the defeated group
Increased Anxiety Via VTA-BLA Synaptic Strengthening

by changes in their subunit composition (37–39). Using opto-
genetic stimulation of VTAGlu fibers, we obtained current-
voltage characteristic curves of isolated AMPAR currents in
VTAGlu-BLA synapses (Figure 5A). CSD produced an increase
in inward rectification, i.e., higher current amplitudes in
hyperpolarized than in depolarized potentials (Figure 5B). The
resulting rectification index was significantly increased after
CSD (Figure 5B). Thus, CSD could increase GluA1 trafficking
that produces GluA2-lacking AMPARs with strong inward
rectification (40,41). Next, we assessed how this synaptic
rearrangement may impact the firing of BLA principal neurons
(Figure 5A, B). Experiments were conducted in current-clamp
mode without glutamatergic receptor antagonists. Opto-
genetic activation of VTAGlu terminals at 20 or 50 Hz resulted
in <20% probability of generating action potentials in BLA
principal neurons from control mice (Figure 5C, D). In CSD
mice, the same protocol reached a significantly higher prob-
ability of action potential firing (Figure 5C, D). We used the
same strategy to optogenetically stimulate mPFCGlu inputs
while recording BLA principal neurons (Figure S6A–C). Opto-
genetic activation of mPFC axonal terminals was sufficient to
induce action potential firing at both 20 and 50 Hz; however,
firing probability was not changed in slices from CSD mice
(Figure S6D).

We obtained structural evidence to test whether CSD would
increase the number of AMPARs containing the GluA1 subunit
using quantitative high-resolution array tomography. We
labeled VTAGlu axons with ChrimsonR-TdTomato and ob-
tained ultrathin serial sections of the BLA. In CSD mice, we
observed significantly more associations between VTAGlu
axon terminals and AMPAR GluA1 in the BLA (Figure 5D, E).
This increment was not due to a CSD-induced change in the
abundance of AMPAR GluA1 subunits. The total amount of
VTAGlu inputs found in the BLA was increased, but the effect
did not reach statistical significance (Figure 5E). In the same
set of serial sections, we evaluated whether cortical synapses
underwent similar adaptations. We counted double-stained
puncta for Vglut1 and Syn as proxies for cortical inputs, in
apposition to AMPAR GluA1 subunits, and did not find sig-
nificant differences between sections from control and CSD
mice (Figure S7). This is consistent with experiments that have
shown that CSD does not potentiate mPFC inputs onto BLA
principal neurons and does not change their capacity to induce
action potentials and with previous studies that have shown
that chronic stress can produce significant remodeling of
dendritic arrangement in the amygdala in an input-specific
manner (16). Overall, these data suggest that CSD potenti-
ates VTAGlu synapses onto BLA principal neurons by
increasing the abundance of postsynaptic GluA1-containing
AMPARs. This synaptic reorganization increases the efficacy

of this input to depolarize BLA neurons into discharging action
potentials.

In Vivo Optogenetic Control of the VTAGlu → BLA Circuit Bidirectionally Modulates Anxiety

We posed the hypothesis that CSD potentiation of VTA-
Glu→BLA synaptic could have a causal connection to
increased anxiety. We performed optogenetic stimulation of
this circuit in vivo for 5 days (Figure 6A). The next day, animals
were placed in a zero maze and allowed to freely explore for 5
minutes. We observed that mice with optogenetic stimulation
of VTAGlu inputs ventured significantly less into and spent less
time in the open arms of the maze, suggesting increased
anxiety, without changes in locomotor activity (Figure 6B).
Mice were then used for ex vivo patch-clamp recordings. BLA
principal neurons receiving VTAGlu inputs showed a signifi-
cantly increased AMPAR/NMDAR ratio, suggesting synaptic
potentiation (Figure 6C). Additionally, we observed increased
inward rectification in VTAGlu inputs from animals expressing
ChR2 (channelrhodopsin-2) compared with controls
(Figure 6D), indicative of GluA2-lacking receptors, presumably
by incorporation of GluA1 subunits. These results showed that
strengthening synaptic inputs from the VTA in the BLA was
sufficient to increase anxiety.

We tested whether inhibiting the VTAGlu → BLA circuit could
blunt anxiety induced by CSD. We expressed halorhodopsin in
VTAGlu neurons and implanted optic fibers over the BLA. Mice
underwent behavioral assessments before and after CSD,
followed by patch-clamp recordings (Figure S8A). During CSD,
yellow light (595 nm) was delivered to activate halorhodopsin.
In the zero maze, control mice showed reduced open arm
exploration after CSD, while NpHR mice spent more time
exploring the open arms without changes in locomotion
(Figure S8B). Patch-clamp recordings of BLA principal neurons
revealed decreased firing probability in CSD mice with NpHR
expression (Figure S8C). These results suggest that inhibiting
VTAGlu → BLA synapses reduces stress-induced anxiety by
decreasing BLA neuron firing.

Trafficking of AMPA GluA1 Receptor Subunits

To test the hypothesis that GluA1 subunit incorporation drives stress-induced maladaptions in VTAGlu inputs and anxiety, we used an intersectional virus strategy. We designed a virus to express the carboxyl cytoplasmic tail of the GluA1 subunits, fused with mCherry, in BLA neurons that received projections from the VTA (Figure 7A, B). This peptide has previously been shown to block trafficking of endogenous GluA1 subunits in a dominant-negative manner (40,42). Expression of GluA1ct blocking peptide in BLA neurons with VTA inputs prevented

<footnote>(t<sub>g</sub> = 2.559, n = 5 control, n = 6 defeat, Student's t test, \*p < .05). (C) Fiber photometry recordings of GCaMP signals over VTAGlu neuron somas. Photo depicts an example of GCaMP8m expression and fiber placement. Representative recordings depicting Ca<sup>2+</sup> responses evoked by defeat bouts. Perievent plot of averaged responses to defeat bouts and signal quantification showing a significant increase in Ca<sup>2+</sup> responses after defeats (t<sub>g</sub> = 4.844, n = 9, paired Student's t test, \*\*p < .01). (D) VGluT2-Cre mice were injected with a retrograde AAV-Flex-TdTomato in the BLA. Three weeks later, mice were subjected to CSD. Twenty-four hours after the last social defeat session, the mice anxious phenotype was confirmed in the zero maze (data not shown), and ex vivo electro-physiological recordings of BLA-projecting VTAGlu neurons was performed. Excitability was increased after CSD (F<sub>6,240</sub> = 3.460, n = 21 control and n = 21 CSD, 2-way repeated-measures analysis of variance followed by Sidak's post hoc test, \*\*\*p < .001). Representative voltage traces are shown for each condition. BLA, basolateral amygdala; CSD, chronic social defeat; Glu, glutamatergic; IVTA, lateral VTA; mVTA, medial VTA; TH, tyrosine hydroxylase; VTA, ventral tegmental area.</footnote>
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**Figure 4.** Converging inputs from mPFC and VTA onto BLA neurons are selectively potentiated by CSD. (A) Strategy used to record calcic responses in BLA neurons in response to mPFC and medial VTA stimulation. An anterograde transsynaptic virus expressing Cre was injected in the mPFC, and a CRE-dependent virus expressing the calcium sensor GCaMP8f was injected in the BLA. Fiber photometry in anesthetized mice was performed 4 weeks later. The optic fiber was placed above the BLA, and 2 stimulating electrodes were placed in the mPFC and medial VTA. (B) Representative example of GCaMP expression and optic fiber placement in the BLA (top left), Cre expression and electrode placement in the mPFC (top right), and electrode placement in the medial VTA (bottom). (C, D) Mean changes in fluorescence following mPFC (C) or medial VTA (D) stimulation in a burst-like pattern (5 pulses of 10 or 20 Hz every 2 s—repeated 100 times) or a continuous-like pattern (30 s at 10 or 20 Hz). Representative traces of mPFC and medial VTA 20-Hz burst-like stimulation are shown, with the calcium-dependent signal (473 nm, light blue) and isobestic signal (405 nM, purple). Scale bar: 100 µm. Arrows illustrate time points of burst-like stimulations. (E) Patch-clamp recordings in BLA principal neurons combined with light-evoked activation of mPFC glutamatergic terminals. AMPAR to NMDAR ratio was not modified after CSD ($t_{15} = 0.5501$, $n = 8$ control and $n = 9$ CSD, Student's *t* test). Representative traces of light-evoked EPSCs in BLA principal neurons at V = +40 mV in the absence or presence of DNQX (10 µM). (F) Patch-clamp recordings in BLA principal neurons combined with light-evoked activation of VTA glutamatergic terminals. The AMPAR to NMDAR ratio was significantly higher after CSD ($t_{14} = 2.247$, $n = 8$ control and $n = 8$ CSD, Student's *t* test, \*p < .05). Representative traces of light-evoked EPSCs in BLA principal neurons at V = +40 mV in the absence or presence of DNQX (10 µM). ACB, nucleus accumbens; AMPAR, AMPAR receptor; BLA, basolateral amygdala; Cg, cingulate cortex; CSD, chronic social defeat; DP, dorsal peduncular area; EPSC, excitatory postsynaptic current; IL, infralimbic cortex; LA, lateral amygdala; LSr, lateral septum; MO, motor cortex; mPFC, medial prefrontal cortex; NMDAR, NMDA receptor; ns, nonsignificant; PL, prelimbic cortex; SNc, substantia nigra pars, compacta; SNr, substantia nigra, pars reticulata; TH, tyrosine hydroxylase; TT, tenia tecta; VTA, ventral tegmental area, WT, wild-type.
Increased Anxiety Via VTA-BLA Synaptic Strengthening

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AAV-DIO-ChR2-mCherry

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**Figure 5.** CSD potentiates VTAGlu-BLA connectivity via the insertion of AMPAR GluA1 subunits. (A) VGluT2-Cre mice were injected with AAV-DIO-ChR2-mCherry in the VTA to perform patch-clamp recordings combined with optogenetic stimulation. (B) Current-voltage curves of AMPAR currents in BLA principal neurons after optogenetic stimulation of VTAGlu terminals. CSD increased rectification of AMPAR currents ($F_{3,63} = 3.591, n = 13$ control and $n = 10$ CSD, 2-way repeated-measures ANOVA followed by Sidak's post hoc test, \*p < .05). Representative current traces at $V = -70, 0, +40,$ and $+60$ mV for each condition. Rectification indices were calculated as current amplitude at $-70$ mV/amplitude at $+60$ mV ($t_{21} = 2.286, n = 13$ control and $n = 10$ CSD, Student's t test, \*p < .05). (C) Optogenetic stimulation of VTAGlu terminals to generate action potentials in BLA principal neurons. Three stimulation trains containing 5 pulses are delivered at 20 Hz (left panel) or 50 Hz (right panel). Exposure to CSD increases the probability of discharge compared with control conditions ($F_{4,152} = 18.96$ and $F_{4,152} = 8.327, n = 18$ control and CSD = 22, 2-way repeated-measures ANOVA followed by Sidak's post hoc test, \*\*p < .001). Representative voltage traces are depicted for each condition. (D) Representative image depicting staining of VTAGlu fibers (violet) and AMPA GluA1 subunits (green) in the BLA using high-resolution array tomography imaging. Yellow arrows: puncta apposition between VTAGlu axon terminals and GluA1-containing postsynaptic receptors. (E) Density of apposed puncta labeled for VTAGlu fibers and GluA1-containing receptors was significantly different in control vs. CSD mice ($t_8 = 2.406, n = 5$ control and $n = 5$ CSD, Student's t test, \*p < .05). All data are expressed as mean ± SEM. AMPAR, AMPA receptor; ANOVA, analysis of variance; BLA, basolateral amygdala; CSD, chronic social defeat; Glu, glutamatergic; VTA, ventral tegmental area.

the increase in anxiety in response to social stress, as depicted by the time spent exploring the open arms of the zero maze (Figure 7C). In the open field, defeated mice with GluA1ct expression spent more time in the center, although this increase was not statistically significant. Notably, locomotor activity remained unchanged (Figure 7D). Patch-clamp recordings were conducted in targeted BLA neurons after behavior. Blocking GluA1 trafficking did not affect basic membrane properties such as resting potential and membrane resistance (Figure 7E) or affect discharge frequency in response to current injections (Figure S9A). We did not observe significant differences in the frequency or amplitude of sEPSCs (Figure S9B) or in the paired-pulse ratio of electrical stimulation responses (Figure S9C). Ratios between AMPAR and NMDAR responses were smaller in GluA1ct neurons, but this effect was

not statistically significant (Figure S9D). However, when we tested synaptic input integration, we observed that GluA1ct-mCherry expression significantly decreased BLA neurons' synaptic firing after CSD compared with controls (Figure 7F). Overall, these results showed that stress-induced anxiety was mediated by integration of GluA1-containing AMPARs in a subset of BLA neurons receiving VTA inputs.

DISCUSSION

Anxiety requires the continuous detection and evaluation of environmental stimuli to generate appropriate motor, automatic, and hormonal responses. Coordinated activity within the amygdala subnuclei is pivotal in processing sensory inputs and identifying them as aversive or potentially dangerous
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**Figure 6.** In vivo potentiation of VTAGlu synapses in the BLA promotes anxiety. (A) Schematic illustration of the experimental timeline and representative image of fiber placement over the BLA. (B) Five-day in vivo optogenetic stimulation protocol significantly decreased exploration time in open arms (*t*<sub>30</sub> = 3.344) and open arm entries (*t*<sub>30</sub> = 2.560) in the zero maze test in ChR2 compared with control mice (*n* = 14 control and *n* = 18 ChR2, Student's *t* test, \* *p* < .05, \*\* *p* < .01). Locomotor activity was not different between the groups (*t*<sub>30</sub> = 0.7098, *p* = .4833). (C) Five-day in vivo optogenetic stimulation produced a significant increase in AMPAR/NMDAR ratio VTAGlu synapses onto BLA principal neurons (*t*<sub>16</sub> = 2.560, *n* = 8 control and *n* = 10 ChR2, Student's *t* test, \* *p* < .05). Representative traces of light-evoked excitatory postsynaptic currents in BLA principal neurons at V = +40 mV in the absence or presence of DNQX (10 µM). (D) Current-voltage curves of AMPAR currents in BLA principal neurons after optogenetic stimulation of VTAGlu terminals. Five-day in vivo optogenetic stimulation produced a significant increase in AMPAR rectification (*F*<sub>3,45</sub> = 3.110, *n* = 7 control and *n* = 10 ChR2, 2-way repeated-measures analysis of variance followed by Sidak's post hoc test, \* *p* < .05). Representative current traces at V = −70, 0, +40, and +60 mV for each condition. Rectification indices were calculated as current amplitude at −70 mV/amplitude at +60 mV (*t*<sub>15</sub> = 2.234, *n* = 7 control and *n* = 10 chronic social defeat, Student's *t* test, \* *p* < .05). AMPAR, AMPA receptor; BLA, basolateral amygdala; ChR2, channelrhodopsin-2; Glu, glutamate; NMDAR, NMDA receptor; ns, nonsignificant; VTA, ventral tegmental area.

(1,43,44). Here, we showed that VTAGlu projections favor connectivity to the central and basolateral parts of the amygdala, both regions that were previously associated with anxiety regulation (17,18). The BLA is an integrative hub that receives inputs from many brain regions and serves roles in the

processing of reward and aversion via functionally distinct cell types and projections (45). It is composed mostly of principal glutamatergic neurons (90%), intermingled with GABAergic interneurons (46,47). Many studies have shown the presence of anxiety-regulating neurons in the BLA (31,48,49), and
Increased Anxiety Via VTA-BLA Synaptic Strengthening

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**Figure 7.** Blockade of AMPAR GluA1 subunit trafficking in VTA-BLA synapses prevents CSD-induced anxiety. (A) Schematic representation showing AMPAR GluA1 subunits in the plasmatic membrane. An 81-amino acid C-terminal domain with mCherry tag is expressed to prevent insertion of AMPAR GluA1 subunits. (B) Schematic and timeline of experiments. Wild-type mice were injected with transsynaptic AAV1-CRE in the VTA and either AAV-DIO-mCherry (control) or AAV-DIOGluA1ct-mCherry (GluA1ct) in the BLA. Representative image showing the expression of GluA1ct-mCherry in BLA neurons that receive synaptic inputs from the VTA (anterior-posterior level –1 mm). (C) In the zero maze, GluA1ct-mCherry expression significantly increased the amount of time spent in open arms ($F_{1,18} = 6.431$) and the number of open arm entries ($F_{1,18} = 2.716$) in CSD mice ($n = 10$ control and $n = 10$ GluA1ct-mCherry, 2-way repeated-measures ANOVA followed by Sidak's post hoc test, **$p < .01$, *$p < .05$). (D) Time spent in the center ($F_{1,18} = 6.431$, $p = .2239$) and total distance traveled ($F_{1,18} = 0.04636$, $p = .8319$) in the open field test ($n = 10$ control and $n = 10$ GluA1ct-mCherry, 2-way repeated-measures ANOVA followed by Sidak's post hoc test). (E) Patch-clamp recordings in mCherry-tagged neurons. Expression of GluA1ct did not affect membrane resting potential or membrane resistance compared with mCherry alone ($n = 15$ control and $n = 14$ GluA1ct, Student's t test, ns). (F) Synaptic stimulation to generate action potentials in BLA principal neurons expressing mCherry (control) or GluA1ct. Three electrical stimulation trains containing 5 pulses are delivered at 50 Hz. Exposure to CSD increases the probability of discharge in controls compared with GluA1ct ($F_{4,124} = 3.951$, $n = 18$ control and $n = 15$ GluA1ct, 2-way repeated-measures ANOVAs followed by Sidak's post hoc test, **$p < .01$). Representative voltage traces are depicted for each condition. All data are expressed as mean ± SEM. AMPAR, AMPA receptor; ANOVA, analysis of variance; BLA, basolateral amygdala; CSD, chronic social defeat; IR, infrared; ns, nonsignificant; Rm, membrane resistance; RMP, resting membrane potential; VTA, ventral tegmental area.

pathological disruption of BLA activity has been linked to increased anxiety (6,7,9). Exposure to several stressors has been shown to increase innate anxiety and conditioned fear responses and to increase the activity and responsiveness in BLA neurons (9,14). Stress induces significant structural changes in amygdala neurons, particularly hypertrophy in the BLA, with increased spine density and dendritic branching in BLA principal neurons after acute and chronic stress (15,50). Because spines serve as locations for glutamatergic synapses, they have the capacity to control the extent of excitatory neurotransmission affecting overall excitability. We observed that $VTAGlu → BLA$ inputs were potentiated after CSD, but not in mPFC$Glu$ inputs. A recent study found no changes in

postsynaptic function in mPFC inputs onto BLA principal neurons after chronic restraint stress (51). However, the authors did find increased presynaptic plasticity favoring glutamatergic release onto BLA neurons (51). In our study, targeted BLA neurons were activated by VTA and PFC electrical stimulation, and optogenetic-assisted patch-clamp recordings confirmed that these inputs may converge in the same neuronal population. However, alterations in postsynaptic functions appear to be restricted to specific synapses, a phenomenon that has been described in other regions (52,53).

Glutamatergic synaptic plasticity has been extensively studied in amygdala regions in the context of fear conditioning and fear extinction (18). The lateral amygdala (LA) and the BLA
play an important role during the formation of Pavlovian conditioned stimulus-unconditioned stimulus associations, involving a considerable rearranging of both NMDARs and AMPARs (18,54). Deletion of AMPAR GluA1 subunits or blocking postsynaptic trafficking and insertion into LA principal neurons affects associative fear learning (40,55). Adaptive regulation of GluA1 AMPAR subunits plays a crucial role in central synaptic plasticity but has also been implicated in pathological conditions such as addiction, alcohol abuse, and pain (37,56,57). Stress exposure can also alter the expression and subcellular localization of GluA1 subunits in several limbic regions including the amygdala (39,58). Here, we showed that CSD increased the availability of GluA1 subunits in VTA → BLA principal neurons and that blocking this insertion was sufficient to prevent the behavioral sequelae of stress. Thus, maladaptive increased expression of GluA1 subunits enhanced AMPAR signaling and strengthened synaptic firing and neuronal excitability, promoting anxiety behaviors.

## Conclusions

The VTA has a canonical role in reward processing and goal-directed behavior, mainly through its dopaminergic projections to the nucleus accumbens (59,60). Recent studies have shown that the VTA also plays a role in signaling aversion and that subpopulations of neurons in this area are readily excited by aversive events such as a footshock, airpuff, or even predator-like stimuli (26,60). This functional divergence could arise from the unique properties in distinct target-specific cell populations within the VTA. Anatomical tracing studies in mice have consistently shown that VTA neurons abundantly innervate the amygdala (28). Recent studies that have focused on VTA dopamine neuron circuits to the amygdala have revealed a role in anxiety regulation (61,62). Concerning VTAGlu population, these neurons are activated by aversive stimuli (26,27), including attacks from conspecifics, as we showed here. Recent studies have shown that activation of VTAGlu neurons is necessary for the expression of defensive behaviors, potentially mediated by projections to the amygdala (27,63). Additionally, we have recently implicated the VTA in the expression of freezing fear responses, serving as a hub between upstream GABAergic inputs from the brainstem and outputs to the amygdala nuclei (64). These data extend the role of the VTA beyond reward processing, demonstrating a primordial role in interpreting environmental threatening stimuli and triggering appropriate defensive responses, extending beyond the dopaminergic population. Environmental challenges that produce lasting maladaptation on this circuit are bound to affect the detection, value, and stress responses to threatening stimuli. We showed here that chronic stress exposure produces overactivation of the VTAGlu → BLA circuit, promoting increased avoidance of open arms, which is indicative of high anxiety.

## ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Université Côte d'Azur, and the Agence Nationale de la Recherche (ANR) (Grant No. ANR-20-CE16-0014 [to SPF and FG]). Additional support was provided by the French government through the France 2030 investment plan managed by the ANR, as part of the Initiative of Excellence of Université Côte d'Azur (Reference No. ANR-15-IDEX-01 [to

JB]), and the Collegium of Advanced Studies, and National Cancer Institute IReSP (Grant No. SPAV1-23-005 [to JB]), the Fédération Pour la Recherche sur le Cerveau and Rotary—Espoir en Tête, and the Fondation pour la Recherche Médicale (Grant Nos. FRM DEQ20180339159 and FRM EQU202403018036 [to JB]). TC is a recipient of a fourth-year Ph.D. fellowship from the Fondation pour la Recherche Médicale (Grant No. FDT202106012968). LR is also a recipient of a Ph.D. fellowship from the Fondation pour la Recherche Médicale (Grant No. FDT202404018356). Experiments done by MFB and SZ were supported by the Intramural Research Program (IRP) of the National Institute on Drug Abuse (NIDA)/National Institutes of Health (NIH).

We acknowledge the resources and services provided by the Histology and Imaging Core, NIDA IRP (NIH). We thank Julius Zhu, Manuel Mameli, and Imane Moutkine for their help to obtain GluA1ct plasmids. We thank the staff from IPMC animal facilities for their technical support and the members of the Physiopathology of Neuronal Circuits team for helpful discussions and technical advice.

The authors report no biomedical financial interests or potential conflicts of interest.

## ARTICLE INFORMATION

From the Université Côte d'Azur, Nice, France (TC, JG-R, LR, HF, JB, SPF); Institut de Pharmacologie Moléculaire & Cellulaire, Centre National de la Recherche Scientifique UMR 7275, Valbonne, France (TC, JG-R, LR, HF, JB, SPF); Institut National de la Santé et de la Recherche Médicale U1323, Bordeaux, France (TC, JG-R, LR, HF, JB, SPF); Université de Bordeaux, Centre National de la Recherche Scientifique, Institut des Maladies Neurodégénérative, UMR 5293, Bordeaux, France (AB, CG, FG); Confocal and Electron Microscopy Core, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland (SZ); Integrative Neuroscience Research Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland (MFB); and Instituto de Fisiologia, Biologia Molecular y Neurociencias, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina (MS-R).

TC and JG-R contributed equally to this work.

JB and SPF are joint last authors.

Address correspondence to Sebastian P. Fernandez, Ph.D., at fernandez@ipmc.cnrs.fr, or Jacques Barik, Ph.D., at barik@ipmc.cnrs.fr.

Received Sep 5, 2024; revised Mar 17, 2025; accepted Apr 6, 2025.

Supplementary material cited in this article is available online at https://doi.org/10.1016/j.biopsych.2025.04.007.

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