# Nuclear Calcium Signaling in D1 Receptor-Expressing Neurons of the Nucleus Accumbens Regulates Molecular, Cellular, and Behavioral Adaptations to Cocaine

Estefani Saint-Jour, Marie-Charlotte Allichon, Andry Andrianarivelo, Enrica Montalban, Claire Martin, Lisa Huet, Nicolas Heck, Anna M Hagenston, Aisha Ravenhorst, Mélanie Marias, et al.

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Estefani Saint-Jour, Marie-Charlotte Allichon, Andry Andrianarivelo, Enrica Montalban, Claire Martin, et al. Nuclear Calcium Signaling in D1 Receptor-Expressing Neurons of the Nucleus Accumbens Regulates Molecular, Cellular, and Behavioral Adaptations to Cocaine. Biological Psychiatry, 2025, 98 (1), pp.34-45. 10.1016/j.biopsych.2025.01.013. hal-05361412

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# Nuclear Calcium Signaling in D₁ Receptor-Expressing Neurons of the Nucleus Accumbens Regulates Molecular, Cellular, and Behavioral Adaptations to Cocaine

Estefani Saint-Jour, Marie-Charlotte Allichon, Andry Andrianarivelo, Enrica Montalban, Claire Martin, Lisa Huet, Nicolas Heck, Anna M. Hagenston, Aisha Ravenhorst, Mélanie Marias, Nicolas Gervasi, Faustine Arrivet, Adèle Vilette, Katleen Pinchaud, Sandrine Betuing, Thomas Lissek, Jocelyne Caboche, Hilmar Bading, and Peter Vanhoutte

## ABSTRACT

**BACKGROUND:** The persistence of cocaine-evoked adaptations relies on gene regulations within the reward circuit, especially in the ventral striatum (i.e., nucleus accumbens [NAc]). Notably, activation of the ERK (extracellular signal-regulated kinase) pathway in the striatum is known to trigger a transcriptional program shaping long-term responses to cocaine. Nuclear calcium signaling has also been shown to control multiple forms of transcription-dependent neuroadaptations, but the dynamics and roles of striatal nuclear calcium signaling in preclinical models of addiction remain unknown.

**METHODS:** A genetically encoded cell type-specific nuclear calcium probe has been developed to monitor calcium dynamics in the nuclei of striatal neurons, including in freely moving mice. A cell type-specific inhibitor of nuclear calcium signaling combined with 3-dimensional imaging of neuronal morphology, immunostaining, and behavior was used to disentangle the roles of nuclear calcium in NAc medium spiny neurons (MSNs) expressing the dopamine D₁ receptor (D1R) or D₂ receptor (D2R) on cocaine-evoked responses.

**RESULTS:** The D1R-mediated potentiation of calcium influx through glutamate NMDA receptors, which shapes cocaine effects, also drives nuclear calcium transients. Fiber photometry revealed that cocaine-treated mice showed a sustained nuclear calcium increase in NAc D1R-MSNs. Disrupting nuclear calcium in D1R-MSNs, but not D2R-MSNs, blocked cocaine-evoked morphological changes of MSNs and gene expression and blunted cocaine's rewarding effects.

**CONCLUSIONS:** Our study unravels the dynamics and roles of cocaine-induced nuclear calcium signaling increases in D1R-MSNs on molecular, cellular, and behavioral adaptations to cocaine and represents a significant breakthrough because it could contribute to the development of innovative strategies with therapeutic potential to alleviate addiction symptoms.

https://doi.org/10.1016/j.biopsych.2025.01.013

Drug addiction is defined by compulsive patterns of drug-seeking and drug-taking behavior despite adverse consequences and recurring episodes of abstinence and relapse. Addictive drugs hijack reward processing by increasing dopamine (DA) in the mesocorticolimbic system (1), which persistently modulates excitatory glutamatergic transmission within the reward circuitry, especially in the ventral part of the striatum, the nucleus accumbens (NAc) (2,3). The NAc is a key target structure of drugs of abuse, notably because it receives converging glutamate inputs from limbic, thalamic, and cortical regions, encoding components of drug-associated stimuli and environment together with DA signals that sense incentive values. Signal integration is achieved by GABAergic

(gamma-aminobutyric acidergic) striatal medium spiny neurons (MSNs) receiving glutamate and DA afferents converging onto their dendritic spines (4,5). Based on the preferential expression of either DA receptor type 1 (D1R) or type 2 (D2R), MSNs primarily form 2 segregated populations (6,7) with distinct roles in controlling addictive-like behavior (8,9), although a fraction of MSNs express both receptors (10). A prevailing hypothesis is that cell type-specific changes in synaptic transmission in the NAc induced by drugs of abuse trigger signaling pathways and downstream genetic and epigenetic responses that likely participate in the transition from casual drug consumption to addiction (8,11). Accordingly, virtually all addictive drugs activate the ERK
(extracellular signal-regulated kinase) pathway in striatal D1R-MSNs (10,12,13). This activation requires the concomitant stimulation of D1R and glutamate receptors of the NMDA subtype (NMDARs) (14,15), which makes ERK activation a coincidence detector of glutamate and DA signaling (16). We previously showed that D1R-mediated facilitation of NMDARs is the trigger for a calcium (Ca²⁺)-dependent activation of ERK by cocaine in the striatum (17). Once activated downstream from D1Rs and NMDARs, ERK drives drug-induced epigenetic and genetic programs with important roles because inhibiting either the potentiation of NMDARs by D1R or ERK activity dampens the rewarding effects of cocaine (15,17) and the reconsolidation of drug-associated memories (18–20).

Cytoplasm-to-nucleus signal propagation is not solely achieved by Ca²⁺-mediated ERK activation. Calcium ions are themselves key actors in cytoplasm-to-nucleus communication because Ca²⁺ signals generated by synaptic activity trigger Ca²⁺ signals within the nucleus (21). Nuclear Ca²⁺ transients are major signaling end points in cytoplasm-to-nucleus communication and function as a master switch for transcription-dependent neuronal plasticity in multiple models of long-term adaptations in the brain (22). For example, nuclear Ca²⁺-dependent signaling sustains acquired neuroprotection in hippocampal neurons (23), an activity-driven form of long-term adaptation in which neurons that have been electrically activated are more resistant to cell death-inducing conditions. Similarly, the consolidation of memories, both in the fruit fly *Drosophila melanogaster* and in rodents, and the development of chronic pain in mice, are critically dependent on nuclear Ca²⁺ signaling (24–26).

Based on multiple similarities in both concepts and mechanisms between cellular processes underlying addiction and other forms of long-lasting neuroadaptations, we investigated the dynamics and roles of nuclear Ca²⁺ in cocaine-mediated adaptations in vivo. Cell type-specific imaging of nuclear Ca²⁺ dynamics revealed that D1R-mediated facilitation of NMDARs, which shapes cocaine-evoked adaptive behavior (14,17,18), triggers a sustained increase in nuclear Ca²⁺ in NAc D1R-MSNs in freely moving mice. The selective inhibition of nuclear Ca²⁺ signaling in D1R-MSNs, but not D2R-MSNs, impaired cocaine-evoked rewarding effects, morphological changes, and immediate early gene (IEG) induction. These findings underscore the cell type-specific role of nuclear Ca²⁺ signals in the striatum in controlling molecular, cellular, and behavioral adaptations induced by cocaine and constitute a significant breakthrough in our understanding of the molecular basis of addiction.

# METHODS AND MATERIALS

## Animals and Treatments

Six-week-old C57BL/6J male mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Mice were housed 4 per cage on a 12-hour light/dark cycle and in stable temperature (22° C) and humidity (60%) conditions with ad libitum access to food and water. They were acclimatized to the animal facility for at least 1 week. All experiments were carried out in accordance with standard ethical guidelines (European Community Council Directive on the Care and Use of Laboratory Animals [86/609/EEC] and the French National Committee [2010/63]). Intraperitoneal (IP) injections of cocaine hydrochloride (Sigma-Aldrich) at

15 mg/kg dissolved in a saline solution (0.9% NaCl wt/vol) were performed in a volume of 10 mL/kg.

## Nuclear Calcium Imaging In Vivo

Stereotaxic injections were performed in the NAc with a serotype 2/1 recombinant AAV (adeno-associated virus) driving expression of the Ca²⁺ probe GCaMP3 targeted to nuclei owing to the fusion of 3 nuclear localization signals (NLSs) (26,27) in a Cre-recombinase-dependent manner (AAV-DIO-GCaMP3-NLS). D1R-MSN-specific expression of GCaMP3-NLS was achieved by co-injecting AAV-DIO-GCaMP3-NLS with a serotype 9 AAV-PPTA-Cre, which drives the expression of the Cre recombinase under the D1R-MSN-specific preprotachykinin (PPTA) promoter (28). Following injections of AAV, a chronically implantable cannula composed of a bare optical fiber and a fiber ferrule was implanted at the location of the viral injection site (1.7 mm rostral to the bregma, 1.2 mm lateral to the midline, and 4.6 mm ventral to the skull). After 3 weeks, mice were habituated to the open field arena. On the test day, the fiber was connected but not switched on for habituation to the open field for at least 30 minutes. Recordings started 10 minutes before the IP injection of saline or cocaine and lasted for 30 to 35 minutes postinjection. For each trial, ΔF/F signal analysis was performed from -5 to +20 minutes relative to the time of injection.

## Cell Type-Specific Inhibition of Nuclear Calcium Signaling

The AAV-DIO-mScarlet-T2A-CaMBP4-HA (AAV-mScarlet-CaMBP4) was injected into the NAc to drive a Cre-recombinase-dependent bicistronic expression of the fluorescent reporter protein mScarlet and CaMBP4 fused to a hemagglutinin (HA) tag. CaMBP4 is a nuclear protein harboring 4 repeats of the rabbit M13 calmodulin-binding protein that binds to Ca²⁺/CaM complexes and inhibits nuclear Ca²⁺/calmodulin-dependent transcription (23,25,29,30). To alter nuclear Ca²⁺ signaling in D1R-MSNs or D2R-MSNs, mice were injected in the NAc with a mixture of AAV-mScarlet-CaMBP4 and AAV-PPTA-Cre (D1R-MSN-specific expression of Cre recombinase) or the AAV-PPE-Cre, which drives the expression of the Cre recombinase under the control of the D2R-MSN-specific prepro-enkephalin (PPE) promoter (31,32). As a control, we co-injected the AAV-DIO-mScarlet-NLS (AAV-mScarlet-NLS), driving the Cre recombinase-dependent expression of mScarlet fused to 3 NLSs, together with AAV-PPTA-Cre or AAV-PPE-Cre. All experiments were carried out 3 weeks after stereotaxic injections.

## Statistical Analysis

Results were analyzed with GraphPad Prism (version 8.0.1). Details about statistical analyses are provided in the figure legends.

See the Supplement for a detailed description of the experimental procedures.

# RESULTS

## Synergistic Action of D1R and NMDAR Stimulation on Nuclear Calcium Transients in Cultured MSNs In Vitro

A prerequisite for the study of striatal nuclear Ca²⁺ signaling function in cocaine-mediated responses in vivo was to monitor
its unknown dynamics in MSNs. Because cocaine-adaptive behaviors critically depend on a $Ca^{2+}$-dependent activation of ERK in NAc D1R-MSNs (8,14,17,18,33), we focused on this MSN subtype. First, we used cultured striatal neurons, a majority of which express the D1R, and stimulated them with a low dose of glutamate (0.3 µM) or the D1R agonist SKF38393 (3 µM) used separately or in combination, a treatment hereafter referred to as co-stimulation. This co-stimulation paradigm has proven to be relevant for identifying signaling events shaping cocaine-

adaptive behavior in vivo (17,34), including the potentiation of NMDAR induced by the D1R stimulation that triggers a $Ca^{2+}$-dependent ERK activation in D1R-MSNs (14,17,18). Therefore, we asked whether D1R-induced facilitation of NMDARs could also yield $Ca^{2+}$ transients in the nucleus of MSNs. Cultured MSNs were infected with AAV-GCaMP3-NLS encoding the calcium probe GCaMP3 targeted to nuclei owing to the fusion of 3 NLSs at its C-terminus end, as already described (26,27) (Figure 1A). As expected, the low dose of glutamate or SKF38393

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Synergistic action of D1R and NMDA receptor stimulation on nuclear calcium transients in cultured medium spiny neurons in vitro. (A) To image nuclear $Ca^{2+}$ signals, cultured striatal neurons were infected with AAV-GCaMP3-NLS, driving the expression of the $Ca^{2+}$ probe GCaMP3 in nuclei (top line panels). The two bottom line panels are representative images of changes of nuclear $Ca^{2+}$ signal intensity ($\Delta$F/F) in basal conditions and during incubations with glutamate (0.3 µM) or the D1R agonist SKF38393 (3 µM), used separately or in combination, in the absence (co-stim) or presence of the NMDA receptor antagonist APV (co-stim + APV; bottom panels). (B) Traces of nuclear $Ca^{2+}$ intensity changes ($\Delta$F/F) before and during the stimulation in each group ($n$ = 15–23 wells per treatment from at least $n$ = 5 independent neuronal cultures; SKF38393: $n$ = 15 wells; glutamate: $n$ = 16 wells, co-stim: $n$ = 23 wells; co-stim + APV: $n$ = 16 wells). (C) Quantifications of the corresponding areas under the curve (arbitrary unit) in each group. One-way analysis of variance followed by a Bonferroni post hoc test: treatment effect, $F_{3,65}$ = 28.36, \*\*\*p < .001. (D) Representative images of GCaMP3-NLS expression (top panels) and ($\Delta$F/F) of nuclear $Ca^{2+}$ levels in cultured medium spiny neurons incubated with either a vehicle solution, the peptide disrupting D1R-NMDA receptor subunit heteromerization (Tat-GluN1C1), or the related control peptide Tat-GluN1C1$\Delta$ 30 minutes prior to and during the co-stim paradigm (bottom panels). (E) Nuclear $Ca^{2+}$ traces obtained in each group ($n$ = 14–21 wells per treatment from at least $n$ = 3 independent neuronal cultures, Tat-GluN1C1 = 15 wells, Tat-GluN1C1$\Delta$ = 15 wells, vehicle = 23 wells). (F) Quantifications of corresponding A.U.C. One-way analysis of variance followed by a Bonferroni post hoc test: peptide effect $F_{2,50}$ = 13.10, \*p < .05; \*\*\*p < .001. Error bars denote SEM. APV, 2-amino-5-phosphonovaleric acid; A.U.C, areas under the curve; D1R, D₁ receptor; n.s., not significant.</figcaption></figure>
applied alone did not impact nuclear Ca²⁺ concentration. In
contrast, the co-stimulation paradigm produced a transient in-
crease in nuclear Ca²⁺ levels, which was blocked by the NMDAR
antagonist APV (2-amino-5-phosphonovaleric acid) (Figure 1A-
C). Because the facilitation of NMDAR functions by DA in D1R-
MSNs relies on the heteromerization of D1Rs with NMDARs
(32,34,35), we also tested whether D1R-NMDAR interactions
contribute to this synergistic action of D1Rs and NMDARs on
nuclear Ca²⁺ signals. This appeared to be true because a cell-
penetrating interfering peptide (TAT-GluN1C1) that disrupts
D1R-NMDAR interactions (34) also blocked nuclear Ca²⁺ tran-
sients induced by the co-stimulation of D1Rs and NMDARs,
whereas the related control peptide (TAT-GluN1C1Δ) (34) had no

effect (Figure 1D–F). These results show that D1R-NMDAR het-
eromerization, which regulates cocaine-evoked responses in vivo
(32,34), is essential to drive striatal nuclear Ca2+ transients in an
in vitro setting.

Cocaine Induces a Sustained Increase of Nuclear
Calcium Levels in D1R-MSNs In Vivo

Whole-cell Ca²⁺ imaging has shown that a single cocaine in-
jection elicits an increase and a decrease in Ca²⁺ responses in
D1R-MSNs and D2R-MSNs, respectively (36). To complete our
in vitro nuclear Ca²⁺ imaging data in a system that more closely
resembles the in vivo scenario, we studied nuclear Ca²⁺

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F

**Figure 2.** Cocaine induces a sustained increase in nuclear calcium levels in D1R-MSNs in vivo. (A) Mice were injected in the NAc with a mixture of AAV-DIO-GCaMP3-NLS and AAV-PPTA to monitor nuclear Ca²⁺ (nucl-Ca²⁺) changes in D1R-MSNs by 2-photon imaging in acute striatum slices incubated with either glutamate (3 µM) or SKF38393 (10 µM) or both in combination. (B) Changes of nuclear Ca²⁺ signals (ΔF/F) before and during 20-minute pharmacological treatments (light blue square) in each group (n = 4–9 slices from 4 to 5 mice, SKF38393; n = 6, glutamate n = 4, co-stim: n = 9). (C) Quantifications of the related areas under the curve (arbitrary unit). One-way analysis of variance followed by a Bonferroni post hoc test: treatment effect, F<sub>2,16</sub> = 15.76, **p** < .01; ***p** < .001. (D) Mice were injected in the NAc with AAV-DIO-GCaMP3-NLS and AAV-PPTA-Cre. Three weeks later, nuclear Ca²⁺ levels in D1R-MSNs were monitored by fiber photometry in freely moving mice first receiving an administration of saline solution and then a single injection of cocaine. (E) Changes of nuclear Ca²⁺ signals (ΔF/F) before and during an intraperitoneal injection of saline solution or cocaine. Two-way analysis of variance shows a significant effect for the interaction between time and treatment: F<sub>22,176</sub> = 10.22, *p* < .0001. Sidak multiple comparison post hoc test: first time point with a significant difference between saline and cocaine treatment is observed 5 minutes post-cocaine injection; \*p* < .05; *n* = 5 mice for each treatment. (F) Quantifications of the related A.U.C. Two-sided Student's *t* test, t<sub>4</sub> = 19.13, \*\*\*p* < .001. Error bars denote SEM. A.U.C, areas under the curve; D1R, D<sub>1</sub> receptor; MSN, medium spiny neuron; NAc, nucleus accumbens.
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**Figure 3.** Nuclear calcium signaling in D1R-MSNs is involved in cocaine's rewarding effects. (A) To evaluate the impact of nuclear Ca²⁺ signaling inhibition on the viability of D1R-MSNs, mice were injected in the nucleus accumbens with a mixture of AAV-hSyn-DIO-mScarlet-T2A-CaMBP4-HA (AAV-mScarlet-CaMBP4; CaMBP4) and AAV-PPTA-Cre. As a control, mice were infected with AAV-hSyn-DIO-mScarlet-NLS-Myc (AAV-mScarlet-NLS; control) and AAV-PPTA-Cre. Neuronal survival of mScarlet-positive D1R-MSNs was analyzed 3 weeks after AAV injections. (B) Illustrative images showing that the AAV-mScarlet-CaMBP4 allows the expression of mScarlet, which was localized in all subcellular compartments, and immunolabeled HA tag, which is fused to CaMBP4, confirmed successful bicistronic expression of both constructs and nuclear targeting of CaMBP4. (C) Representative images of mScarlet expression, Hoechst counterstaining, and NeuN immunolabeling obtained from mice infected with AAV-mScarlet-NLS and PPTA-Cre (top panels; control) or AAV-mScarlet-CaMBP4 and PPTA-Cre (bottom panels, CaMBP4). Quantifications of mScarlet-positive D1R-MSN viability based on nuclear counterstaining with Hoechst (D) and (E) the coexpression of NeuN. Results are expressed as ratios compared with the control group (n = 3 mice per group). D/Hoechst: 2-sided Student's t test, t₂ = 0.3619, *p* = .7521. E/NeuN: 2-sided Student's t test, t₂ = 3.384, *p* = .0773. (F–H) Mice co-infected in the NAc with AAV-PPTA-Cre and either AAV-mScarlet-CaMBP4 (CaMBP4) or AAV-mScarlet-NLS (control) were evaluated 3 weeks following viral infection for CPP and basal locomotor activity. (I) Schematic of the experimental timeline. (J) CPP scores of mice infected in D1R-MSNs with AAV-mScarlet-NLS (control, outlined orange) or AAV-
dynamics ex vivo in striatal slices in which D1R- and D2R-MSNs are mostly segregated (28). Nuclear Ca²⁺ imaging was performed in acute striatal slices prepared from adult mice and incubated either with glutamate (3 µM) or SKF38393 (10 µM) or both. In this model, the co-stimulation paradigm triggers some of the cocaine-induced adaptations in D1R-MSNs, including ERK activation, downstream IEG induction, and increased dendritic spine density (28). For cell type-specific monitoring of nuclear Ca²⁺ in D1R-MSNs, mice were injected in the NAc with a mixture of the AAV-PPTA-Cre, driving the expression of the Cre recombinase under the D1R-MSN-specific preprotachykinin promoter (28,31,32,37), and AAV-DIO-GCaMP3-NLS. Two-photon imaging revealed that the co-stimulation of D1Rs and NMDARs induced a progressive and sustained nuclear Ca²⁺ rise in D1R-MSNs, while glutamate or SKF38393 alone did not impact nuclear Ca²⁺ levels (Figure 2A-C). Because a single cocaine injection has been shown to trigger an increased frequency of Ca²⁺ transients in D1R-MSNs using whole-cell Ca²⁺ imaging in freely moving animals (38), we used the same viral approach to probe whether this increased occurrence of Ca²⁺ events could affect the nuclear Ca²⁺ pool in D1R-MSNs of freely moving mice. To this end, we used fiber photometry to monitor NAc nuclear Ca²⁺ dynamics before and after a saline or a cocaine injection (Figure 2D). We found that a single cocaine injection triggered an increase in nuclear Ca²⁺ levels in D1R-MSNs that reached significance as soon as 5 minutes after cocaine injection compared with saline-treated mice. This rise in nuclear Ca²⁺ then progressively increases during the first 10 minutes postinjection and reached a plateau between 10 and 20 minutes postinjection (Figure 2E, F and Figure S1), showing that cocaine also induced rapid and sustained increases of nuclear Ca²⁺ levels in D1R-MSNs in vivo.

## Nuclear Calcium Signaling in D1R-MSNs Is Involved in the Rewarding Effects of Cocaine

To evaluate the function of nuclear Ca²⁺ signals in D1R-MSNs specifically as pertains to cocaine-induced adaptations, we used the CaMBP4 protein that contains 4 repeats of the M13 CaM binding motif derived from the rabbit skeletal muscle myosin light chain kinase (30). Because CaMBP4 bears several NLSs (39), it localizes to the nucleus, where it binds with a high affinity to nuclear Ca²⁺/CaM complexes and inhibits nuclear Ca²⁺/CaM-dependent transcription (23,25,29,30). To selectively interfere with nuclear Ca²⁺ signaling in D1R-MSNs, we designed and co-injected into the NAc an AAV vector (AAV-DIO-mScarlet-CaMBP4) that drives the Cre-recombinase-dependent expression of the fluorescent reporter protein mScarlet and CaMBP4

fused to an HA tag with AAV-PPTA-Cre (Figure 3A). As a control, we used the AAV-DIO-mScarlet-NLS that drives the Cre-dependent nuclear expression of mScarlet owing to its fusion with NLS repeats. As expected, D1R-MSNs infected with AAV-DIO-mScarlet-CaMBP4 displayed a somatic and dendritic expression of mScarlet, whereas HA immunostaining confirmed the nuclear localization of CaMBP4 (Figure 3B). Because nuclear Ca²⁺ signaling has been shown to control a genetic program involved in neuroprotection of hippocampal neurons (23), we first assessed the viability of NAc D1R-MSNs expressing CaMBP4. Morphological analyses of nuclei counterstained with Hoechst, as well as the expression of the nuclear neuronal marker NeuN (Figure 3C), revealed that disrupting nuclear Ca²⁺ signaling did not affect D1R-MSN survival in vivo (Figure 3D, E). Then we tested whether nuclear Ca²⁺ in NAc D1R-MSNs could contribute to the rewarding effects of cocaine using the conditioned place preference (CPP) paradigm (Figure 3F). As expected, mice infected with the control AAV developed a significant cocaine-induced CPP. In contrast, the expression of CaMBP4 in D1R-MSNs inhibited CPP to cocaine (Figure 3G) while preserving basal locomotion (Figure 3H) and cocaine-induced psychomotor sensitization (Figure 3I-K), demonstrating that activation of nuclear Ca²⁺-dependent signaling triggered by cocaine in D1R-MSNs selectively regulated the rewarding properties of this drug.

## Nuclear Calcium-Dependent Signaling in D1R-MSNs Controls Cocaine-Mediated IEG Induction and Morphological Changes of MSNs

Multiple lines of evidence support the idea that nuclear Ca²⁺ signaling is a main route linking changes in synaptic activity to transcription in various models of long-lasting neuronal adaptations (22). Thus, we interrogated its functional role on cocaine-mediated IEG expression, focusing on c-Fos and Arc, the induction of which in D1R-MSNs is key for the development of enduring behavioral adaptations to cocaine (8,40,41). Mice injected in the NAc with the mixture of AAV to alter nuclear Ca²⁺ signaling in D1R-MSNs described above received a single injection of saline or cocaine and were perfused 1 hour later to study IEG expression levels in mScarlet-expressing D1R-MSNs (Figure 4A). We found that mScarlet-positive D1R-MSNs infected with the control virus displayed a significant induction of c-Fos and Arc proteins in response to cocaine, which was blocked in the presence of CaMBP4 (Figure 4A-D), showing that activation of nuclear Ca²⁺ signaling pathway controlled cocaine-evoked expression of neuronal plasticity-related IEGs.

mScarlet-CaMBP4 (CaMBP4, filled orange) and treated with saline or cocaine. Two-way analysis of variance virus × treatment interaction F₁,₄₈ = 4.8, \*p < .05. Bonferroni multiple comparison test: \*\*\*p < .0001; \*p < .001; n = 11–15 mice per group. (H) Basal locomotor activity of mice expressing AAV-mScarlet-NLS (control) or AAV-mScarlet-CaMBP4 (CaMBP4) in D1R-MSNs was automatically measured during the 20-minute pretest session of the CPP and represented as a cumulative sum of beam breaks during the entire session. n = 16 mice per group. Two-sided Student's *t* test, t₁₅ = 1.662, p = .1173. (I) Schematic representation of the experimental timeline of viral infections and cocaine (15 mg/kg) injections to study the impact of inhibiting nuclear Ca²⁺ signaling in D1R-MSNs on the development of locomotor sensitization. (J) Inhibition of nuclear Ca²⁺ signaling in D1R-MSNs with CaMBP4 does not alter the development of cocaine-induced locomotor sensitization compared with the control group infected with the related control virus (AAV-mScarlet-NLS). Two-way analysis of variance: virus effect, F₁,₂₂ = 0.5064; n = 11–12 mice per group. (K) Sensitization index of cocaine-treated mice infected with CaMBP4 (filled orange) and related control mice (outlined orange). One-sample Student's *t* test: t₀ = 2.291, \*\*\*p < .01 for cocaine control vs. 1; t₈ = 4.594, \*p < .05 for cocaine CaMBP4 vs. 1. Two-sided Student's *t* test, t₁₇ = 0.9356, p = .3626. Error bars denote SEM. CPP, conditioned place preference; DIR, D₁ receptor; HA, hemagglutinin; MSN, medium spiny neuron; n.s., not significant; PPTA, preprotachykinin.
<figure><img src="image_18.png" /><figcaption>**Figure 4.** Nuclear calcium-dependent signaling in D1R-MSN controls cocaine-mediated immediate early gene induction and morphological changes of MSNs. (A) To characterize the impact of inhibiting nuclear Ca²⁺ signaling on cocaine-triggered immediate early gene induction in D1R-MSNs, mice were injected in the NAc with a mixture of AAV-hSyn-DIO-mScarlet-T2A-CaMBP4-HA (AAV-mScarlet-CaMBP4; CaMBP4) or the related control virus, AAV-hSyn-</figcaption></figure>
Acute or repeated cocaine administration also triggers an increase in dendritic spine density that is confined to D1R-MSNs (28,42), leading to an enhancement of glutamatergic connectivity to NAc D1R-MSNs (28,43). Because nuclear Ca²⁺ signaling controls neuronal differentiation and spine density in the hippocampus (25,44), we analyzed the contribution of nuclear Ca²⁺ signaling to cocaine-evoked morphological changes of MSNs. Mice expressing CaMBP4 in D1R-MSNs and related control animals were exposed to 5 daily saline or cocaine injections and perfused 24 hours after the last injections to analyze dendritic spine density (Figure 4E). To ensure accurate detection of dendritic spines in both AAV-mScarlet-CaMBP4 and AAV-mScarlet-NLS-expressing neurons, we chose a gene gun-mediated delivery of the green lipophilic plasma membrane dye DIO to stochastically label dendritic fragments of both D1R- and D2R-MSN subtypes upon inhibition of nuclear Ca²⁺ in D1R-MSNs in the mScarlet-positive viral injection site (Figure 4F). We found that mice infected with the control AAV in D1R-MSNs displayed a significant cocaine-mediated increase of dendritic spine density, which was blocked by CaMBP4 expression in D1R-MSNs (Figure 4G). Further analysis revealed that expressing CaMBP4 in D1R-MSNs similarly affected both thin and mushroom spines (Figure S2). These results establish that the increase in dendritic spine density induced by cocaine in D1R-MSNs required the activation of nuclear Ca²⁺ signaling in this MSN subtype.

## Inhibition of Nuclear Calcium Signaling in D2R-MSNs Does Not Impact Cellular and Behavioral Adaptations Induced by Cocaine

Because Ca²⁺ signals are differentially modulated by cocaine in D1R- and D2R-MSN subpopulations and play distinct roles in the formation of associations between cocaine and contextual cues (36,38), we also studied the consequences of inhibiting nuclear Ca²⁺ signaling in D2R-MSNs. We verified

that disruption of nuclear Ca²⁺-dependent signaling in NAc D2R-MSNs preserved their viability (Figure 5A–C) and spared basal locomotor activity (Figure 5D, E). In direct contrast to what we observed for D1R-MSNs, the inhibition of nuclear Ca²⁺-dependent signaling in D2R-MSNs altered neither the rewarding properties of cocaine (Figure 5F) nor the morphological adaptations induced by cocaine (Figure 5G, H). All together, these data demonstrate that the recruitment of nuclear Ca²⁺-dependent signaling in NAc D1R-MSNs, but not D2R-MSNs, regulates gene expression and dendritic spine density specifically in this MSN subpopulation, as well as the rewarding effects of cocaine.

## DISCUSSION

Nuclear Ca²⁺ signaling is a main route for cytoplasm-to-nucleus communication and plays critical roles in many forms of transcription-dependent neuronal adaptations, including long-term synaptic plasticity, memory consolidation, neuroprotection of hippocampal neurons, and the development of chronic pain (22). Because striatal signaling is a main driver of pathological drug-adaptive behavior (8,11), we investigated, in a cell type-specific manner, the unknown dynamics and roles of nuclear Ca²⁺ signals in preclinical models of cocaine exposure. We underline the contribution of nuclear Ca²⁺-dependent signaling in NAc D1R-MSNs by showing that cocaine triggers a rapid and sustained nuclear Ca²⁺ increase in NAc D1R-MSNs that shapes cocaine-evoked molecular, cellular, and behavioral responses.

As a first step, nuclear Ca²⁺ dynamics were monitored in cultured MSNs incubated with a D1R agonist and a low dose of glutamate, which recapitulates several signaling events triggered by cocaine in D1R-MSNs in vivo (17,18), including a D1R-mediated potentiation of NMDAR-dependent Ca²⁺ influx triggering the activation of ERK in D1R-MSNs, which is critical for the sensitizing and rewarding effects of this drug in vivo (17). We showed that the cross-talk between D1R- and NMDAR-dependent signaling not only elicited ERK activation

DIO-mScarlet-NLS-Myc (AAV-mScarlet-NLS; control), with AAV-PPTA-Cre. Three weeks after viral infections, mice were habituated for 3 days and then subjected to a single injection of saline or cocaine and perfused 1 hour postinjection. Bottom panels show illustrative images of mScarlet fluorescence and c-Fos immunostaining performed from control mice and mice expressing CaMBP4 in D1R-MSNs and treated with saline or cocaine. (B) Quantifications of mScarlet- and c-Fos-positive D1R-MSNs in each group. Control saline group: *n* = 4 mice, *n* = 2671 infected D1R-MSNs analyzed; control cocaine group: *n* = 6 mice, *n* = 4407 infected D1R-MSNs; CaMBP4 saline group: *n* = 4 mice, *n* = 781 infected D1R-MSNs; CaMBP4 cocaine group: *n* = 6 mice, *n* = 822 infected D1R-MSNs. Two-way ANOVA followed by a Bonferroni post hoc test: treatment effect: *F*<sub>1,16</sub> = 6.693, ***p* < .001 saline vs. cocaine in control groups; virus effect: *F*<sub>1,16</sub> = 33.33, ***p* < .001 cocaine control vs. cocaine CaMBP4 (*n* = 4–6 mice per group). (C) Representative pictures of mScarlet fluorescence and Arc immunostaining performed from control mice and mice expressing CaMBP4 in D1R-MSNs and treated with saline or cocaine. (D) Quantifications of mScarlet- and Arc-positive D1R-MSNs in each group. Control saline group: *n* = 4 mice, *n* = 844 infected D1R-MSNs analyzed; control cocaine group: *n* = 6 mice, *n* = 1106 infected D1R-MSNs analyzed; CaMBP4 saline group: *n* = 4 mice, *n* = 335 infected D1R-MSNs; CaMBP4 cocaine group: *n* = 6 mice, *n* = 406 infected D1R-MSNs. Two-way ANOVA followed by a Bonferroni post hoc test: treatment effect: *F*<sub>1,15</sub> = 6.935, ***p* < .01 saline vs. cocaine in control groups; virus effect: *F*<sub>1,15</sub> = 42.22, ***p* < .001 cocaine control vs. cocaine CaMBP4 (*n* = 4–6 mice per group). (E) To analyze the role of nuclear Ca²⁺ signaling in D1R-MSNs on cocaine-mediated changes of dendritic spine density in MSNs, mice were injected in the NAc with a mixture of AAV-mScarlet-CaMBP4 (CaMBP4) or the related control virus (AAV-mScarlet-NLS; control), with AAV-PPTA-Cre. Three weeks after viral infections, mice were habituated for 3 days and then subjected to daily injections of saline or cocaine for 5 days in a new cage. As shown in the right panels, dendritic spine density was studied via the stochastic labeling of both MSN subtypes with the lipophilic plasma membrane dye DIO (green) in the area of viral infection. (F) Three-dimensional rendering of MSN dendritic fragments obtained from control mice and mice expressing CaMBP4 in D1R-MSNs treated with saline or cocaine (*n* = 18–53 dendritic fragments from 4 to 6 mice per group). (G) Nuclear Ca²⁺ inhibition in D1R-MSNs fully blocks cocaine-induced increase in dendritic spine density of MSNs. Mixed-effect ANOVA with virus and treatment as fixed effects and mice tested as a random effect followed by a post hoc test conducted with the Wald method. Saline vs. cocaine in the control group: *p* < .001, Wald statistic = 6.1197; saline vs. cocaine in the CaMBP4 group: *p* = .90372, Wald statistic = 0.12124; control vs. CaMBP4 in the saline group: *p* = .34629, Wald statistic = 0.94575; control vs. CaMBP4 in the cocaine group: *p* < .001, Wald statistic = 5.0004. Error bars denote SEM. ***p* < .001. ANOVA, analysis of variance; coc, cocaine; D1R, D₁ receptor; hab., habituated; MSN, medium spiny neuron; NAc, nucleus accumbens; n.s., not significant; sal, saline.
<figure><img src="image_19.png" /><figcaption>**Figure 5.** Inhibition of nuclear calcium signaling in D2R-MSNs does not impact cellular and behavioral adaptations induced by cocaine. (A) Top: experimental timeline to study the impact of inhibiting nuclear Ca²⁺ signaling on the viability of D2R-MSNs. Mice were injected in the nucleus accumbens with a mixture of AAV-mScarlet-CaMBP4 (CaMBP4) and AAV-PPE-Cre. As a control, mice were infected with AAV-mScarlet-NLS (control) and AAV-PPE-Cre, which drives the expression of the Cre recombinase in D2R-MSNs. Bottom: representative images of mScarlet expression, Hoechst counterstaining, and NeuN immunolabeling obtained from mice infected with the control virus (top panels) or the CaMBP4 virus (bottom panels) in D2R-MSNs. Quantifications of mScarlet-positive D2R-MSN viability based on a nuclear counterstaining with Hoechst (B) and (C) the expression of NeuN. Results are expressed as ratios compared with the control group (n = 4 mice per group). B/Hoechst: Two-sided Student's t test, t₃ = 0.8847, *p* = .4415. C/NeuN: Two-sided Student's t test, t₃ = 0.5600, *p* = .6145. (D) Experimental timeline and viral infections used to study the role of nuclear Ca²⁺ signaling in D2R-MSNs on locomotor activity and cocaine-induced CPP. (E) Basal locomotor activity of control mice and mice expressing CaMBP4 in D2R-MSNs. Two-sided Student's t test, t₁₅ = 1.006, *p* = .3305; n = 16 per group. (F) CPP scores of mice infected in D2R-MSNs with AAV-mScarlet-NLS (control) or AAV-mScarlet-CaMBP4 (CaMBP4) and treated with saline or cocaine. Two-way analysis of variance: virus effect: F₁,₂₆ = 1.4, *p* = .2457; treatment effect: F₁,₂₆ = 18, *p* < .0001, Bonferroni multiple comparison: **p** < .001; *n* = 7-8 mice per group. (G) Top: Experimental timeline to analyze the role of nuclear Ca²⁺ signaling in D2R-MSNs on cocaine-mediated changes of MSN dendritic spine density. Mice were injected in the NAc with a mixture of AAV-mScarlet-CaMBP4 (CaMBP4) or the related control virus (AAV-mScarlet-</figcaption></figure>
but also generated NMDAR-dependent nuclear Ca²⁺ transients (Figure 1). Disrupting D1R-NMDAR heteromerization, which controls behavioral adaptations to cocaine (34), also prevented nuclear Ca²⁺ increases induced downstream from D1Rs and NMDARs, raising the prospect that nuclear Ca²⁺ signals in D1R-MSNs may participate in drug-evoked responses in vivo. Cell type-specific nuclear Ca²⁺ imaging highlighted that an acute cocaine injection induced a rapid and progressive increase in nuclear Ca²⁺ in NAc D1R-MSNs in vivo in freely moving mice (Figure 2), with kinetics paralleling those reported for whole-cell Ca²⁺ in D1R-MSNs after acute cocaine administration in anesthetized mice (36). In contrast, in D2R-MSNs, cocaine and cocaine-associated cues have previously been shown to decrease Ca²⁺ levels in this cell type (36,38). Because autonomous nuclear Ca²⁺ transients, independently of a cytosolic Ca²⁺ rise, have not yet been reported (22), it is unlikely that the pharmacological effects of cocaine would increase nuclear Ca²⁺ in D2R-MSNs. Accordingly, we found that inhibiting nuclear Ca²⁺/CaM signaling in D2R-MSNs with CaMBP4 did not impact the cocaine-induced adaptations analyzed in the current work (Figure 5). These findings support the idea that nuclear Ca²⁺/CaM signaling is not recruited in D2R-MSNs in response to cocaine or, alternatively, that it does not play any functionally relevant role in the cocaine-induced responses studied in the current study. All together, these data suggest that cocaine-evoked nuclear Ca²⁺ increases may originate from a D1R-mediated potentiation of Ca²⁺ influx through NMDARs in D1R-MSNs that subsequently invades the nucleus as a consequence of voltage-gated calcium channel activation by propagating excitatory postsynaptic potentials, as has already been proposed (22).

To study the role of nuclear Ca²⁺ signaling in NAc MSNs, we used a cell type-specific AAV-mediated expression of the CaMBP4 protein, which has been shown to efficiently inhibit nuclear Ca²⁺/CaM complexes both in vitro and in vivo (23–26). A limitation of this strategy is that it preserves nuclear Ca²⁺ functions that are calmodulin independent. Nuclear Ca²⁺ ions are able to directly bind to the EF motifs of the transcription repressor DREAM. Upon a rise in nuclear Ca²⁺ levels, the direct binding of nuclear Ca²⁺ ions to DREAM relieves its binding to DNA and promotes the activation of DREAM-responsive element-driven transcription in a calmodulin-independent manner (45,46). Although overexpressing CaMBP4 allowed us to show that nuclear Ca²⁺ in D1R-MSNs is key for some cocaine-evoked adaptations, we might have underestimated the full impact of nuclear Ca²⁺ signaling by using this interfering approach.

Because nuclear Ca²⁺ has been shown to promote acquired neuroprotection in cultured hippocampal neurons (23), we assessed whether nuclear Ca²⁺ blockade in vivo could alter the MSN survival rate in naïve mice expressing CaMBP4 in either NAc D1R-MSNs or D2R-MSNs. We found that the

AAV-mediated long-term expression of CaMBP4 was not detrimental for either MSN subtype. This is likely due to the low basal activity of MSNs that did not yield spontaneous nuclear Ca²⁺ transients in freely moving naïve mice (see Figure 2), as opposed to cultured hippocampal neurons that display spontaneous bursts of activity (47), which may trigger neuroprotective spontaneous nuclear Ca²⁺ transients. Accordingly, inhibiting nuclear Ca²⁺ signaling in both MSN subtypes spared basal locomotor activity. By contrast, blocking cocaine-induced nuclear Ca²⁺ signaling in D1R-MSNs impaired the development of cocaine-induced CPP, whereas the same manipulation in D2R-MSNs did not have an impact on the rewarding effects of cocaine. These observations show that disrupting nuclear Ca²⁺ signaling in NAc D1R-MSNs alters the association between the rewarding effects of the drug and the drug-paired environment, which further supports an important role for nuclear Ca²⁺ in associative learning and memory (24,25) in the current context of a psychiatric disorder.

Behavioral adaptations to cocaine are accompanied by an enhancement of dendritic spine density selectively in D1R-MSNs (28,42). This leads to the increased formation of glutamatergic synapses onto D1R-MSNs (28), which is likely involved in the long-term effects of cocaine on glutamate transmission onto D1R-MSNs and drug-adaptive behavior (18,48). The inhibition of nuclear Ca²⁺ signaling in either D1R-MSNs or D2R-MSNs did not impact dendritic spine density in saline-treated mice. By contrast, the increase in dendritic spine density induced by cocaine in D1R-MSNs required the activation of nuclear Ca²⁺ signaling in this MSN subtype, which is consistent with previous findings showing that the constitutive activation of nuclear Ca²⁺-CaMKIV signaling increases spine density in hippocampal neurons (44).

At the cellular and molecular level, disrupting nuclear Ca²⁺ signaling in D1R-MSNs fully blocked both cocaine-induced induction of the IEG c-Fos and Arc and the increase in dendritic spine density of NAc MSNs (Figure 4). Similarly, we previously showed that the pharmacological inhibition of ERK or its downstream targets also fully hindered the induction of these IEGs (15,40,49), as well as the increase in dendritic spines in D1R-MSNs in response to cocaine (28).

Notably, CaMBP4 expression was detected via HA immunostaining in exclusively nuclear locations (Figure 3B). Furthermore, global inhibition of Ca²⁺/CaM-dependent kinases with the CaMK inhibitor KN62 did not impair ERK activation downstream from D1Rs and NMDARs in mouse MSNs (Figure S3). Moreover, inhibitors of ERK prevent the development of both cocaine-induced locomotor sensitization and CPP (15,17,18), whereas we showed here that the inhibition of nuclear Ca²⁺ signaling selectively impaired CPP but preserved locomotor sensitization (Figure 3). Accordingly, we can rule out the possibility that CaMBP4 inhibits ERK activation as a result of an artifactual inhibition of dendritic and cytosolic Ca²⁺/

NLS; control), with AAV-PPE-Cre. Three weeks after viral infections, mice were habituated for 3 days and then subjected to a daily injection of saline or cocaine for 5 days in a new cage. Bottom: Three-dimensional rendering of MSN dendritic fragments obtained from control mice and mice expressing CaMBP4 in D2R-MSNs treated with saline or cocaine (n = 20–34 dendritic fragments from 4 to 6 mice per group). (H) Mixed-effect analysis of variance with virus and treatment as fixed effects and mice tested as a random effect followed by a post hoc test conducted with the Wald method. Saline vs. cocaine in the control group: *p* < .001, Wald statistic = 4.379; saline vs. cocaine in the CaMBP4 group: *p* < .001, Wald statistic = 4.0779; control vs. CaMBP4 in the saline group: *p* = .26026, Wald statistic = 1.1313; control vs. CaMBP4 in the cocaine group: *p* = .23109, Wald statistic = 1.2039. Error bars denote SEM. **\*\*p* < .001. coc, cocaine; CPP, conditioned place preference; D2R, D₂ receptor; hab, habituated; MSN, medium spiny neuron; n.s., not significant; PPE, preproenkephalin; sal, saline.
calmodulin/CaMK resulting from a leakage of CaMBP4 outside
nuclei. Instead, it seems that both nuclear Ca²⁺ and ERK
signaling are required for the full expression of molecular,
cellular, and behavioral changes induced by cocaine.

The selective implication of nuclear Ca²⁺ signaling down-
stream from D1Rs and NMDARs in the rewarding effects of
cocaine may seem counterintuitive because both D1R- and
NMDAR-dependent signaling are involved in both cocaine-
induced locomotor sensitization and CPP. However, multiple
pathways are recruited in D1R-MSNs in response to cocaine
as a consequence of concomitant D1R and NMDAR stimula-
tion, including the cAMP/PKA/DARPP32 and ERK pathways
(8,18), as well as nuclear Ca²⁺ signaling (the current work). The
role of the cAMP/PKA/DARPP32 and ERK pathways in con-
trolling cocaine-evoked locomotor sensitization and CPP has
been well documented (18). The fact that blockade of nuclear
Ca²⁺ signaling with CaMBP4, which does not impact other
pathways downstream from D1Rs and NMDARs (see
Figure S3), supports that the transcriptional program driven by
nuclear Ca²⁺ in response to cocaine selectively shapes the
rewarding effects of this drug. This is consistent with our
previous observation that the IEG *Npas4*, which is induced in
MSNs downstream from nuclear Ca²⁺ signaling, is not
involved in the sensitizing effects of cocaine (50) but instead
controls cocaine-induced CPP (51).

Conclusions

Therefore, our study unravels a functional role of nuclear Ca²⁺ signaling in D1R-MSNs, but not D2R-MSNs, on molecular, cellular, and behavioral adaptations to cocaine. These important findings constitute a significant breakthrough in the understanding of the molecular bases of cocaine-induced adaptations and highlight the potential benefit of targeting nuclear Ca²⁺ signaling not only in the field of addiction, but also potentially for multiple neuropsychiatric disorders associated with an imbalance of DA and glutamate transmission.

ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported by the Centre National de la Recherche Scientifique; the Institut National de la Santé et de la Recherche Médicale National; Sorbonne Université, Faculté des Sciences et Ingénierie; the Agence Nationale pour la Recherche and the German Research Foundation (Grant No. ANR-15-CE16-001 [to PV]) and German Research Foundation (Grant No. BA 1007/11-1[to HB] and Grant No. ANR-18-CE37-0003-02 [to PV]); the Fondation pour la Recherche Médicale (Grant No. DPA20140629798 [to JC]); and the Institut de Recherche en Santé Publique Aviesan APP-addiction 2019 (Grant No. 201146-00 [to PV]). AA, LH, and AV are recipients of Ph.D. fellowships from the French Ministry of Research. ES-J and M-CA are recipients of a fourth-year Ph.D. fellowship from the Fondation Pour la Recherche Médicale.

ES-J performed most calcium imaging studies, stereotaxic injections, behavioral assessments, immunohistochemistry, and confocal and 2-photon imaging experiments with the help of AA, M-CA, MM, AV, and SB. Viruses were designed by PV, HB, and AMH and produced by AMH, AR, and TL. NG supported ES-J in the analysis and quantifications of nuclear calcium signals on cultured neurons and striatal slices. Fiber photometry experiments were performed by EM and CM with the help of KP. Dendritic spine analysis was performed by FA, LH, and NH. Western blot experiments were performed by AMH and AR. Data analysis was completed by ES-J, M-CA, HB, JC, and PV. Experimental design was realized by ES-J, AA, JC, HB, AMH, and PV. The manuscript was written by HB, JC, and PV and was edited by all co-authors.

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

ARTICLE INFORMATION

From the Sorbonne University, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Institut of Biology Paris-Seine, Center for Neuroscience at Sorbonne University, Paris, France (ES-J, M-CA, AA, LH, NH, MM, FA, AV, KP, SB, JC, PV); Université Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, Unité Mixte de Recherche 8251, Centre National de la Recherche Scientifique, Paris, France (EM, CM); Heidelberg University, Interdisciplinary Center for Neurosciences, Institute of Neurobiology, Heidelberg, Germany (AMH, AR, TL, HB); and Center for Interdisciplinary Research in Biology, College de France, Centre National de la Recherche Scientifique UMR 7241, Institut National de la Santé et de la Recherche Médicale U1050, Paris Science et Lettre Research University, Paris, France (NG).

Address correspondence to Peter Vanhoutte, Ph.D., at peter.vanhoutte@ sorbonne-universite.fr.

Received Jun 28, 2024; revised Jan 10, 2025; accepted Jan 15, 2025.

Supplementary material cited in this article is available online at https://doi.org/10.1016/j.biopsych.2025.01.013.

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