# Characterization of mice with cell type-specific Gnal loss of function provides insights on GNAL-linked dystonia

Sophie Longueville, Ruiyi Yuan, Claire Naon, Emmanuel Valjent, Assunta Pelosi, Emmanuel Roze, Louise-Laure Mariani, Jean-Antoine Girault, Denis Hervé

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Sophie Longueville, Ruiyi Yuan, Claire Naon, Emmanuel Valjent, Assunta Pelosi, et al.. Characterization of mice with cell type-specific Gnal loss of function provides insights on GNAL-linked dystonia. Neurobiology of Disease, 2025, 215, pp.107071. 10.1016/j.nbd.2025.107071. hal-05274227

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# Characterization of mice with cell type-specific *Gnal* loss of function provides insights on *GNAL*-linked dystonia

Sophie Longueville<sup>a,b,c,d</sup>, Ruiyi Yuan<sup>a,b,c,d</sup>, Claire Naon<sup>e</sup>, Emmanuel Valjent<sup>e</sup>, Assunta Pelosi<sup>d</sup>,
Emmanuel Roze<sup>d</sup>, Louise-Laure Mariani<sup>b,d</sup>, Jean-Antoine Girault<sup>a,b,c,d</sup>, Denis Hervé<sup>a,b,c,d,*</sup>

## ARTICLE INFO

**Keywords:**

GNAL
Gaαolf
Dystonia
Striatum
Dopamine
Adenosine
D1 receptor
A2A receptor
Adenylyl-cyclase
Motor control
Psychostimulant

## ABSTRACT

Isolated dystonia can be caused by loss-of-function mutations in the *GNAL* gene (DYT-GNAL/DYT25). This gene encodes the αolf subunit of the heterotrimeric Golf protein, which, with β2γ7 subunits, mediates the stimulatory coupling of dopamine D1 and adenosine A2A receptors to adenylyl-cyclase. These receptors are expressed in distinct striatal projection neurons (SPNs) with complementary functions in motor behavior. To dissect the specific roles of Gaolf in the two main populations of SPNs, we generated and characterized mice in which *Gnal* was conditionally deleted in neurons expressing either D1 receptors (D1-SPNs) or A2A receptors (A2A-SPNs). Our results confirm the critical role of Gaolf in regulating adenylyl-cyclase 5 and its coupling with D1 and A2A receptors. Mice with a selective loss of Gaolf in D1-SPNs show nocturnal hyperactivity, deficits in motor performances, but no overt abnormal movements or generalized motor disability. Our experiments also reveal that Gαolf in D1-SPNs is required locomotor responses induced by some, but not all, D1 agonists or psychostimulants. Selective loss of Gaolf in A2A-SPNs does not affect motor abilities or learning but strikingly increases spontaneous locomotor activity. Hyperactivity is not further enhanced by psychostimulant drugs (cocaine, D-amphetamine, methylphenidate) or a selective A2 agonist, KW6002, but is paradoxically reduced by caffeine. Our study identifies specific roles of Gaolf downstream of D1 and A2A receptors in the control of motor behavior and drug responses, highlighting their respective individual contribution in dysfunctional striatal signaling, including dystonia.

## 1. Introduction

Dystonia is a heterogeneous neurological entity characterized by sustained or intermittent abnormal movements and/or postures that are typically patterned and repetitive (Albanese, 2025). Dystonia has multiple causes and a broad phenomenological spectrum from adult-onset focal dystonia to infant-onset generalized dystonia, including tremulous and jerky forms. Converging evidence emphasizes the important

role of dysfunctions of the striatum in many patients with dystonia (Bhatia and Marsden, 1994; Neychev et al., 2011). Numerous defects in dopamine-related striatal neurotransmission have been associated with dystonia (Ribot et al., 2019). They include disruption of dopamine synthesis or transport, altered development or survival of dopaminergic neurons, and modified intracellular signal transduction downstream of dopamine receptors (Mencacci et al., 2020). Alterations in neuron-specific intracellular signaling in the striatum, particularly those

Abbreviations: A2AR, A2A receptor; AC, adenylyl-cyclase; AC5, adenylyl-cyclase 5; D1R, D1 receptor; D2R, D2 receptor; DARPP-32, dopamine and cAMP-regulated phospho-protein 32 kDa; DYT-GNAL, dystonia linked to *GNAL* gene; F, female; *Gnal*-A2AcKO, *Gnal* conditional knockout in A2A receptor-expressing neurons; *Gnal*-D1cKO, *Gnal* conditional knockout in D1 receptor-expressing neurons; GPCR, G protein-coupled receptor; ip, intraperitoneal; KO, knockout; cKO, conditional knockout; M, male; ns, not significant; SPN, striatal projection neuron; A2A-SPN, A2A receptor-expressing striatal projection neuron; D1-SPN, D1 receptor-expressing striatal projection neuron; dSPN, striatal projection neuron of direct pathway; iSPN, striatal projection neuron of indirect pathway; WT, wild-type.

<footnote><sup>a</sup> Inserm UMR-S 1270, Paris 75005, France</footnote>

<footnote><sup>b</sup> Sorbonne Université, Faculty of Sciences and Engineering, Paris 75005, France</footnote>

<footnote><sup>c</sup> Institut du Fer à Moulin, 17 rue du Fer à Moulin, Paris 75005, France</footnote>

<footnote><sup>d</sup> Sorbonne Université, Institut du Cerveau, Inserm, CNRS, AP-HP, Institut de Neurologie, Hôpital de la Salpetrière, Paris 75013, France</footnote>

<footnote><sup>e</sup> Institut de Neurosciences, Inserm, Montpellier University, Montpellier 34000, France</footnote>

<footnote>* Corresponding author at: INSERM 17 rue du Fer à Moulin, 75005 Paris, France.</footnote>

<footnote>E-mail address: denis.herve@inserm.fr (D. Hervé).</footnote>
involved in dopamine transmission, can cause various hyperkinetic
motor disorders with dystonic components. These disorders may result
from pathogenic variants in several genes, including ADCY5 encoding
the adenyllyl-cyclase 5 (AC5) (Chang, 2016; Chen et al., 2014), PPP1R1B
encoding the dopamine and cAMP-regulated phosphoprotein 32 kDa
(DARPP-32) (Khan et al., 2022), GNAO1 encoding the α₀ subunit of G
protein (Ananth et al., 2016) or several genes encoding phosphodies-
terases (Erro et al., 2021; Miyatake et al., 2018; Niccolini et al., 2018;
Salpietro et al., 2018). GNAL-linked dystonia (DYT-GNAL or DYT25),
typically an adult-onset isolated dystonia, is caused by pathogenic var-
iants of GNAL (Fuchs et al., 2013; Goodchild et al., 2013), a gene
encoding the Gα₀lf subunit that is highly expressed in striatal neurons
(Drinnan et al., 1991; Herve et al., 1993). The Gα₀lf subunit has a high
sequence homology with the ubiquitously expressed Gαs protein, coded
by the GNAS gene, which also stimulates adenyllyl-cyclase (AC) (Jones
and Reed, 1989; Liu et al., 2001; Xie et al., 2015). In the principal
neurons of the striatum, the striatal projection neurons (SPNs), Gα₀lf is
much more expressed than Gαs and plays a crucial role in the positive
coupling of dopamine D1 (D1R) and adenosine A2A (A2AR) receptors
with AC (Corvol et al., 2001; Zhuang et al., 2000). In some populations
of GABAergic interneurons and in cholinergic interneurons Gα₀lf and Gαs
are co-expressed (Herve et al., 2001; Millett et al., 2024). Careful
analysis of the function of Gα₀lf proteins carrying pathogenic variants
has shown the diversity of mechanisms leading to the disruption of G
protein-coupled receptor (GPCR) signaling (Masuho et al., 2018).
Importantly, variants generating truncated and non-functional forms of
Gα₀lf can cause DYT-GNAL dystonia (Fuchs et al., 2013; Kumar et al.,
2014). Therefore, mouse models in which a mutation in Gnal sequence
prevents Gα₀lf expression correctly mimic the alterations present in some
patients. These models have a substantial limitation because homozy-
gous invalidation of Gnal (Gnal⁻¹⁻²⁻³⁻⁴) leads to a very high lethality in the
first days after birth, caused in part by anosmia, which disrupts feeding
reflexes in newborns (Belluscio et al., 1998). Heterozygous Gnal
knockout (KO) rodents (Gnal⁺⁻/⁻) are fully viable and reproduce the
genetic condition seen in the human disease, in whom most pathogenic
variants are autosomal dominant (Masuho et al., 2018). In the striatum
of Gnal⁺⁻/⁻ mice, basal and dopamine- or adenosine-induced AC activ-
ities are reduced (Corvol et al., 2001). In Gnal⁺⁻/⁻ rats, several signaling
proteins are altered including AC5 and dopamine D2 receptors (D2R) (El
Atiallah et al., 2024). While both mice and rats do not show spontaneous
abnormal movements reminiscent of dystonia, they may present alter-
ations in motor coordination and motor learning (Pelosi et al., 2017; Yu-
Taeger et al., 2020). In Gnal⁺⁻/⁻ mice, abnormal movements are trig-
gered by oxotremorine, a muscarinic agonist, revealing their vulnera-
bility to dystonic-type manifestations (Pelosi et al., 2017).

Although Gα₀lf is expressed outside the striatum, in the cerebellum and cerebral cortex (Millett et al., 2024; Vemula et al., 2013), its specific role in striatal dysfunction appears important in DYT-GNAL. A recent study has shown that the selective conditional Gnal KO in the striatum at embryonic or adult stages leads to abnormal postures, reminiscent of human symptoms (Chambers, 2024). In the striatum, there are two main populations of SPNs. One population expresses the dopamine D1R and forms the direct pathway (dSPNs) projecting directly to the basal ganglia output structures, namely the internal globus pallidus and the substantia nigra pars reticulata. The other population expresses D2Rs and A2ARs, and is the first relay in the indirect pathway (iSPNs) also reaching the output structures (Gerfen and Surmeier, 2011). The two populations of SPNs have complementary functions (Cui et al., 2013), with dSPNs stimulating spontaneous and acquired motor behaviors and iSPNs inhibiting competing motor actions, which promote the emergence of context-adapted responses (Durieux et al., 2009; Hikida et al., 2010; Kravitz et al., 2012; Nelson and Kreitzer, 2014). These processes may be disrupted in dystonia leading to maladaptive motor command and, subsequently, abnormal movements and postures (Mink, 2003). Here, we address the role of Gα₀lf in each population of SPN in mouse motor behavior, to better understand the pathophysiology of DYT-GNAL. We

also explore how its absence in dSPNs or iSPNs affects responses to drugs
acting on dopamine or adenosine signaling, to test their possible effects
in this disease.

## 2. Materials and methods

2.1. Generation of Gnal conditional KO mice

A targeting vector with the exons 3 and 4 of Gnal flanked by LoxP sites was introduced by homologous recombination in C57BL/6N embryonic stem cells and chimeric mice were produced at Phenomin (Strasbourg, France). The male (M) chimeric mice were bred with female (F) Flp deleter mice to excise the FRT-flanked selection cassette (FRT-Neo-FRT) and generate F1 heterozygotes with the floxed Gnal allele (Gnal<sup>f/f</sup>). To generate conditional KO (cKO) mice used in this study, Gnal<sup>f/f</sup> homozygotes were bred to either Drd1-Cre (Tg(Drd1-Cre) EY262<sup>Gsat</sup>) (Gong et al., 2007) or Adora2a-Cre (Tg(Adora2a-cre)<sup>2MDkde</sup>) (Durieux et al., 2009) mice, expressing Cre recombinase under the control of D1R (Drd1) and A2AR (Adora2a) gene promoters, kindly provided by Pr Paul Greengard (Rockefeller University) and Dr. Alban de Kerchove d'Exaerde (Université libre de Bruxelles), respectively. These crosses generated Gnal-D1cKO and Gnal-A2AcKO mice with Gnal KO in neurons expressing D1R and A2AR, respectively. Mice bearing Gnal<sup>f/f</sup> genotype and Cre-expressing transgene were crossed with Gnal<sup>f/f</sup> mice to obtain M and F experimental animals. For genotyping, DNA was extracted from a tail biopsy taken between postnatal days 4 and 7 and processed for PCR using primers to test the Cre transgene (Nestin CreER F: ATTTGCCTGCATTACCGGTC; Nestin CreER R: ATCAACGTTTTC TTTTCGG, annealing temperature 57 °C) as well as floxed and wild-type (WT) Gnal (Gnal-Lf: CCACAGTGTCAAACTGCCTGTATCGC Gnal-Lr: CAAGGCATCTGCCAAGTTTTTACCC; annealing temperature 62 °C). PCR fragments were resolved in 2 or 3 % (w/v) agarose gel. In several litters, pup survival and weight were assessed daily. In all experiments, mice with the Gnal<sup>f/f</sup> or Gnal<sup>f/f</sup> genotypes that lack the Cre transgene were considered as WT mice because Gα₀lf expression in Gnal<sup>f/f</sup> mice was not different from that in Gnal<sup>+/-</sup> mice (see below). Throughout the manuscript the mice with conditionally deleted alleles are termed Gnal-D1cKO<sup>+/-</sup> or Gnal-A2AcKO<sup>+/-</sup> for heterozygous, and Gnal-D1cKO<sup>-/-</sup> or Gnal-A2AcKO<sup>-/-</sup> for homozygous mice, depending on the Cre driver line used.

The mice were maintained in a 12-h light/dark cycle, in stable conditions of temperature (21 ± 1 °C), with access to food and water *ad libitum*, and housed 2 to 6 per cage. All animal procedures used in the present study were approved by the Ethical committee for animal experiments (CEEA Charles Darwin) and by the *Ministère de l'Éducation Nationale de l'Enseignement Supérieur de la Recherche, France* (projects APAFIS #8196 and #37798). The laboratory animal facility was approved to carry out animal experiments by the *Sous-Direction de la Protection Sanitaire et de l'Environnement de la Préfecture de Police* (licence D75-05-22). The total number of mice used in the experiments was 507, including 215 F and 292 M, 176 WT, 80 Gnal-D1cKO⁺⁻/⁻, 87 Gnal-A2AcKO⁺⁻/⁻, 81 Gnal-D1cKO⁻/⁻ and 83 Gnal-A2AcKO⁻/⁻.

2.2. Drugs

Selective A2AR antagonist KW6002 (istradefylline, #5147), non-selective adenosine antagonist caffeine (#2793), selective A2AR agonist CGS21680 hydrochloride (#1063), and the selective D1R agonists, SKF81297 hydrobromide (#1447), dihydrexidine hydrochloride (#0884) and, SKF83822 hydrobromide (#2075) were purchased from Tocris. Cocaine hydrochloride was provided by Cooper (Melun, France) and D-amphetamine sulfate and methylphenidate hydrochloride by Sigma. KW6002, SKF81297, dihydrexidine, SKF83822 and CGS21680, used for investigating their effects on locomotion activity, were dissolved in a solution containing 5 % dimethylsulfoxide (DMSO), 5 % Tween20, 15 % polyethylene glycol (both vol/vol) in H₂O. Caffeine, D-
amphetamine, cocaine and methylphenidate were dissolved in 9 g.L⁻¹ NaCl. For cAMP assays, SKF81297 hydrobromide (0.1 mM) was dissolved in H₂O and CGS21680 hydrochloride (30 mM) in DMSO. Drugs were then diluted in H₂O to obtain the appropriate concentrations.

## 2.3. Western blot analysis

Mice (*Gnal-D1cKO*⁻/⁻, *n* = 8; *Gnal-D1cKO*⁺/⁻, *n* = 8; *Gnal-A2AcKO*⁻/⁻, *n* = 6; *Gnal-A2AcKO*⁺/⁻, *n* = 6, WT, *n* = 14; age 6–9 months) were killed by decapitation, and their brain was quickly dissected out, frozen in 2-methylbutane (Sigma) cooled at –30 °C in dry ice and then stored at –80 °C. Olfactory bulbs were removed and frozen in dry ice. Using a Cryostat (Leica), frozen brains were cut into 210 µm-thick slices from which the dorsal striatum and nucleus accumbens were punched out using cold stainless-steel cannulas (1.4 mm for dorsal striatum and nucleus accumbens). The samples were sonicated in 10 g. L⁻¹ sodium dodecyl sulfate (SDS) and heated at 100 °C for 5 min. Aliquots (5 µL) of homogenate were used for measuring protein concentration using a bicinchoninic acid (BCA) assay kit (Thermo technologies). Equal amounts of protein (20 µg) were separated in 15–10 % (w/v) pre-casted gel (Bio-Rad) in the presence of SDS and electrophoretically transferred to nitrocellulose membranes (Trans-Blot Turbo Transfer, Bio-Rad). The membranes were immunoblotted using the following primary antibodies: rabbit polyclonal affinity-purified *Ga*α₀lf and *Ga*ₛ (1:1000) (Herve et al., 2001), AC5 (Millipore MAPS2049, 1:500), mouse monoclonal DARPP-32 (1:1000) (gift of Pr P. Greengard), *Gβ*₂ (Sigma AV48278, 1:1000), rabbit polyclonal *Gy*₇ (1:1000) (gift of Pr Robishaw), mouse monoclonal D1R (1:1000) (gift of Pr Luedtke), chicken polyclonal TH (Aves, #TYH, 1:1000), D2R (Frontier Institute, D2-Rb-Af960, 1:500), A2AR (Frontier Institute, A2A-GoAf700, 1:500) and mouse or rabbit polyclonal actin (Sigma-Aldrich, A5441, A2066, 1:5000) and IRDye 800CW- or 700CW-conjugated anti-mouse, anti-chicken, anti-goat and anti-rabbit IgG (1:5000) (Rockland Immunochemical) as secondary antibodies. Their binding was quantified using an Odyssey–LI-COR infrared fluorescent detection system (LI-COR).

## 2.4. Measures of AC activity

Mice (*Gnal-D1cKO*⁻/⁻, *n* = 5; *Gnal-A2AcKO*⁻/⁻, *n* = 3; WT, *n* = 8; age 4.5–6.5 months) were killed by cervical dislocation, and the brains were quickly removed from the skull. The right and left striata were dissected with glass manipulators on ice and homogenized in 600 µL of a cold buffer containing 2 mM Tris-maleate (pH 7.2), 2 mM EGTA, and 300 mM sucrose, using a Potter-Elvehjem apparatus. AC activity was measured by incubating tissue homogenates (6–12 µg of protein) for 10 min at 30 °C in 30 µL of a reaction mix containing 25 mM Tris-maleate (pH 7.2), 0.5 mM ATP, 1 mM MgSO₄, 0.33 mM EGTA, 0.1 mM papaverine, 2 µg. mL⁻¹ adenosine deaminase, 10 mM creatine phosphate, 0.3 mg.L⁻¹ creatine kinase, 1 µM GTP and 50 mM sucrose. For every homogenate, AC activities were determined in the presence of vehicle or various concentrations of SKF81297 or CGS21680. The reaction was stopped by adding 20 µL of cold lysis buffer (Cisbio kit) and cooling the tube on ice. The levels of cAMP formed were determined in 1/20 of reaction volume by cAMP–Gs Dynamic kit according to the manufacturer's protocol (Cisbio Bioassays). Förster resonance energy transfer (FRET) in plate wells was quantified (330 nm excitation, 620 nm and 665 nm emission of respective fluorophores) and analyzed using a Homogeneous Time Resolved Fluorescence (HTRF) program on a Mithras LB940 instrument (Berthold). The ratio obtained from Signal 665 nm/Signal 620 nm was used to interpolate cAMP concentrations from a cAMP standard curve obtained from the same plate and calculated with GraphPad Prism (GraphPad Software, La Jolla, CA). Protein concentration in tissue homogenates was measured using a bicinchoninic acid (BCA) assay (Micro BCA protein assay kit, Thermo Scientific). AC activity was expressed in pmoles of cAMP formed per min per mg of protein and normalized as %

of basal activity in WT striatum.

## 2.5. Behavioral tests

Behavioral experiments were conducted using mutant and control littermates of both sexes (age indicated in figure legends for each test) between 8 am and 6 pm.

**Locomotor activity in a circular corridor.** Locomotor activity was measured in a circular corridor (or circular maze, corridor width 5 cm, external diameter 17 cm) with four infrared beams placed at every 90° angle (Imetronic, Pessac, France) in a low luminosity environment. Counts were incremented by consecutive interruptions of two adjacent beams (1/4 turn). Mice were habituated to handling 3 days before testing. In one experiment, animals were placed in the circular maze at 11 am, for 28 h to assess their circadian rhythm of activity with free access to water and food throughout the test. In other experiments to test drug responses, mice were habituated to the device during 3 sessions of 90 min (1 session/day). On day 1, they were just placed in the maze. On days 2 and 3, they additionally received an intraperitoneal (ip) injection of saline 30 min after the start of the recording. We used the locomotor activity during the first 30 min (before saline injection) to evaluate response to a novel environment and habituation. The following days, drug response was assessed by comparing locomotor activities after vehicle and drug administrations on two consecutive days. Recording lasted 90 or 120 min depending on the drug tested, and vehicle and drug were administered ip 30 min after the start of the recording. The drug doses used were selected based on previously published studies: 5 mg. Kg⁻¹ SKF81297 and dihydrexidine (Yano et al., 2018), 0.5 mg.Kg⁻¹ SKF83822 (Brunori et al., 2021; O'Sullivan et al., 2004); 0.5 mg.Kg⁻¹ CGS21680 (Ledent et al., 1997), 3.3 mg.Kg⁻¹ KW6002 (Xiao et al., 2006), 15 mg.Kg⁻¹ caffeine (El Yacoubi et al., 2000), 20 mg.Kg⁻¹ cocaine (Corvol et al., 2007), 2.5 mg.Kg⁻¹ D-amphetamine and 10 mg. Kg⁻¹ methylphenidate (Pascoli et al., 2005).

**Rotarod and Open-field activity.** Mutant and WT littermates (see figure legends for the number and sex of animals) were trained for 7 days on the Rotarod (BIOSEB, model LE8200), with 7 trials per day, spaced by 5-min intervals using a procedure adapted from (Varani, 2024). The trial started by placing mice on a rod with a rotation accelerating from 4 to 40 rpm over 5 min and ended when either the mice fell off the rod or 300 s elapsed. During the 5-min intervals, animals were placed in home cages. Before and 45 min after the 7 trials, animals were placed in Open-field boxes (50 × 50 × 30 cm) made of white Plexiglas and allowed to freely explore it within 5 min. Locomotion was tracked using a ceiling-mounted camera (Panasonic WV BP332) and ViewPoint videotrack. The distance traveled was measured to verify the absence of gross locomotor deficit in the tested mice. The Rotarod was cleaned using 30 % ethanol (vol/vol) between subjects.

**Pole test.** Motor function was assessed using Pole test in mutant and WT littermates of either sex. The device consisted of a 50 cm high, 1 cm diameter vertical pole covered with elastic adhesive tape to allow the animals to grip it. The pole was placed in the middle of a cage. The mouse was placed at the top of the pole, head up, and the mouse turning and descent were videotaped. On the video movies, we measured the time to turn completely downward (Tturn) and that to descend to the floor (Tdown). The maximum time for Tturn was set to 120 s. After this time, the mice were gently pushed by hand to begin the descent. The test consisted of 5 trials per animal and the mean values of Tturn and Tdown were measured. Since there was no significant difference of Tdown between groups of mutant and WT mice, we have only used results on Tturn. The pole was carefully cleaned after testing between mice.

**Grid test.** Mice were placed on a cage grid lid, then the grid was inverted, and mice were allowed to hang with the grid maintained about 30 cm above the home cage. The mice were videotaped, and we measured the time (in s) the mice took to fall off the grid. The average time in 3 trials was used to assess neuromuscular strength.

**Grip test.** Neuromuscular functions were also evaluated in groups of
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>

D

<figure><img src="image_7.png" /></figure>

E

<figure><img src="image_8.png" /></figure>

F

<figure><img src="image_9.png" /></figure>

(caption on next page)
**Fig. 1.** Adenylyl-cyclase responses to D1 or A2A agonists in the dorsal striatum of *Gnal* cKO mice. A-B) cAMP formation was measured in the presence or absence of various concentrations of the D1 agonist SKF81297 (A) or A2A agonist CGS21680 (B) in striatal homogenates from *Gnal*-D1cKO<sup>-/-</sup> mice (n = 3, 3 F) and WT littermates (n = 3, 2 F, 1 M, age, 4.5-6.5 months). In *Gnal*-D1cKO<sup>-/-</sup> mice, the adenyllyl-cyclase response to the D1 agonist SKF81297 was abolished (A), while that to A2A agonist CGS21680 remained significant (B). Two-way ANOVA: Responses to SKF81297 (A): Genotype factor, $F_{1,82} = 502, p < 0.001$; SKF81297 factor, $F_{10,82} = 3.08, p < 0.001$; Interaction, $F_{10,82} = 7.34, p < 0.01$. Responses to CGS21680 (B): Genotype factor, $F_{1,56} = 326, p < 0.001$; CGS21680 factor, $F_{7,56} = 59.1, p < 0.001$, Interaction, $F_{7,56} = 2.26, p < 0.05$. C) cAMP production under basal conditions or in the presence of a maximum concentration of CGS 21680 (CGS, 10 µM) was reduced in *Gnal*-D1cKO<sup>-/-</sup> mice (n = 5, 3 F, 2 M) compared to WT littermates (n = 5, 2 F, 3 M) but the difference between CGS-stimulated and basal productions (CGS - Basal) was not significantly different. Comparisons of mutant and WT mice: Basal, Unpaired t-test, $t_{27} = 11.9, p < 0.001$; CGS21680, Mann-Whitney test, U = 10, p < 0.001; CGS-Basal, Unpaired t-test, $t_{28} = 1.40$, ns. D-E) cAMP formation was measured in the presence or absence of various concentrations of A2A agonist CGS21680 (D) or D1 agonist SKF81297 (E) in the striatal homogenates of *Gnal*-A2AcKO<sup>-/-</sup> (n = 1, 1 F) and WT mice (n = 1, 1 F, 6 months). In *Gnal*-A2AcKO<sup>-/-</sup> mice, the adenyllyl-cyclase response to the A2A agonist CGS21680 was abolished, while that to the D1 agonist SKF81297 remained significant. Two-way ANOVA. Response to CGS21680 (D): Genotype factor, $F_{1,17} = 673, p < 0.001$; CGS21680 factor, $F_{7,17} = 20.4, p < 0.001$; Interaction, $F_{7,17} = 18.4, p < 0.001$. Response to SKF81297 (E): Genotype factor, $F_{1,20} = 114, p < 0.001$; SKF81297 factor, $F_{7,20} = 6.66, p < 0.001$, Interaction, $F_{7,20} = 0.616$, ns. F) cAMP production under basal conditions or in the presence of a maximum concentration of SKF81297 (SKF, 0.3 µM) was reduced in *Gnal*-A2AcKO<sup>-/-</sup> mice (n = 2, 2 F) compared to WT littermates (n = 2, 2 F) but the difference between CGS-stimulated and basal productions (CGS - Basal) was not significantly different. Comparisons of *Gnal*-A2AcKO<sup>-/-</sup> and WT mice: Basal, Mann Whitney test, U = 0, p < 0.01; SKF81297, Mann Whitney test, U = 4, ns; SKF-Basal, Mann Whitney test, U = 11, ns. The data of cAMP production were expressed as % of basal activity evaluated in WT mice and are pooled from 3 (A, B) or 2 (E, F) separate experiments. The mean ± SD of basal adenyllyl-cyclase activity was 106 ± 55 (A-C) and 86 ± 31 (D-F) pmoles of cAMP formed.min<sup>-1</sup>.mg<sup>-1</sup> protein. All the statistical results are shown in Supplementary Tables. The dose-response curves were best-fitted with GraphPad Prism software. **p < 0.01 and ***, p < 0.001, significantly different from WT littermates.

tyrosine hydroxylase, a marker of dopamine terminals, were not modified (Figs. 2 and S1A) provided strong evidence that the number and trophicity of striatal SPNs and their dopamine innervation were not altered in mutant mice.

In *Gnal*-A2AcKO<sup>-/-</sup> and *Gnal*-A2AcKO<sup>+/-</sup> mice, $Ga_{01f}$ levels were decreased, the effect being more marked in homozygotes (−59 %, p < 0.01) than in heterozygotes (−24 %, ns) (Fig. 3). We also observed reductions in AC5 levels which can account for the decreased baseline AC activity measured in striatal tissue homogenates (see Fig. 1D). D2R and A2AR levels tended to slightly decrease (Fig. S3). No changes in D1R, $Ga_{5}$, and $G\beta_{2}$ levels were observed (Fig. 3). The lack of alteration in DARPP-32 and

tyrosine hydroxylase levels indicated that SPNs and striatal dopamine innervation were not grossly altered in *Gnal*-A2AcKO mice (Fig. 3).

### 3.3. *Gnal* cKO in D1-SPNs impairs survival of pups at weaning

Genotyping of pups from crosses between *Gnal*-D1cKO<sup>-/-</sup> and *Gnal*<sup>f/f</sup> mice did not differ significantly from the expected Mendelian distribution a few days after birth (4-7 days) (90 *Gnal*<sup>+/-</sup>, 91 *Gnal*<sup>+/-</sup>, 98 *Gnal*-D1cKO<sup>+/-</sup>, 70 *Gnal*-D1cKO<sup>-/-</sup>, $\chi^{2} = 0.173$, ns). This excluded a major in utero or early postnatal mortality in mice lacking $Ga_{01f}$ in D1-SPNs. In contrast, we observed an increase in lethality in *Gnal*-D1cKO<sup>-/-</sup> from 21 days, around the weaning period, until the 40th day (Fig. 4A). Ultimately, 34 % of *Gnal*-D1cKO<sup>-/-</sup> pups died before 60 days of age. When we retrospectively examined the body weight curve of pups dead before 60 days (Fig. 4B), we observed that their weight gain stopped around 15 days and even dropped in some individuals, suggesting a feeding defect. Interestingly, the other part of the population of *Gnal*-D1cKO<sup>-/-</sup> pups, which survived beyond 60 days, showed a normal weight gain. When we determined the weight of adult mice, we observed no significant difference between the different genotypes in either sex (Fig. 4C). These data indicated that *Gnal*-D1cKO<sup>-/-</sup> animals surviving beyond 60 days had a normal development and maintained a body weight similar to that of WT mice, suggesting a normal feeding.

### 3.4. *Gnal* cKO in D1-SPNs increases nocturnal locomotor activity and impairs motor performance

When we examined locomotor activity of *Gnal*-D1cKO<sup>-/-</sup> mice in a circular corridor for 28 h, they exhibited locomotor hyperactivity during the night period (Fig. 4D). When they were tested during the day, *Gnal*-D1cKO<sup>-/-</sup> mice also showed increased activity when placed in the circular corridor for 30 min (Fig. 4E). Although locomotor activity was reduced by daily repetition of the test, it remained significantly increased compared to WT mice (*Gnal*-D1cKO<sup>-/-</sup> vs WT, p < 0.001 in

the 3 days, Fig. 4E). *Gnal*-D1cKO<sup>+/-</sup> mice exhibited locomotor activities similar to those of WT mice.

We then tested motor performance and motor learning in *Gnal*-D1cKO<sup>-/-</sup>, *Gnal*-D1cKO<sup>+/-</sup> and WT mice in an accelerating Rotarod. The mice were tested for 7 days with 7 sessions per day (Fig. 4F). The performances of *Gnal*-D1cKO<sup>-/-</sup> mice were much lower than those of WT or *Gnal*-D1cKO<sup>+/-</sup> mice. However, as indicated by their locomotor hyperactivity, this deficiency could not be attributed to a generalized impairment of the ability to move. The disturbance in Rotarod performance of *Gnal*-D1cKO<sup>-/-</sup> mice remained significant until the 6th day of testing (ANOVA indicated differences between *Gnal*-D1cKO<sup>-/-</sup> and WT from Day 1 to Day 5, p < 0.01; Day 6, p < 0.05) but we observed a progressive increase in performance. On day 7, their scores, although still lower, did not differ significantly from those of WT mice (Fig. 4F). The *Gnal*-D1cKO<sup>-/-</sup> mice were thus capable of motor learning. *Gnal*-D1cKO<sup>+/-</sup> mice, which displayed a motor performance similar to WT mice on Day 1, had a longer latency to fall from the accelerating Rotarod than WT, a difference which was significant from Day 5 to 7 (*Gnal*-D1cKO<sup>+/-</sup> vs. WT, Day 5 and Day 6, p < 0.05; Day 7, p < 0.01). Visual observation of *Gnal*-D1cKO<sup>+/-</sup> mice in this task showed them to appear more focused and perseverant than WT, possibly explaining the longer time spent remaining on the rod. In this experiment, the scores of WT mice increased significantly between days 1 and 4, then plateaued thereafter (Fig. 4G). In contrast, in heterozygous and homozygous mice, performances increased significantly between days 4 and 7 (Fig. 4G).

Because the *Gnal*-D1cKO<sup>-/-</sup> mice performed poorly on the Rotarod we specifically examined their motor skills and strength in several other tests. In the Pole test, *Gnal*-D1cKO<sup>-/-</sup> mice took a significantly longer time than WT to turn around and get off the pole (*Gnal*-D1cKO<sup>+/-</sup> vs WT, p < 0.05; Fig. 4H). Visual observations indicated that mutant mice had great difficulty in initiating the relatively complex actions of turning around at the top of the pole, whereas their descent was quick. *Gnal*-D1cKO<sup>+/-</sup> mice performed like WT in this test (Fig. 4H). In the Grid and Grip tests that evaluate neuromuscular strength in mice (Fig. 4I and J), there was no significant deficit in *Gnal*-D1cKO<sup>-/-</sup>, showing the absence of significant neuromuscular weakness that could explain the poor motor performances in the Rotarod and Pole tests. Because hindlimb clasping, a phenotype observed in a wide variety of neuropathologies in mice (Lalonde and Strazielle, 2011), has been described in a recent study of mice lacking *Gnal* expression in all striatal neurons (Chambers, 2024), we examined this response in our mutant mice. *Gnal*-D1cKO<sup>-/-</sup> and *Gnal*-D1cKO<sup>+/-</sup> mice showed no hindlimb clasping when suspended by the tail (Fig. S4A). In addition, visual observation of these mice did not indicate any obvious abnormalities in posture or spontaneous movements compared to WT mice.

In the Open-field, *Gnal*-D1cKO<sup>-/-</sup> mice visited the central area of the
<figure><img src="image_10.png" /><figcaption>**Fig. 2.** Molecular characterization of *Gnal*-D1cKO<sup>-/-</sup> and *Gnal*-D1cKO<sup>+/-</sup> mice.</figcaption></figure>

Representative immunoblots and quantification of Gα<sub>olf</sub>, adenyllyl-cyclase 5 (AC5), DARPP-32, tyrosine hydroxylase (TH), β2 (Gβ2), γ7, (Gγ7) and αs (Gαs) subunits of G proteins and, D1 dopamine receptor (D1R) in dorsal striatum homogenates of *Gnal*-D1cKO<sup>-/-</sup> (*n* = 8, 8 F), *Gnal*-D1cKO<sup>+/-</sup> (*n* = 8, 8 F) and WT (*n* = 8, 8 F) littermates (age, 6–8.5 months). We quantified the long isoform of Gαs, the more highly expressed of the two isoforms in the brain. Data corresponded to the ratio between the immunofluorescence of the indicated protein and actin, expressed as a % of the mean value in WT samples. Means ± SD are indicated in the scatter dot plots. Partial or complete loss of *Gnal* expression in D1-SPNs reduced significantly the levels of Gα<sub>olf</sub>, but also those of AC5. No significant change was observed in Gβ2 and Gγ7 levels, two proteins associated with Gα<sub>olf</sub>. Gαs was increased. DARPP-32, D1R and TH (markers of SPNs, D1-SPNs and dopamine terminals, respectively) levels were not significantly altered. Protein levels were compared using one-way ANOVA followed by Bonferroni's multiple comparison test or Kruskal-Wallis test followed Dunn's multiple comparison test depending on whether data distribution was normal or not (see the detailed results in Supplementary Tables). Gα<sub>olf</sub>, one-way ANOVA, F<sub>2,18</sub> = 19.2, *p* < 0.001; AC5, Kruskal-Wallis test, H<sub>2</sub> = 6.13, *p* < 0.05; DARPP-32, one-way ANOVA, F<sub>2,18</sub> = 0.105, ns; TH, one-way ANOVA, F<sub>2,18</sub> = 0.299, ns; Gβ2, Kruskal-Wallis test, H<sub>2</sub> = 0.845, ns; Gαs, long isoform, one-way ANOVA, F<sub>2,21</sub> = 9.29, *p* < 0.01; Gγ7, one-way ANOVA, F<sub>2,19</sub> = 1.82, ns; D1R, one-way ANOVA, F<sub>2,23</sub> = 1.46, ns. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* , *p* < 0.001.

arena more frequently than WT or *Gnal*-D1cKO<sup>+/-</sup> mice (*Gnal*-D1cKO<sup>-/-</sup> vs. WT, *p* < 0.01, Fig. 4K). However, the total distance traveled was similar in the three groups of mice (total distance as % of WT mean: *Gnal*-D1cKO<sup>-/-</sup>, 110 ± 12; *Gnal*-D1cKO<sup>+/-</sup>, 99 ± 7; WT, 100 ± 7; one-way ANOVA, F(2, 27) = 0.43, ns, mean ± SEM, *n* = 10 in each group). In this experimental condition in which locomotor activity was measured during 20 min in an open arena, we did not detect in the *Gnal*-D1cKO<sup>-/-</sup> mice the locomotor hyperactivity previously observed in a circular maze with a low intensity light environment (see above). The more frequent presence of *Gnal*-D1cKO<sup>-/-</sup> mice in the central area of the Open-field could indicate that these mice were less anxious than WT mice. However, when we subjected the mice to a test specifically evaluating risk

assessment (elevated O maze, Fig. 4L), no significant difference could be detected between the three groups of mice. This indicated that the deficits found on the Rotarod in *Gnal*-D1cKO<sup>-/-</sup> mice were not related to elevated risk threshold assessment.

### 3.5. *Gnal* cKO in A2A-SPNs results in high locomotor hyperactivity without impaired motor performance

When we monitored the survival curve of pups from mating of *Gnal*-A2AcKO<sup>+/-</sup> mice with *Gnal*<sup>f/f</sup> mice, we did not observe any particular mortality in any of the resulting genotypes (Fig. 5A). Adult *Gnal*-A2AcKO<sup>-/-</sup> male mice displayed a slight body weight deficit compared
<figure><img src="image_11.png" /><figcaption>Fig. 3. Molecular characterization of *Gnal*-A2AcKO<sup>-/-</sup> and *Gnal*-A2AcKO<sup>+/-</sup> mice.</figcaption></figure>

Representative immunoblots and quantification of Gα<sub>olf</sub>, adenylate cyclase 5 (AC5), DARPP-32, tyrosine hydroxylase (TH), β2 (Gβ2) and αs (Gαs) subunits of G proteins and, D1 receptor (D1R) in dorsal striatum homogenates of *Gnal*-A2AcKO<sup>-/-</sup> (*n* = 6, 6 M), *Gnal*-A2AcKO<sup>+/-</sup> (*n* = 6, 6 F) and WT (*n* = 6, 3 M, 3 F) littermates (age, 3–4 months). We quantified the long isoform of Gαs. Data corresponded to the ratio between the immunofluorescence of the indicated protein and actin, expressed as a % of the mean value in WT samples. Means ± SD are indicated in the scatter dot plots. Partial or complete loss of Gnal expression in D1-SPNs reduced the levels of Gαolf, but also those of AC5. By contrast, no significant change was observed in levels of G protein subunits, Gβ2 and Gαs levels. DARPP-32, D1R or TH (markers of SPNs, D1-SPNs and dopamine terminals, respectively) levels were not significantly altered. Protein levels were compared using Kruskal-Wallis test followed Dunn's multiple comparison test (see the detailed results in Supplementary Tables). Gαolf: H₂ = 11.9, *p* < 0.01; AC5: H₂ = 9.69, *p* < 0.01; DARPP-32: H₂ = 0.971, ns; TH: H₂ = 4.35, ns; Gαs long isoform: H₂ = 0.547, ns; Gβ2: H₂ = 2.14, ns; D1R: H₂ = 1.12, ns. ** *p* < 0.01.

to the other groups of mice, but this deficit was not observed in F
(Fig. 5B).

When we examined locomotor activity in a circular corridor for 28 h, we noticed that *Gnal*-A2AcKO<sup>-/-</sup> mice exhibited a strong permanent locomotor hyperactivity that was particularly pronounced during the night phase (Genotype, Time, and Genotype X Time were significant in ANOVA, *p* < 0.01, *p* < 0.001 and *p* < 0.001, respectively, Fig. 5C). Although the high physical expenditure in these mice could account for the lower body weight of male *Gnal*-A2AcKO<sup>-/-</sup> mice, this is unlikely because female *Gnal*-A2AcKO<sup>-/-</sup> mice which were more hyperactive than male (Fig. S5) showed no body weight difference with WT mice. When WT and *Gnal*-A2AcKO<sup>+/-</sup> mice were placed every day for three consecutive days in a circular maze during daytime, they displayed an exploration activity within the first 30 min of exposure in this novel environment on Day 1, but gradually habituated to the circular maze so

that their activity decreased on day 2 and 3 (Fig. 5D). In contrast, the activity of *Gnal*-A2AcKO<sup>-/-</sup> mice that was not significantly different from WT or *Gnal*-A2AcKO<sup>+/-</sup> mice on Day 1 (*Gnal*-A2AcKO<sup>-/-</sup> vs. WT: +35 %, ns; Fig. 5D), increased progressively on Day 2 and 3, to be about 3-fold higher than in WT mice on Day 3 (*Gnal*-A2AcKO<sup>-/-</sup> vs. WT: +122 % and +211 % on Day 2 and 3, respectively, *p* < 0.001; Fig. 5D). This indicates that *Gnal*-A2AcKO<sup>-/-</sup> mice displayed constant locomotor hyperactivity with a lack of habituation.

We then examined motor abilities and learning in the accelerating Rotarod using multiple sessions as described above (see 3.4). Both *Gnal*-A2AcKO<sup>-/-</sup> and *Gnal*-A2AcKO<sup>+/-</sup> mice increased their performance over time, showing motor learning similar to WT mice (Trial effect was significant, *p* < 0.001, but not genotype and Trial X Genotype in ANOVA, Fig. 5E). In this experiment, the progression of performance was similar in the three genotypes (Fig. 5E and F). *Gnal*-A2AcKO<sup>-/-</sup> and
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<caption>**Fig. 4.** Survival and motor behavior of *Gnal-D1cKO* mice.</caption>

A) Survival curve of littermate mice with different genotypes (WT, *n* = 155; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 86; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 57) within the first 60 days after birth. A portion of *Gnal-D1cKO<sup>-/-</sup>* mice died around and after postnatal day 21, corresponding to the weaning period (vertical dashed line indicates day 21). This was not the case in littermates with WT and *Gnal-D1cKO<sup>+/-</sup>*. The difference was significant according to log-rank (Mantel-Cox) test: *χ*² = 93.1, df = 3, *p* < 0.001. B) Weight curve of littermate mice (WT, *n* = 51; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 43; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 34). For *Gnal-D1cKO<sup>-/-</sup>* mice, data for mice alive (*n* = 24) and dead (*n* = 10) at postnatal day 60 were separated. C) Weight of M and F adult mice with different genotypes (WT, *n* = 29 M, 12 F; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 25 M, 12 F; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 23 M, 16 F; age, M 2–8 months, F 2–5 months). Two-way ANOVA: Genotype factor, F<sub>2,113</sub> = 0.544, ns; Sex factor, F<sub>1,113</sub> = 129, *p* < 0.001; Interaction, F<sub>2,113</sub> = 0.639, ns. D) Spontaneous locomotor activity during 28 h indicating hyperactivity in *Gnal-D1cKO<sup>-/-</sup>* mice, the circadian rhythm remaining unaltered (WT, *n* = 5 M, *Gnal-D1cKO<sup>+/-</sup>*, *n* = 5 M, *Gnal-D1cKO<sup>-/-</sup>*, *n* = 5 M; age, 3–6 months). Repeated measures two-way ANOVA followed by Bonferroni's multiple comparison test: Genotype factor: F<sub>2,324</sub> = 4.37, *p* < 0.05; Time factor: F<sub>27,324</sub> = 9.80, *p* < 0.001; Interaction, F<sub>54,324</sub> = 3.38, *p* < 0.001. E) Hyperactivity was also observed in these mutant mice when exploratory activity was assessed in a novel environment (circular maze) or during sessions on the 2 following days (WT, *n* = 30, 8 F, 22 M; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 28, 8 F, 20 M; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 29, 12 F, 17 M, age, 4–10 months). Repeated measures two-way ANOVA followed by Bonferroni's multiple comparison test: Genotype factor, F<sub>2,168</sub> = 22.2, *p* < 0.001; Day factor, F<sub>2,168</sub> = 99.1, *p* < 0.001; Interaction, F<sub>4,168</sub> = 0.659, ns. *** *p* < 0.001. F) Motor performance and motor learning measured with accelerating Rotarod. Mean ± SEM of latency to fall (in seconds, s) in each session (WT, *n* = 9, 4 F, 5 M; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 8, 5 F, 3 M; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 9, 6 F, 3 M; age, 2–5 months). For each day, repeated measures two-way ANOVA. *Gnal-D1cKO<sup>-/-</sup>* vs. WT, Genotype factor: Day 1, F<sub>1,96</sub> = 10.6, *p* < 0.01; Day 2, F<sub>1,96</sub> = 60.1, *p* < 0.001; Day 3, F<sub>1,96</sub> = 16.3, *p* < 0.001; Day 4, F<sub>1,96</sub> = 15.6, *p* < 0.001; Day 5, F<sub>1,96</sub> = 16.2, *p* < 0.001; Day 6, F<sub>1,96</sub> = 4.8, *p* < 0.05; Day 7, F<sub>1,96</sub> = 1.77, ns. *Gnal-D1cKO<sup>+/-</sup>* vs. WT, Genotype factor: Day 1, F<sub>1,90</sub> = 0.37, ns; Day 2, F<sub>1,90</sub> = 3.85, ns; Day 3, F<sub>1,90</sub> = 1.91, ns; Day 4, F<sub>1,90</sub> = 3.01, ns; Day 5, F<sub>1,90</sub> = 6.53, *p* < 0.05; Day 6, F<sub>1,90</sub> = 5.75, *p* < 0.05; Day 7, F<sub>1,90</sub> = 9.56, *p* < 0.01 (see complete results in Supplementary Tables). * *p* < 0.05, ** *p* < 0.01, *** *p* < 0.001, genotype factor between mutant and WT littermates, ns, not significant. G) Motor performance at days 1, 4, and 7 in the accelerating Rotarod test. Each point is the average fall latency over 7 sessions shown in F. Data were analyzed by repeated measures two-way ANOVA followed by Bonferroni's multiple comparison test: Genotype factor, F<sub>2,46</sub> = 18.6, *p* < 0.001; Day factor, F<sub>2,46</sub> = 49.0, *p* < 0.001; Interaction, F<sub>4,46</sub> = 4.00, *p* < 0.01. * *p* < 0.05, ** *p* < 0.01, *** *p* < 0.001, different between days. H. Motor skills measured by the pole test (WT, *n* = 10, 4 F, 6 M; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 10, 4 F, 6 M; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 10, 4 F, 6 M; age, 2–4 months). Time to turn completely downward at the top of the pole was measured and each point is the mean of 3 trials. *Gnal-D1cKO<sup>-/-</sup>* mice showed a deficiency compared to littermates. Kruskal-Wallis test: H<sub>2</sub> = 9.27, *p* < 0.01. I and J) Assessment of muscular strength measured by grid test (I) and grip strength (J) (same animals as in H). I, data are the time (in sec) before mice fall when they hang from a grid. J. Each point corresponds to the average grip strength (maximal force in g, 5 trials/mouse) divided by the mouse weight (in g). No genotype difference in performances in these two tests. Kruskal-Wallis test: Grid test, H<sub>2</sub> = 0.250, ns; Grip strength, H<sub>2</sub> = 0.560, ns. K. and L. Evaluation of anxiety in mutant mice compared to WT littermates. In K, % distance traveled in the center of an open field (same animals as in H). Kruskal-Wallis test, H<sub>2</sub> = 9.86, *p* < 0.01. In L, risk assessment was evaluated by the time spent by mice in open arms of an elevated O-maze. (WT, *n* = 11, 4 F, 7 M; *Gnal-D1cKO<sup>+/-</sup>*, *n* = 11, 5 F, 6 M; *Gnal-D1cKO<sup>-/-</sup>*, *n* = 12, 5 F, 7 M, age, 5.5–8 months). Kruskal Wallis test, H<sub>2</sub> = 2.17, ns. H-L, Kruskal-Wallis test was followed by Dunn's multiple comparison tests (see Supplementary Tables). * *p* < 0.05, ** *p* < 0.01.

*Gnal-A2AcKO<sup>+/-</sup>* mice showed no overt abnormal movements or hindlimb clasping when suspended by the tail (Fig. S4B). Grip strength of mutant mice was not significantly different from that measured in WT mice (Fig. 5G). In the elevated O-maze, the time spent in the open arm was similar in heterozygous and homozygous mutant mice as in WT, suggesting that the hyperactivity of *Gnal-A2AcKO<sup>-/-</sup>* mice did not result from an altered risk assessment (Fig. 5H).

### 3.6. *Gnal cKO* in *D1-SPNs* selectively reduces responses to *D1* receptor agonists

Because the absence of *Gnal* expression in D1-SPNs abolished AC response to a D1R agonist in striatal homogenates, we investigated behavioral responses to two D1R agonists, SKF81297 (5 mg.Kg⁻¹) and dihydrexidine (5 mg.Kg⁻¹). These two agonists increased locomotion in WT and *Gnal-D1cKO<sup>+/-</sup>* mice (SKF81297, WT, +479 % *p* < 0.001, *Gnal-D1cKO<sup>+/-</sup>*, +248 %, *p* < 0.01; dihydrexidine, WT, +263 %, *p* < 0.01; *Gnal-D1cKO<sup>+/-</sup>* +248 %, *p* < 0.01, Fig. 6A-B). In contrast the locomotor activity was not significantly increased by SKF81297 or dihydrexidine in mice lacking *Gα*<sub>olf</sub> in D1-SPNs (SKF81297, *Gnal-D1cKO<sup>-/-</sup>*, +93 %, ns; dihydrexidine, *Gnal-D1cKO<sup>-/-</sup>* + 47 %, ns, Fig. 6A-B). Surprisingly, locomotor response to another D1R agonist, SKF83822 (0.5 mg.Kg⁻¹), which is reported to be specifically associated with AC activation (Undie et al., 1994), appeared not to depend on *Gα*<sub>olf</sub> expression (Fig. S6).

We also investigated whether locomotor responses to A2AR agents were altered in mice with reduced or no *Gnal* expression in D1-SPNs. The decrease in locomotor activity in response to administration of a selective A2AR agonist, CGS21680 (0.5 mg.Kg⁻¹), was very small (−27 %, ns) in WT mice but was significant in *Gnal-D1cKO<sup>+/-</sup>* (−74 %, *p* < 0.05) and *Gnal-D1cKO<sup>-/-</sup>* (−71 %, *p* < 0.001) mice (Fig. 6C), which had some basal hyperactivity. Response to A2AR agonist thus appeared to be somehow enhanced in these mice. When we treated mice with a selective A2AR antagonist, KW6002 (a.k.a. istradefylline, 3.3 mg.Kg⁻¹), the increase in locomotor activity within 30 min of injection was similar in WT (+289 %, *p* < 0.05), *Gnal-D1cKO<sup>+/-</sup>* (+310 %, *p* < 0.01) and *Gnal-D1cKO<sup>-/-</sup>* (+188 %, *p* < 0.001) mice (Fig. 6D). These results clearly indicated that the absence of *Gnal* expression in D1-SPNs significantly

reduced locomotor responses to D1R agonists but not to an A2AR antagonist.

### 3.7. *Gnal cKO* in *D1-SPNs* diversely impairs responses to psychostimulants

Psychostimulant drugs increase dopamine, noradrenaline, and serotonin in the synaptic extracellular space through various mechanisms (Docherty and Alsupyani, 2021). We tested the consequences of *Gnal* KO in D1-SPNs on responses to three psychostimulants, cocaine, D-amphetamine and methylphenidate. In *Gnal-D1cKO<sup>+/-</sup>* mice, the locomotor response to cocaine (20 mg.Kg⁻¹) was decreased compared to WT (Fig. 7A). In *Gnal-D1cKO<sup>-/-</sup>* mice, the progressive decrease in locomotor activity observed after vehicle treatment was prevented, leading to a relative increase in locomotor activity that remained significant (ANOVA, Treatment factor, *p* < 0.05, Fig. 7A). As expected, we observed a strong locomotor hyperactivity in response to 2.5 mg.Kg⁻¹ D-amphetamine in WT animals (16.4-fold increase, *p* < 0.001, Fig. 7B right panel). With this high dose, large responses were also observed in *Gnal-D1cKO<sup>+/-</sup>* (18.7-fold increase, *p* < 0.001) and *Gnal-D1cKO<sup>-/-</sup>* mice (7.4-fold increase, *p* < 0.001) and there was no effect of genotype in the ANOVA (Fig. 7B right panel, Genotype and Genotype X D-amphetamine not significant). Methylphenidate (10 mg.Kg⁻¹) increased locomotor activity similarly in WT and *Gnal-D1cKO<sup>+/-</sup>* (27- and 34-fold increases, respectively, *p* < 0.001, Fig. 7C right panel). In contrast, there was virtually no response to methylphenidate in *Gnal-D1cKO<sup>-/-</sup>* mice (Fig. 7C right panel). These results show that *Gnal* KO in D1-SPNs had contrasted effects on locomotor responses to psychostimulants depending on the drug used. They provide evidence that the D1R/*Gα*<sub>olf</sub>-dependent activation of AC contributes to the locomotor responses to psychostimulants but that other mechanisms are also involved, which appear to differ between drugs.

### 3.8. *Gnal cKO* in *A2A-SPNs* alters responses to A2AR antagonists and a D1 agonist

The A2AR agonist CGS21680 (0.5 mg.Kg⁻¹) reduced locomotion in
A

<figure><img src="image_24.png" /><figcaption>Survival</figcaption></figure>

B

<figure><img src="image_25.png" /><figcaption>Body weight</figcaption></figure>

C

<figure><img src="image_26.png" /><figcaption>24-h activity</figcaption></figure>

<figure><img src="image_27.png" /></figure>

D

<figure><img src="image_28.png" /><figcaption>Repeated activity</figcaption></figure>

E

<figure><img src="image_29.png" /><figcaption>Accelerating Rotarod</figcaption></figure>

F

<figure><img src="image_30.png" /><figcaption>Accelerating
Rotarod</figcaption></figure>

G

<figure><img src="image_31.png" /><figcaption>Grip test</figcaption></figure>

H

<figure><img src="image_32.png" /><figcaption>O-maze</figcaption></figure>

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<caption>**Fig. 5.** Survival and motor behavior of *Gnal-A2AcKO<sup>-/-</sup>* and *Gnal-A2AcKO<sup>+/-</sup>* mice.</caption>

A) Survival curve of *Gnal-A2AcKO<sup>-/-</sup>* (*n* = 19) and *Gnal-A2AcKO<sup>+/-</sup>* (*n* = 25) mice compared to WT (*n* = 24) littermates within the first 60 days after birth. Postnatal day 21 (vertical dashed line) corresponds to the weaning period. No significant difference was detected using log-rank (Mantel-Cox) test: *χ*² = 2.80, ns, df = 2 (see Supplementary Tables). B) Weight of M and F adult mice with different genotypes (WT, *n* = 46, 21 F, 25 M; *Gnal-A2AcKO<sup>-/-</sup>*, *n* = 47, 25 F, 22 M; age, 2–6 months). Two-way ANOVA (Genotype factor, F<sub>2,135</sub> = 3.83, *p* < 0.05; Sex factor, F<sub>1,135</sub> = 242, *p* < 0.001; Interaction, F<sub>2,135</sub> = 3.54, *p* < 0.05) followed by Bonferroni's multiple comparison test (see Supplementary Tables). * *p* < 0.05, ** *p* < 0.01. C) Spontaneous locomotor activity over 28 h indicating a hyperactivity in *Gnal-A2AcKO<sup>-/-</sup>* mice (WT, *n* = 5, 5 M; *Gnal-A2AcKO<sup>+/-</sup>*, *n* = 5, 5 M; *Gnal-A2AcKO<sup>-/-</sup>*, *n* = 5, 5 M; age, 3.0–6.0 months), the circadian rhythm remaining unaltered. Repeated measures two-way ANOVA: Genotype factor, F<sub>2,324</sub> = 10.8, *p* < 0.01; Time factor, F<sub>27,324</sub> = 6.51, *p* < 0.001; Interaction, F<sub>54,324</sub> = 3.73, *p* < 0.001. D) Activity measured in a circular maze for 30 min on 3 consecutive days (WT, *n* = 29, 13 F, 16 M; *Gnal-A2AcKO<sup>+/-</sup>*, *n* = 30, 16 F, 14 M; *Gnal-A2AcKO<sup>-/-</sup>*, *n* = 27, 14 F, 13 M; age, 3–6 months). Repeated measures two-way ANOVA (Genotype factor, F<sub>2,166</sub> = 10.3, *p* < 0.001; Day factor, F<sub>2,166</sub> = 12.4, *p* < 0.001; Interaction, F<sub>4,166</sub> = 7.76, *p* < 0.001) followed by Bonferroni's multiple comparison test. ** *p* < 0.01, *** *p* < 0.001 (see Supplementary Tables). E) Motor performance and motor learning measured by the Rotarod. Mean ± SEM of latency to fall (in s) in each session (WT, *n* = 9, 2 F, 7 M; *Gnal-A2AcKO<sup>+/-</sup>*, *n* = 9, 4 F, 5 M; *Gnal-A2AcKO<sup>-/-</sup>*, *n* = 9, 6 F, 3 M; age, 2–5 months). Repeated measures two-way ANOVA (Genotype factor: F<sub>2,1104</sub> = 1.40, ns; Trial factor, F<sub>48,1104</sub> = 29.8, *p* < 0.001, Interaction, F<sub>94,1104</sub> = 0.697, ns) followed by Bonferroni's multiple comparison test (see Supplementary Tables). ns, not significant. F) Motor learning on days 4 and 7 in the Rotarod. Each point is the average fall latency over 7 sessions shown in (E). Repeated measures two-way ANOVA (Genotype factor, F<sub>2,46</sub> = 1.58, ns; Day factor, F<sub>2,46</sub> = 140, *p* < 0.001; Interaction, F<sub>4,46</sub> = 0.492, ns) followed by Bonferroni's multiple comparison test (see Supplementary Tables). ** *p* < 0.01, *** *p* < 0.001, different between days. G) Assessment of neuromuscular strength by the grip test (WT, *n* = 8, 6 F, 2 M; *Gnal-A2AcKO<sup>+/-</sup>*, *n* = 9, 5 F, 4 M; *Gnal-A2AcKO<sup>-/-</sup>*, *n* = 10, 4 F, 6 M, age, 2–2.5 months). Each point corresponds to the average grip strength (maximal force in g, 5 trials/mouse) divided by the mouse weight (in g). There was no significant difference between genotypes. Kruskal-Wallis test: *H*<sub>2</sub> = 1.70, ns. H) Risk assessment was evaluated by the time spent by mice in open arms of an elevated O-maze (WT, *n* = 12, 7 F, 5 M; *Gnal-A2AcKO<sup>+/-</sup>*, *n* = 10, 6 F, 4 M; *Gnal-A2AcKO<sup>-/-</sup>*, *n* = 11, 5 F, 6 M; age, 5–6 months). Kruskal-Wallis test: *H*<sub>2</sub> = 1.86. For detailed results see Supplementary Tables.

mice with the three different genotypes, including the *Gnal-A2AcKO<sup>-/-</sup>* mutants that showed strong basal hyperlocomotion (–83 %, –82 %, and –51 % in WT, *Gnal-A2AcKO<sup>+/-</sup>* and *Gnal-A2AcKO<sup>-/-</sup>*), respectively, Fig. 8A). The A2A antagonist KW6002 (3.3 mg.Kg⁻¹) increased locomotor activity in WT animals (+389 %, *p* < 0.001, Fig. 8B left and right panels). This effect was diminished in *Gnal-A2AcKO<sup>+/-</sup>* (+179 %, ns) and absent in *Gnal-A2AcKO<sup>-/-</sup>* (–3 %, ns, Fig. 8B right panel). Caffeine is an antagonist of adenosine receptors with a fairly high affinity, but not selective for A2AR (Fredholm et al., 1999). In WT mice, caffeine (15 mg. Kg⁻¹) increased the locomotor activity (+305 %, *p* < 0.001, Fig. 8C). The time-course of caffeine response and its 30-min response in *Gnal-A2AcKO<sup>+/-</sup>* indicated that its effect was diminished and variable (not significant difference with saline response in Fig. 8C right panel). Interestingly, *Gnal-A2AcKO<sup>-/-</sup>* mice showed a paradoxical effect following caffeine administration, with a reduction in their basal hyperactivity (–42 %, *p* < 0.001, Fig. 8C right panel). *Gnal-A2AcKO<sup>-/-</sup>* mice that were highly hyperactive prior to the injection also exhibited a paradoxical decreased activity following administration of the D1 agonist SKF81297 (5 mg.Kg⁻¹) (–49 %, *p* < 0.05, Fig. 8D right panel) whereas WT and *Gnal-A2AcKO<sup>+/-</sup>* mice exhibited a hyperactive response (Fig. 8D). These results revealed a paradoxical effect of several drugs (caffeine and SKF81297), which increased locomotor activity in WT mice but decreased the spontaneous hyperactivity of *Gnal-A2AcKO<sup>-/-</sup>* mice.

### 3.9. Locomotor hyperactivity induced by *Gnal* cKO in *A2A*-SPNs occludes psychostimulant effects

The hyperlocomotor effects of cocaine (20 mg.Kg⁻¹) were similar between WT and *Gnal-A2AcKO<sup>+/-</sup>* mice (+351 % and + 394+, respectively, *p* < 0.05, Fig. 9A). However, in *Gnal-A2AcKO<sup>-/-</sup>* mice that were already hyperactive at baseline, no further increase in locomotion was observed following cocaine administration (Fig. 9A). Similar results were observed in response to D-amphetamine (2.5 mg.Kg⁻¹, Fig. 9B) and methylphenidate (10 mg.Kg⁻¹, Fig. 9C) with comparable locomotor activation in WT (D-amphetamine 8.1-fold increase, *p* < 0.001, methylphenidate 44-fold increase, *p* < 0.05) and *Gnal-A2AcKO<sup>+/-</sup>* mice (D-amphetamine, 8.8-fold increase, *p* < 0.001, methylphenidate 15-fold increase, *p* < 0.01) (right panels Fig. 9B and C). In *Gnal-A2AcKO<sup>-/-</sup>* mice, which were hyperactive, no additional effects were seen (Fig. 9B and C). These results were consistent for the different psychostimulants. Hence, haplo-deficiency of *Gnal* in A2A-SPNs did not modify the response to psychostimulants. In contrast, the locomotor hyperactivity resulting from the absence of *Gnal* expression could not be further increased by psychostimulants, suggesting an occlusion mechanism.

## 4. Discussion

The heterotrimeric G protein that stimulates cAMP production in striatal principal neurons is for the most part comprised of *Gα*<sub>olf</sub> subunit associated with *Gβ*<sub>2</sub> and *Gγ*<sub>7</sub> (Schwindinger et al., 2010; Xie et al., 2015). In our study we used conditional genetic manipulation to dissect the functions of *Gα*<sub>olf</sub> in the two major populations of SPNs. The current work represents an extension and clarification of previous studies using heterozygous *Gnal* full KO mice and rats (Herve, 2011, Corvol et al., 2001, Pelosi et al., 2017, Yu-Taeger et al., 2020) and mice with *Gnal* cKO targeted to all striatal neurons without distinction of cell types (Chambers, 2024). Cell type-specific deletion of *Gnal* abolishes cAMP production stimulated by D1R or A2AR activation in the corresponding SPN population. The absence of *Gα*<sub>olf</sub> expression in D1-SPNs or A2A-SPNs results in differential behavioral effects. *Gnal*-D1cKO induces a significant loss of motor performance with mild locomotor hyperactivity, whereas the absence of *Gα*<sub>olf</sub> in A2A-SPNs makes mice very hyperactive without significant loss of motor skills. These effects observed in a rodent model parse the consequences of *Gnal* decreased expression in each population of SPNs (see summary in Table 1), whose combination might contribute to dystonia in patients. In addition, we report that both *Gnal*-D1cKO and *Gnal*-A2AcKO mice have abnormal responses to drugs interfering with dopamine or adenosine transmission.

### 4.1. Role of *Gα*<sub>olf</sub> in SPNs

The absence of *Gα*<sub>olf</sub> in D1-SPNs or A2A-SPNs results in a marked reduction in AC5 levels in the striatum, supporting results obtained in heterozygous *Gnal* full KO (El Atiallah et al., 2024; Xie et al., 2015). These effects mirror those observed after AC5 deletion, which leads to a pronounced reduction in *Gα*<sub>olf</sub> levels in the striatum (Iwamoto et al., 2004; Xie et al., 2015). In vivo, AC5 has been shown to form a stable complex with the heteromeric G protein subunits *Gα*<sub>olf</sub>/β<sub>2</sub>/γ<sub>7</sub> (Herve, 2011; Schwindinger et al., 2010; Xie et al., 2015) even in the absence of receptor stimulation. The AC5-*Gα*<sub>olf</sub> complex has been proposed to form a pre-coupled “signalosome” stabilized by phosducin-like protein 1 (PhLP1), a Gβ chaperone (Xie et al., 2015). In this model, AC5 activation upon GPCR stimulation results from rearrangement of different subunits rather than their dissociation. The formation of this oligomeric complex protects its various components from proteolysis, an effect that likely allows their precise stoichiometry in the cell. As a result, the absence of *Gα*<sub>olf</sub> or AC5 expression increases the degradation of their partner and strongly reduces their cellular concentrations [see review in (Herve, 2011)]. In *Gnal* cKO mice, we observed a strong reduction in basal AC activity in the striatum, as we previously observed in full KO mice
A SKF81297 5 mg/kg

<figure><img src="image_33.png" /></figure>

B Dihydrexidine 5 mg/kg

<figure><img src="image_34.png" /></figure>

C CGS21680 0.5 mg/kg

<figure><img src="image_35.png" /></figure>

D KW6002 3.3 mg/kg

<figure><img src="image_36.png" /></figure>

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**Fig. 6.** Effects of D1R or A2AR ligands on locomotion in *Gnal-D1cKO* mice. A-D) Time course of locomotor activity (3 left panels), before and after an ip injection of the D1R agonist SKF81297 (SKF, 5 mg.Kg⁻¹) (A), D1R agonist dihydrexidine (DHX, 5 mg.Kg⁻¹) (B), A2AR agonist CGS21680 (CGS, 0.5 mg.Kg⁻¹) (C) or A2AR antagonist KW6002 (KW or istrafedylline, 3.3 mg.Kg⁻¹) (D) in WT (*n* = 9, 4 F, 5 M), *Gnal-D1cKO⁺/⁺* (*n* = 9, 2 F, 7 M) and *Gnal-D1cKO⁻/⁻* (*n* = 10, 6 F, 4 M) mice (age, 4–6 months). In all cases, comparisons were made with locomotion time course after vehicle administration. Each point is the mean ± SEM of the number of ¼ turns in a circular corridor during 5 min. Time-courses of responses to drug and vehicle were compared using repeated measures two-way ANOVA. A) WT: SKF81297: F₁,₁₇ = 19.2, *p* < 0.001; Time: F₁,₂₇₂ = 5.78, *p* < 0.001; Interaction, F₁,₂₇₂ = 7.40, *p* < 0.05. *Gnal-D1cKO⁺/⁺*: SKF81297, F₁,₂₇₂ = 7.39, *p* < 0.05, Time, F₁,₂₇₂ = 6.34; Interaction, F₁,₂₇₂ = 5.17, *p* < 0.001. *Gnal-D1cKO⁻/⁻*: SKF81297, F₁,₂₇₂ = 7.29, *p* < 0.05; Time, F₁,₂₇₂ = 6.41, *p* < 0.001; Interaction, F₁,₂₇₂ = 1.11, ns. B) WT: Dihydrexidine, F₁,₂₇₂ = 17.5, *p* < 0.001; Time: F₁,₂₇₂ = 10.7, *p* < 0.001; Interaction, F₁,₂₇₂ = 2.32, *p* < 0.01. *Gnal-D1cKO⁺/⁺*: Dihydrexidine, F₁,₂₇₂ = 4.84, *p* < 0.05, Time, F₁,₂₇₂ = 15.4, *p* < 0.001; Interaction, F₁,₂₇₂ = 4.51, *p* < 0.001. *Gnal-D1cKO⁻/⁻*: Dihydrexidine, F₁,₂₇₂ = 1.59, ns; Time, F₁,₂₇₂ = 9.25, *p* < 0.001; Interaction, F₁,₂₇₂ = 1.13, ns. C) WT: CGS21680, F₁,₁₇₆ = 0.738, ns; Time: F₁,₁₇₆ = 23.2, *p* < 0.001; Interaction, F₁,₁₇₆ = 2.98, *p* < 0.01. *Gnal-D1cKO⁺/⁺*: CGS21680, F₁,₁₇₆ = 5.56, *p* < 0.05; Time: F₁,₁₇₆ = 5.56, *p* < 0.001; Interaction, F₁,₁₇₆ = 0.838, ns. *Gnal-D1cKO⁻/⁻*: CGS21680, F₁,₁₇₆ = 7.80, *p* < 0.05; Time: F₁,₁₇₆ = 25.7, *p* < 0.001; Interaction, F₁,₁₇₆ = 0.607, ns. D) WT: KW6002, F₁,₁₇₆ = 7.16, *p* < 0.05; Time: F₁,₁₇₆ = 2.71, *p* < 0.01; Interaction, F₁,₁₇₆ = 1.96, *p* < 0.05. *Gnal-D1cKO⁺/⁺*: KW6002, F₁,₁₇₆ = 7.40, *p* < 0.05; Time: F₁,₁₇₆ = 1.82, ns; Interaction, F₁,₁₇₆ = 2.56, *p* < 0.01. *Gnal-D1cKO⁻/⁻*: KW6002, F₁,₁₇₆ = 7.86, *p* < 0.05; Time: F₁,₁₇₆ = 8.31, *p* < 0.001; Interaction, F₁,₁₇₆ = 1.78, ns. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables). A-D, right panels, cumulative responses on 30 min after drug or vehicle administration from the time courses on the left. Graphs represent scatter dot plots with mean ± SD. Responses to drug and vehicle in mouse groups with different genotypes were compared using repeated measures two-way ANOVA; A) SKF81297: F₁,₂₃ = 42.3, *p* < 0.001; Genotype: F₂,₂₃ = 2.59, ns; Interaction: F₂,₂₃ = 4.64, *p* < 0.05. B) Dihydrexidine: F₁,₂₄ = 22.1, *p* < 0.001; Genotype: F₂,₂₄ = 0.83, ns; Interaction: F₂,₂₄ = 2.37, ns. C) CGS21680: F₁,₂₄ = 30.9, *p* < 0.001; Genotype: F₂,₂₄ = 2.48, ns; Interaction: F₂,₂₄ = 5.07, *p* < 0.05. D) KW6002: F₁,₂₄ = 44.8, *p* < 0.001; Genotype: F₂,₂₄ = 2.57, ns; Interaction: F₂,₂₄ = 0.496, ns. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables). * *p* < 0.05, ** *p* < 0.01, *** *p* < 0.001.

(Corvol et al., 2001), which may result from the decrease in AC5. Gγ7 may play a critical role in the hierarchical assembly of the Gα<sub>olf</sub>/β<sub>2</sub>/γ<sub>7</sub> complex, because Gγ7 is required for the stabilization of Gα<sub>olf</sub> or Gβ<sub>2</sub> (Schwindinger et al., 2010) whereas the presence of Gα<sub>olf</sub> is not required for Gγ7 or Gβ<sub>2</sub> stabilization. This is consistent with a model in which heterotrimer formation begins with the interaction of Gγ with Gβ to form a stable Gβγ dimer that then associates with an α subunit (Brunori et al., 2021; Pelletier et al., 2024). As a result, the absence of Gα<sub>olf</sub> expression leaves Gβ<sub>2</sub>/γ<sub>7</sub> levels unchanged in cells and Gβγ dimer-dependent signaling may remain efficient or be constitutively active due to the loss of control in the heterotrimeric complex. This may contribute to the phenotype of mutant mice and requires further exploration.

The *Drd1*-Cre line we used has been shown to recombine floxed genes in D1-SPNs (Bateup et al., 2010; Brunori et al., 2021; Lobo et al., 2010), as well as in other regions expressing D1R, including the cerebral cortex, hippocampus, olfactory bulb, thalamus, but not the cerebellum (Gong et al., 2007; Brunori et al., 2021). There is therefore a possibility that Gα<sub>olf</sub> disappears in these regions and that this effect contributes to the phenotype of *Gnal-D1cKO<sup>-/-</sup>* or *Gnal-D1cKO<sup>+</sup>/<sup>-</sup>* mice. However, in the olfactory bulb Gα<sub>olf</sub> levels were not reduced in the mutant mice, suggesting that the cells expressing Gα<sub>olf</sub> did not express Cre. In the cerebral cortex, despite a significant expression of *Gnal* mRNA (Millett et al., 2024), we did not detect Gα<sub>olf</sub> by immunoblotting. Moreover, in a recent translome analysis of D1R-expressing neurons in the cortex, we found that Gα<sub>s</sub>-encoding mRNAs were 5.5 times more abundant than *Gnal* mRNAs (Montalban et al., 2022). This high expression of Gα<sub>s</sub> would likely compensate for the absence of Gα<sub>olf</sub>. In the cerebellum, D1R expression has only been reported in Bergman glial cells (Cutando et al., 2021), unlike Gα<sub>olf</sub> which is expressed in Purkinje cells (Belluscio et al., 1998; Millett et al., 2024; Vemula et al., 2013). It is therefore unlikely that Gα<sub>olf</sub> expression is affected in the cerebellum of our *Gnal-D1cKO* mice. In the *Adora2a*-Cre line, it has been shown that genetic recombination is highly selective for SPNs expressing enkephalin (Durieux et al., 2009). The piriform cortex is the only region where recombinant neurons have been observed (Durieux et al., 2009).

In the striatum, selective *Gnal-D1cKO* results in complete loss of AC response to the D1 agonist SKF81297, demonstrating a necessary role for Gα<sub>olf</sub> in coupling D1R to AC. Gα<sub>s</sub> is also expressed in the striatum, mainly in cholinergic and GABAergic interneurons and in afferent fibers, but very little in SPNs (Herve et al., 2001; Millett et al., 2024; Montalban et al., 2022). The absence of AC response to the A2A agonist CGS21680 in *Gnal-A2AcKO<sup>-/-</sup>* mice demonstrates that Gα<sub>olf</sub> is also essential for coupling A2AR to AC. In the striatum of *Gnal-D1cKO<sup>-/-</sup>* mice, the response of AC to an A2AR agonist was not significantly altered and in *Gnal-A2AcKO<sup>-/-</sup>* mice, AC response to a D1R agonist remained unchanged. These results are consistent with the overall dichotomy of SPNs

in the dorsal striatum (Gerfen and Surmeier, 2011), in spite of the existence of a minor population expressing both D1R and D2R in rodents (Bertran-Gonzalez et al., 2008; Bonnavion et al., 2024).

## 4.2. *Gnal cKO* in *D1-SPN* impairs motor abilities

The absence of Gα<sub>olf</sub> expression in D1-SPNs results in poor performances in the Rotarod and pole tests. This phenotype cannot be attributed to a generalized motor deficit since mutant mice exhibit high locomotor activity especially during the night and do not show any muscular weakness. Mutant mice did not show any sign of abnormal anxiety or risk assessment that could bias the results of the Rotarod and pole test. In the Rotarod, *Gnal-D1cKO<sup>-/-</sup>* mice are initially markedly impaired but capable of some motor learning with the repetition of training sessions. Mice with constitutive deletion of D1Rs show similar deficits in the Rotarod (Karasinska et al., 2005; Kobayashi et al., 2004; Short et al., 2006; Wall et al., 2011) in the first days of testing, but are also able to increase their performance after multiple learning sessions (Nakamura et al., 2014). Mice lacking AC5 expression also show reduced motor performance in the Rotarod and pole test although some motor learning can still be observed (Iwamoto et al., 2003). Together these data support an important role in motor behavior of the D1R/ Gα<sub>olf</sub>/AC5 complex in D1-SPNs. Yet this motor deficit may be partially compensated by other neural circuits and/or intracellular signaling pathways, allowing learning after prolonged training. Poor performance in the Rotarod test with apparently aberrant motor learning have been observed in other animal models of dystonia (reviewed in (Richter and Richter, 2014)). This phenotype may represent a consistent species-specific modeling of human dystonic symptoms in mice. In contrast, *Gnal-A2AcKO<sup>-/-</sup>* mice do not show any impairment in motor performance or learning in the Rotarod test. This is consistent with data showing that ablation of A2A-SPNs has less effect in the Rotarod than ablation of D1-SPNs (Durieux et al., 2012).

## 4.3. *Gnal cKO* in *D1-SPN* impairs responses to drugs

*Gnal-D1cKO* has contrasting effects on locomotor responses to various D1 agonists. Responses to SKF81297 or dihydrexidine are completely or largely abolished, whereas SKF83822-induced hyperactivity is preserved. Contrasting alterations are also observed in responses to cocaine, D-amphetamine, and methylphenidate. *Gnal-D1cKO<sup>-/-</sup>* mice do not respond to methylphenidate while they exhibit decreased responses to cocaine and virtually no alteration in D-amphetamine effects. It is therefore clear that part of the locomotor response to these drugs depends on the presence of Gα<sub>olf</sub> in D1-SPNs, but that another part does not, the proportion between the two varying from one drug to another.
A Cocaine 20 mg/kg

<figure><img src="image_37.png" /></figure>

B D-amphetamine 2.5 mg/kg

<figure><img src="image_38.png" /></figure>

C Methylphenidate 10 mg/kg

<figure><img src="image_39.png" /></figure>

(caption on next page)
**Fig. 7.** Effects of psychostimulants on locomotion in *Gnal-D1cKO<sup>-/-</sup>* and *Gnal-D1cKO<sup>+/-</sup>* mice. A-C) Time course of locomotor activity (3 left panels) before and after an ip injection of cocaine (Coc, 20 mg.Kg⁻¹; WT *n* = 9, 4 F, 5 M, *Gnal-D1cKO<sup>+/-</sup>* *n* = 9, 6 F, 3 M, *Gnal-D1cKO<sup>-/-</sup>* *n* = 9, 6 F, 3 M, age, 3–6 months) (A), D-amphetamine (Amph, 2.5 mg.Kg⁻¹; WT *n* = 10, 10 M, *Gnal-D1cKO<sup>+/-</sup>* *n* = 9, 9 M; *Gnal-D1cKO<sup>-/-</sup>* *n* = 9, 9 M; age, 7–10 months) (B), or methylphenidate (MPH, 0.5 mg.Kg⁻¹; WT *n* = 11, 5 F, 6 M, *Gnal-D1cKO<sup>+/-</sup>* *n* = 10, 4 F, 6 M, *Gnal-D1cKO<sup>-/-</sup>* *n* = 5, 4 F, 1 M; age, 10–12 months) (C). In all cases, comparisons were made with time courses after vehicle administration. Each point is the mean ± SEM of the number of ¼ turns in a circular corridor during 5 min. Time-courses of responses to drug and vehicle were compared using repeated measures two-way ANOVA. A) WT: Cocaine: *F*<sub>1,110</sub> = 10.6, *p* < 0.01; Time: *F*<sub>11,110</sub> = 4.90, *p* < 0.001, Interaction: *F*<sub>11,110</sub> = 6.22, *p* < 0.001. *Gnal-D1cKO<sup>+/-</sup>*: Cocaine: *F*<sub>1,176</sub> = 5.53, *p* < 0.05; Time: *F*<sub>11,176</sub> = 5.00, *p* < 0.001, Interaction: *F*<sub>11,176</sub> = 5.53, *p* < 0.001. *Gnal-D1cKO<sup>-/-</sup>*: Cocaine: *F*<sub>1,176</sub> = 4.69, *p* < 0.01; Time: *F*<sub>11,176</sub> = 1.14, ns, Interaction: *F*<sub>11,176</sub> = 1.48, ns. B) WT: D-amphetamine: *F*<sub>1,306</sub> = 135, *p* < 0.001; Time: *F*<sub>17,306</sub> = 12.9, *p* < 0.001, Interaction: *F*<sub>17,306</sub> = 12.7, *p* < 0.001. *Gnal-D1cKO<sup>+/-</sup>*: D-amphetamine: *F*<sub>1,272</sub> = 92.1, *p* < 0.001; Time: *F*<sub>17,272</sub> = 8.91, *p* < 0.001, Interaction: *F*<sub>17,272</sub> = 10.6, *p* < 0.001. *Gnal-D1cKO<sup>-/-</sup>*: D-amphetamine: *F*<sub>1,306</sub> = 41.2, *p* < 0.001; Time: *F*<sub>17,306</sub> = 11.4, *p* < 0.001; Interaction: *F*<sub>17,306</sub> = 10.5, *p* < 0.001. C) WT: Methylphenidate: *F*<sub>1,340</sub> = 45.2, *p* < 0.001; Time: *F*<sub>17,340</sub> = 1.57, ns; Interaction: *F*<sub>17,340</sub> = 2.11, *p* < 0.01. *Gnal-D1cKO<sup>+/-</sup>*: Methylphenidate: *F*<sub>1,306</sub> = 22.9, *p* < 0.001; Time: *F*<sub>17,306</sub> = 13.1, *p* < 0.001, Interaction: *F*<sub>17,306</sub> = 13.1, *p* < 0.001. *Gnal-D1cKO<sup>-/-</sup>*: Methylphenidate: *F*<sub>1,238</sub> = 5.89, *p* < 0.05; Time: *F*<sub>17,238</sub> = 4.13, *p* < 0.001; Interaction: *F*<sub>17,238</sub> = 0.974, ns. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables. A-C, right panels, cumulative responses on 30 min after drug or vehicle administration from the time courses on the left. Graphs represent scatter dot plots with mean ± SD. Responses to drug and vehicle in mouse groups with different genotypes were compared using repeated measures two-way ANOVA. A) Cocaine: *F*<sub>1,21</sub> = 65.7, *p* < 0.001; Genotype: *F*<sub>2,21</sub> = 2.76, ns; Interaction: *F*<sub>2,21</sub> = 5.81, *p* < 0.001. B) D-amphetamine: *F*<sub>1,25</sub> = 268, *p* < 0.001; Genotype: *F*<sub>2,25</sub> = 3.15, ns; Interaction: *F*<sub>2,25</sub> = 1.90, ns. C) Methylphenidate: *F*<sub>1,26</sub> = 41.1, *p* < 0.001; Genotype: *F*<sub>2,26</sub> = 5.05, *p* < 0.05; Interaction: *F*<sub>2,26</sub> = 6.47, *p* < 0.01. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables. *p* < 0.05, **p* < 0.01, ***p* < 0.001.

Responses to SKF81297 or cocaine are largely mediated by dopamine D1Rs, since they are completely abolished by disruption of *Drd1* gene (Xu et al., 1994a; Xu et al., 1994b). In contrast, the absence of D1Rs only attenuates responses to D-amphetamine (Crawford et al., 1997; Xu et al., 2000). Deletion of *Gγ7* in D1-SPNs, which strongly reduces *Gα*<sub>olf</sub> expression in the striatum, does not significantly alter locomotor responses to the D1R agonists SKF83822 and SKF83959, or to D-amphetamine (Brunori et al., 2021). As discussed above, it is unlikely that *Gα*<sub>s</sub> in SPNs is responsible for the partly preserved responses to D1 agonists in *Gnal-D1cKO<sup>-/-</sup>* mice. However, D1R activates several cAMP-independent signaling pathways, including β-arrestins (Conroy et al., 2015; Kaya et al., 2020; Urs et al., 2011), *Gq*/Phospholipase C (Jin et al., 2003; Lee et al., 2014; Medvedev et al., 2013; Undie and Friedman, 1990), tyrosine kinase Src leading to NMDA receptor phosphorylation (Kaya et al., 2020; Pascoli et al., 2011) and D1R heteromerization with NMDA receptor subunits (Andrianarivelo et al., 2021; Cahill et al., 2014; Fiorentini et al., 2003; Lee et al., 2002). These alternative signaling pathways could mediate responses to D1R-stimulating drugs in the absence of *Gα*<sub>olf</sub>. In addition, different D1R agonists exhibit biases in their abilities to activate different signaling pathways downstream of D1Rs (Conroy et al., 2015; Jin et al., 2003; Jones-Tabah et al., 2021; Martini et al., 2019; Yano et al., 2018) which could be a factor explaining different consequences of *Gα*<sub>olf</sub> absence in D1-SPNs and would require further investigation.

### 4.4. *Gnal cKO* in *A2A-SPN* induces a strong locomotor hyperactivity

Disruption of *Gnal* in A2A-SPNs leads to strong spontaneous locomotor hyperactivity, especially during the nocturnal phases corresponding to wakefulness periods in mice. During the night the locomotor activity of *Gnal-A2AcKO<sup>-/-</sup>* mice was ~15-fold higher than in WT animals. Importantly, this hyperactivity was prolonged and even tended to increase in a familiar environment, in contrast to what is observed in WT mice. This hyperactivity is similar to that observed when A2A-SPNs, particularly those in the dorsomedial region of striatum, are selectively destroyed by diphtheria toxin (Durieux et al., 2009; Durieux et al., 2012). However, *Gnal-A2AcKO<sup>-/-</sup>* mice do not show any evidence of A2A-SPN degeneration since the levels of DARPP-32, a specific marker of SPNs, are normal in these mice. Spontaneous locomotor hyperactivity is also observed when *Gγ7* or DARPP-32 is lacking in A2A-SPNs (Bateup et al., 2010; Brunori et al., 2021). This locomotor hyperactivity could be the consequence of disrupted A2AR activity. However, complete A2AR deletion or selective A2AR deletion in the striatum does not significantly alter spontaneous locomotor activity (Ledent et al., 1997; Shen et al., 2008). Although A2AR is probably the most abundantly expressed receptor stimulating AC in A2A-SPNs other receptors may compensate for its absence. It is also possible that hyperlocomotion is related to a

decrease in basal cAMP levels, rather than to impairment of cAMP-dependent signaling of A2AR. In support of this hypothesis, in *Gnal-A2AcKO<sup>-/-</sup>* mice, the selective A2AR antagonist KW6002 (istradefyl-line) loses its hyperlocomotor effect and caffeine decreases locomotion, results reminiscent of those observed in mutant mice lacking A2AR (Ledent et al., 1997) or *Gγ7* in A2A-SPNs (Brunori et al., 2021). Interestingly, the effects of KW6002 and caffeine are reduced in *Gnal-A2AcKO<sup>+/-</sup>* mice compared to WT mice, confirming similar results in mice with constitutive *Gnal* hemizygoty (Herve et al., 2001). This clearly shows that the locomotor psychostimulant effects of caffeine and KW6002 are mediated by the decrease in A2AR-dependent cAMP production. In the absence of *Gα*<sub>olf</sub> in A2A-SPNs, all other psychostimulant drugs tested lose their effects on locomotor activity, which appear to be occluded by the preexisting hyperactivity of the *Gnal-A2AcKO<sup>-/-</sup>* mice. In contrast, the D1R agonist, SKF81297 acquires a hypo-locomotor effect, the mechanism of which would require further investigation.

### 4.5. *Gnal KO* in D1-SPNs increases mortality around weaning

We observed an abnormally high mortality rate of *Gnal-D1cKO<sup>-/-</sup>* mice around weaning. Approximately a third of homozygotes died during this period. Those mice ceased gaining weight around day 15, a period when mothers gradually stop nursing their pups. The other remaining mutant mice were apparently not affected during this period and had normal development and body weight similar to that of WT mice as adults. The most likely hypothesis is that the gradual transition from milk feeding by the mother to a more solid and diverse diet (weaning) is disrupted in some mutant mice. In mice with a constitutive deletion of *Gnal*, a very high newborn lethality was observed at an earlier time, from the first days after birth. This mortality was attributed to the early anosmia of these mice, caused by a loss of olfactory receptor signaling (Belluscio et al., 1998), depriving the pups from the olfactory signals necessary for suckling. However, *Gα*<sub>olf</sub> deficits in striatal neurons could also contribute to the lethality in pups since very high infant mortality was also reported in a model of conditioned deletion in all striatal neurons from the embryonic stages (Chambers, 2024). Mutant mice with severe dopamine deficiency that were indistinguishable from their WT littermates by postnatal week 1, became hypoactive and stopped feeding by postnatal days 10–15 (Zhou and Palmiter, 1995). A similar phenotype and lethality were observed in mice lacking ERK1/2 in the D1-SPNs (Hutton et al., 2017). In our *Gnal-D1cKO*, mortality was less massive, affecting only 1/3 of mutant mice but the developmental halt appeared at the same period as that observed in dopamine or ERK1/2-deficient mice. This P10-P15 period corresponds to a time when both *Gα*<sub>olf</sub> and AC5 expression increase sharply in the developing striatum (Iwamoto et al., 2004; Rius et al., 1994). *Gα*<sub>olf</sub> expression is very low at birth and reaches its maximum at P21 corresponding to weaning. At
A CGS21680 0.5 mg/kg

<figure><img src="image_40.png" /></figure>

B KW6002 3.3 mg/kg

<figure><img src="image_41.png" /></figure>

C Caffeine 15 mg/kg

<figure><img src="image_42.png" /></figure>

D SKF81297 5 mg/kg

<figure><img src="image_43.png" /></figure>

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**Fig. 8.** Effects of D1R or A2AR ligands on locomotion in *Gnal-A2AcKO<sup>-/-</sup>* and *Gnal-A2AcKO<sup>+/</sup>* mice. A-D) Time course of locomotor activity (3 left panels) before and after an ip injection of the A2A agonist CGS21680 (CGS, 0.5 mg.Kg⁻¹) (A), A2A antagonist KW6002 (KW or istradefylline, 3.3 mg.Kg⁻¹) (B), non-selective adenosine receptor antagonist caffeine (15 mg.Kg⁻¹)(C) or D1 agonist SKF81297 (SKF, 5 mg.Kg⁻¹) (D) (WT, n = 10, 6 F, 4 M; *Gnal-A2AcKO<sup>-/-</sup>*, n = 9, 5 F, 4 M; *Gnal-A2AcKO<sup>-/-</sup>*, n = 9, 5 F, 4 M; age, 3–5 months). In all cases, comparisons were made with time courses after vehicle administration. Each point is the mean ± SEM of the number of ¼ turns in a circular corridor during 5 min. Time-courses of responses to drug and vehicle were compared using repeated measures two-way ANOVA. A) WT: CGS21680, F₁,₃₀₆ = 5.62, *p* < 0.05; Time: F₁₇,₃₀₆ = 27.6, *p* < 0.001; Interaction, F₁₇,₃₀₆ = 2.35, *p* < 0.01. *Gnal-A2AcKO<sup>-/-</sup>*: CGS21680, F₁,₂₇₂ = 6.95, *p* < 0.05; Time: F₁₇,₂₇₂ = 9.30, *p* < 0.001; Interaction, F₁₇,₂₇₂ = 0.660, ns. *Gnal-A2AcKO<sup>-/-</sup>*: CGS21680, F₁,₂₇₂ = 3.97, ns; Time: F₁₇,₂₇₂ = 3.23, *p* < 0.001; Interaction, F₁₇,₂₇₂ = 2.84, *p* < 0.001. B) WT: KW6002, F₁,₃₀₆ = 12.7, *p* < 0.01; Time: F₁₇,₃₀₆ = 1.18, ns; Interaction, F₁₇,₃₀₆ = 2.27, *p* < 0.01. *Gnal-A2AcKO<sup>+/</sup>*: KW6002, F₁,₂₇₂ = 19.8, *p* < 0.001; Time: F₁₇,₂₇₂ = 1.87, *p* < 0.05; Interaction, F₁₇,₂₇₂ = 2.22, *p* < 0.01. *Gnal-A2AcKO<sup>-/-</sup>*: KW6002, F₁,₂₇₂ = 0.159, ns; Time: F₁₇,₂₇₂ = 11.6, *p* < 0.001; Interaction, F₁₇,₂₇₂ = 1.35, ns. C) WT: Caffeine, F₁,₃₀₆ = 24.4 *p* < 0.001; Time: F₁₇,₃₀₆ = 1.96, *p* < 0.05; Interaction, F₁₇,₃₀₆ = 3.52, *p* < 0.001. *Gnal-A2AcKO<sup>+/</sup>*: Caffeine, F₁,₂₇₂ = 9.08, *p* < 0.01; Time: F₁₇,₂₇₂ = 1.28, ns; Interaction, F₁₇,₂₇₂ = 0.8331, ns. *Gnal-A2AcKO<sup>-/-</sup>*: Caffeine, F₁,₂₇₂ = 3.98, ns; Time: F₁₇,₂₇₂ = 13.77, *p* < 0.001; Interaction, F₁₇,₂₇₂ = 1.22, ns. D) WT: SKF81297, F₁,₃₀₆ = 2.10, ns; Time: F₁₇,₃₀₆ = 0.828, ns; Interaction, F₁₇,₃₀₆ = 4.10, *p* < 0.001. *Gnal-A2AcKO<sup>+/</sup>*: SKF81297, F₁,₂₇₂ = 7.10, *p* < 0.05; Time: F₁₇,₂₇₂ = 0.629, ns; Interaction, F₁₇,₂₇₂ = 1.56, ns. *Gnal-A2AcKO<sup>-/-</sup>*: SKF81297, F₁,₂₇₂ = 3.03, ns; Time: F₁₇,₂₇₂ = 12.2, *p* < 0.001; Interaction, F₁₇,₂₇₂ = 0.999, ns. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables. A-D, the right panels, cumulative responses on 30 min after drug or vehicle administration from the time courses on the left. Graphs represent scatter dot plots with mean ± SD. Responses to drug and vehicle in mouse groups with different genotypes were compared using repeated measures two-way. A) CGS21680: F₁,₂₅ = 2.33, *p* < 0.01; Genotype: F₂,₂₅ = 12.8, *p* < 0.001; Interaction: F₂,₂₅ = 2.13, ns. B) KW6006: F₁,₂₅ = 15.1, *p* < 0.001; Genotype: F₂,₂₅ = 12.2, *p* < 0.001; Interaction: F₂,₂₅ = 5.84, *p* < 0.01. C) Caffeine: F₁,₂₅ = 1.06, ns; Genotype: F₂,₂₅ = 25.0, *p* < 0.001; Interaction: F₂,₂₅ = 23.7, *p* < 0.001. D) SKF81297: F₁,₂₆ = 0.63, ns; Genotype: F₂,₂₆ = 14.8, *p* < 0.001; Interaction: F₂,₂₆ = 6.04, *p* < 0.001. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables. *p* < 0.05, **p* < 0.01, ***p* < 0.001.

birth, $G\alpha_s$ expression exceeds that of $G\alpha_{olf}$ but the ratio reverses during the second postnatal week. It is therefore likely that functional D1-SPN disruption only begins to be significant from this second week, possibly impairing motivational and learning processes important for feeding. Our results suggest that D1-SPNs play an important role in these behavioral adaptations at weaning. The reasons why this lethal phenotype and the prior loss of weight appeared in only a third of the mice remain to be determined. We can speculate that compensatory mechanisms are insufficient at this critical period of life in some pups, possibly due to environmental factors such as position in litter or epigenetic regulations. In contrast, it is striking that *Gnal-A2AcKO<sup>-/-</sup>* mice have normal viability, revealing a non-critical role of A2A-SPNs around weaning.

## 4.6. Limitations of the study

This study is the first general exploration of *Gnal* cKO examining the effect of the selective deletion of *Gnal* in each of the two specific populations of SPNs. We did not observe spontaneous dystonic movement but further exploration with recording of muscles and the use of drugs potentially triggering dystonia, such as muscarinic agonists (Pelosi et al., 2017) will be important as well as characterization of potential dysfunctions of the cerebello-thalamic pathways (Aissa et al., 2022). It is well established that *GNAL*-linked dystonia typically appears in adulthood with dystonic symptoms spreading with age in a subset of patients (Fuchs et al., 2013). Additionally, clinical data indicate that prevalence of the disease may be higher in females compared to males (Kilic-Berkmen et al., 2024). These data suggest that age and sex may influence the phenotypic expression of *Gnal* deficiency in *Gnal*-cKO mice. In the current study we used groups of young adults and more mature adults (less than 12 months old) of both sexes and observed no trends indicating age- or sex-related differences. However, to rigorously assess these potential influences, larger cohorts of animals stratified by age and sex would be required. Concerning the exploration of responses to pharmacological agents, the use of dose-response curves and blockade of responses with specific antagonists would be informative to address the apparent heterogeneity of the consequences of the absence of $G\alpha_{olf}$ in D1-SPNs and A2A-SPNs on responses to D1 agonists and psychostimulants. Because of their high hyperactivity, *Gnal-A2AcKO* mice could be a mouse model of ADHD. Although we observed a decreased locomotion in response to a D1 agonist, SKF81297, our results with methylphenidate or D-amphetamine were not conclusive. This would require further investigation including exploration of possible attention disturbances and of effects of various doses and duration of treatment with methylphenidate and D-amphetamine.

## 4.7. Conclusions

Our study shows that $G\alpha_{olf}$-mediated striatal signaling in selective populations of SPNs differentially controls motor behavior and drug responses in mice. Our results confirm that $G\alpha_{olf}$ is critical for the activation of cAMP production induced by stimulation of D1R in D1-SPNs and A2AR in A2A-SPNs and support the role of $G\alpha_{olf}$/β2/γ7 heterotrimers in stabilizing AC5 signalosome (Herve, 2011; Schwindinger et al., 2010; Xie et al., 2015). We propose that the observed phenotypes arise from the destabilization of this signalosome, leading to decreased basal cAMP production and a loss of receptor-mediated stimulation, particularly affecting D1R and A2AR. $G\alpha_{olf}$ deficiency in D1-SPNs induces significant disturbances in motor control that could contribute to *GNAL*-related dystonic disorders in humans. In contrast, when $G\alpha_{olf}$ is lacking in A2A-SPNs, mice become highly hyperactive without overt motor coordination impairment. This hyperactivity may result from a disinhibition of movement which might also contribute to the development of dystonic symptoms in patients with DYT-*GNAL*. The hypothesis of joint involvement of D1-SPNs and A2A-SPNs is consistent with findings of a recent study (Chambers, 2024) in which selective suppression of *Gnal* in the striatum, affecting both D1-SPNs and A2A-SPNs, as well as interneurons, induced abnormal movements in mice, reminiscent of human dystonic symptoms. In that study the phenotype was more pronounced when *Gnal* KO was induced in adulthood using a viral approach, suggesting that compensatory processes can take place during the development of the motor system to mitigate the consequence of the striatal dysfunction associated with *GNAL* suppression. In addition, in the study of (Chambers, 2024), *Gnal* disruption affects all types of striatal neurons, notably cholinergic neurons and parvalbumin-positive GABAergic neurons that express $G\alpha_{olf}$ in addition to $G\alpha_s$ (Herve et al., 2001; Millett et al., 2024). The absence of $G\alpha_{olf}$ in these cholinergic and GABAergic neurons may also contribute to the development of the observed dystonic-like phenotype.

Interestingly, locomotor responses to various D1 agonists and psychostimulants are differentially affected by the absence of $G\alpha_{olf}$ in D1-SPNs, showing that these responses are only partially dependent on $G\alpha_{olf}$-dependent D1R signaling. Our results suggest the possibility that alternative cAMP-independent D1R-effectors contribute to the effects of psychostimulant drugs. Thus, we identify $G\alpha_{olf}$ as an important, but not exclusive, mediator of D1R and A2AR signaling, which would make it an interesting target for the development of modulatory drugs as new therapeutic options for dystonia but also other neurological or psychiatric diseases, such as Parkinson's disease and drug addiction.
<figure><img src="image_44.png" /><figcaption>**Fig. 9.** Effects of psychostimulants on locomotion in *Gnal-A2AcKO<sup>-/-</sup>* and *Gnal-A2AcKO<sup>+/-</sup>* mice.</figcaption></figure>

A-C) Time course of locomotor activity (3 left panels) before and after an ip injection of cocaine (Coc, 20 mg.Kg⁻¹; WT, n = 9, 2 F, 7 M; *Gnal-A2AcKO<sup>+/-</sup>*; n = 9, 4 F, 5 M; *Gnal-A2AcKO<sup>-/-</sup>*; n = 9, 6 F, 3 M; age 3.5-6 months) (A), D-amphetamine (Amph, 2.5 mg.Kg⁻¹; WT, n = 20, 11 F, 9 M; *Gnal-A2AcKO<sup>+/-</sup>*; n = 21, 12 F, 9 M; *Gnal-A2AcKO<sup>-/-</sup>*; n = 17, 8 F, 9 M, age, 3.5-8 months) (B), or methylphenidate (MPH, 0.5 mg.Kg⁻¹; WT, n = 7, 1 F, 6 M; *Gnal-A2AcKO<sup>+/-</sup>*; n = 9, 3 F, 6 M; *Gnal-A2AcKO<sup>-/-</sup>*; n = 4, 1 F, 3 M; age, 9-11 months) (C). In all cases, comparisons were made with time courses after vehicle administration. Each point is the mean ± SEM of the number of ¼ turns in a circular corridor during 5 min. Time-courses of responses to drug and vehicle were compared using repeated measures two-way ANOVA. A) WT: Cocaine: *F*<sub>1,176</sub> = 7.19, *p* < 0.05; Time: *F*<sub>11,176</sub> = 2.04, *p* < 0.05, Interaction: *F*<sub>11,176</sub> = 1.34, ns. *Gnal-A2AcKO<sup>+/-</sup>*: Cocaine: *F*<sub>1,176</sub> = 6.60, *p* < 0.05; Time: *F*<sub>11,176</sub> = 1.97, *p* < 0.05, Interaction: *F*<sub>11,176</sub> = 1.96, *p* < 0.05. *Gnal-A2AcKO<sup>-/-</sup>*: Cocaine: *F*<sub>1,154</sub> = 0.443, ns; Time: *F*<sub>11,154</sub> = 0.982, ns, Interaction: *F*<sub>11,154</sub> = 1.13, ns. B) WT: D-amphetamine: *F*<sub>1,578</sub> = 60.8, *p* < 0.001; Time: *F*<sub>17,578</sub> = 10.9, *p* < 0.001, Interaction: *F*<sub>17,578</sub> = 15.9, *p* < 0.001. *Gnal-A2AcKO<sup>+/-</sup>*: D-amphetamine: *F*<sub>1,680</sub> = 26.5, *p* < 0.001; Time: *F*<sub>17,680</sub> = 14.5, *p* < 0.001, Interaction: *F*<sub>17,680</sub> = 21.2, *p* < 0.001. *Gnal-A2AcKO<sup>-/-</sup>*: D-amphetamine: *F*<sub>1,510</sub> = 0.536, ns; Time: *F*<sub>17,510</sub> = 0.808, ns; Interaction: *F*<sub>17,510</sub> = 0.496, ns. C) WT: Methylphenidate: *F*<sub>1,204</sub> = 3.75, *p* < 0.001; Time: *F*<sub>17,204</sub> = 2.57, *p* < 0.001. *Gnal-A2AcKO<sup>+/-</sup>*: Methylphenidate: *F*<sub>1,272</sub> = 10.2, *p* < 0.01; Time: *F*<sub>17,272</sub> = 2.57, *p* < 0.001, Interaction: *F*<sub>17,272</sub> = 2.99, *p* < 0.001. *Gnal-A2AcKO<sup>-/-</sup>*: Methylphenidate: *F*<sub>1,102</sub> = 0.589, ns; Time: *F*<sub>17,104</sub> = 1.57, ns; Interaction: *F*<sub>17,104</sub> = 1.09, ns. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables). A-C) in the right panels, cumulative responses on 30 min after drug or vehicle administration from the time courses on the left. Graphs represent scatter dot plots with mean ± SD. Responses to drug and vehicle in mouse groups with different genotypes were compared using repeated measures two-way ANOVA. A) Cocaine: *F*<sub>1,23</sub> = 1.34, *p* < 0.01; Genotype: *F*<sub>2,23</sub> = 1.07, ns; Interaction: *F*<sub>2,23</sub> = 0.973, ns. B) D-amphetamine: *F*<sub>1,52</sub> = 64.4, *p* < 0.001; Genotype: *F*<sub>2,52</sub> = 7.61, *p* < 0.01; Interaction: *F*<sub>2,52</sub> = 23.0, *p* < 0.001. C) Methylphenidate: *F*<sub>1,17</sub> = 16.3, *p* < 0.001; Genotype: *F*<sub>2,17</sub> = 0.81, ns; Interaction: *F*<sub>2,17</sub> = 1.28, ns. ANOVAs were followed by Bonferroni's multiple comparison test (see Supplementary Tables. * *p* < 0.05, ** *p* < 0.01, *** *p* < 0.001.
<table><caption>**Table 1**
Genotype and phenotype of Gnal conditional mutant mice.</caption><thead><tr><th>Gnal genotype</th><th>+/f</th><th>f/f</th><th>+/f</th><th>f/f</th></tr></thead><tbody><tr><td>Cre</td><td>Drd1-Cre</td><td>Drd1-Cre</td><td>Adora2a-Cre</td><td>Adora2a-Cre</td></tr><tr><td>Abbreviation</td><td>Gnal-D1cKO<sup>+/-</sup></td><td>Gnal-D1cKO<sup>-/-</sup></td><td>Gnal-A2AcKO<sup>+/-</sup></td><td>Gnal-A2AcKO<sup>-/-</sup></td></tr><tr><td>Basal AC activity</td><td>nt</td><td>Decreased (-60 %)</td><td>nt</td><td>Decreased (-55 %)</td></tr><tr><td>D1 agonist AC stimulation</td><td>nt</td><td>Blocked</td><td>nt</td><td>Normal</td></tr><tr><td>A2A agonist AC stimulation</td><td>nt</td><td>Normal</td><td>nt</td><td>Blocked</td></tr><tr><td>Gα<sub>olf</sub> protein</td><td>-39 %</td><td>-71 %</td><td>-24 %</td><td>-59 %</td></tr><tr><td>AC5 protein</td><td>-24 %</td><td>-37 %</td><td>-24 %</td><td>-37 %</td></tr><tr><td>Lethality at weaning</td><td>No</td><td>34 %</td><td>No</td><td>No</td></tr><tr><td>24-h locomotor activity</td><td>Unchanged</td><td>Increased at night</td><td>Small increase / night</td><td>Increased / 24 h</td></tr><tr><td>Exploration activity in novel environment</td><td>Unchanged</td><td>Increase activity, reduced habituation</td><td>Unchanged</td><td>Increased activity, Absence of habituation</td></tr><tr><td>Rotarod performance</td><td>Enhanced</td><td>Decreased</td><td>Unchanged</td><td>Unchanged</td></tr><tr><td>Drug responses compared to WT:</td><td></td><td></td><td></td><td></td></tr><tr><td>SKF81297 (D1 ago)</td><td>Unchanged</td><td>Blocked</td><td>Unchanged</td><td>Decrease of basal hyperlocomotion</td></tr><tr><td>Dihydrexidine (D1 ago)</td><td>Unchanged</td><td>Blocked</td><td>nt</td><td>nt</td></tr><tr><td>CGS21680 (A2A ago)</td><td>Unchanged</td><td>Unchanged</td><td>Unchanged</td><td>Present</td></tr><tr><td>KW6002 (A2A antag)</td><td>Unchanged</td><td>Unchanged</td><td>Slight diminution</td><td>Occluded</td></tr><tr><td>Caffeine</td><td>nt</td><td>nt</td><td>Slight diminution</td><td>Decrease of basal hyperlocomotion</td></tr><tr><td>Cocaine</td><td>Slight diminution</td><td>Slight modification</td><td>Unchanged</td><td>Occlusion</td></tr><tr><td>D-amphetamine</td><td>Unchanged</td><td>Unchanged</td><td>Unchanged</td><td>Occlusion</td></tr><tr><td>Methylphenidate</td><td>Unchanged</td><td>Blocked</td><td>Unchanged</td><td>No response</td></tr></tbody></table>

nt, not tested.

CRediT authorship contribution statement

**Sophie Longueville:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation.
**Ruiyi Yuan:** Methodology, Investigation.
**Claire Naon:** Methodology, Investigation.
**Emmanuel Valjent:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation.
**Assunta Pelosi:** Writing – review & editing, Methodology, Investigation.
**Emmanuel Roze:** Writing – review & editing, Supervision, Resources, Funding acquisition.
**Louise-Laure Mariani:** Writing – review & editing, Supervision, Resources, Methodology, Investigation, Funding acquisition.
**Jean-Antoine Girault:** Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.
**Denis Hervé:** Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Funding

This work was supported by INSERM, Sorbonne Université, Paris Brain Institute (Big Brain Theory grant 2021) to LLM and ER, and grants from Agence Nationale de la Recherche (ANR-16-37-0003CE) to DH, grants from Agence Nationale de la Recherche (ANR-16-16-0018CE) and Fondation pour la Recherche Médicale (FRM # DPA20140629798) to JAG and grant from Dystonia Medical Research Foundation, Merz Pharma, Aguettant and Enjoy Sharing to ER.

Declaration of competing interest

Emmanuel Roze received honorarium for speech from Orkyn, Aguettant, Elivie, Merz-Pharma, Janssen, Teva, Everpharma and for participating in advisory boards from Merz-Pharma, Elivie, Teva, and BIAL. He received research support from Merz-Pharma, Orkyn, Elivie,

Everpharma, Aguettant.

Louise-Laure Mariani declares no competing interests related to the present work. Disclosures of financial interests unrelated to the present work: received research support grants from the CNES, INSERM, the French Parkinson Association, Fondation of France, Paris Brain Institute, Axa foundation; received honorarium for scientific advice or lectures for Digital Medical Hub, Elivie, Sanofi-Genzyme, Accure therapeutics, Owickin, Emeis, Paris Brain Institute and received travel funding from the Movement Disorders Society, ANAINF, Merck, Merz Pharma, Medtronic, Teva and AbbVie, and is a co-inventor of a patent outside of the submitted work.

Ruiyi Yuan received a PhD grant from the ADCY5.org patient association and the Fondation pour la Recherche Médicale during the current work, has been employed by LEO Pharma and is currently employed by Quinten Health outside of the submitted work.

The other authors declare no competing interest.

Acknowledgments

We thank the *Mouse breeding and phenotyping facility* for animal care of the Institut du Fer à Moulin (IFM). Part of the animal work was conducted at the Paris Brain Institute Pheno-ICMice Core Facility. The Core is supported by "Investissements d'avenir" (ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS) and the "Fondation pour la Recherche Médicale". We gratefully acknowledge Nadege Sarrazin for assistance with animal behavior facility use. This work benefited from equipment and services from the iGenSeq core facility (Genotyping and Sequencing) at the Paris Brain Institute.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2025.107071.
# Data availability

Data will be made available upon reasonable request.

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