The spinal cord (SC) serves as the primary relay for sensory information originating in the periphery and transmitted to the brain for processing. Sensitive primary afferent fibers project to the dorsal horn, which contains a highly diverse array of neurons forming a complex network of excitatory and inhibitory circuits. Previous studies have indicated that this neuronal network can be modulated by the monoaminergic system, particularly through the spinal dopaminergic circuit, partly via dopamine D2 receptors (D2R). However, the identity of the cells expressing D2R within the spinal cord remains largely unknown. By combining whole-mount immunostaining, volume imaging and Ribotag methodology, we analyzed the distribution and characterized the molecular identity of D2R-expressing cells of the mouse spinal cord. Our study revealed that D2R are expressed by neurons, but not glial cells, distributed preferentially in the dorsal horn of the spinal cord. Furthermore, SC D2R neurons were not motorneurons but instead belong to molecularly distinct classes of excitatory and inhibitory neuronal populations. By providing a detailed molecular characterization of D2R-expressing cells in the spinal cord, the present work lays the foundation for more targeted investigations into the specific functional roles of D2Rs in sensory information processing.

The sodium leak channel NALCN is an important modulator of cell excitability. Studies so far demonstrated the critical role of this highly conserved channel in the generation and maintenance of pacemaker activity in cells with spontaneous firing, such as cardiomyocytes, adrenal cells, or neurons. However, the physiological importance of NALCN for neurons with no spontaneous firing has been largely overlooked and remains unknown. Yet, Drd2-expressing striatal projection neurons (SPNs) show an enriched expression of NALCN while they are highly hyperpolarized neurons. Considering that pathogenic variants of NALCN in human result in severe pathological conditions with symptoms that include cognitive and motor impairments, we hypothesized that NALCN in Drd2-SPNs was necessary for their correct signal integration and consequently striatal-associated behaviors. Here, we investigated the impact of NALCN deletion in Drd2-SPNs in both male and female mice. Unexpectedly, we found that only male mice with deletion of NALCN in Drd2-expressing neurons exhibited enhanced locomotor responses to novel environment and reduced motivation in food-seeking tasks, while female mice were unaffected in their behavior. Similarly, electrophysiological recordings of SPNs revealed significant sex differences, with male SPNs lacking NALCN exhibiting altered membrane properties and increased excitability, while females showed only subtle changes. Finally, we found that eticlopride-induced catalepsy and signaling events were differently altered by NALCN deletion in Drd2-SPNs male and female mice. This work constitutes the first evidence that NALCN in Drd2-SPNs participates to striatal function and may be a key modulator of response to antidopaminergic treatments, with significant sex differences.

Parvalbumin (PV) interneurons in the dorsal striatum (DS) are fast-spiking GABAergic cells critical for feedforward inhibition and synaptic integration within basal ganglia circuits. Despite their well-characterized electrophysiological roles, their molecular identity remains incompletely defined. Using the Ribotag approach in Pvalb-Cre mice, we profiled the translatome of DS PV interneurons and identified over 2,700 transcripts significantly enriched (fold-change > 1.5) in this population. Our data validate established PV markers and reveal a distinct molecular signature of DS PV neurons compared to PV interneurons from the nucleus accumbens. Gene ontology analyses highlight prominent expression of genes related to extracellular matrix components, cell adhesion molecules, synaptic organization, ion channels, and neurotransmitter receptors, particularly those mediating glutamatergic and GABAergic signaling. Notably, perineuronal net markers were robustly expressed in DS PV interneurons and confirmed by immunofluorescence. Transcriptomic analysis of DS PV neurons following repeated d-amphetamine exposure identified Gm20683 as the only differentially expressed transcript between treated groups. Furthermore, RNAseq analysis of mice subjected to an operant behavior paradigm with two types of food reward (high-palatable diet or standard chow) identified over 1,000 and 100 genes enriched in DS PV neurons from standard and high-palatable masters, respectively. These findings provide a comprehensive molecular profile of DS PV interneurons, distinguishing them from other striatal PV populations, and reveal specific gene expression changes associated with psychostimulant exposure and reward-driven behaviors. Our findings deepen insight into the molecular mechanisms of PV interneuron activity in striatal circuits and their potential roles in neuropsychiatric, motor and reward-related disorders. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND: Adolescent cannabinoid exposure can have long-lasting effects on the brain, particularly in the prefrontal cortex, where the reelin protein plays a crucial role in neural organization. Chronic cannabinoid exposure leads to reelin deficiency and behavioral abnormalities, but the underlying mechanisms remain unclear. With the increasing use of synthetic cannabinoids (SCs) among young people, understanding these effects is crucial. METHODS: We examined the cellular and synaptic consequences of initial SC exposure in adolescent male mice 1 day after a single in vivo exposure to WIN 55,212-2. Our approach combined immunohistochemistry, Western blots, conditional CB1 receptor (CB1R) knockout mouse lines, quantitative polymerase chain reaction, and ex vivo electrophysiology to investigate the effects of SC on reelin expression and synaptic plasticity. Additionally, singlemolecule fluorescent in situ hybridization profiling was used to identify cellular coexpression patterns of reelin and RESULTS: Our findings indicate that a single exposure to SC decreased reelin expression in specific prefrontal cortex layers accompanied by disrupted proteolytic fragmentation but not changes in messenger RNA expression. Singlemolecule fluorescent in situ hybridization profiling revealed a strong coexpression of CB1R and reelin. Furthermore, our pharmacological and genetic approaches demonstrated that CB1Rs in GABAergic (gamma-aminobutyric acidergic) neurons mediate the SC-induced decrease in reelin. This decrease in reelin results in a reduction in long-term potentiation, phenocopying reelin haploinsufficient mice. Notably, we restored long-term potentiation by infusing reelin bilaterally, establishing a functional link between reelin depletion and synaptic deficits. CONCLUSIONS: These findings provide new insights into the neural consequences of adolescent cannabinoid consumption and highlight the critical role of reelin in the cellular mechanisms associated with SC initiation during adolescence.

The detection of cytosolic dsDNA by the cyclic GMP-AMP synthase (cGAS) is tightly regulated to avoid pathological inflammatory responses. Here, we show that the methyl-CpG-binding protein 2 (MeCP2), a major transcriptional regulator, controls dsDNA-associated inflammatory responses. The presence of cytosolic dsDNA promotes MeCP2 export from the nucleus to the cytosol where it interacts with dsDNA, dampening detection by cGAS. MeCP2 export partially phenocopies MeCP2 deficiency, leading to innate immune activation and enforcing an antiviral state. Finally, MeCP2 displacement from the nucleus following dsDNA stimulation disrupts its canonical function, leading to the reactivation of otherwise repressed genes, such as endogenous retroelements. Re-expression of the latter leads to the accumulation of DNA species feeding cGAS-dependent signalling. We thus establish a direct role of MeCP2 in the regulation of the breadth and nature of dsDNA-associated inflammatory responses and suggest targeting dsDNA-associated pathways or endogenous retroelements as therapeutic options for patients with MeCP2 deficiency.

The detection of cytosolic dsDNA is tightly regulated to avoid pathological inflammatory responses. A major pathway involved in their detection relies on the cyclic GMP-AMP synthase (cGAS) that triggers activation of the Stimulator of interferon genes (STING) which subsequently drives the expression of inflammatory genes and type I Interferons (IFNs). Here, we show that the methyl-CpG-binding protein 2 (MECP2), a major transcriptional regulator, controls dsDNA-associated inflammatory responses. We show that the presence of cytosolic dsDNA promotes MECP2 export from the nucleus to the cytosol where it interacts with dsDNA, dampening cGAS activation. Our data also indicate that MECP2 export from the nucleus partially phenocopies MECP2 deficiency, leading to the expression of inflammatory and interferon stimulated genes, enforcing an antiviral state. Finally, we also show that MECP2 displacement from the nucleus following dsDNA stimulation is sufficient to disrupt its canonical function, leading to the reactivation of otherwise repressed genes, such endogenous retroelements of the Long interspersed nuclear element-1 (LINE-1) family. Re-expression of the latter led to the accumulation of DNA species feeding cGAS-dependent signaling and can be dampened by reverse transcriptase inhibitors. We thus establish a previously unforeseen direct role of MECP2 in the regulation of the breadth and nature of dsDNA-associated inflammatory responses. Furthermore, our results suggest that targeting dsDNA-associated pathways or pharmacological inhibition of LINE-1 may bear therapeutic hopes for Rett syndrome (RTT) patients that present with MECP2 deficiency. ### Competing Interest Statement The authors have declared no competing interest.

Nicotine activates ventral tegmental area (VTA) dopaminergic (DA) neurons projecting to the nucleus accumbens (NAc) to drive its reinforcing effects. Simultaneously, nicotine inhibits those projecting to the amygdala (Amg) to mediate anxiety-like behavior through a process that remains unknown. Here, we show that in male mice, NAc- and Amg-projecting DA neurons respond with similar polarities to ethanol and nicotine, suggesting a shared network-based mechanism underlying the inhibitory effect of these otherwise pharmacologically-distinct drugs. Selective activation of NAc-projecting DA neurons, using genetic or optogenetic strategies, produced inhibition of Amg-projecting DA neurons, through a GABAergic feedback loop. Furthermore, optogenetically silencing this feedback loop prevented nicotine from inducing both inhibition of DA neurons and anxiety-like behavior. Therefore, nicotine-induced inhibition of the VTA-Amg DA pathway results from a VTA-NAc inhibitory feedback loop, mediating anxiety-like behavior.

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 Gαolf 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 Gαolf in regulating adenylyl-cyclase 5 and its coupling with D1 and A2A receptors. Mice with a selective loss of Gαolf 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 Gαolf 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 Gαolf 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.

Objective Absence epilepsy is characterized by brief but frequent seizures with loss of consciousness. Existing treatments, which come with heavy side effects, are only partially effective and do not address the associated comorbidities, including cognitive and social deficits. A tripartite link between seizures, cognitive deficits, and diet has been established. Intermittent fasting (IF) is a regime where daily periods of fasting are alternated with periods of food intake. To date, the effects of IF on infantile epilepsy have not been addressed. We evaluated the therapeutic potential of a daily, 1-month protocol of IF on an established model of generalized absence epilepsy, the Grm7AAA knock-in mouse.Methods To assess the effects of IF in mice, we combined electroencephalography, behavior analysis, high-throughput RNA sequencing, and histology approaches.Results We demonstrate for the first time that daily IF reduces social recognition deficits and epileptic seizures. These effects are associated with the remodeling of the cerebral vascular system. We identified a deregulation of genes involved in vascularization, reflected in the malformation of blood vessels in one of the key brain areas of the absence seizure circuit, the thalamus. Along with its antiseizure effects, we show that IF is able to counteract both abnormal gene expression and vessel morphology. Finally, we demonstrate that IF successfully reduces both seizure frequency and social interaction deficits in the AY-9944 mouse, a preclinical model of atypical absence seizures, found in more severe epileptic syndromes.Significance Taken together, this study is the first to demonstrate the positive and multiscale effects of IF in the treatment of absence epilepsy.

Spatiomolecular mapping of hippocampal dopamine D2 receptor neurons revealed that in addition to hilar mossy cells, hippocampal somatostatin interneurons and, to a lesser extent, parvalbumin interneurons expressed dopamine D2 receptor. However, the consequence of dopamine D2 receptor activation on hippocampal somatostatin interneurons and parvalbumin interneurons is unknown. By combining pharmacological approaches and patch-clamp recordings in organotypic hippocampal slices from control mice or mice lacking Drd2 selectively in somatostatin or parvalbumin interneurons, we found that dopamine D2 receptor activation increases excitability in somatostatin interneurons while it decreases it in parvalbumin interneurons in the CA1 area. These changes depend on voltage-gated K channels and rely on distinct intracellular pathways, involving the non-canonical β-arrestin-dependent pathway in somatostatin interneurons and the G protein-dependent pathway in parvalbumin interneurons. Finally, our study unveils that activation of dopamine D2 receptor in somatostatin interneurons modulates methacholine-induced rhythmic synaptic activity at 4-15 Hz.

Parvalbumin (PV) interneurons in the dorsal striatum (DS) are fast-spiking GABAergic cells critical for feedforward inhibition and synaptic integration within basal ganglia circuits. Despite their well-characterized electrophysiological roles, their molecular identity remains incompletely defined. Using the Ribotag approach in Pvalb-Cre mice, we profiled the translatome of DS PV interneurons and identified over 2,700 transcripts significantly enriched (fold-change > 1.5) in this population. Our data validate established PV markers and reveal a distinct molecular signature of DS PV neurons compared to PV interneurons from the nucleus accumbens. Gene ontology analyses highlight prominent expression of genes related to extracellular matrix components, cell adhesion molecules, synaptic organization, ion channels, and neurotransmitter receptors, particularly those mediating glutamatergic and GABAergic signaling. Notably, perineuronal net markers were robustly expressed in DS PV interneurons and confirmed by immunofluorescence. Transcriptomic analysis of DS PV neurons following repeated d-amphetamine exposure identified Gm20683 as the only differentially expressed transcript between treated groups. Furthermore, RNAseq analysis of mice subjected to an operant behavior paradigm with two types of food reward (high-palatable diet or standard chow) identified over 1,000 and 100 genes enriched in DS PV neurons from standard and high-palatable masters, respectively. These findings provide a comprehensive molecular profile of DS PV interneurons, distinguishing them from other striatal PV populations, and reveal specific gene expression changes associated with psychostimulant exposure and reward-driven behaviors. Our findings deepen insight into the molecular mechanisms of PV interneuron activity in striatal circuits and their potential roles in neuropsychiatric, motor and reward-related disorders.

BACKGROUND:Highly palatable food triggers behavioral alterations reminiscent of those induced by addictive drugs. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the effects of highly palatable food on feeding behavior are poorly understood.METHODS:We studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral effects and dendritic spine modifications in the NAc. We compared the translating mRNA in NAc neurons identified by the type of dopamine receptors they express, depending on the type of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn (Neurochondrin).RESULTS:Operant conditioning for highly palatable food increases motivation for food even in well-fed mice. In control mice, free access to regular or highly palatable food results in increased weight as compared to regular food only. Highly palatable food increases spine density in the NAc. In animals trained for highly palatable food, translating mRNAs are modified in NAc dopamine D2-receptor-expressing neurons, mostly corresponding to striatal projection neurons, but not in those expressing D1-receptors. Knock-out of Ncdn, an abundant down-regulated gene, opposes the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting down-regulation may be a compensatory mechanism.CONCLUSIONS:Our results emphasize the importance of mRNA alterations D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

The insular cortex (or insula), and particularly its anterior region, plays a crucial role in the control of emotional valence and anxiety (Etkin & Wager, 2007; Mendez-Ruette et al., 2019; Nicolas et al., 2023). While dopamine neurotransmission is known to modulate anxiety levels in humans (Hjorth et al., 2021) and animal models (de la Mora et al., 2010; Bananej et al., 2012; Zarrindast & Khakpai, 2015; DeGroot et al., 2020; Godino et al., 2023), its regulatory effects on the anterior insula remained unexplored. Here, using a multifaceted approach, we uncovered how dopamine shapes anterior insula function in anxiety and valence processing. First, we revealed a high density of neurons expressing type-1 dopamine receptors (D1) in the insula, particularly important in the anterior insula, and seven times greater than the density of neurons expressing type-2 dopamine receptors (D2). Few neurons co-expressed Drd1 and Drd2 mRNAs in the anterior and posterior insula, and the density of Drd1+ neurons in the anterior insula was twice higher among inhibitory neurons than excitatory neurons. Second, we found that pharmacological activation of D1 in the anterior insula is anxiogenic, suggesting a direct link between insular dopamine signaling and anxiety-related behaviors. Using fiber-photometry recordings, we identified that the amplitude of dopamine release onto D1+ neurons in the anterior insula while mice were in anxiogenic spaces or receiving mild foot shocks was both positively correlated with mice level of trait anxiety. Population dynamics and deep-learning analyses of anterior insula single-unit recordings uncovered distinct coding patterns of anxiety-provoking and safe environments, as well as tastants of positive and negative valence. Remarkably, systemic D1 activation, which heightens anxiety-related behaviors, dampens this coding dichotomy by increasing coding variability for protected spaces while increasing the coding reliability for anxiogenic spaces. Interestingly, the coding reliability of anxiogenic areas was positively correlated with mice level of trait anxiety, and we observed a trend towards a positive correlation between the coding reliability of a negative tastants, and mice level of anxiety. Altogether, our findings provide a new model of neural population coding of anxiety and emotional valence and unravel D1-dependent coding mechanisms in the mouse anterior insula. ### Competing Interest Statement The authors have declared no competing interest.

The sodium leak channel NALCN is an important modulator of neuronal excitability, yet its specific role in striatal medium-sized spiny neurons remains largely unexplored. In this study, considering that Nalcn transcripts are enriched in the dorsal and ventral striatum of Drd2-SPNs, we investigated the functional impact of NALCN deletion in Drd2-expressing SPNs in both male and female mice. Electrophysiological recordings revealed significant sex differences, with male SPNs exhibiting altered membrane properties and increased excitability, while females showed more subtle changes. Interestingly, eticlopride-induced intracellular signaling was selectively enhanced in female SPNs lacking NALCN. Behaviorally, male mice exhibited reduced motivation in food-seeking tasks and impaired discrimination of threat cues. Our findings uncover an important, sex-specific role for NALCN in regulating striatal function and behavior and underscore its significance in maintaining normal striatal function. ### Competing Interest Statement The authors have declared no competing interest.

Salience attributed to stimuli predicting rewarding or aversive outcomes is critical for adaptive behavior. Dopamine (DA)-neurons play a central role in this process by modulating responses to both rewarding and aversive cues. DA-neurons are tightly and readily modulated by DA D2 autoreceptors (autoD2Rs), but their role in regulating responses to aversive stimuli remains unclear. In this study, we investigated the role of autoD2R in regulating the activity of VTA DA-neurons in response to salient aversive stimuli. Using Drd2Slc6a3 mice, in which Drd2 is selectively deleted in DA-neurons, we observed enhanced excitatory and inhibitory responses of VTA DA-neurons to aversive stimuli, suggesting that autoD2R acts as a critical regulatory brake. Importantly, this modulation occurred independently of either the pacemaker activity of DA-neurons or their coupling to the non-selective sodium leak channel NALCN. Behaviorally, Drd2Slc6a3 male mice showed enhanced discrimination between threat-predicting and non-predicting cues that persisted during extinction learning, highlighting a sex-biased role of autoD2R in threat processing. Our results provide new mechanistic insights through which autoD2R influence behavioral responses to aversive stimuli, with implications for understanding neuropsychiatric disorders characterized by maladaptive threat processing. ### Competing Interest Statement The authors have declared no competing interest.

Comorbidity between psychiatric traits is thought to involve overlapping pleiotropic effects from sets of genes. Notably, substance abuse is a shared comorbid condition among various neurodevelopmental disorders with externalizing symptoms such as autism spectrum disorder and attention-deficit hyperactivity disorder, thus hinting at the nucleus accumbens (NAc) as a site for predisposition underlying convergence of genetic influences in reward-related comorbidity. Here, we identify the autism-related gene encoding the adhesion G protein-coupled receptor (aGPCR) Latrophilin-1/ADGRL1 as an essential transducer of reward mechanisms in the NAc. We found that ADGRL1 mRNA is ubiquitously expressed throughout major NAc neuronal populations in mice. A mouse model of pan-neuronal Adgrl1 deficiency in the NAc displayed cocaine-seeking impairments in adult individuals denoting its role in drug-induced reinforcement and reward. Connecting molecular pathways of cocaine-induced learning, we uncover that ADGRL1 constitutes a functional receptor for autism-related cocaine effector molecule hevin/SPARCL1. Indeed, hevin interacts with membrane-expressed ADGRL1 and induces its internalization while stabilizing its uncleaved fraction. Moreover, hevin alters the formation of intercellular adhesion contacts mediated by ADGRL1 and Neurexin-1. Importantly, the functional constitutive coupling between ADGRL1 and various G protein pathways is selectively modulated by hevin stimulation with a bias toward Gi3, Gs, and G13 proteins. These findings unveil the dual role of ADGRL1 and hevin as genetic risk factors for both psychiatric disorders and substance abuse to define the molecular etiology of comorbidity. ### Competing Interest Statement The authors have declared no competing interest.

The Edinger–Westphal nucleus (EW) is a midbrain nucleus composed of a preganglionic, cholinergic subpopulation and a densely clustered peptidergic subpopulation (EWcp). The EWcp is one of the few brain regions that show consistent induction of FOS following voluntary alcohol intake. Previous results in rodents point to urocortin 1 (UCN1) as one of the peptides most involved in the control of ethanol intake and preference. Notably, the functions described for UCN1, such as reward processing, stress coping or the regulation of feeding behavior are similar to those described for the neuropeptide neuromedin U (NMU). Interestingly, NMU has been recently associated with the modulation of alcohol-related behaviors. However, little is known about the expression and functionality of NMU neurons in alcohol-responsive areas. In this study, we used the recently developed Nmu-Cre knock-in mouse model to examine the expression of NMU in the subaqueductal paramedian zone comprising the EWcp. We delved into the characterization and co-expression of NMU with other markers already described in the EWcp. Moreover, using FOS as a marker of neuronal activity, we tested whether NMU neurons were sensitive to acute alcohol administration. Overall, we provided novel insights on NMU expression and functionality in the EW region. We showed the presence of NMU within a subpopulation of UCN1 neurons in the EWcp and demonstrated that this partial co-expression does not interfere with the responsivity of UCN1-containing cells to alcohol. Moreover, we proposed that the UCN1 content in these neurons may be influenced by sex.