Dopamine D2 receptors in WFS1-neurons regulate food-seeking and avoidance behaviors.

The selection and optimization of appropriate adaptive responses depends on interoceptive and exteroceptive stimuli as well as on the animal's ability to switch from one behavioral strategy to another. Although growing evidence indicate that dopamine D2R-mediated signaling events ensure the selection of the appropriate strategy for each specific situation, the underlying neural circuits through which they mediate these effects are poorly characterized. Here, we investigated the role of D2R signaling in a mesolimbic neuronal subpopulation expressing the Wolfram syndrome 1 (Wfs1) gene. This subpopulation is located within the nucleus accumbens, the central amygdala, the bed nucleus of the stria terminalis, and the tail of the striatum, all brain regions critical for the regulation of emotions and motivated behaviors. Using a mouse model carrying a temporally controlled deletion of D2R in WFS1-neurons, we demonstrate that intact D2R signaling in this neuronal population is necessary to regulate homeostasis-dependent food-seeking behaviors in both male and female mice. In addition, we found that reduced D2R signaling in WFS1-neurons impaired active avoidance learning and innate escape responses. Collectively, these findings identify a yet undocumented role for D2R signaling in WFS1-neurons as a novel effector through which dopamine optimizes appetitive behaviors and regulates defensive behaviors.

Imaging and Genetic Tools for the Investigation of the Endocannabinoid System in the CNS.

As central nervous system (CNS)-related disorders present an increasing cause of global morbidity, mortality, and high pressure on our healthcare system, there is an urgent need for new insights and treatment options. The endocannabinoid system (ECS) is a critical network of endogenous compounds, receptors, and enzymes that contribute to CNS development and regulation. Given its multifaceted involvement in neurobiology and its significance in various CNS disorders, the ECS as a whole is considered a promising therapeutic target. Despite significant advances in our understanding of the ECS's role in the CNS, its complex architecture and extensive crosstalk with other biological systems present challenges for research and clinical advancements. To bridge these knowledge gaps and unlock the full therapeutic potential of ECS interventions in CNS-related disorders, a plethora of molecular-genetic tools have been developed in recent years. Here, we review some of the most impactful tools for investigating the neurological aspects of the ECS. We first provide a brief introduction to the ECS components, including cannabinoid receptors, endocannabinoids, and metabolic enzymes, emphasizing their complexity. This is followed by an exploration of cutting-edge imaging tools and genetic models aimed at elucidating the roles of these principal ECS components. Special emphasis is placed on their relevance in the context of CNS and its associated disorders.

Vestibular CCK signaling drives motion sickness-like behavior in mice.

Travel can induce motion sickness (MS) in susceptible individuals. MS is an evolutionary conserved mechanism caused by mismatches between motion-related sensory information and past visual and motion memory, triggering a malaise accompanied by hypolocomotion, hypothermia, hypophagia, and nausea. Vestibular nuclei (VN) are critical for the processing of movement input from the inner ear. Motion-induced activation of VN neurons recapitulates MS-related signs. However, the genetic identity of VN neurons mediating MS-related autonomic and aversive responses remains unknown. Here, we identify a central role of cholecystokinin (CCK)-expressing VN neurons in motion-induced malaise. Moreover, we show that CCK VN inputs onto the parabrachial nucleus activate Calca-expressing neurons and are sufficient to establish avoidance to novel food, which is prevented by CCK-A receptor antagonism. These observations provide greater insight into the neurobiological regulation of MS by identifying the neural substrates of MS and providing potential targets for treatment.

Cerebellar dopamine D2 receptors regulate social behaviors.

The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.

Regulation of GluA1 phosphorylation by d-amphetamine and methylphenidate in the cerebellum.

Prescription stimulants, such as d-amphetamine or methylphenidate are used to treat suffering from attention-deficit hyperactivity disorder (ADHD). They potently release dopamine (DA) and norepinephrine (NE) and cause phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 in the striatum. Whether other brain regions are also affected remains elusive. Here, we demonstrate that d-amphetamine and methylphenidate increase phosphorylation at Ser845 (pS845-GluA1) in the membrane fraction of mouse cerebellum homogenate. We identify Bergmann glial cells as the source of pS845-GluA1 and demonstrate a requirement for intact NE release. Consequently, d-amphetamine-induced pS845-GluA1 was prevented by ß1-adenoreceptor antagonist, whereas the blockade of DA D1 receptor had no effect. Together, these results indicate that NE regulates GluA1 phosphorylation in Bergmann glial cells in response to prescription stimulants.

Mammalian Target of Rapamycin-RhoA Signaling Impairments in Direct Striatal Projection Neurons Induce Altered Behaviors and Striatal Physiology in Mice.

BACKGROUND: As an integrator of molecular pathways, mTOR (mammalian target of rapamycin) has been associated with diseases including neurodevelopmental, psychiatric, and neurodegenerative disorders such as autism spectrum disorder, schizophrenia, and Huntington's disease. An important brain area involved in all these diseases is the striatum. However, the mechanisms behind how mTOR is involved in striatal physiology and its relative role in distinct neuronal populations in these striatal-related diseases still remain to be clarified. METHODS: Using Drd1-Cre mTOR-conditional knockout male mice, we combined behavioral, biochemical, electrophysiological, and morphological analysis aiming to untangle the role of mTOR in direct pathway striatal projection neurons and how this would impact on striatal physiology. RESULTS: Our results indicate deep behavioral changes in absence of mTOR in Drd1-expressing neurons such as decreased spontaneous locomotion, impaired social interaction, and decreased marble-burying behavior. These alterations were accompanied by a Kv1.1-induced increase in the fast phase of afterhyperpolarization and coincident decreased distal spine density in striatal direct pathway striatal projection neurons. The physiological changes were mechanistically independent of protein synthesis but sensitive to pharmacological blockade of transforming protein RhoA activity. CONCLUSIONS: These results identify mTOR signaling as an important regulator of striatal functions through an intricate mechanism involving RhoA and culminating in Kv1.1 overfunction, which could be targeted to treat striatal-related monogenic disorders associated with the mTOR signaling pathway.

Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum.

Action control is a key brain function determining the survival of animals in their environment. In mammals, neurons expressing dopamine D2 receptors (D2R) in the dorsal striatum (DS) and the nucleus accumbens (Acb) jointly but differentially contribute to the fine regulation of movement. However, their region-specific molecular features are presently unknown. By combining RNAseq of striatal D2R neurons and histological analyses, we identified hundreds of novel region-specific molecular markers, which may serve as tools to target selective subpopulations. As a proof of concept, we characterized the molecular identity of a subcircuit defined by WFS1 neurons and evaluated multiple behavioral tasks after its temporally-controlled deletion of D2R. Consequently, conditional D2R knockout mice displayed a significant reduction in digging behavior and an exacerbated hyperlocomotor response to amphetamine. Thus, targeted molecular analyses reveal an unforeseen heterogeneity in D2R-expressing striatal neuronal populations, underlying specific D2R's functional features in the control of specific motor behaviors.

Hypothalamic dopamine signaling regulates brown fat thermogenesis.

Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.

Dopamine signaling in the striatum.

The striatum integrates dopamine-mediated reward signals to generate appropriate behavior in response to glutamate-mediated sensory cues. Such associative learning relies on enduring neural plasticity in striatal GABAergic spiny projection neurons which, when altered, can lead to the development of a wide variety of pathological states. Considerable progress has been made in our understanding of the intracellular signaling mechanisms in dopamine-related behaviors and pathologies. Through the prism of the regulation of histone H3 and ribosomal protein S6 phosphorylation, we review how dopamine-mediated signaling events regulate gene transcription and mRNA translation. Particularly, we focus on the intracellular cascades controlling these phosphorylations downstream of the modulation of dopamine receptors by psychostimulants, antipsychotics and l-DOPA. Finally, we highlight the importance to precisely determine in which neuronal populations these signaling events occur in order to understand how they participate in remodeling neural circuits and altering dopamine-related behaviors.

Cartography of hevin-expressing cells in the adult brain reveals prominent expression in astrocytes and parvalbumin neurons.

Hevin, also known as SPARC-like 1, is a member of the secreted protein acidic and rich in cysteine family of matricellular proteins, which has been implicated in neuronal migration and synaptogenesis during development. Unlike previously characterized matricellular proteins, hevin remains strongly expressed in the adult brain in both astrocytes and neurons, but its precise pattern of expression is unknown. The present study provides the first systematic description of hevin mRNA distribution in the adult mouse brain. Using isotopic in situ hybridization, we showed that hevin is strongly expressed in the cortex, hippocampus, basal ganglia complex, diverse thalamic nuclei and brainstem motor nuclei. To identify the cellular phenotype of hevin-expressing cells, we used double fluorescent in situ hybridization in mouse and human adult brains. In the mouse, hevin mRNA was found in the majority of astrocytes but also in specific neuronal populations. Hevin was expressed in almost all parvalbumin-positive projection neurons and local interneurons. In addition, hevin mRNA was found in: (1) subsets of other inhibitory GABAergic neuronal subtypes, including calbindin, cholecystokinin, neuropeptide Y, and somatostatin-positive neurons; (2) subsets of glutamatergic neurons, identified by the expression of the vesicular glutamate transporters VGLUT1 and VGLUT2; and (3) the majority of cholinergic neurons from motor nuclei. Hevin mRNA was absent from all monoaminergic neurons and cholinergic neurons of the ascending pathway. A similar cellular profile of expression was observed in human, with expression of hevin in parvalbumin interneurons and astrocytes in the cortex and caudate nucleus as well as in cortical glutamatergic neurons. Furthermore, hevin transcript was enriched in ribosomes of astrocytes and parvalbumin neurons providing a direct evidence of hevin mRNAs translation in these cell types. This study reveals the unique and complex expression profile of the matricellular protein hevin in the adult brain. This distribution is compatible with a role of hevin in astrocytic-mediated adult synaptic plasticity and in the regulation of network activity mediated by parvalbumin-expressing neurons.

Hippocampal Protein Kinase C Signaling Mediates the Short-Term Memory Impairment Induced by Delta9-Tetrahydrocannabinol.

Cannabis affects cognitive performance through the activation of the endocannabinoid system, and the molecular mechanisms involved in this process are poorly understood. Using the novel object-recognition memory test in mice, we found that the main psychoactive component of cannabis, delta9-tetrahydrocannabinol (THC), alters short-term object-recognition memory specifically involving protein kinase C (PKC)-dependent signaling. Indeed, the systemic or intra-hippocampal pre-treatment with the PKC inhibitors prevented the short-term, but not the long-term, memory impairment induced by THC. In contrast, systemic pre-treatment with mammalian target of rapamycin complex 1 inhibitors, known to block the amnesic-like effects of THC on long-term memory, did not modify such a short-term cognitive deficit. Immunoblot analysis revealed a transient increase in PKC signaling activity in the hippocampus after THC treatment. Thus, THC administration induced the phosphorylation of a specific Ser residue in the hydrophobic-motif at the C-terminal tail of several PKC isoforms. This significant immunoreactive band that paralleled cognitive performance did not match in size with the major PKC isoforms expressed in the hippocampus except for PKCθ. Moreover, THC transiently enhanced the phosphorylation of the postsynaptic calmodulin-binding protein neurogranin in a PKC dependent manner. These data demonstrate that THC alters short-term object-recognition memory through hippocampal PKC/neurogranin signaling.

Cell Type-Specific mRNA Dysregulation in Hippocampal CA1 Pyramidal Neurons of the Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is a genetic disorder due to the silencing of the Fmr1 gene, causing intellectual disability, seizures, hyperactivity, and social anxiety. All these symptoms result from the loss of expression of the RNA binding protein fragile X mental retardation protein (FMRP), which alters the neurodevelopmental program to abnormal wiring of specific circuits. Aberrant mRNAs translation associated with the loss of Fmr1 product is widely suspected to be in part the cause of FXS. However, precise gene expression changes involved in this disorder have yet to be defined. The objective of this study was to identify the set of mistranslated mRNAs that could contribute to neurological deficits in FXS. We used the RiboTag approach and RNA sequencing to provide an exhaustive listing of genes whose mRNAs are differentially translated in hippocampal CA1 pyramidal neurons as the integrative result of FMRP loss and subsequent neurodevelopmental adaptations. Among genes differentially regulated between adult WT and Fmr1-/y mice, we found enrichment in FMRP-binders but also a majority of non-FMRP-binders. Interestingly, both up- and down-regulation of specific gene expression is relevant to fully understand the molecular deficiencies triggering FXS. More importantly, functional genomic analysis highlighted the importance of genes involved in neuronal connectivity. Among them, we show that Klk8 altered expression participates in the abnormal hippocampal dendritic spine maturation observed in a mouse model of FXS.

Ribosomal Protein S6 Phosphorylation Is Involved in Novelty-Induced Locomotion, Synaptic Plasticity and mRNA Translation.

The phosphorylation of the ribosomal protein S6 (rpS6) is widely used to track neuronal activity. Although it is generally assumed that rpS6 phosphorylation has a stimulatory effect on global protein synthesis in neurons, its exact biological function remains unknown. By using a phospho-deficient rpS6 knockin mouse model, we directly tested the role of phospho-rpS6 in mRNA translation, plasticity and behavior. The analysis of multiple brain areas shows for the first time that, in neurons, phospho-rpS6 is dispensable for overall protein synthesis. Instead, we found that phospho-rpS6 controls the translation of a subset of mRNAs in a specific brain region, the nucleus accumbens (Acb), but not in the dorsal striatum. We further show that rpS6 phospho-mutant mice display altered long-term potentiation (LTP) in the Acb and enhanced novelty-induced locomotion. Collectively, our findings suggest a previously unappreciated role of phospho-rpS6 in the physiology of the Acb, through the translation of a selective subclass of mRNAs, rather than the regulation of general protein synthesis.

Anatomical and molecular characterization of dopamine D1 receptor-expressing neurons of the mouse CA1 dorsal hippocampus.

In the hippocampus, a functional role of dopamine D1 receptors (D1R) in synaptic plasticity and memory processes has been suggested by electrophysiological and pharmacological studies. However, comprehension of their function remains elusive due to the lack of knowledge on the precise localization of D1R expression among the diversity of interneuron populations. Using BAC transgenic mice expressing enhanced green fluorescent protein under the control of D1R promoter, we examined the molecular identity of D1R-containing neurons within the CA1 subfield of the dorsal hippocampus. In agreement with previous findings, our analysis revealed that these neurons are essentially GABAergic interneurons, which express several neurochemical markers, including calcium-binding proteins, neuropeptides, and receptors among others. Finally, by using different tools comprising cell type-specific isolation of mRNAs bound to tagged-ribosomes, we provide solid data indicating that D1R is present in a large proportion of interneurons expressing dopamine D2 receptors. Altogether, our study indicates that D1Rs are expressed by different classes of interneurons in all layers examined and not by pyramidal cells, suggesting that CA1 D1R mostly acts via modulation of GABAergic interneurons.

Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum.

Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum.

PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons.

Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency.

drd2-cre:ribotag mouse line unravels the possible diversity of dopamine d2 receptor-expressing cells of the dorsal mouse hippocampus.

Increasing evidences suggest that dopamine facilitates the encoding of novel memories by the hippocampus. However, the role of dopamine D2 receptors (D2R) in such regulations remains elusive due to the lack of the precise identification of hippocampal D2R-expressing cells. To address this issue, mice expressing the ribosomal protein Rpl22 tagged with the hemagglutinin (HA) epitope were crossed with Drd2-Cre mice allowing the selective expression of HA in D2R-containing cells (Drd2-Cre:RiboTag mice). This new transgenic model revealed a more widespread pattern of D2R-expressing cells identified by HA immunoreactivity than the one initially reported in Drd2-EGFP mice, in which the hilar mossy cells were the main neuronal population detectable. In Drd2-Cre:RiboTag mice, scattered HA/GAD67-positive neurons were detected throughout the CA1/CA3 subfields, being preferentially localized in stratum oriens and stratum lacunosum-moleculare. At the cellular level, HA-labeled cells located in CA1/CA3 subfields co-localized with calcium-binding proteins (parvalbumin, calbindin, and calretinin), neuropeptides (neuropeptide Y, somatostatin), and other markers (neuronal nitric oxide synthase, mGluR1α, reelin, coupTFII, and potassium channel-interacting protein 1). These results suggest that in addition to the glutamatergic hilar mossy cells, D2R-expressing cells constitute a subpopulation of GABAergic hippocampal interneurons.

Ribosomal Protein S6 Phosphorylation in the Nervous System: From Regulation to Function.

Since the discovery of the phosphorylation of the 40S ribosomal protein S6 (rpS6) about four decades ago, much effort has been made to uncover the molecular mechanisms underlying the regulation of this post-translational modification. In the field of neuroscience, rpS6 phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signaling activation or as a marker for neuronal activity. Nevertheless, its biological role in neurons still remains puzzling. Here we review the pharmacological and physiological stimuli regulating this modification in the nervous system as well as the pathways that transduce these signals into rpS6 phosphorylation. Altered rpS6 phosphorylation observed in various genetic and pathophysiological mouse models is also discussed. Finally, we examine the current state of knowledge on the physiological role of this post-translational modification and highlight the questions that remain to be addressed.

Sex-dependent psychoneuroendocrine effects of THC and MDMA in an animal model of adolescent drug consumption.

Ecstasy is a drug that is usually consumed by young people at the weekends and frequently, in combination with cannabis. In the present study we have investigated the long-term effects of administering increasing doses of delta-9-tetrahydrocannabinol [THC; 2.5, 5, 10 mg/kg; i.p.] from postnatal day (pnd) 28 to 45, alone and/or in conjunction with 3,4-methylenedioxymethamphetamine [MDMA; two daily doses of 10 mg/kg every 5 days; s.c.] from pnd 30 to 45, in both male and female Wistar rats. When tested one day after the end of the pharmacological treatment (pnd 46), MDMA administration induced a reduction in directed exploration in the holeboard test and an increase in open-arm exploration in an elevated plus maze. In the long-term, cognitive functions in the novel object test were seen to be disrupted by THC administration to female but not male rats. In the prepulse inhibition test, MDMA-treated animals showed a decrease in prepulse inhibition at the most intense prepulse studied (80 dB), whereas in combination with THC it induced a similar decrease at 75 dB. THC decreased hippocampal Arc expression in both sexes, while in the frontal cortex this reduction was only evident in females. MDMA induced a reduction in ERK1/2 immunoreactivity in the frontal cortex of male but not female animals, and THC decreased prepro-orexin mRNA levels in the hypothalamus of males, although this effect was prevented when the animals also received MDMA. The results presented indicate that adolescent exposure to THC and/or MDMA induces long-term, sex-dependent psychophysiological alterations and they reveal functional interactions between the two drugs.