Neuralized1 activates CPEB3: a function for nonproteolytic ubiquitin in synaptic plasticity and memory storage.

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), a regulator of local protein synthesis, is the mouse homolog of ApCPEB, a functional prion protein in Aplysia. Here, we provide evidence that CPEB3 is activated by Neuralized1, an E3 ubiquitin ligase. In hippocampal cultures, CPEB3 activated by Neuralized1-mediated ubiquitination leads both to the growth of new dendritic spines and to an increase of the GluA1 and GluA2 subunits of AMPA receptors, two CPEB3 targets essential for synaptic plasticity. Conditional overexpression of Neuralized1 similarly increases GluA1 and GluA2 and the number of spines and functional synapses in the hippocampus and is reflected in enhanced hippocampal-dependent memory and synaptic plasticity. By contrast, inhibition of Neuralized1 reduces GluA1 and GluA2 levels and impairs hippocampal-dependent memory and synaptic plasticity. These results suggest a model whereby Neuralized1-dependent ubiquitination facilitates hippocampal plasticity and hippocampal-dependent memory storage by modulating the activity of CPEB3 and CPEB3-dependent protein synthesis and synapse formation.

Impact of dietary vitamin A on striatal function in adult rats.

The striatum is a brain structure involved in the control of voluntary movement. Striatum contains high amounts of retinoic acid, the active metabolite of vitamin A, as well as retinoid receptors, RARß and RXRγ. Previous studies revealed that disruption of retinoid signaling initiated during development is deleterious for striatal physiology and related motor functions. However, the alteration of retinoid signaling, and the importance of vitamin A supply during adulthood on striatal physiology and function has never been established. In the present study, we investigated the impact of vitamin A supply on striatal function. Adult Sprague-Dawley rats were fed with three specific diets, either sub-deficient, sufficient, or enriched in vitamin A (0.4, 5, and 20 international units [IU] of retinol per g of diet, respectively) for 6 months. We first validated that vitamin A sub-deficient diet in adult rats constitutes a physiological model of retinoid signaling reduction in the striatum. We then revealed subtle alterations of fine motor skills in sub-deficient rats using a new behavioral apparatus specifically designed to test forepaw reach-and-grasp skills relying on striatal function. Finally, we showed using qPCR analysis and immunofluorescence that the striatal dopaminergic system per se was not affected by vitamin A sub-deficiency at adult age. Rather, cholinergic synthesis in the striatum and µ-opioid receptor expression in striosomes sub-territories were the most affected by vitamin A sub-deficiency starting at adulthood. Taken together these results revealed that retinoid signaling alteration at adulthood is associated with motor learning deficits together with discrete neurobiological alterations in the striatum.

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of ß-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

Microarray and Mass Spectrometry-Based Methodology for Lipid Profiling of Tissues and Cell Cultures.

The recent developments in mass spectrometry have revealed the importance of lipids as biomarkers in the context of different diseases and as indicators of the cell's homeostasis. However, further advances are required to unveil the complex relationships between lipid classes and lipid species with proteins. Here, we present a new methodology that combines microarrays with mass spectrometry to obtain the lipid fingerprint of samples of a different nature in a standardized and fast way, with minimal sample consumption. As a proof of concept, we use the methodology to obtain the lipid fingerprint of 20 rat tissues and to create a lipid library for tissue classification. Then, we combine those results with immunohistochemistry and enzymatic assays to unveil the relationship between some lipid species and two enzymes. Finally, we demonstrate the performance of the methodology to explore changes in lipid composition of the nucleus accumbens from mice subjected to two lipid diets.

The effect of high-fat diet consumption on appetitive instrumental behavior in rats.

Evidence now indicates that the chronic consumption of high-calorie foods, such as a high-fat diet (HFD), is associated with impaired control over food-seeking, yet the extent of this alteration is not fully understood. Using different reinforcement schedules, we evaluated whether HFD intake from weaning to adulthood modifies instrumental responding and induces a shift from goal-directed actions to habitual responding. We first observed reduced instrumental performance and motivation for a food reward in HFD-fed rats trained under schedules of reinforcement that facilitate habitual responding [Random Interval (RI)]. However, this deficit was alleviated if rats trained under RI were subsequently trained with reinforcement schedules that promote goal-directed strategies [Random Ratio (RR)]. Using an outcome devaluation procedure, we then demonstrated that consumption of a HFD promoted habitual behavior in rats trained under RI but not RR schedules. Finally, extended HFD exposure did not interfere with the ability of RR training to overcome impaired RI instrumental performance and to favor goal-directed behavior. These results indicate that chronic consumption of a HFD changes the co-ordination of goal-directed actions and habits and that alteration of food-seeking may be reversed under particular behavioral conditions.

Inflammation early in life is a vulnerability factor for emotional behavior at adolescence and for lipopolysaccharide-induced spatial memory and neurogenesis alteration at adulthood.

BACKGROUND: The postnatal period is a critical time window during which inflammatory events have significant and enduring effects on the brain, and as a consequence, induce alterations of emotional behavior and/or cognition later in life. However, the long-term effect of neonatal inflammation on behavior during adolescence, a sensitive period for the development of neurodevelopmental psychiatric disorders, has been little studied. In this study, we examined whether an early-life inflammatory challenge could alter emotional behaviors and spatial memory at adolescence and adulthood and whether stress axis activity, inflammatory response and neurogenesis were affected. METHODS: Lipopolysaccharide (LPS, 100 µg/kg) was administered to mice on postnatal day (PND) 14 and cytokine expression was measured in the plasma and in brain structures 3 hours later. Anxiety-like and depressive-like behavior (measured in the novelty-suppressed feeding test and the forced swim test, respectively) and spatial memory (Y-maze test) were measured at adolescence (PND30) and adulthood (PND90). Hypothalamic-pituitary-adrenal (HPA) axis activity (plasma corticosterone and glucocorticoid receptors in the hippocampus and prefrontal cortex) was measured at adulthood. In addition, the impact of a novel adult LPS challenge (100 µ/kg) was measured on spatial memory (Y-maze test), neurogenesis (doublecortin-positive cell numbers in the hippocampus) and plasma cytokine expression. RESULTS: First, we show in PND14 pups that a peripheral administration of LPS induced the expression of pro- and anti-inflammatory cytokines in the plasma and brain structures that were studied 3 hours after administration. Anxiety-like behavior was altered in adolescent, but not in adult, mice, whereas depressive-like behavior was spared at adolescence and increased at adulthood. This was accompanied by a decreased phosphorylation of the glucocorticoid receptor in the prefrontal cortex, with no effect on corticosterone levels. Second, neonatal LPS treatment had no effect on spatial memory in adolescence and adulthood. However, a second challenge of LPS in adulthood impaired spatial memory performance and neurogenesis and increased circulating levels of CCL2. CONCLUSIONS: Our study shows for the first time, in mice, that a peripheral LPS treatment at PND14 differentially alters emotional behaviors, but not spatial memory, at adolescence and adulthood. The behavioral effect of LPS at PND14 could be attributed to HPA axis deregulation and neurogenesis impairment.

Obesogenic diet induces circuit-specific memory deficits in mice.

Obesity is associated with neurocognitive dysfunction, including memory deficits. This is particularly worrisome when obesity occurs during adolescence, a maturational period for brain structures critical for cognition. In rodent models, we recently reported that memory impairments induced by obesogenic high-fat diet (HFD) intake during the periadolescent period can be reversed by chemogenetic manipulation of the ventral hippocampus (vHPC). Here, we used an intersectional viral approach in HFD-fed male mice to chemogenetically inactivate specific vHPC efferent pathways to nucleus accumbens (NAc) or medial prefrontal cortex (mPFC) during memory tasks. We first demonstrated that HFD enhanced activation of both pathways after training and that our chemogenetic approach was effective in normalizing this activation. Inactivation of the vHPC-NAc pathway rescued HFD-induced deficits in recognition but not location memory. Conversely, inactivation of the vHPC-mPFC pathway restored location but not recognition memory impairments produced by HFD. Either pathway manipulation did not affect exploration or anxiety-like behaviour. These findings suggest that HFD intake throughout adolescence impairs different types of memory through overactivation of specific hippocampal efferent pathways and that targeting these overactive pathways has therapeutic potential.

Chemogenetic activation or inhibition of histaminergic neurons bidirectionally modulates recognition memory formation and retrieval in male and female mice.

Several lines of evidence demonstrate that the brain histaminergic system is fundamental for cognitive processes and the expression of memories. Here, we investigated the effect of acute silencing or activation of histaminergic neurons in the hypothalamic tuberomamillary nucleus (TMNHA neurons) in vivo in both sexes in an attempt to provide direct and causal evidence of the necessary role of these neurons in recognition memory formation and retrieval. To this end, we compared the performance of mice in two non-aversive and non-rewarded memory tests, the social and object recognition memory tasks, which are known to recruit different brain circuitries. To directly establish the impact of inactivation or activation of TMNHA neurons, we examined the effect of specific chemogenetic manipulations during the formation (acquisition/consolidation) or retrieval of recognition memories. We consistently found that acute chemogenetic silencing of TMNHA neurons disrupts the formation or retrieval of both social and object recognition memory in males and females. Conversely, acute chemogenetic activation of TMNHA neurons during training or retrieval extended social memory in both sexes and object memory in a sex-specific fashion. These results suggest that the formation or retrieval of recognition memory requires the tonic activity of histaminergic neurons and strengthen the concept that boosting the brain histaminergic system can promote the retrieval of apparently lost memories.

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.

Nucleus accumbens D1- and D2-expressing neurons control the balance between feeding and activity-mediated energy expenditure.

Accumulating evidence points to dysregulations of the Nucleus Accumbens (NAc) in eating disorders (ED), however its precise contribution to ED symptomatic dimensions remains unclear. Using chemogenetic manipulations in male mice, we found that activity of dopamine D1 receptor-expressing neurons of the NAc core subregion facilitated effort for a food reward as well as voluntary exercise, but decreased food intake, while D2-expressing neurons have opposite effects. These effects are congruent with D2-neurons being more active than D1-neurons during feeding while it is the opposite during running. Chronic manipulations of each subpopulations had limited effects on energy balance. However, repeated activation of D1-neurons combined with inhibition of D2-neurons biased behavior toward activity-related energy expenditure, whilst the opposite manipulations favored energy intake. Strikingly, concomitant activation of D1-neurons and inhibition of D2-neurons precipitated weight loss in anorexia models. These results suggest that dysregulations of NAc dopaminoceptive neurons might be at the core of EDs.

The Addiction-Susceptibility TaqIA/Ankk1 Controls Reward and Metabolism Through D2 Receptor-Expressing Neurons.

BACKGROUND: A large body of evidence highlights the importance of genetic variants in the development of psychiatric and metabolic conditions. Among these, the TaqIA polymorphism is one of the most commonly studied in psychiatry. TaqIA is located in the gene that codes for the ankyrin repeat and kinase domain containing 1 kinase (Ankk1) near the dopamine D2 receptor (D2R) gene. Homozygous expression of the A1 allele correlates with a 30% to 40% reduction of striatal D2R, a typical feature of addiction, overeating, and other psychiatric pathologies. The mechanisms by which the variant influences dopamine signaling and behavior are unknown. METHODS: Here, we used transgenic and viral-mediated strategies to reveal the role of Ankk1 in the regulation of activity and functions of the striatum. RESULTS: We found that Ankk1 is preferentially enriched in striatal D2R-expressing neurons and that Ankk1 loss of function in the dorsal and ventral striatum leads to alteration in learning, impulsivity, and flexibility resembling endophenotypes described in A1 carriers. We also observed an unsuspected role of Ankk1 in striatal D2R-expressing neurons of the ventral striatum in the regulation of energy homeostasis and documented differential nutrient partitioning in humans with or without the A1 allele. CONCLUSIONS: Overall, our data demonstrate that the Ankk1 gene is necessary for the integrity of striatal functions and reveal a new role for Ankk1 in the regulation of body metabolism.

In vivo imaging of the actin polymerization state with two-photon fluorescence anisotropy.

Using two-photon fluorescence anisotropy imaging of actin-GFP, we have developed a method for imaging the actin polymerization state that is applicable to a broad range of experimental systems extending from fixed cells to live animals. The incorporation of expressed actin-GFP monomers into endogenous actin polymers enables energy migration FRET (emFRET, or homoFRET) between neighboring actin-GFPs. This energy migration reduces the normally high polarization of the GFP fluorescence. We derive a simple relationship between the actin-GFP fluorescence polarization anisotropy and the actin polymer fraction, thereby enabling a robust means of imaging the actin polymerization state with high spatiotemporal resolution and providing what to the best of our knowledge are the first direct images of the actin polymerization state in live, adult brain tissue and live, intact Drosophila larvae.

A synaptomic analysis reveals dopamine hub synapses in the mouse striatum.

Dopamine transmission is involved in reward processing and motor control, and its impairment plays a central role in numerous neurological disorders. Despite its strong pathophysiological relevance, the molecular and structural organization of the dopaminergic synapse remains to be established. Here, we used targeted labelling and fluorescence activated sorting to purify striatal dopaminergic synaptosomes. We provide the proteome of dopaminergic synapses with 57 proteins specifically enriched. Beyond canonical markers of dopamine neurotransmission such as dopamine biosynthetic enzymes and cognate receptors, we validated 6 proteins not previously described as enriched. Moreover, our data reveal the adhesion of dopaminergic synapses to glutamatergic, GABAergic or cholinergic synapses in structures we named "dopamine hub synapses". At glutamatergic synapses, pre- and postsynaptic markers are significantly increased upon association with dopamine synapses. Dopamine hub synapses may thus support local dopaminergic signalling, complementing volume transmission thought to be the major mechanism by which monoamines modulate network activity.

Combined Gene Expression and Chromatin Immunoprecipitation From a Single Mouse Hippocampus.

All neuronal cells hold the same genetic information but vary by their structural and functional plasticity depending on the brain area and environmental influences. Such variability involves specific gene regulation, which is driven by transcription factors (TFs). In the field of neuroscience, epigenetics is the main mechanism that has been investigated to understand the dynamic modulation of gene expression by behavioral responses, stress responses, memory processes, etc. Nowadays, gene expression analyzed by real-time quantitative PCR and TF binding estimated by chromatin immunoprecipitation (ChIP) enables one to dissect this regulation. Because of the wide range of transgenic models, as well as cost-effective aspects, mouse models are widely used neuroscience. Thus, we have set up a protocol that allows extraction of both RNA for gene expression analysis and chromatin for ChIP experiment from a single mouse hippocampus. Using such protocols, information regarding gene expression and regulatory molecular mechanisms from the same animal can be integrated and correlated with neurobiological and behavioral outcomes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Hippocampus isolation from mouse brain Basic Protocol 2: RNA extraction and gene expression analysis from a mouse half hippocampus Basic Protocol 3: ChIP from one hemisphere side mouse hippocampus.

Cell-Type-Specific Adaptions in Striatal Medium-Sized Spiny Neurons and Their Roles in Behavioral Responses to Drugs of Abuse.

Drug addiction is defined as a compulsive pattern of drug-seeking- and taking- behavior, with recurrent episodes of abstinence and relapse, and a loss of control despite negative consequences. Addictive drugs promote reinforcement by increasing dopamine in the mesocorticolimbic system, which alters excitatory glutamate transmission within the reward circuitry, thereby hijacking reward processing. Within the reward circuitry, the striatum is a key target structure of drugs of abuse since it is at the crossroad of converging glutamate inputs from limbic, thalamic and cortical regions, encoding components of drug-associated stimuli and environment, and dopamine that mediates reward prediction error and incentive values. These signals are integrated by medium-sized spiny neurons (MSN), which receive glutamate and dopamine axons converging onto their dendritic spines. MSN primarily form two mostly distinct populations based on the expression of either DA-D1 (D1R) or DA-D2 (D2R) receptors. While a classical view is that the two MSN populations act in parallel, playing antagonistic functional roles, the picture seems much more complex. Herein, we review recent studies, based on the use of cell-type-specific manipulations, demonstrating that dopamine differentially modulates dendritic spine density and synapse formation, as well as glutamate transmission, at specific inputs projecting onto D1R-MSN and D2R-MSN to shape persistent pathological behavioral in response to drugs of abuse. We also discuss the identification of distinct molecular events underlying the detrimental interplay between dopamine and glutamate signaling in D1R-MSN and D2R-MSN and highlight the relevance of such cell-type-specific molecular studies for the development of innovative strategies with potential therapeutic value for addiction. Because drug addiction is highly prevalent in patients with other psychiatric disorders when compared to the general population, we last discuss the hypothesis that shared cellular and molecular adaptations within common circuits could explain the co-occurrence of addiction and depression. We will therefore conclude this review by examining how the nucleus accumbens (NAc) could constitute a key interface between addiction and depression.

Disrupting D1-NMDA or D2-NMDA receptor heteromerization prevents cocaine's rewarding effects but preserves natural reward processing.

Addictive drugs increase dopamine in the nucleus accumbens (NAc), where it persistently shapes excitatory glutamate transmission and hijacks natural reward processing. Here, we provide evidence, from mice to humans, that an underlying mechanism relies on drug-evoked heteromerization of glutamate N-methyl-d-aspartate receptors (NMDAR) with dopamine receptor 1 (D1R) or 2 (D2R). Using temporally controlled inhibition of D1R-NMDAR heteromerization, we unraveled their selective implication in early phases of cocaine-mediated synaptic, morphological, and behavioral responses. In contrast, preventing D2R-NMDAR heteromerization blocked the persistence of these adaptations. Interfering with these heteromers spared natural reward processing. Notably, we established that D2R-NMDAR complexes exist in human samples and showed that, despite a decreased D2R protein expression in the NAc, individuals with psychostimulant use disorder display a higher proportion of D2R forming heteromers with NMDAR. These findings contribute to a better understanding of molecular mechanisms underlying addiction and uncover D2R-NMDAR heteromers as targets with potential therapeutic value.

Endocytosis controls glutamate-induced nuclear accumulation of ERK.

Nuclear translocation of activated extracellular signal-regulated kinases (ERK) in neurons is critical for gene regulations underlying long-term neuronal adaptation and memory formation. However, it is unknown how activated ERK travel from the post-synaptic elements where their activation occurs, to the nucleus where they translocate to exert their transcriptional roles. In cultured neurons, we identified endocytosis as a prime event in glutamate-induced nuclear trafficking of ERK2. We show that glutamate triggers a rapid recruitment of ERK2 to a protein complex comprising markers of the clathrin-dependent endocytotic and AMPA/glutamate receptor subtype. Inhibition of endocytosis results in a neuritic withholding of activated ERK2 without modification of ERK2 activity. As a consequence, endocytosis blockade alters ERK-dependent nuclear events, such as mitogen and stressed-activated kinase-1 (MSK-1) activation, histone H3 phosphorylation and gene regulations. Our data provide the first evidence that the endocytic pathway controls ERK nuclear translocation and ERK-dependent gene regulations induced by glutamate.

From Signaling Molecules to Circuits and Behaviors: Cell-Type-Specific Adaptations to Psychostimulant Exposure in the Striatum.

Addiction is characterized by a compulsive pattern of drug seeking and consumption and a high risk of relapse after withdrawal that are thought to result from persistent adaptations within brain reward circuits. Drugs of abuse increase dopamine (DA) concentration in these brain areas, including the striatum, which shapes an abnormal memory trace of drug consumption that virtually highjacks reward processing. Long-term neuronal adaptations of gamma-aminobutyric acidergic striatal projection neurons (SPNs) evoked by drugs of abuse are critical for the development of addiction. These neurons form two mostly segregated populations, depending on the DA receptor they express and their output projections, constituting the so-called direct (D1 receptor) and indirect (D2 receptor) SPN pathways. Both SPN subtypes receive converging glutamate inputs from limbic and cortical regions, encoding contextual and emotional information, together with DA, which mediates reward prediction and incentive values. DA differentially modulates the efficacy of glutamate synapses onto direct and indirect SPN pathways by recruiting distinct striatal signaling pathways, epigenetic and genetic responses likely involved in the transition from casual drug use to addiction. Herein we focus on recent studies that have assessed psychostimulant-induced alterations in a cell-type-specific manner, from remodeling of input projections to the characterization of specific molecular events in each SPN subtype and their impact on long-lasting behavioral adaptations. We discuss recent evidence revealing the complex and concerted action of both SPN populations on drug-induced behavioral responses, as these studies can contribute to the design of future strategies to alleviate specific behavioral components of addiction.

Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay.

Combining immunological and molecular biological methods, the antibody-based proximity ligation assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and sectioning for PLA_BF Basic Protocol 2: PLA_BF staining of brain tissue Basic Protocol 3: Image acquisition and result analysis Support Protocol: Luxol fast blue/cresyl violet staining.