RESUMEN
The combination of fluorogenic probes (fluorogens) and self-labeling protein tags represent a promising tool for imaging biological processes with high specificity but it requires the adequation between the fluorogen and its target to ensure a good activation of its fluorescence. In this work, we report a strategy to develop molecular rotors that specifically target HaloTag with a strong enhancement of their fluorescence. The divergent design facilitates the diversification of the structures to tune the photophysical and cellular properties. Four bright fluorogens with emissions ranging from green to red were identified and applied in wash-free live cell imaging experiments with good contrast and selectivity. A HaloTag mutant adapted from previous literature reports was also tested and shown to further improve the brightness and reaction rate of the most promising fluorogen of the series both inâ vitro and in cells. This work opens new possibilities to develop bright chemogenetic reporters with diverse photophysical and biological properties by exploring a potentially large chemical space of simple dipolar fluorophores in combination with protein engineering.
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Colorantes Fluorescentes , Ingeniería de Proteínas , Colorantes Fluorescentes/química , HumanosRESUMEN
Current antidepressants act principally by blocking monoamine reuptake by high-affinity transporters in the brain. However, these antidepressants show important shortcomings such as slow action onset and limited efficacy in nearly a third of patients with major depression disorder. Here, we report the development of a prodrug targeting organic cation transporters (OCT), atypical monoamine transporters recently implicated in the regulation of mood. Using molecular modeling, we designed a selective OCT2 blocker, which was modified to increase brain penetration. This compound, H2-cyanome, was tested in a rodent model of chronic depression induced by 7-week corticosterone exposure. In male mice, prolonged administration of H2-cyanome induced positive effects on several behaviors mimicking symptoms of depression, including anhedonia, anxiety, social withdrawal, and memory impairment. Importantly, in this validated model, H2-cyanome compared favorably with the classical antidepressant fluoxetine, with a faster action on anhedonia and better anxiolytic effects. Integrated Z-scoring across these depression-like variables revealed a lower depression score for mice treated with H2-cyanome than for mice treated with fluoxetine for 3 weeks. Repeated H2-cyanome administration increased ventral tegmental area dopaminergic neuron firing, which may underlie its rapid action on anhedonia. H2-cyanome, like fluoxetine, also modulated several intracellular signaling pathways previously involved in antidepressant response. Our findings provide proof-of-concept of antidepressant efficacy of an OCT blocker, and a mechanistic framework for the development of new classes of antidepressants and therapeutic alternatives for resistant depression and other psychiatric disturbances such as anxiety.
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Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Depresión/tratamiento farmacológico , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Anhedonia/efectos de los fármacos , Animales , Antidepresivos/administración & dosificación , Antidepresivos/farmacocinética , Ansiedad/tratamiento farmacológico , Modelos Animales de Enfermedad , Fluoxetina/uso terapéutico , Humanos , Masculino , Memoria/efectos de los fármacos , RatonesRESUMEN
ß-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice ß-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide ß-catenin enrichment mapping, we identify Dicer1-important in small RNA (for example, microRNA) biogenesis--as a ß-catenin target gene that mediates resilience. Small RNA profiling after excising ß-catenin from nucleus accumbens in the context of chronic stress reveals ß-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish ß-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.
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ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica , MicroARNs/genética , Resiliencia Psicológica , Ribonucleasa III/genética , Estrés Fisiológico/genética , beta Catenina/metabolismo , Adaptación Fisiológica/genética , Animales , ARN Helicasas DEAD-box/metabolismo , Depresión/fisiopatología , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Neuronas/metabolismo , Ribonucleasa III/metabolismo , Transducción de Señal , beta Catenina/genéticaRESUMEN
Cre/loxP recombination is a widely used approach to study gene function in vivo, using mice models expressing the Cre recombinase under the control of specific promoters or through viral delivery of Cre-expressing constructs. A profuse literature on transgenic mouse lines points out the deleterious effects of Cre expression in various cell types and tissues, presumably by acting on illegitimate loxP-like sites present in the genome. However, most studies reporting the consequences of Cre-lox gene invalidation often omit adequate controls to exclude the potential toxic effects of Cre, compromising the interpretation of data. In this study, we report the anatomical, neurochemical, and behavioral consequences in mice of adeno-associated virus (AAV)-mediated Cre expression in the dopaminergic nuclei substantia nigra, at commonly used viral titers (3 × 109 genome copies/0.3 µL or 2 × 109 genome copies/0.6 µL). We found that injecting AAV-eGFP-Cre into the SN engendered drastic and reproducible modifications of behavior, with increased basal locomotor activity as well as impaired locomotor response to cocaine compared to AAV-eGFP-injected controls. Cre expression in the SN induced a massive decrease in neuronal populations of both pars compacta and pars reticulata and dopamine depletion in the nigrostriatal pathway. This anatomical injury was associated with typical features of programmed cell death, including an increase in DNA break markers, evidence of apoptosis, and disrupted macroautophagy. These observations underscore the need for careful control of Cre toxicity in the brain and the reassessment of previous studies. In addition, our findings suggest that Cre-mediated ablation may constitute an efficient tool to explore the function of specific cell populations and areas in the brain, and the impact of neurodegeneration in these populations.
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Integrasas , Neuronas/patología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Animales , Apoptosis/efectos de los fármacos , Dependovirus , Dopamina/metabolismo , Vectores Genéticos , Integrasas/administración & dosificación , Integrasas/genética , Integrasas/toxicidad , Locomoción/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Several subtypes of modulatory neurons co-express vesicular glutamate transporters (VGLUTs) in addition to their cognate vesicular transporters. These neurons are believed to establish new forms of neuronal communication. The atypical VGLUT3 is of particular interest since in the striatum this subtype is found in tonically active cholinergic interneurons (TANs) and in a subset of 5-HT fibers. The striatum plays a major role in psychomotor effects induced by amphetamine. Whether and how VGLUT3-operated glutamate/ACh or glutamate/5HT co-transmissions modulates psychostimulants-induced maladaptive behaviors is still unknown. Here, we investigate the involvement of VGLUT3 and glutamate co-transmission in amphetamine-induced psychomotor effects and stereotypies. Taking advantage of constitutive and cell-type specific VGLUT3-deficient mouse lines, we tackled the hypothesis that VGLUT3 could gate psychomotor effects (locomotor activity and stereotypies) induced by acute or chronic administration of amphetamine. Interestingly, VGLUT3-null mice demonstrated blunted amphetamine-induced stereotypies as well as reduced striatal ∆FosB expression. VGLUT3-positive varicosities within the striatum arise in part from 5HT neurons. We tested the involvement of VGLUT3 deletion in serotoninergic neurons in amphetamine-induced stereotypies. Mice lacking VGLUT3 specifically in 5HT fibers showed no alteration to amphetamine sensitivity. In contrast, specific deletion of VGLUT3 in cholinergic neurons partially phenocopied the effects observed in the constitutive knock-out mice. Our results show that constitutive deletion of VGLUT3 modulates acute and chronic locomotor effects induced by amphetamine. They point to the fact that the expression of VGLUT3 in multiple brain areas is pivotal in gating amphetamine-induced psychomotor adaptations. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Anfetamina/farmacología , Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Locomoción/efectos de los fármacos , Animales , Encéfalo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Epigenetic mechanisms, which control chromatin structure and function, mediate changes in gene expression that occur in response to diverse stimuli. Recent research has established that environmental events and behavioral experience induce epigenetic changes at particular gene loci and that these changes help shape neuronal plasticity and function and hence behavior. Some of these changes can be stable and can even persist for a lifetime. Increasing evidence supports the hypothesis that aberrations in chromatin remodeling and subsequent effects on gene expression within limbic brain regions contribute to the pathogenesis of depression and other stress-related disorders such as post-traumatic stress disorder and other anxiety syndromes. Likewise, the gradually developing but persistent therapeutic effects of antidepressant medications may be achieved in part via epigenetic mechanisms. This review discusses recent advances in our understanding of the epigenetic regulation of stress-related disorders and focuses on three distinct aspects of stress-induced epigenetic pathology: the effects of stress and antidepressant treatment during adulthood, the lifelong effects of early-life stress on subsequent stress vulnerability, and the possible transgenerational transmission of stress-induced abnormalities.
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Antidepresivos/farmacología , Depresión/tratamiento farmacológico , Depresión/genética , Animales , Antidepresivos/farmacocinética , Antidepresivos/uso terapéutico , Metilación de ADN , Depresión/metabolismo , Epigénesis Genética , Expresión Génica , HumanosRESUMEN
Regulator of G protein signaling 4 (Rgs4) is a signal transduction protein that controls the function of monoamine, opiate, muscarinic, and other G protein-coupled receptors via interactions with Gα subunits. Rgs4 is expressed in several brain regions involved in mood, movement, cognition, and addiction and is regulated by psychotropic drugs, stress, and corticosteroids. In this study, we use genetic mouse models and viral-mediated gene transfer to examine the role of Rgs4 in the actions of antidepressant medications. We first analyzed human postmortem brain tissue and found robust up-regulation of RGS4 expression in the nucleus accumbens (NAc) of subjects receiving standard antidepressant medications that target monoamine systems. Behavioral studies of mice lacking Rgs4, including specific knockdowns in NAc, demonstrate that Rgs4 in this brain region acts as a positive modulator of the antidepressant-like and antiallodynic-like actions of several monoamine-directed antidepressant drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors, and norepinephrine reuptake inhibitors. Studies using viral-mediated increases in Rgs4 activity in NAc further support this hypothesis. Interestingly, in prefrontal cortex, Rgs4 acts as a negative modulator of the actions of nonmonoamine-directed drugs that are purported to act as antidepressants: the N-methyl-D-aspartate glutamate receptor antagonist ketamine and the delta opioid agonist (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide. Together, these data reveal a unique modulatory role of Rgs4 in the brain region-specific actions of a wide range of antidepressant drugs and indicate that pharmacological interventions at the level of RGS4 activity may enhance the actions of such drugs used for the treatment of depression and neuropathic pain.
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Antidepresivos/farmacología , Depresión/tratamiento farmacológico , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Neuralgia/tratamiento farmacológico , Proteínas RGS/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Conducta Animal , Encéfalo/patología , Desipramina/farmacología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Trastornos del Humor/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Decreased medial prefrontal cortex (mPFC) neuronal activity is associated with social defeat-induced depression- and anxiety-like behaviors in mice. However, the molecular mechanisms underlying the decreased mPFC activity and its prodepressant role remain unknown. We show here that induction of the transcription factor ΔFosB in mPFC, specifically in the prelimbic (PrL) area, mediates susceptibility to stress. ΔFosB induction in PrL occurred selectively in susceptible mice after chronic social defeat stress, and overexpression of ΔFosB in this region, but not in the nearby infralimbic (IL) area, enhanced stress susceptibility. ΔFosB produced these effects partly through induction of the cholecystokinin (CCK)-B receptor: CCKB blockade in mPFC induces a resilient phenotype, whereas CCK administration into mPFC mimics the anxiogenic- and depressant-like effects of social stress. We previously found that optogenetic stimulation of mPFC neurons in susceptible mice reverses several behavioral abnormalities seen after chronic social defeat stress. Therefore, we hypothesized that optogenetic stimulation of cortical projections would rescue the pathological effects of CCK in mPFC. After CCK infusion in mPFC, we optogenetically stimulated mPFC projections to basolateral amygdala or nucleus accumbens, two subcortical structures involved in mood regulation. Stimulation of corticoamygdala projections blocked the anxiogenic effect of CCK, although no effect was observed on other symptoms of social defeat. Conversely, stimulation of corticoaccumbens projections reversed CCK-induced social avoidance and sucrose preference deficits but not anxiogenic-like effects. Together, these results indicate that social stress-induced behavioral deficits are mediated partly by molecular adaptations in mPFC involving ΔFosB and CCK through cortical projections to distinct subcortical targets.
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Trastornos de Ansiedad/fisiopatología , Colecistoquinina/fisiología , Trastorno Depresivo/fisiopatología , Corteza Prefrontal/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Receptor de Colecistoquinina B/fisiología , Animales , Ansiolíticos/farmacología , Trastornos de Ansiedad/patología , Mapeo Encefálico , Enfermedad Crónica , Trastorno Depresivo/patología , Indoles/farmacología , Sistema Límbico/citología , Sistema Límbico/efectos de los fármacos , Sistema Límbico/fisiología , Masculino , Meglumina/análogos & derivados , Meglumina/farmacología , Ratones , Ratones Endogámicos C57BL , Vías Nerviosas/fisiología , Corteza Prefrontal/citología , Corteza Prefrontal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/genética , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/genética , Predominio Social , Estrés Psicológico/patología , Estrés Psicológico/fisiopatologíaRESUMEN
The mechanisms underlying the enduring neurobiological consequences of antidepressant exposure during adolescence are poorly understood. Here, we assessed the long-term effects of exposure to fluoxetine (FLX), a selective serotonin reuptake inhibitor, during adolescence on behavioral reactivity to emotion-eliciting stimuli. We administered FLX (10 mg/kg, bi-daily, for 15 d) to male adolescent [postnatal day 35 (P35) to P49] C57BL/6 mice. Three weeks after treatment (P70), reactivity to aversive stimuli (i.e., social defeat stress, forced swimming, and elevated plus maze) was assessed. We also examined the effects of FLX on the expression of extracellular signal-regulated kinase (ERK) 1/2-related signaling within the ventral tegmental area (VTA) of adolescent mice and Sprague Dawley rats. Adolescent FLX exposure suppressed depression-like behavior, as measured by the social interaction and forced swim tests, while enhancing anxiety-like responses in the elevated plus maze in adulthood. This complex behavioral profile was accompanied by decreases in ERK2 mRNA and protein phosphorylation within the VTA, while stress alone resulted in opposite neurobiological effects. Pharmacological (U0126) inhibition, as well as virus-mediated downregulation of ERK within the VTA mimicked the antidepressant-like profile observed after juvenile FLX treatment. Conversely, overexpression of ERK2 induced a depressive-like response, regardless of FLX pre-exposure. These findings demonstrate that exposure to FLX during adolescence modulates responsiveness to emotion-eliciting stimuli in adulthood, at least partially, via long-lasting adaptations in ERK-related signaling within the VTA. Our results further delineate the role ERK plays in regulating mood-related behaviors across the lifespan.
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Antidepresivos de Segunda Generación/uso terapéutico , Reacción de Prevención/efectos de los fármacos , Depresión/tratamiento farmacológico , Fluoxetina/uso terapéutico , Factores de Edad , Animales , Antidepresivos de Segunda Generación/farmacología , Reacción de Prevención/fisiología , Depresión/enzimología , Depresión/psicología , Fluoxetina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiologíaRESUMEN
Drugs of abuse induce neuroplasticity in the natural reward pathway, specifically the nucleus accumbens (NAc), thereby causing development and expression of addictive behavior. Recent evidence suggests that natural rewards may cause similar changes in the NAc, suggesting that drugs may activate mechanisms of plasticity shared with natural rewards, and allowing for unique interplay between natural and drug rewards. In this study, we demonstrate that sexual experience in male rats when followed by short or prolonged periods of loss of sex reward causes enhanced amphetamine reward, indicated by sensitized conditioned place preference for low-dose (0.5 mg/kg) amphetamine. Moreover, the onset, but not the longer-term expression, of enhanced amphetamine reward was correlated with a transient increase in dendritic spines in the NAc. Next, a critical role for the transcription factor ΔFosB in sex experience-induced enhanced amphetamine reward and associated increases in dendritic spines on NAc neurons was established using viral vector gene transfer of the dominant-negative binding partner ΔJunD. Moreover, it was demonstrated that sexual experience-induced enhanced drug reward, ΔFosB, and spinogenesis are dependent on mating-induced dopamine D1 receptor activation in the NAc. Pharmacological blockade of D1 receptor, but not D2 receptor, in the NAc during sexual behavior attenuated ΔFosB induction and prevented increased spinogenesis and sensitized amphetamine reward. Together, these findings demonstrate that drugs of abuse and natural reward behaviors act on common molecular and cellular mechanisms of plasticity that control vulnerability to drug addiction, and that this increased vulnerability is mediated by ΔFosB and its downstream transcriptional targets.
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Anfetamina/administración & dosificación , Plasticidad Neuronal/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Recompensa , Conducta Sexual Animal/fisiología , Animales , Conducta Adictiva/metabolismo , Conducta Adictiva/psicología , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Antagonistas de Dopamina/farmacología , Femenino , Masculino , Plasticidad Neuronal/efectos de los fármacos , Ratas , Receptores Dopaminérgicos/metabolismo , Conducta Sexual Animal/efectos de los fármacosRESUMEN
The transcription factor ΔFosB and the brain-enriched calcium/calmodulin-dependent protein kinase II (CaMKIIα) are induced in the nucleus accumbens (NAc) by chronic exposure to cocaine or other psychostimulant drugs of abuse, in which the two proteins mediate sensitized drug responses. Although ΔFosB and CaMKIIα both regulate AMPA glutamate receptor expression and function in NAc, dendritic spine formation on NAc medium spiny neurons (MSNs), and locomotor sensitization to cocaine, no direct link between these molecules has to date been explored. Here, we demonstrate that ΔFosB is phosphorylated by CaMKIIα at the protein-stabilizing Ser27 and that CaMKII is required for the cocaine-mediated accumulation of ΔFosB in rat NAc. Conversely, we show that ΔFosB is both necessary and sufficient for cocaine induction of CaMKIIα gene expression in vivo, an effect selective for D1-type MSNs in the NAc shell subregion. Furthermore, induction of dendritic spines on NAc MSNs and increased behavioral responsiveness to cocaine after NAc overexpression of ΔFosB are both CaMKII dependent. Importantly, we demonstrate for the first time induction of ΔFosB and CaMKII in the NAc of human cocaine addicts, suggesting possible targets for future therapeutic intervention. These data establish that ΔFosB and CaMKII engage in a cell-type- and brain-region-specific positive feedforward loop as a key mechanism for regulating the reward circuitry of the brain in response to chronic cocaine.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Trastornos Relacionados con Cocaína/patología , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Núcleo Accumbens/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Adolescente , Adulto , Anciano , Análisis de Varianza , Animales , Benzazepinas/farmacología , Calcio/metabolismo , Inmunoprecipitación de Cromatina , Trastornos Relacionados con Cocaína/metabolismo , Estudios de Cohortes , Antagonistas de Dopamina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Núcleo Accumbens/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Proto-Oncogénicas c-fos/genética , Ratas , Salicilamidas/farmacología , Serina/metabolismo , Adulto JovenRESUMEN
The transcription factor, ΔFosB, is robustly and persistently induced in striatum by several chronic stimuli, such as drugs of abuse, antipsychotic drugs, natural rewards, and stress. However, very few studies have examined the degree of ΔFosB induction in the two striatal medium spiny neuron (MSN) subtypes. We make use of fluorescent reporter BAC transgenic mice to evaluate induction of ΔFosB in dopamine receptor 1 (D1) enriched and dopamine receptor 2 (D2) enriched MSNs in ventral striatum, nucleus accumbens (NAc) shell and core, and in dorsal striatum (dStr) after chronic exposure to several drugs of abuse including cocaine, ethanol, Δ(9)-tetrahydrocannabinol, and opiates; the antipsychotic drug, haloperidol; juvenile enrichment; sucrose drinking; calorie restriction; the serotonin selective reuptake inhibitor antidepressant, fluoxetine; and social defeat stress. Our findings demonstrate that chronic exposure to many stimuli induces ΔFosB in an MSN-subtype selective pattern across all three striatal regions. To explore the circuit-mediated induction of ΔFosB in striatum, we use optogenetics to enhance activity in limbic brain regions that send synaptic inputs to NAc; these regions include the ventral tegmental area and several glutamatergic afferent regions: medial prefrontal cortex, amygdala, and ventral hippocampus. These optogenetic conditions lead to highly distinct patterns of ΔFosB induction in MSN subtypes in NAc core and shell. Together, these findings establish selective patterns of ΔFosB induction in striatal MSN subtypes in response to chronic stimuli and provide novel insight into the circuit-level mechanisms of ΔFosB induction in striatum.
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Cuerpo Estriado/citología , Dopaminérgicos/farmacología , Emociones/efectos de los fármacos , Optogenética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Antidepresivos/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Dronabinol/farmacología , Ambiente , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/clasificación , Neuronas/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/genéticaRESUMEN
Fluorescent protein-based pH biosensors enable the tracking of pH changes during protein trafficking and, in particular, exocytosis. The recent development of chemogenetic reporters combining synthetic fluorophores with self-labeling protein tags offers a versatile alternative to fluorescent proteins that combines the diversity of chemical probes and indicators with the selectivity of the genetic encoding. However, this hybrid protein labeling strategy does not avoid common drawbacks of organic fluorophores such as the risk of off-target signal due to unbound molecules. Here, we describe a novel fluorogenic and chemogenetic pH sensor based on a cell-permeable molecular pH indicator called pHluo-Halo-1, whose fluorescence can be locally activated in cells by reaction with HaloTag, ensuring excellent signal selectivity in wash-free imaging experiments. pHluo-Halo-1 was selected out of a series of four fluorogenic molecular rotor structures based on protein chromophore analogues. It displays good pH sensitivity with a pKa of 6.3 well-suited to monitor pH variations during exocytosis and an excellent labeling selectivity in cells. It was applied to follow the secretion of CD63-HaloTag fusion proteins using TIRF microscopy. We anticipate that this strategy based on the combination of a tunable and chemically accessible fluorogenic probe with a well-established protein tag will open new possibilities for the development of versatile alternatives to fluorescent proteins for elucidating the dynamics and regulatory mechanisms of proteins in living cells.
RESUMEN
The molecular mechanism underlying induction by cocaine of ΔFosB, a transcription factor important for addiction, remains unknown. Here, we demonstrate a necessary role for two transcription factors, cAMP response element binding protein (CREB) and serum response factor (SRF), in mediating this induction within the mouse nucleus accumbens (NAc), a key brain reward region. CREB and SRF are both activated in NAc by cocaine and bind to the fosB gene promoter. Using viral-mediated Cre recombinase expression in the NAc of single- or double-floxed mice, we show that deletion of both transcription factors from this brain region completely blocks cocaine induction of ΔFosB in NAc, whereas deletion of either factor alone has no effect. Furthermore, deletion of both SRF and CREB from NAc renders animals less sensitive to the rewarding effects of moderate doses of cocaine when tested in the conditioned place preference (CPP) procedure and also blocks locomotor sensitization to higher doses of cocaine. Deletion of CREB alone has the opposite effect and enhances both cocaine CPP and locomotor sensitization. In contrast to ΔFosB induction by cocaine, ΔFosB induction in NAc by chronic social stress, which we have shown previously requires activation of SRF, is unaffected by the deletion of CREB alone. These surprising findings demonstrate the involvement of distinct transcriptional mechanisms in mediating ΔFosB induction within this same brain region by cocaine versus stress. Our results also establish a complex mode of regulation of ΔFosB induction in response to cocaine, which requires the concerted activities of both SRF and CREB.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Núcleo Accumbens/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Respuesta Sérica/metabolismo , Análisis de Varianza , Animales , Proteína de Unión a CREB/deficiencia , Inmunoprecipitación de Cromatina , Condicionamiento Operante/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Núcleo Accumbens/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Factor de Respuesta Sérica/deficiencia , Estrés Psicológico/genética , Estrés Psicológico/metabolismo , Estrés Psicológico/fisiopatología , Transducción GenéticaRESUMEN
ΔFosB, a Fosb gene product, is induced in nucleus accumbens (NAc) and caudate-putamen (CPu) by repeated exposure to drugs of abuse such as cocaine. This induction contributes to aberrant patterns of gene expression and behavioral abnormalities seen with repeated drug exposure. Here, we assessed whether a remote history of cocaine exposure in rats might alter inducibility of the Fosb gene elicited by subsequent drug exposure. We show that prior chronic cocaine administration, followed by extended withdrawal, increases inducibility of Fosb in NAc, as evidenced by greater acute induction of ΔFosB mRNA and faster accumulation of ΔFosB protein after repeated cocaine reexposure. No such primed Fosb induction was observed in CPu; in fact, subsequent acute induction of ΔFosB mRNA was suppressed in CPu. These abnormal patterns of Fosb expression are associated with chromatin modifications at the Fosb gene promoter. Prior chronic cocaine administration induces a long-lasting increase in RNA polymerase II (Pol II) binding at the Fosb promoter in NAc only, suggesting that Pol II "stalling" primes Fosb for induction in this region upon reexposure to cocaine. A cocaine challenge then triggers the release of Pol II from the gene promoter, allowing for more rapid Fosb transcription. A cocaine challenge also decreases repressive histone modifications at the Fosb promoter in NAc, but increases such repressive marks and decreases activating marks in CPu. These results provide new insight into the chromatin dynamics at the Fosb promoter and reveal a novel mechanism for primed Fosb induction in NAc upon reexposure to cocaine.
Asunto(s)
Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Epigénesis Genética/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/genética , Animales , Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Expresión Génica/efectos de los fármacos , Masculino , Actividad Motora/efectos de los fármacos , Núcleo Accumbens/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Excessive inhibition of brain neurons in primary or slice cultures can induce homeostatic intrinsic plasticity, but the functional role and underlying molecular mechanisms of such plasticity are poorly understood. Here, we developed an ex vivo locus coeruleus (LC) slice culture system and successfully recapitulated the opiate-induced homeostatic adaptation in electrical activity of LC neurons seen in vivo. We investigated the mechanisms underlying this adaptation in LC slice cultures by use of viral-mediated gene transfer and genetic mutant mice. We found that short-term morphine treatment of slice cultures almost completely abolished the firing of LC neurons, whereas chronic morphine treatment increased LC neuronal excitability as revealed during withdrawal. This increased excitability was mediated by direct activation of opioid receptors and up-regulation of the cAMP pathway and accompanied by increased cAMP response-element binding protein (CREB) activity. Overexpression of a dominant negative CREB mutant blocked the increase in LC excitability induced by morphine- or cAMP-pathway activation. Knockdown of CREB in slice cultures from floxed CREB mice similarly decreased LC excitability. Furthermore, the ability of morphine or CREB overexpression to up-regulate LC firing was blocked by knockout of the CREB target adenylyl cyclase 8. Together, these findings provide direct evidence that prolonged exposure to morphine induces homeostatic plasticity intrinsic to LC neurons, involving up-regulation of the cAMP-CREB signaling pathway, which then enhances LC neuronal excitability.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Locus Coeruleus/efectos de los fármacos , Morfina/farmacología , Neuronas/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Adenilil Ciclasas/genética , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Técnicas de Inactivación de Genes , Homeostasis/efectos de los fármacos , Locus Coeruleus/metabolismo , Locus Coeruleus/fisiología , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Astrocytic-secreted matricellular proteins have been shown to influence various aspects of synaptic function. More recently, they have been found altered in animal models of psychiatric disorders such as drug addiction. Hevin (also known as Sparc-like 1) is a matricellular protein highly expressed in the adult brain that has been implicated in resilience to stress, suggesting a role in motivated behaviors. To address the possible role of hevin in drug addiction, we quantified its expression in human postmortem brains and in animal models of alcohol abuse. Hevin mRNA and protein expression were analyzed in the postmortem human brain of subjects with an antemortem diagnosis of alcohol use disorder (AUD, n = 25) and controls (n = 25). All the studied brain regions (prefrontal cortex, hippocampus, caudate nucleus and cerebellum) in AUD subjects showed an increase in hevin levels either at mRNA or/and protein levels. To test if this alteration was the result of alcohol exposure or indicative of a susceptibility factor to alcohol consumption, mice were exposed to different regimens of intraperitoneal alcohol administration. Hevin protein expression was increased in the nucleus accumbens after withdrawal followed by a ethanol challenge. The role of hevin in AUD was determined using an RNA interference strategy to downregulate hevin expression in nucleus accumbens astrocytes, which led to increased ethanol consumption. Additionally, ethanol challenge after withdrawal increased hevin levels in blood plasma. Altogether, these results support a novel role for hevin in the neurobiology of AUD.
Asunto(s)
Alcoholismo , Adulto , Ratones , Humanos , Animales , Encéfalo/metabolismo , ARN Mensajero/metabolismo , Consumo de Bebidas Alcohólicas , EtanolRESUMEN
Selective serotonin reuptake inhibitors (SSRI) are common first-line treatments for major depression. However, a significant number of depressed patients do not respond adequately to these pharmacological treatments. In the present preclinical study, we demonstrate that organic cation transporter 2 (OCT2), an atypical monoamine transporter, contributes to the effects of SSRI by regulating the routing of the essential amino acid tryptophan to the brain. Contrarily to wild-type mice, OCT2-invalidated mice failed to respond to prolonged fluoxetine treatment in a chronic depression model induced by corticosterone exposure recapitulating core symptoms of depression, i.e., anhedonia, social withdrawal, anxiety, and memory impairment. After corticosterone and fluoxetine treatment, the levels of tryptophan and its metabolites serotonin and kynurenine were decreased in the brain of OCT2 mutant mice compared to wild-type mice and reciprocally tryptophan and kynurenine levels were increased in mutants' plasma. OCT2 was detected by immunofluorescence in several structures at the blood-cerebrospinal fluid (CSF) or brain-CSF interface. Tryptophan supplementation during fluoxetine treatment increased brain concentrations of tryptophan and, more discreetly, of 5-HT in wild-type and OCT2 mutant mice. Importantly, tryptophan supplementation improved the sensitivity to fluoxetine treatment of OCT2 mutant mice, impacting chiefly anhedonia and short-term memory. Western blot analysis showed that glycogen synthase kinase-3ß (GSK3ß) and mammalian/mechanistic target of rapamycin (mTOR) intracellular signaling was impaired in OCT2 mutant mice brain after corticosterone and fluoxetine treatment and, conversely, tryptophan supplementation recruited selectively the mTOR protein complex 2. This study provides the first evidence of the physiological relevance of OCT2-mediated tryptophan transport, and its biological consequences on serotonin homeostasis in the brain and SSRI efficacy.
Asunto(s)
Trastorno Depresivo Mayor , Transportador 2 de Cátion Orgánico , Inhibidores Selectivos de la Recaptación de Serotonina , Animales , Ratones , Anhedonia , Antidepresivos/uso terapéutico , Encéfalo/metabolismo , Corticosterona/farmacología , Trastorno Depresivo Mayor/tratamiento farmacológico , Fluoxetina/farmacología , Quinurenina/metabolismo , Transportador 2 de Cátion Orgánico/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Serotonina/metabolismo , Triptófano/metabolismoRESUMEN
The molecular mechanisms underlying stress- and drug-induced neuronal adaptations are incompletely understood. One molecule implicated in such adaptations is ΔFosB, a transcription factor that accumulates in the rodent nucleus accumbens (NAc), a key brain reward region, in response to either chronic stress or repeated exposure to drugs of abuse. The upstream transcriptional mechanisms controlling ΔFosB induction by these environmental stimuli remain elusive. Here, we identify the activity-dependent transcription factor, serum response factor (SRF), as a novel upstream mediator of stress-, but not cocaine-, induced ΔFosB. SRF is downregulated in NAc of both depressed human patients and in mice chronically exposed to social defeat stress. This downregulation of SRF is absent in resilient animals. Through the use of inducible mutagenesis, we show that stress-mediated induction of ΔFosB, which occurs predominantly in resilient mice, is dependent on SRF expression in this brain region. Furthermore, NAc-specific genetic deletion of SRF promotes a variety of prodepressant- and proanxiety-like phenotypes and renders animals more sensitive to the deleterious effects of chronic stress. In contrast, we demonstrate that SRF does not play a role in ΔFosB accumulation in NAc in response to chronic cocaine exposure. Furthermore, NAc-specific knock-out of SRF has no effect on cocaine-induced behaviors, indicating that chronic social defeat stress and repeated cocaine exposure regulate ΔFosB accumulation and behavioral sensitivity through independent mechanisms.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/fisiología , Resiliencia Psicológica/efectos de los fármacos , Factor de Respuesta Sérica/fisiología , Medio Social , Estrés Psicológico/psicología , Adulto , Anciano , Animales , Antidepresivos/sangre , Ansiedad/metabolismo , Ansiedad/psicología , Western Blotting , Cocaína/farmacología , Depresión/metabolismo , Depresión/psicología , Femenino , Desamparo Adquirido , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Actividad Motora/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Respuesta Sérica/genética , Conducta SocialRESUMEN
Brain stimulation and imaging studies in humans have highlighted a key role for the prefrontal cortex in clinical depression; however, it remains unknown whether excitation or inhibition of prefrontal cortical neuronal activity is associated with antidepressant responses. Here, we examined cellular indicators of functional activity, including the immediate early genes (IEGs) zif268 (egr1), c-fos, and arc, in the prefrontal cortex of clinically depressed humans obtained postmortem. We also examined these genes in the ventral portion of the medial prefrontal cortex (mPFC) of mice after chronic social defeat stress, a mouse model of depression. In addition, we used viral vectors to overexpress channel rhodopsin 2 (a light-activated cation channel) in mouse mPFC to optogenetically drive "burst" patterns of cortical firing in vivo and examine the behavioral consequences. Prefrontal cortical tissue derived from clinically depressed humans displayed significant reductions in IEG expression, consistent with a deficit in neuronal activity within this brain region. Mice subjected to chronic social defeat stress exhibited similar reductions in levels of IEG expression in mPFC. Interestingly, some of these changes were not observed in defeated mice that escape the deleterious consequences of the stress, i.e., resilient animals. In those mice that expressed a strong depressive-like phenotype, i.e., susceptible animals, optogenetic stimulation of mPFC exerted potent antidepressant-like effects, without affecting general locomotor activity, anxiety-like behaviors, or social memory. These results indicate that the activity of the mPFC is a key determinant of depression-like behavior, as well as antidepressant responses.