RESUMEN
The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain-binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to â¼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Homeostasis , Motivación , Dominios PDZ , Recompensa , Animales , Sitios de Unión , Cocaína/administración & dosificación , Condicionamiento Operante , Masculino , Ratones , Unión Proteica , AutoadministraciónRESUMEN
As the main input structure of the basal ganglia (BG), the striatum collects and integrates information from several brain areas and funnels them forward to other BG nuclei. The striatal projection neurons are medium-sized spiny neurons classified in two main subpopulations, based on their neurochemical characterization and projection targets. These subpopulations are segregated into two distinct circuits, the direct and the indirect pathway, which originate in the striatum and interconnect the BG, ultimately reaching their output nuclei. In this review, we discuss current opinions on the striatal circuit and present different strategies to decipher this circuit complexity by utilizing cell ablation, opto- and chemogenetics, tetanus toxin-induced neuronal silencing, and calcium imaging techniques. We also describe genetically encoded biosensors to monitor signaling dynamics in the striatal circuit with high spatial and temporal resolution by targeting both glutamate and dopamine transmission together with downstream signaling effectors. Recent findings revealing transcriptional, functional diversity, and regionally distinct signaling properties of spiny projection neurons argue that refined interrogation will be pertinent for a deeper understanding of this circuit. Moreover, future mapping the G-protein-coupled receptor repertoire in SPNs will potentially enable pathway-specific modulation of SPN activity and provide a novel framework for targeting BG diseases. Overall, these tools will be critical for designing next-generation treatments for BG diseases.
Asunto(s)
Cuerpo Estriado/fisiología , Técnicas Genéticas , Vías Nerviosas/fisiología , Animales , HumanosRESUMEN
Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signalling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways.
Asunto(s)
Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Homeostasis/fisiología , Animales , Conducta Animal , Neuronas Dopaminérgicas/metabolismo , Integrasas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Serotonergic neurotransmission is terminated by reuptake of extracellular serotonin (5-HT) by the high-affinity serotonin transporter (SERT). Selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine or escitalopram inhibit SERT and are currently the principal treatment for depression and anxiety disorders. In addition, SERT is a major molecular target for psychostimulants such as cocaine and amphetamines. Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. The interaction between SERT and αCaMKII was verified using biochemical assays and FRET analysis and colocalization of the two molecules was confirmed in primary serotonergic neurons in culture. Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to 3,4-methylenedioxymethamphetamine (also known as "ecstasy") and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII. This finding supports the concept that uptake and efflux at monoamine transporters are asymmetric processes that can be targeted separately. Ultimately, this may provide a molecular mechanism for putative drug developments to treat amphetamine addiction.
Asunto(s)
Anfetamina/farmacología , Antidepresivos/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Cocaína/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Anfetamina/metabolismo , Animales , Antidepresivos/metabolismo , Células Cultivadas , Cocaína/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Ratas Sprague-DawleyRESUMEN
Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.
Asunto(s)
Intolerancia a la Glucosa/metabolismo , Trastornos del Crecimiento/metabolismo , Proteínas Nucleares/deficiencia , Vesículas Secretoras/metabolismo , Animales , Autoantígenos/fisiología , Células COS , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , Chlorocebus aethiops , Drosophila melanogaster , Femenino , Expresión Génica , Regulación de la Expresión Génica , Glucosa/metabolismo , Intolerancia a la Glucosa/genética , Trastornos del Crecimiento/genética , Hormona del Crecimiento/deficiencia , Hormona del Crecimiento/metabolismo , Homeostasis , Insulina/metabolismo , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Hipófisis/metabolismo , Unión Proteica , Transporte de Proteínas , Ratas , Imagen de Lapso de Tiempo , Red trans-Golgi/metabolismoRESUMEN
Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000-30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1-enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1-eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1-eGFP and wild-type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Neuronas Dopaminérgicas/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Conducta Animal/fisiología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Sinaptosomas/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca(2+)-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects.
Asunto(s)
Anfetamina/farmacología , Péptidos de Penetración Celular/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/farmacología , Dopamina/metabolismo , Anfetamina/efectos adversos , Animales , Bencilaminas/farmacología , Péptidos de Penetración Celular/metabolismo , Estimulantes del Sistema Nervioso Central/efectos adversos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/farmacocinética , Humanos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Dominios PDZ , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacologíaRESUMEN
Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat-eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway.
Asunto(s)
Neuronas Dopaminérgicas , Receptores Acoplados a Proteínas G , Animales , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Neuronas Dopaminérgicas/metabolismo , Ratones , Ingestión de Alimentos , Ingestión de Líquidos , Ratones Endogámicos C57BL , Masculino , Mesencéfalo/metabolismoRESUMEN
Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in an enriched environment for two weeks after permanent middle cerebral artery occlusion, we found increased sigma-1 receptor expression in peri-infarct areas. Treatment of rats subjected to permanent or transient middle cerebral artery occlusion with 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor activation increased the levels of the synaptic protein neurabin and neurexin in membrane rafts in the peri-infarct area, while sigma-1 receptor silencing prevented sigma-1 receptor-mediated neurite outgrowth in primary cortical neuronal cultures. In astrocytic cultures, oxygen and glucose deprivation induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection.
Asunto(s)
Encéfalo/metabolismo , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Plasticidad Neuronal/fisiología , Receptores sigma/metabolismo , Recuperación de la Función/fisiología , Animales , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Caveolina 1/genética , Caveolina 1/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ambiente , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glucosa/deficiencia , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Movimiento/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Plasticidad Neuronal/efectos de los fármacos , Neuronas/citología , Neuronas/metabolismo , Nootrópicos/farmacología , Nootrópicos/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Transporte de Proteínas/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Ratas , Ratas Endogámicas SHR , Receptores sigma/genética , Recuperación de la Función/efectos de los fármacos , Estadísticas no Paramétricas , Transfección/métodos , Receptor Sigma-1RESUMEN
The striatum collects and integrates information from many different areas of the brain and propels this forward to the basal ganglia (BG) output structures. In this way, the striatum is playing a pivotal role in control of voluntary movements and is implicated in debilitating movement disorders such as Parkinson's disease. The functional backbone of the striatum is represented by direct pathway (dSPN) Drd1-expressing and indirect pathway (iSPN) Drd2-expressing spiny projection neurons (SPN), exerting opposite effects on movement. In rodent models of striatal function, unilateral dopamine deprivation is known to induce ipsilateral rotational behavior. To further study imbalance of the BG circuit and striatal domain influence on behavioral outcome, we employed a viral approach based on tetanus toxin light chain (TeLC) activity for permanent inhibition of dSPN activity in dorsomedial striatum (DMS). Cre-dependent TeLC injected unilaterally into the DMS of Drd1-Cre mice resulted in robust expression of TeLC in the dSPN cell populations as shown by immunohistochemistry. In the TeLC expressing mice, but not in control mice, we observed ipsilateral rotations that were enhanced upon administration of amphetamine to augment striatal dopamine levels. We argue that the observed single turns of ipsilateral rotations occur because of TeLC-mediated silencing of dSPN activity in one hemisphere, causing unresponsiveness to dopamine transients during movement initiation. This evokes a temporal BG circuit imbalance manifested as short bursts of rotations, particular evident during extrinsic dopaminergic modulation.
Asunto(s)
Ganglios Basales , Cuerpo Estriado , Animales , Interneuronas , Ratones , Neostriado , NeuritasRESUMEN
Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Trastornos Mentales/genética , Neuronas/metabolismo , Trastornos Parkinsonianos/genética , Adulto , Animales , Conducta Animal , Transporte Biológico , Células Cultivadas , Bases de Datos Genéticas , Drosophila , Exoma , Femenino , Humanos , Hipocinesia/diagnóstico por imagen , Hipocinesia/genética , Hipocinesia/metabolismo , Masculino , Trastornos Mentales/metabolismo , Mesencéfalo/metabolismo , Ratones , Persona de Mediana Edad , Actividad Motora/genética , Mutación , Trastornos Parkinsonianos/diagnóstico por imagen , Trastornos Parkinsonianos/metabolismo , Fenotipo , Transmisión Sináptica , Tomografía Computarizada de Emisión de Fotón Único , TransfecciónRESUMEN
The striatum of the basal ganglia is pivotal for voluntary movements and is implicated in debilitating movement disorders such as Parkinsonism and dystonia. Striatum projects to downstream nuclei through direct (dSPN) and indirect (iSPN) pathway projection neurons thought to exert opposite effects on movement. In rodent models of striatal function, unilateral dopamine deprivation induces ipsiversive rotational behavior. The dSPNs of the dorsal striatum are believed to engage distinct motor programs but underlying mechanisms remain unclear. Here, we show by employing chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] that unilateral inhibition of dorsomedial dSPNs is sufficient to selectively impair contraversive movement and elicit ipsiversive rotational behavior in mice. Adeno-associated virus (AAV) encoding Cre-dependent Gi-coupled DREADD was injected unilaterally into the dorsomedial striatum of Drd1-Cre mice, resulting in expression of the modified human M4 muscarinic receptor (hM4Di) in â¼20% of dorsostriatal dSPNs. Upon hM4Di activation, a striking positive linear correlation was found between turn ratio and viral expression, which corroborates a relationship between unilateral inhibition of dorsomedial dSPNs and rotational behavior. Bursts of ipsiversive rotations were interspersed with normal ambulation. However, partial unilateral inhibition of â¼20% of dorsostriatal dSPNs did not affect horizontal and vertical locomotion or forelimb use preference. Overall, our results substantiate a unique role of dSPNs in promoting response bias in rotational behavior and show this to be a highly sensitive measure of dSPN performance.
Asunto(s)
Drogas de Diseño/farmacología , Neostriado/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Animales , Ganglios Basales/metabolismo , Conducta Animal , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neostriado/citología , Neostriado/efectos de los fármacos , Vías Nerviosas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/metabolismo , Receptor Muscarínico M4/metabolismo , Receptores de Dopamina D1/metabolismo , RotaciónRESUMEN
The precise regulation of synaptic dopamine (DA) content by the dopamine transporter (DAT) ensures the phasic nature of the DA signal, which underlies the ability of DA to encode reward prediction error, thereby driving motivation, attention, and behavioral learning. Disruptions to the DA system are implicated in a number of neuropsychiatric disorders, including attention deficit hyperactivity disorder (ADHD) and, more recently, Autism Spectrum Disorder (ASD). An ASD-associated de novo mutation in the SLC6A3 gene resulting in a threonine to methionine substitution at site 356 (DAT T356M) was recently identified and has been shown to drive persistent reverse transport of DA (i.e. anomalous DA efflux) in transfected cells and to drive hyperlocomotion in Drosophila melanogaster. A corresponding mutation in the leucine transporter, a DAT-homologous transporter, promotes an outward-facing transporter conformation upon substrate binding, a conformation possibly underlying anomalous dopamine efflux. Here we investigated in vivo the impact of this ASD-associated mutation on DA signaling and ASD-associated behaviors. We found that mice homozygous for this mutation display impaired striatal DA neurotransmission and altered DA-dependent behaviors that correspond with some of the behavioral phenotypes observed in ASD.
Asunto(s)
Trastorno Autístico/metabolismo , Conducta Animal , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Mutación Missense , Transmisión Sináptica , Sustitución de Aminoácidos , Animales , Trastorno Autístico/genética , Trastorno Autístico/patología , Trastorno Autístico/fisiopatología , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Dopamina/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ratones , Ratones MutantesRESUMEN
Injury to the brain (e.g., stroke) results in a disruption of neuronal connectivity and loss of fundamental sensori-motor functions. The subsequent recovery of certain functions involves structural rearrangements in areas adjacent to the infarct. This remodeling of the injured brain requires trafficking of macromolecular components including cholesterol and phospholipids, a transport carried out by apolipoproteins including apolipoprotein D (apoD). We investigated the changes in the levels of apoD mRNA and protein, and its cellular localization during a recovery period up to 30 days after experimental stroke in the rat brain. In the core of the brain infarct, apoD immunoreactivity but not mRNA increased in dying pyramidal neurons, indicative of cellular redistribution of lipids. During 2 to 7 days of recovery after stroke, the apoD levels increased in the peri-infarct and white matter areas in cells identified as mature oligodendrocytes. The apoD expressing cells were conspicuously located along the rim of the infarct, suggesting a role for apoD in tissue repair. Furthermore, housing animals in an enriched environment improved sensori-motor function and increased the apoD levels. Our data strongly suggest that apoD is involved in regenerative processes and scar formation in the peri-infarct area presumably by enhancing lipid trafficking.
Asunto(s)
Apolipoproteínas D/fisiología , Oligodendroglía/metabolismo , Regeneración , Accidente Cerebrovascular/patología , Animales , Apolipoproteínas D/análisis , Apolipoproteínas D/genética , Transporte Biológico , Metabolismo de los Lípidos , Oligodendroglía/fisiología , ARN Mensajero/análisis , RatasRESUMEN
Protein interacting with C-kinase 1 (PICK1) is a widely expressed scaffold protein known to interact via its PSD-95/discs-large/ZO-1 (PDZ)-domain with several membrane proteins including the dopamine (DA) transporter (DAT), the primary target for cocaine's reinforcing actions. Here, we establish the importance of PICK1 for behavioral effects observed after both acute and repeated administration of cocaine. In PICK1 knock-out (KO) mice, the acute locomotor response to a single injection of cocaine was markedly attenuated. Moreover, in support of a role for PICK1 in neuroadaptive changes induced by cocaine, we observed diminished cocaine intake in a self-administration paradigm. Reduced behavioral effects of cocaine were not associated with decreased striatal DAT distribution and most likely not caused by the â¼30% reduction in synaptosomal DA uptake observed in PICK1 KO mice. The PICK1 KO mice demonstrated preserved behavioral responses to DA receptor agonists supporting intact downstream DA receptor signaling. Unexpectedly, we found a prominent increase in striatal DA content and levels of striatal tyrosine hydroxylase (TH) in PICK1 KO mice. Chronoamperometric recordings showed enhanced DA release in PICK1 KO mice, consistent with increased striatal DA pools. Viral-mediated knock-down (KD) of PICK1 in cultured dopaminergic neurons increased TH expression, supporting a direct cellular effect of PICK1. In summary, in addition to demonstrating a key role of PICK1 in mediating behavioral effects of cocaine, our data reveal a so far unappreciated role of PICK1 in DA homeostasis that possibly involves negative regulation of striatal TH levels.
Asunto(s)
Proteínas Portadoras/metabolismo , Cocaína/administración & dosificación , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Homeostasis/efectos de los fármacos , Proteínas Nucleares/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Refuerzo en Psicología , Transducción de Señal/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Dopamine plays a key role in the cellular and behavioral responses to drugs of abuse, but the implication of metabotropic regulatory input to dopaminergic neurons on acute drug effects and subsequent drug-related behavior remains unclear. Here, we used chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] to modulate dopamine signaling and activity before cocaine administration in mice. We show that chemogenetic inhibition of dopaminergic ventral tegmental area (VTA) neurons differentially affects locomotor and reward-related behavioral responses to cocaine. Stimulation of Gi-coupled DREADD (hM4Di) expressed in dopaminergic VTA neurons persistently reduced the locomotor response to repeated cocaine injections. An attenuated locomotor response was seen even when a dual-viral vector approach was used to restrict hM4Di expression to dopaminergic VTA neurons projecting to the nucleus accumbens. Surprisingly, despite the attenuated locomotor response, hM4Di-mediated inhibition of dopaminergic VTA neurons did not prevent cocaine sensitization, and the inhibitory effect of hM4Di-mediated inhibition was eliminated after withdrawal. In the conditioned place-preference paradigm, hM4Di-mediated inhibition did not affect cocaine-induced place preference; however, the extinction period was extended. Also, hM4Di-mediated inhibition had no effect on preference for a sugar-based reward over water but impaired motivation to work for the same reward in a touchscreen-based motivational assay. In addition, to support that VTA dopaminergic neurons operate as regulators of reward motivation toward both sugar and cocaine, our data suggest that repeated cocaine exposure leads to adaptations in the VTA that surmount the ability of Gi-signaling to suppress and regulate VTA dopaminergic neuronal activity.
Asunto(s)
Cocaína/administración & dosificación , Neuronas Dopaminérgicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Locomoción , Recompensa , Área Tegmental Ventral/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Condicionamiento Clásico , Neuronas Dopaminérgicas/efectos de los fármacos , Comportamiento de Búsqueda de Drogas , Femenino , Masculino , Ratones , Motivación , Transducción de Señal , Área Tegmental Ventral/efectos de los fármacosRESUMEN
The genomic response following brain ischemia is very complex and involves activation of both protective and detrimental signaling pathways. Immediate early genes (IEGs) represent the first wave of gene expression following ischemia and are induced in extensive regions of the ischemic brain including cerebral cortex and hippocampus. Brain-derived neurotrophic factor (BDNF), Neuritin and Activity-regulated cytoskeleton-associated protein (Arc) belong to a subgroup of immediate early genes implicated in synaptic plasticity known as effector immediate early genes. Here, we investigated the spatial and temporal activation pattern for these genes during the first 24 h of reperfusion following 2-h occlusion of the middle cerebral artery. Neuritin showed a persistent activation in frontal-cingulate cortex while Arc displayed a biphasic response. Also, in dentate gyrus, activation was observed at 0-6 h of reperfusion for Neuritin and 0-12 h of reperfusion for Arc while BDNF was induced 0-9 h of reperfusion. Our study demonstrates a rapid and long-term activation of effector immediate early genes in distinct brain areas following ischemic injury in rat. Effector gene activation may be part of long-term synaptic responses of ischemic brain tissue.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Infarto Encefálico/patología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Giro Dentado/metabolismo , Proteínas Musculares/metabolismo , Neuropéptidos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Infarto Encefálico/etiología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Factor Neurotrófico Derivado del Encéfalo/genética , Proteínas Ligadas a GPI , Regulación de la Expresión Génica/fisiología , Hibridación in Situ/métodos , Masculino , Proteínas Musculares/genética , Neuropéptidos/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Activación TranscripcionalRESUMEN
Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson's disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases. Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas. DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay. Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Sinaptosomas/metabolismo , Animales , Homeostasis , Masculino , RatonesRESUMEN
The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show that overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication of apoptosis was the finding that one of the key enzymes in the apoptotic process, caspase-3, was induced when DFMO was omitted from the growth medium. Furthermore, inhibition of the caspase activity significantly reduced the recruitment of cells to the sub-G1 fraction. In conclusion, deregulation of polyamine homeostasis may negatively affect cell proliferation and eventually lead to cell death by apoptosis if putrescine levels become too high.
Asunto(s)
Apoptosis/efectos de los fármacos , Putrescina/farmacología , Animales , Células CHO , Caspasa 3 , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Eflornitina/farmacología , Inhibidores Enzimáticos , Fase G1/efectos de los fármacos , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Putrescina/biosíntesisRESUMEN
Addiction to psychostimulants (ie, amphetamines and cocaine) imposes a major socioeconomic burden. Prevention and treatment represent unmet medical needs, which may be addressed, if the mechanisms underlying psychostimulant action are understood. Cocaine acts as a blocker at the transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), but amphetamines are substrates that do not only block the uptake of monoamines but also induce substrate efflux by promoting reverse transport. Reverse transport has been a focus of research for decades but its mechanistic basis still remains enigmatic. Recently, transporter-interacting proteins were found to regulate amphetamine-triggered reverse transport: calmodulin kinase IIα (αCaMKII) is a prominent example, because it binds the carboxyl terminus of DAT, phosphorylates its amino terminus, and supports amphetamine-induced substrate efflux in vitro. Here, we investigated whether, in vivo, the action of amphetamine was contingent on the presence of αCaMKII by recording the behavioral and neurochemical effects of amphetamine. Measurement of dopamine efflux in the dorsal striatum by microdialysis revealed that amphetamine induced less dopamine efflux in mice lacking αCaMKII. Consistent with this observation, the acute locomotor responses to amphetamine were also significantly blunted in αCaMKII-deficient mice. In addition, while the rewarding properties of amphetamine were preserved in αCaMKII-deficient mice, their behavioral sensitization to amphetamine was markedly reduced. Our findings demonstrate that amphetamine requires the presence of αCaMKII to elicit a full-fledged effect on DAT in vivo: αCaMKII does not only support acute amphetamine-induced dopamine efflux but is also important in shaping the chronic response to amphetamine.