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The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.
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Alcoholismo/genética , Polimorfismo de Nucleótido Simple/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Adolescente , Femenino , Genotipo , Humanos , MasculinoRESUMEN
Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme-coated microelectrodes and the risk for light-induced artifacts. In this study, we establish a method for the combination of in vivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme-coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinson's disease, has recently proven opotogenetically targetable in Pitx2-Cre-transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2-eYFP constructs into the STN, amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi-step analysis approach based on self-referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of in vivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate-based approaches in neuroscience.
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Globo Pálido/metabolismo , Ácido Glutámico/análisis , Optogenética/métodos , Animales , Ratones , Ratones Transgénicos , Microelectrodos , Optogenética/instrumentaciónRESUMEN
Alcohol use disorder is the outcome of both genetic and environmental influences and their interaction via epigenetic mechanisms. The neurotransmitter glutamate is an important regulator of reward circuits and implicated in adaptive changes induced by ethanol intake. The present study aimed at investigating corticolimbic and corticostriatal genetic signatures focusing on the glutamatergic phenotype in relation to early-life stress (ELS) and consequent adult ethanol consumption. A rodent maternal separation model was employed to mimic ELS, and a free-choice paradigm was used to assess ethanol intake in adulthood. Gene expression levels of the Vesicular Glutamate Transporters (Vglut) 1, 2 and 3, as well as two key regulators of DNA methylation, DNA (cytosine-5)-methyltransferase 1 (Dnmt1) and methyl-CpG-binding protein 2 (Mecp2), were analyzed. Brain regions of interest were the ventral tegmental area (VTA), nucleus accumbens (Acb), medial prefrontal cortex (mPFC) and dorsal striatum (dStr), all involved in mediating aspects of ethanol reward. Region-specific Vglut, Dnmt1 and Mecp2 expression patterns were observed. ELS was associated with down-regulated expression of Vglut2 in the VTA and mPFC. Rats exposed to ELS were more sensitive to ethanol-induced changes in Vglut expression in the VTA, Acb, and dStr and in Dnmt1 and Mecp2 expression in the striatal regions. These findings suggest long-term glutamatergic and DNA methylation neuroadaptations as a consequence of ELS, and show an association between voluntary drinking in non-preferring, non-dependent, rodents and different Vglut, Dnmt1 and Mecp2 expression depending on early-life history.
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Depresores del Sistema Nervioso Central/farmacología , Cuerpo Estriado/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Etanol/farmacología , Expresión Génica/efectos de los fármacos , Sistema Límbico/efectos de los fármacos , Privación Materna , Terminales Presinápticos/efectos de los fármacos , Estrés Psicológico/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Conducta de Elección , Cuerpo Estriado/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/genética , Femenino , Ácido Glutámico/metabolismo , Sistema Límbico/metabolismo , Masculino , Proteína 2 de Unión a Metil-CpG/efectos de los fármacos , Proteína 2 de Unión a Metil-CpG/genética , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Terminales Presinápticos/metabolismo , Ratas , Ratas Wistar , Recompensa , Estrés Psicológico/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/efectos de los fármacos , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/efectos de los fármacos , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteínas de Transporte Vesicular de Glutamato/efectos de los fármacos , Proteínas de Transporte Vesicular de Glutamato/genéticaRESUMEN
The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.
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Ácido Glutámico/metabolismo , Hipercinesia/metabolismo , Núcleo Subtalámico/metabolismo , Transmisión Sináptica/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Animales , Dopamina/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Proteínas de Homeodominio/metabolismo , Hipercinesia/etiología , Inmunohistoquímica , Hibridación in Situ , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.
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Ácido Glutámico/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Proteínas de Transporte Vesicular de Glutamato/fisiología , Animales , Encéfalo/citología , Encéfalo/metabolismo , Proteínas de Transporte Vesicular de Glutamato/clasificaciónRESUMEN
Presynaptic proteins orchestrate an intricate interplay of dynamic interactions in order to regulate quantal exocytosis of transmitter-filled vesicles, and their dysregulation might cause neurological and neuropsychiatric dysfunction. Mice carrying a spatiotemporal restriction in the expression of the Vesicular glutamate transporter 2 (Vglut2; aka Slc17a6) in the cortex, amygdala and hippocampal subiculum from the third postnatal week show a strong anxiolytic phenotype and certain behavioral correlates of schizophrenia. To further understand the molecular consequences of this targeted deletion of Vglut2, we performed an unbiased microarray analysis comparing gene expression levels in the subiculum of these conditional Vglut2 knockout mice (Vglut2f/f;CamKII cKO) to those in control littermates. Expression of Unc13C (Munc13-3), a member of the Unc/Munc family, previously shown to be important for glutamatergic transmission, was identified to be significantly down-regulated. Subsequent analysis by quantitative RT-PCR revealed a 50% down-regulation of Munc 13-1, the gene encoding the Unc/Munc subtype described as an essential component in the majority of glutamtergic synapses in the hippocampus. Genes encoding additional components of the presynaptic machinery were also found regulated, including Rab3A, RIM1α, as well as Syntaxin1 and Synaptobrevin. Altered expression levels of these genes were further found in the amygdala and in the retrosplenial group of the cortex, additional regions in which Vglut2 was conditionally targeted. These findings suggest that expression levels of Vglut2 might be important for the maintenance of gene expression in the presynaptic machinery in the adult mouse brain. Synapse 68:624-633, 2014. © 2014 Wiley Periodicals, Inc.
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Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Na(v)1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Na(v)1.8(Cre)-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2(f/f);Na(v)1.8(Cre) mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Na(v)1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.
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Hiperalgesia/metabolismo , Modelos Neurológicos , Dolor/metabolismo , Células Receptoras Sensoriales/metabolismo , Sustancia P/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Análisis de Varianza , Animales , Cartilla de ADN/genética , Genotipo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The subthalamic nucleus (STN) is a major neuromodulation target for the alleviation of neurological and neuropsychiatric symptoms using deep brain stimulation (DBS). STN-DBS is today applied as treatment in Parkinson´s disease, dystonia, essential tremor, and obsessive-compulsive disorder (OCD). STN-DBS also shows promise as a treatment for refractory Tourette syndrome. However, the internal organization of the STN has remained elusive and challenges researchers and clinicians: How can this small brain structure engage in the multitude of functions that renders it a key hub for therapeutic intervention of a variety of brain disorders ranging from motor to affective to cognitive? Based on recent gene expression studies of the STN, a comprehensive view of the anatomical and cellular organization, including revelations of spatio-molecular heterogeneity, is now possible to outline. In this review, we focus attention to the neurobiological architecture of the STN with specific emphasis on molecular patterns discovered within this complex brain area. Studies from human, non-human primate, and rodent brains now reveal anatomically defined distribution of specific molecular markers. Together their spatial patterns indicate a heterogeneous molecular architecture within the STN. Considering the translational capacity of targeting the STN in severe brain disorders, the addition of molecular profiling of the STN will allow for advancement in precision of clinical STN-based interventions.
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Enfermedad de Parkinson , Núcleo Subtalámico , Animales , Humanos , Encéfalo , Enfermedad de Parkinson/terapia , InvestigadoresRESUMEN
Neuromodulation such as deep brain stimulation (DBS) is advancing as a clinical intervention in several neurological and neuropsychiatric disorders, including Parkinson's disease, dystonia, tremor, and obsessive-compulsive disorder (OCD) for which DBS is already applied to alleviate severely afflicted individuals of symptoms. Tourette syndrome and drug addiction are two additional disorders for which DBS is in trial or proposed as treatment. However, some major remaining obstacles prevent this intervention from reaching its full therapeutic potential. Side-effects have been reported, and not all DBS-treated individuals are relieved of their symptoms. One major target area for DBS electrodes is the subthalamic nucleus (STN) which plays important roles in motor, affective and associative functions, with impact on for example movement, motivation, impulsivity, compulsivity, as well as both reward and aversion. The multifunctionality of the STN is complex. Decoding the anatomical-functional organization of the STN could enhance strategic targeting in human patients. The STN is located in close proximity to zona incerta (ZI) and the para-subthalamic nucleus (pSTN). Together, the STN, pSTN and ZI form a highly heterogeneous and clinically important brain area. Rodent-based experimental studies, including opto- and chemogenetics as well as viral-genetic tract tracings, provide unique insight into complex neuronal circuitries and their impact on behavior with high spatial and temporal precision. This research field has advanced tremendously over the past few years. Here, we provide an inclusive review of current literature in the pre-clinical research fields centered around STN, pSTN and ZI in laboratory mice and rats; the three highly heterogeneous and enigmatic structures brought together in the context of relevance for treatment strategies. Specific emphasis is placed on methods of manipulation and behavioral impact.
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Estimulación Encefálica Profunda , Trastornos Mentales , Núcleo Subtalámico , Zona Incerta , Núcleo Subtalámico/fisiología , Animales , Estimulación Encefálica Profunda/métodos , Zona Incerta/fisiología , Trastornos Mentales/terapia , Humanos , Enfermedades del Sistema Nervioso/terapia , RoedoresRESUMEN
Recent studies have proposed that glutamate corelease by mesostriatal dopamine (DA) neurons regulates behavioral activation by psychostimulants. How and when glutamate release by DA neurons might play this role remains unclear. Considering evidence for early expression of the type 2 vesicular glutamate transporter in mesencephalic DA neurons, we hypothesized that this cophenotype is particularly important during development. Using a conditional gene knock-out approach to selectively disrupt the Vglut2 gene in mouse DA neurons, we obtained in vitro and in vivo evidence for reduced growth and survival of mesencephalic DA neurons, associated with a decrease in the density of DA innervation in the nucleus accumbens, reduced activity-dependent DA release, and impaired motor behavior. These findings provide strong evidence for a functional role of the glutamatergic cophenotype in the development of mesencephalic DA neurons, opening new perspectives into the pathophysiology of neurodegenerative disorders involving the mesostriatal DA system.
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Supervivencia Celular/fisiología , Neuronas Dopaminérgicas/metabolismo , Ácido Glutámico/metabolismo , Mesencéfalo/metabolismo , Anfetamina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Estimulantes del Sistema Nervioso Central/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Ácido Glutámico/genética , Masculino , Mesencéfalo/efectos de los fármacos , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The "One neuron-one neurotransmitter" concept has been challenged frequently during the last three decades, and the coexistence of neurotransmitters in individual neurons is now regarded as a common phenomenon. The functional significance of neurotransmitter coexistence is, however, less well understood. Several studies have shown that a subpopulation of dopamine (DA) neurons in the ventral tegmental area (VTA) expresses the vesicular glutamate transporter 2 (VGLUT2) and has been suggested to use glutamate as a cotransmitter. The VTA dopamine neurons project to limbic structures including the nucleus accumbens, and are involved in mediating the motivational and locomotor activating effects of psychostimulants. To determine the functional role of glutamate cotransmission by these neurons, we deleted VGLUT2 in DA neurons by using a conditional gene-targeting approach in mice. A DAT-Cre/Vglut2Lox mouse line (Vglut2(f/f;DAT-Cre) mice) was produced and analyzed by in vivo amperometry as well as by several behavioral paradigms. Although basal motor function was normal in the Vglut2(f/f;DAT-Cre) mice, their risk-taking behavior was altered. Interestingly, in both home-cage and novel environments, the gene targeted mice showed a greatly blunted locomotor response to the psychostimulant amphetamine, which acts via the midbrain DA system. Our results show that VGLUT2 expression in DA neurons is required for normal emotional reactivity as well as for psychostimulant-mediated behavioral activation.
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Conducta Animal/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Dopamina/metabolismo , Neuronas , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Anfetamina/farmacología , Animales , Conducta Animal/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Ácido Glutámico/metabolismo , Masculino , Mesencéfalo/citología , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Distribución Aleatoria , Asunción de Riesgos , Factores Sexuales , Proteína 2 de Transporte Vesicular de Glutamato/genéticaRESUMEN
Ventral tegmental area (VTA) dopamine (DA) neurons are implicated in reward processing, motivation, reward prediction error, and in substance use disorder. Recent studies have identified distinct neuronal subpopulations within the VTA that can be clustered based on their molecular identity, neurotransmitter profile, physiology, projections and behavioral role. One such subpopulation is characterized by expression of the NeuroD6 gene, and projects primarily to the nucleus accumbens medial shell. We recently showed that optogenetic stimulation of these neurons induces real-time place preference while their targeted deletion of the Vmat2 gene caused altered response to rewarding substances, including ethanol and psychostimulants. Based on these recent findings, we wanted to further investigate the involvement of the NeuroD6-positive VTA subpopulation in reward processing. Using the same NeuroD6Cre+/wt ;Vmat2flox/flox mice as in our prior study, we now addressed the ability of the mice to process sucrose reward. In order to assess appetitive behavior and motivation to obtain sucrose reward, we tested conditional knockout (cKO) and control littermate mice in an operant sucrose self-administration paradigm. We observed that cKO mice demonstrate higher response rates to the operant task and consume more sucrose rewards than control mice. However, their motivation to obtain sucrose is identical to that of control mice. Our results highlight previous observations that appetitive behavior and motivation to obtain rewards can be served by distinct neuronal circuits, and demonstrate that the NeuroD6 VTA subpopulation is involved in mediating the former, but not the latter. Together with previous studies on the NeuroD6 subpopulation, our findings pinpoint the importance of unraveling the molecular and functional role of VTA subpopulations in order to better understand normal behavior and psychiatric disease.
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The subthalamic nucleus (STN) is critical for behavioral control; its dysregulation consequently correlated with neurological and neuropsychiatric disorders, including Parkinson's disease. Deep brain stimulation (DBS) targeting the STN successfully alleviates parkinsonian motor symptoms. However, low mood and depression are affective side effects. STN is adjoined with para-STN, associated with appetitive and aversive behavior. DBS aimed at STN might unintentionally modulate para-STN, causing aversion. Alternatively, the STN mediates aversion. To investigate causality between STN and aversion, affective behavior is addressed using optogenetics in mice. Selective promoters allow dissociation of STN (e.g., Pitx2) vs. para-STN (Tac1). Acute photostimulation results in aversion via both STN and para-STN. However, only STN stimulation-paired cues cause conditioned avoidance and only STN stimulation interrupts on-going sugar self-administration. Electrophysiological recordings identify post-synaptic responses in pallidal neurons, and selective photostimulation of STN terminals in the ventral pallidum replicates STN-induced aversion. Identifying STN as a source of aversive learning contributes neurobiological underpinnings to emotional affect.
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Estimulación Encefálica Profunda , Enfermedad de Parkinson , Trastornos Parkinsonianos , Núcleo Subtalámico , Animales , Ratones , Reacción de Prevención , Estimulación Encefálica Profunda/métodos , Enfermedad de Parkinson/terapiaRESUMEN
Effective neural stimulation for the treatment of severe psychiatric disorders needs accurate characterisation of surgical targets. This is especially true for the medial subthalamic region (MSR) which contains three targets: the anteromedial STN for obsessive compulsive disorder (OCD), the medial forebrain bundle (MFB) for depression and OCD, and the "Sano triangle" for pathological aggressiveness. Blocks containing the subthalamic area were obtained from two human brains. After obtaining 11.7-Tesla MRI, blocks were cut in regular sections for immunohistochemistry. Fluorescent in situ hybridisation was performed on the macaque MSR. Electron microscopic observation for synaptic specialisation was performed on human and macaque subthalamic fresh samples. Images of human brain sections were reconstructed in a cryoblock which was registered on the MRI and histological slices were then registered. The STN contains glutamatergic and fewer GABAergic neurons and has no strict boundary with the adjacent MSR. The anteromedial STN has abundant dopaminergic and serotoninergic innervation with very sparse dopaminergic neurons. The MFB is composed of dense anterior dopaminergic and posterior serotoninergic fibres, and fewer cholinergic and glutamatergic fibres. Medially, the Sano triangle presumably contains orexinergic terminals from the hypothalamus, and neurons with strong nuclear oestrogen receptor-alpha staining with a decreased anteroposterior and mediolateral gradient of staining. These findings provide new insight regarding MSR cells and their fibre specialisation, forming a transition zone between the basal ganglia and the limbic systems. Our 3D reconstruction enabled us to visualize the main histological features of the three targets which should enable better targeting and understanding of neuromodulatory stimulation results in severe psychiatric conditions.
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Ganglios Basales , Sistema Límbico , Humanos , Animales , Encéfalo , Haz Prosencefálico Medial , Dopamina , MacacaRESUMEN
Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.
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The mesostriatal dopamine (DA) system contributes to several aspects of responses to rewarding substances and is implicated in conditions such as drug addiction and eating disorders. A subset of DA neurons has been shown to express the type 2 Vesicular glutamate transporter (Vglut2) and may therefore corelease glutamate. In the present study, we analyzed mice with a conditional deletion of Vglut2 in DA neurons (Vglut2(f/f;DAT-Cre)) to address the functional significance of the glutamate-DA cophenotype for responses to cocaine and food reinforcement. Biochemical parameters of striatal DA function were also examined by using DA receptor autoradiography, immediate-early gene quantitative in situ hybridization after cocaine challenge, and DA-selective in vivo chronoamperometry. Mice in which Vglut2 expression had been abrogated in DA neurons displayed enhanced operant self-administration of both high-sucrose food and intravenous cocaine. Furthermore, cocaine seeking maintained by drug-paired cues was increased by 76%, showing that reward-dependent plasticity is perturbed in these mice. In addition, several lines of evidence suggest that adaptive changes occurred in both the ventral and dorsal striatum in the absence of VGLUT2: DA receptor binding was increased, and basal mRNA levels of the DA-induced early genes Nur77 and c-fos were elevated as after cocaine induction. Furthermore, in vivo challenge of the DA system by potassium-evoked depolarization revealed less DA release in both striatal areas. This study demonstrates that absence of VGLUT2 in DA neurons leads to perturbations of reward consumption as well as reward-associated memory, features of particular relevance for addictive-like behavior.
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Conducta Adictiva/fisiopatología , Cocaína/administración & dosificación , Señales (Psicología) , Inhibidores de Captación de Dopamina/administración & dosificación , Dopamina/metabolismo , Mesencéfalo/citología , Neuronas/metabolismo , Sacarosa/administración & dosificación , Proteína 2 de Transporte Vesicular de Glutamato/deficiencia , Análisis de Varianza , Animales , Autorradiografía , Conducta Adictiva/genética , Conducta Animal , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/inmunología , Condicionamiento Operante/efectos de los fármacos , Condicionamiento Operante/fisiología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Técnicas Electroquímicas/métodos , Preferencias Alimentarias/efectos de los fármacos , Preferencias Alimentarias/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Cloruro de Potasio/farmacología , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Receptores Dopaminérgicos/metabolismo , Esquema de Refuerzo , Recompensa , Autoadministración/métodosRESUMEN
Despite the increasing use of genetically modified mice to investigate the dopamine (DA) system, little is known about the ultrastructural features of the striatal DA innervation in the mouse. This issue is particularly relevant in view of recent evidence for expression of the vesicular glutamate transporter 2 (VGLUT2) by a subset of mesencephalic DA neurons in mouse as well as rat. We used immuno-electron microscopy to characterize tyrosine hydroxylase (TH)-labeled terminals in the core and shell of nucleus accumbens and the neostriatum of two mouse lines in which the Vglut2 gene was selectively disrupted in DA neurons (cKO), their control littermates, and C57BL/6/J wild-type mice, aged P15 or adult. The three regions were also examined in cKO mice and their controls of both ages after dual TH-VGLUT2 immunolabeling. Irrespective of the region, age and genotype, the TH-immunoreactive varicosities appeared similar in size, vesicular content, percentage with mitochondria, and exceedingly low frequency of synaptic membrane specialization. No dually labeled axon terminals were found at either age in control or in cKO mice. Unless TH and VGLUT2 are segregated in different axon terminals of the same neurons, these results favor the view that the glutamatergic cophenotype of mesencephalic DA neurons is more important during the early development of these neurons than for the establishment of their scarce synaptic connectivity. They also suggest that, in mouse even more than rat, the mesostriatal DA system operates mainly through non-targeted release of DA, diffuse transmission and the maintenance of an ambient DA level.
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Cuerpo Estriado/citología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/deficiencia , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/ultraestructura , Núcleo Accumbens/citología , Proteína 2 de Transporte Vesicular de Glutamato/deficiencia , Factores de Edad , Animales , Animales Recién Nacidos , Cuerpo Estriado/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica , Núcleo Accumbens/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Tirosina 3-Monooxigenasa/ultraestructuraRESUMEN
The subthalamic nucleus (STN) is critical for the execution of intended movements. Loss of its normal function is strongly associated with several movement disorders, including Parkinson's disease for which the STN is an important target area in deep brain stimulation (DBS) therapy. Classical basal ganglia models postulate that two parallel pathways, the direct and indirect pathways, exert opposing control over movement, with the STN acting within the indirect pathway. The STN is regulated by both inhibitory and excitatory input, and is itself excitatory. While most functional knowledge of this clinically relevant brain structure has been gained from pathological conditions and models, primarily parkinsonian, experimental evidence for its role in normal motor control has remained more sparse. The objective here was to tease out the selective impact of the STN on several motor parameters required to achieve intended movement, including locomotion, balance and motor coordination. Optogenetic excitation and inhibition using both bilateral and unilateral stimulations of the STN were implemented in freely-moving mice. The results demonstrate that selective optogenetic inhibition of the STN enhances locomotion while its excitation reduces locomotion. These findings lend experimental support to basal ganglia models of the STN in terms of locomotion. In addition, optogenetic excitation in freely-exploring mice induced self-grooming, disturbed gait and a jumping/escaping behavior, while causing reduced motor coordination in advanced motor tasks, independent of grooming and jumping. This study contributes experimentally validated evidence for a regulatory role of the STN in several aspects of motor control.
Asunto(s)
Locomoción/fisiología , Corteza Motora/fisiología , Vías Nerviosas/fisiología , Núcleo Subtalámico/fisiología , Animales , Ganglios Basales/fisiología , Estimulación Encefálica Profunda/métodos , Ratones Endogámicos C57BL , Neuronas/fisiología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatologíaRESUMEN
DNA methylation and gene expression can be altered by early life stress (ELS) and/or ethanol consumption. The present study aimed to investigate whether DNA methylation of the Vesicular Glutamate Transporters (Vglut)1-3 is related to previously observed Vglut1-3 transcriptional differences in the ventral tegmental area (VTA), nucleus accumbens (Acb), dorsal striatum (dStr) and medial prefrontal cortex (mPFC) of adult rats exposed to ELS, modelled by maternal separation, and voluntary ethanol consumption. Targeted next-generation bisulfite sequencing was performed to identify the methylation levels on 61 5'-cytosine-phosphate-guanosine-3' sites (CpGs) in potential regulatory regions of Vglut1, 53 for Vglut2, and 51 for Vglut3. In the VTA, ELS in ethanol-drinking rats was associated with Vglut1-2 CpG-specific hypomethylation, whereas bidirectional Vglut2 methylation differences at single CpGs were associated with ELS alone. Exposure to both ELS and ethanol, in the Acb, was associated with lower promoter and higher intronic Vglut3 methylation; and in the dStr, with higher and lower methylation in 26% and 43% of the analyzed Vglut1 CpGs, respectively. In the mPFC, lower Vglut2 methylation was observed upon exposure to ELS or ethanol. The present findings suggest Vglut1-3 CpG-specific methylation signatures of ELS and ethanol drinking, underlying previously reported Vglut1-3 transcriptional differences in the mesocorticolimbic brain.