RESUMEN
Midbrain dopamine neurons, which can be regulated by neuropeptides and hormones, play a fundamental role in controlling cognitive processes, reward mechanisms, and motor functions. The hormonal actions of insulin-like growth factor 1 (IGF-1) produced by the liver have been well described, but the role of neuronally derived IGF-1 remains largely unexplored. We discovered that dopamine neurons secrete IGF-1 from the cell bodies following depolarization, and that IGF-1 controls release of dopamine in the ventral midbrain. In addition, conditional deletion of dopamine neuron-derived IGF-1 in adult mice leads to decrease of dopamine content in the striatum and deficits in dopamine neuron firing and causes reduced spontaneous locomotion and impairments in explorative and learning behaviors. These data identify that dopamine neuron-derived IGF-1 acts as a regulator of dopamine neurons and regulates dopamine-mediated behaviors.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Locomoción/genética , Mesencéfalo/fisiología , Animales , Cognición/fisiología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiología , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/fisiología , Conducta Exploratoria/fisiología , Hormonas/metabolismo , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Aprendizaje/fisiología , Locomoción/fisiología , Mesencéfalo/metabolismo , Ratones , Neuropéptidos/genéticaRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.
Asunto(s)
Envejecimiento/metabolismo , Antiparkinsonianos/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Levodopa/farmacología , Mutación , Enfermedad de Parkinson/genética , Potenciales de Acción , Envejecimiento/patología , Sustitución de Aminoácidos , Animales , Muerte Celular/genética , Cromosomas Artificiales Bacterianos/química , Cromosomas Artificiales Bacterianos/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Masculino , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Regiones Promotoras Genéticas , Dominios Proteicos , Ratas , Ratas Transgénicas , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
Midbrain dopaminergic (mDA) neurons are implicated in cognitive functions, neuropsychiatric disorders, and pathological conditions; hence understanding genes regulating their homeostasis has medical relevance. Transcription factors FOXA1 and FOXA2 (FOXA1/2) are key determinants of mDA neuronal identity during development, but their roles in adult mDA neurons are unknown. We used a conditional knockout strategy to specifically ablate FOXA1/2 in mDA neurons of adult mice. We show that deletion of Foxa1/2 results in down-regulation of tyrosine hydroxylase, the rate-limiting enzyme of dopamine (DA) biosynthesis, specifically in dopaminergic neurons of the substantia nigra pars compacta (SNc). In addition, DA synthesis and striatal DA transmission were reduced after Foxa1/2 deletion. Furthermore, the burst-firing activity characteristic of SNc mDA neurons was drastically reduced in the absence of FOXA1/2. These molecular and functional alterations lead to a severe feeding deficit in adult Foxa1/2 mutant mice, independently of motor control, which could be rescued by L-DOPA treatment. FOXA1/2 therefore control the maintenance of molecular and physiological properties of SNc mDA neurons and impact on feeding behavior in adult mice.
Asunto(s)
Dopamina/metabolismo , Conducta Alimentaria , Factor Nuclear 3-alfa del Hepatocito/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Neuronas/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Eliminación de Gen , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/genética , Ratones , Ratones Noqueados , Neuronas/citología , ARN Mensajero/genéticaRESUMEN
Corticostriatal regulation of striatal dopamine (DA) transmission has long been postulated, but ionotropic glutamate receptors have not been localized directly to DA axons. Striatal cholinergic interneurons (ChIs) are emerging as major players in striatal function, and can govern DA transmission by activating nicotinic receptors (nAChRs) on DA axons. Cortical inputs to ChIs have historically been perceived as sparse, but recent evidence indicates that they strongly activate ChIs. We explored whether activation of M1/M2 corticostriatal inputs can consequently gate DA transmission, via ChIs. We reveal that optogenetic activation of channelrhodopsin-expressing corticostriatal axons can drive striatal DA release detected with fast-scan cyclic voltammetry and requires activation of nAChRs on DA axons and AMPA receptors on ChIs that promote short-latency action potentials. By contrast, DA release driven by optogenetic activation of intralaminar thalamostriatal inputs involves additional activation of NMDA receptors on ChIs and action potential generation over longer timescales. Therefore, cortical and thalamic glutamate inputs can modulate DA transmission by regulating ChIs as gatekeepers, through ionotropic glutamate receptors. The different use of AMPA and NMDA receptors by cortical versus thalamic inputs might lead to distinct input integration strategies by ChIs and distinct modulation of the function of DA and striatum.
RESUMEN
The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 µm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade.
Asunto(s)
Corteza Cerebral/citología , Cuerpo Estriado/efectos de los fármacos , GMP Cíclico/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Biofisica , Cuerpo Estriado/citología , AMP Cíclico/metabolismo , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microdiálisis , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/genética , Ratas , Ratas Sprague-DawleyRESUMEN
The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice (SNCA-OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA-OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA-OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA-OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.
Asunto(s)
Envejecimiento/metabolismo , Cuerpo Estriado/metabolismo , Neuronas Dopaminérgicas/metabolismo , Trastornos Parkinsonianos/metabolismo , Sustancia Negra/metabolismo , Transmisión Sináptica , Envejecimiento/patología , Animales , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales Bacterianos/metabolismo , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Neuronas Dopaminérgicas/patología , Humanos , Ratones , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/fisiopatología , Sustancia Negra/patología , Sustancia Negra/fisiopatología , alfa-Sinucleína/biosíntesis , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5-10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca-/-) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca-/- animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca-/- mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca-/- mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD.
Asunto(s)
Ganglios Basales/metabolismo , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Norepinefrina/metabolismo , alfa-Sinucleína/genética , Factores de Edad , Animales , Cromosomas Artificiales Bacterianos , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Núcleos Septales/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Nicotine is the primary psychoactive component of tobacco. Its reinforcing and addictive properties depend on nicotinic acetylcholine receptors (nAChRs) located within the mesolimbic axis originating in the ventral tegmental area (VTA). The roles and oligomeric assembly of subunit α4- and subunit α6-containing nAChRs in dopaminergic (DAergic) neurons are much debated. Using subunit-specific knockout mice and targeted lentiviral re-expression, we have determined the subunit dependence of intracranial nicotine self-administration (ICSA) into the VTA and the effects of nicotine on dopamine (DA) neuron excitability in the VTA and on DA transmission in the nucleus accumbens (NAc). We show that the α4 subunit, but not the α6 subunit, is necessary for ICSA and nicotine-induced bursting of VTA DAergic neurons, whereas subunits α4 and α6 together regulate the activity dependence of DA transmission in the NAc. These data suggest that α4-dominated enhancement of burst firing in DA neurons, relayed by DA transmission in NAc that is gated by nAChRs containing α4 and α6 subunits, underlies nicotine self-administration and its long-term maintenance.
Asunto(s)
Neuronas/metabolismo , Nicotina/metabolismo , Receptores Nicotínicos/metabolismo , Área Tegmental Ventral/metabolismo , Análisis de Varianza , Animales , Autorradiografía , Dopamina/metabolismo , Electrofisiología , Vectores Genéticos/genética , Lentivirus , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotina/farmacología , Receptores Nicotínicos/genéticaRESUMEN
The synucleins (α, ß, and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signaling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4- to 14-month-old) animals using a combination of behavioral, biochemical, histological, and electrochemical techniques. Adult triple-synuclein-null (TKO) mice displayed no overt phenotype and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover, and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms that differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson's disease may ultimately inform on the selectivity of the disease process.
Asunto(s)
Cuerpo Estriado/fisiología , Sustancia Negra/fisiología , alfa-Sinucleína/deficiencia , Sinucleína beta/deficiencia , gamma-Sinucleína/deficiencia , Animales , Dopamina/fisiología , Masculino , Mesencéfalo/citología , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/clasificación , Neuronas/metabolismo , Neuronas/fisiología , Neurotransmisores/genética , Neurotransmisores/metabolismo , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , alfa-Sinucleína/genética , Sinucleína beta/genética , gamma-Sinucleína/genéticaRESUMEN
Striatal dopamine (DA) and acetylcholine (ACh) regulate motivated behaviors and striatal plasticity. Interactions between these neurotransmitters may be important, through synchronous changes in parent neuron activities and reciprocal presynaptic regulation of release. How DA signaling is regulated by striatal muscarinic receptors (mAChRs) is unresolved; contradictory reports indicate suppression or facilitation, implicating several mAChR subtypes on various neurons. We investigated whether mAChR regulation of DA signaling varies with presynaptic activity and identified the mAChRs responsible in sensorimotor- versus limbic-associated striatum. We detected DA in real time at carbon fiber microelectrodes in mouse striatal slices. Broad-spectrum mAChR agonists [oxotremorine-M, APET (arecaidine propargyl ester tosylate)] decreased DA release evoked by low-frequency stimuli (1-10 Hz, four pulses) but increased the sensitivity of DA release to presynaptic activity, even enhancing release by high frequencies (e.g., >25 Hz for four pulses). These bidirectional effects depended on ACh input to striatal nicotinic receptors (nAChRs) on DA axons but not GABA or glutamate input. In caudate-putamen (CPu), knock-out of M(2)- or M(4)-mAChRs (not M(5)) prevented mAChR control of DA, indicating that M(2)- and M(4)-mAChRs are required. In nucleus accumbens (NAc) core or shell, mAChR function was prevented in M(4)-knock-outs, but not M(2)- or M(5)-knock-outs. These data indicate that striatal mAChRs, by inhibiting ACh release from cholinergic interneurons and thus modifying nAChR activity, offer variable control of DA release probability that promotes how DA release reflects activation of dopaminergic axons. Furthermore, different coupling of striatal M(2)/M(4)-mAChRs to the control of DA release in CPu versus NAc suggests targets to influence DA/ACh function differentially between striatal domains.
Asunto(s)
Acetilcolina/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Interneuronas/metabolismo , Receptores Muscarínicos/metabolismo , Transmisión Sináptica/fisiología , Animales , Ganglios Basales/citología , Ganglios Basales/efectos de los fármacos , Ganglios Basales/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Electrofisiología , Interneuronas/citología , Interneuronas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Muscarínicos/farmacología , Neostriado/citología , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Núcleo Accumbens/citología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M4/efectos de los fármacos , Receptor Muscarínico M4/genética , Receptor Muscarínico M4/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/genética , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Striatal dopamine transporters (DAT) powerfully regulate dopamine signaling, and can contribute risk to degeneration in Parkinson's disease (PD). DATs can interact with the neuronal protein α-synuclein, which is associated with the etiology and molecular pathology of idiopathic and familial PD. Here, we tested whether DAT function in governing dopamine (DA) uptake and release is modified in a human-α-synuclein-overexpressing (SNCA-OVX) transgenic mouse model of early PD. Using fast-scan cyclic voltammetry (FCV) in ex vivo acute striatal slices to detect DA release, and biochemical assays, we show that several aspects of DAT function are promoted in SNCA-OVX mice. Compared to background control α-synuclein-null mice (Snca-null), the SNCA-OVX mice have elevated DA uptake rates, and more pronounced effects of DAT inhibitors on evoked extracellular DA concentrations ([DA]o) and on short-term plasticity (STP) in DA release, indicating DATs play a greater role in limiting DA release and in driving STP. We found that DAT membrane levels and radioligand binding sites correlated with α-synuclein level. Furthermore, DAT function in Snca-null and SNCA-OVX mice could also be promoted by applying cholesterol, and using Tof-SIMS we found genotype-differences in striatal lipids, with lower striatal cholesterol in SNCA-OVX mice. An inhibitor of cholesterol efflux transporter ABCA1 or a cholesterol chelator in SNCA-OVX mice reduced the effects of DAT-inhibitors on evoked [DA]o. Together these data indicate that human α-synuclein in a mouse model of PD promotes striatal DAT function, in a manner supported by extracellular cholesterol, suggesting converging biology of α-synuclein and cholesterol that regulates DAT function and could impact DA function and PD pathophysiology.
RESUMEN
Striatal dopamine (DA) is critical for action and learning. Recent data show that DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be determined by plasma membrane GABA uptake transporters (GATs) located on astrocytes and neurons. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs are downregulated, tonic GABAergic inhibition of DA release augmented, and nigrostriatal GABA co-release attenuated. These data define previously unappreciated and important roles for GATs and astrocytes in supporting DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Regulación hacia Abajo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Trastornos Parkinsonianos/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Astrocitos/metabolismo , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Glutamato Descarboxilasa/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Núcleo Accumbens/metabolismoRESUMEN
Parkinson's disease (PD) affects millions of patients worldwide and is characterized by alpha-synuclein aggregation in dopamine neurons. Molecular tweezers have shown high potential as anti-aggregation agents targeting positively charged residues of proteins undergoing amyloidogenic processes. Here we report that the molecular tweezer CLR01 decreased aggregation and toxicity in induced pluripotent stem cell-derived dopaminergic cultures treated with PD brain protein extracts. In microfluidic devices CLR01 reduced alpha-synuclein aggregation in cell somas when axonal terminals were exposed to alpha-synuclein oligomers. We then tested CLR01 in vivo in a humanized alpha-synuclein overexpressing mouse model; mice treated at 12 months of age when motor defects are mild exhibited an improvement in motor defects and a decreased oligomeric alpha-synuclein burden. Finally, CLR01 reduced alpha-synuclein-associated pathology in mice injected with alpha-synuclein aggregates into the striatum or substantia nigra. Taken together, these results highlight CLR01 as a disease-modifying therapy for PD and support further clinical investigation.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/administración & dosificación , Neuronas Dopaminérgicas/efectos de los fármacos , Organofosfatos/administración & dosificación , Enfermedad de Parkinson/tratamiento farmacológico , Sustancias Protectoras/administración & dosificación , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Humanos , Masculino , Ratones , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Agregado de Proteínas/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
The cyclic nucleotide phosphodiesterase 10A (PDE10A) is highly expressed in striatal medium-sized spiny projection neurons (MSNs), apparently playing a critical role in the regulation of both cGMP and cAMP signaling cascades. Genetic disruption or pharmacological inhibition of PDE10A reverses behavioral abnormalities associated with subcortical hyperdopaminergia. Here, we investigate the effect of PDE10A inhibition on the activity of MSNs using single-unit extracellular recordings performed in the dorsal striatum of anesthetized rats. Antidromic stimulation of the substantia nigra pars reticulata was used to identify striatonigral (SNr+) MSNs. Intrastriatal infusion of the selective PDE10A inhibitors papaverine or TP-10 [2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid] by reverse microdialysis did not affect spontaneous firing but robustly increased measures of cortically evoked spike activity in a stimulus intensity-dependent manner. Systemic administration of TP-10 also increased cortically evoked spike activity in a stimulus intensity- and dose-dependent manner. A robust increase in cortically evoked activity was apparent in SNr- MSNs (primarily striatopallidal). It is interesting that TP-10 administration did not affect cortically evoked activity in SNr+ MSNs. However, TP-10 administration increased the incidence of antidromically activated (i.e., SNr+) MSNs. These findings indicate that inhibition of striatal PDE10A activity increases the responsiveness of MSNs to depolarizing stimuli. Furthermore, given the lack of effect of TP-10 on SNr+ MSNs, we speculate that PDE10A inhibition may have a greater facilitatory effect on corticostriatal synaptic activity in striatopallidal MSNs. These data support further investigation of selective targeting of PDE signaling pathways in MSN subpopulations because this may represent a promising novel approach for treating brain disorders involving dysfunctional glutamatergic and dopaminergic neurotransmission.
Asunto(s)
Cuerpo Estriado/fisiología , Neuronas/fisiología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Estimulación Eléctrica , Electrofisiología/métodos , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/fisiología , Globo Pálido/efectos de los fármacos , Globo Pálido/fisiología , Masculino , Microdiálisis , Neuronas/efectos de los fármacos , Papaverina/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/fisiologíaRESUMEN
Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 or TSC2, which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. Here we use cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. We show that loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor. Our results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC.
Asunto(s)
Cognición/fisiología , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Animales , Axones/patología , Conducta Animal , Cuerpo Celular/patología , Cuerpo Estriado/patología , Neuronas Dopaminérgicas/patología , Técnicas de Inactivación de Genes , Hipertrofia , Ratones , Motivación , Plasticidad Neuronal/genética , Transducción de Señal , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/psicología , Proteína 1 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Mesostriatal dopaminergic neurons possess extensively branched axonal arbours. Whether action potentials are converted to dopamine output in the striatum will be influenced dynamically and critically by axonal properties and mechanisms that are poorly understood. Here, we address the roles for mechanisms governing release probability and axonal activity in determining short-term plasticity of dopamine release, using fast-scan cyclic voltammetry in the ex vivo mouse striatum. We show that brief short-term facilitation and longer short term depression are only weakly dependent on the level of initial release, i.e. are release insensitive. Rather, short-term plasticity is strongly determined by mechanisms which govern axonal activation, including K+-gated excitability and the dopamine transporter, particularly in the dorsal striatum. We identify the dopamine transporter as a master regulator of dopamine short-term plasticity, governing the balance between release-dependent and independent mechanisms that also show region-specific gating.
Asunto(s)
Axones/metabolismo , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Animales , Transporte Biológico , Inhibidores de Captación de Dopamina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad Neuronal/fisiologíaRESUMEN
Parkinson's disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta (SNc) and accumulation of α-synuclein. Impaired autophagy has been implicated and activation of autophagy proposed as a treatment strategy. We generate a human α-synuclein-expressing mouse model of PD with macroautophagic failure in dopamine neurons to understand the interaction between impaired macroautophagy and α-synuclein. We find that impaired macroautophagy generates p62-positive inclusions and progressive neuron loss in the SNc. Despite this parkinsonian pathology, motor phenotypes accompanying human α-synuclein overexpression actually improve with impaired macroautophagy. Real-time fast-scan cyclic voltammetry reveals that macroautophagy impairment in dopamine neurons increases evoked extracellular concentrations of dopamine, reduces dopamine uptake, and relieves paired-stimulus depression. Our findings show that impaired macroautophagy paradoxically enhances dopamine neurotransmission, improving movement while worsening pathology, suggesting that changes to dopamine synapse function compensate for and conceal the underlying PD pathogenesis, with implications for therapies that target autophagy.
Asunto(s)
Autofagia , Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Dopamina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Transmisión Sináptica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
The substantia nigra pars reticulata (SNr) forms a principal output from the basal ganglia. It also receives significant histamine (HA) input from the tuberomammillary nucleus whose functions in SNr remain poorly understood. One identified role is the regulation of serotonin (5-HT) neurotransmission via the HA-H(3) receptor. Here we have explored regulation by another HA receptor expressed in SNr, the H(2)-receptor (H(2)R), by monitoring electrically evoked 5-HT release with fast-scan cyclic voltammetry at carbon-fiber microelectrodes in SNr in rat brain slices. Selective H(2)R antagonists (inverse agonists) ranitidine and tiotidine enhanced 5-HT release while the agonist amthamine suppressed release. The 'neutral' competitive antagonist burimamide alone was without effect but prevented ranitidine actions indicating that inverse agonist effects result from constitutive H(2)R activity independent of HA tone. H(2)R control of 5-HT release was most apparent (from inverse agonist effects) at lower frequencies of depolarization (< or = 20 Hz), and prevailed in the presence of antagonists of GABA, glutamate or H(3)-HA receptors. These data reveal that H(2)Rs in SNr are constitutively active and inhibit 5-HT release through H(2)Rs on 5-HT axons. These data may have therapeutic implications for Parkinson's disease, when SNr HA levels increase, and for neuropsychiatric disorders in which 5-HT is pivotal.
Asunto(s)
Neuronas/metabolismo , Receptores Histamínicos H2/metabolismo , Serotonina/metabolismo , Sustancia Negra/metabolismo , Animales , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Masculino , Microelectrodos , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores Histamínicos H2/efectos de los fármacos , Sustancia Negra/efectos de los fármacosRESUMEN
The gaseous neurotransmitter nitric oxide plays an important role in the modulation of corticostriatal synaptic transmission. This study examined the impact of frontal cortex stimulation on striatal nitric oxide efflux and neuron activity in urethane-anesthetized rats using amperometric microsensor and single-unit extracellular recordings, respectively. Systemic administration of the neuronal nitric oxide synthase inhibitor 7-nitroindazole decreased spontaneous spike activity without affecting activity evoked by single-pulse stimulation of the ipsilateral cortex. Train (30 Hz) stimulation of the contralateral frontal cortex transiently increased nitric oxide efflux in a robust and reproducible manner. Evoked nitric oxide efflux was attenuated by systemic administration of 7-nitroindazole and the non-selective nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester. Train stimulation of the contralateral cortex, in a manner identical to that used to evoke nitric oxide efflux, had variable effects on spike activity assessed during the train stimulation trial, but induced a short-term depression of cortically evoked activity in the first post-train stimulation trial. Interestingly, 7-nitroindazole potently decreased cortically evoked activity recorded during the train stimulation trial. Moreover, the short-term depression of spike activity induced by train stimulation was enhanced following pretreatment with 7-nitroindazole and attenuated after systemic administration of the dopamine D2 receptor antagonist eticlopride. These results demonstrate that robust activation of frontal cortical afferents in the intact animal activates a powerful nitric oxide-mediated feed-forward excitation which partially offsets concurrent D2 receptor-mediated short-term inhibitory influences on striatal neuron activity. Thus, nitric oxide signaling is likely to play an important role in the integration of corticostriatal sensorimotor information in striatal networks.
Asunto(s)
Corteza Cerebral/fisiología , Cuerpo Estriado/fisiología , Retroalimentación Fisiológica/fisiología , Neuronas/fisiología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Animales , Estimulación Eléctrica/métodos , Masculino , Vías Nerviosas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Striatal dopamine (DA) is a major player in action selection and reinforcement. DA release is under strong local control by striatal ACh acting at axonal nicotinic ACh receptors (nAChRs) on DA axons. Striatal nAChRs have been shown to control how DA is released in response to ascending activity from DA neurons, and they also directly drive DA release following synchronized activity in a small local cholinergic network. The source of striatal ACh has been thought to arise solely from intrinsic cholinergic interneurons (ChIs), but recent findings have identified a source of cholinergic inputs to striatum from brainstem nuclei, the pedunculopontine nucleus (PPN) and laterodorsal tegmentum (LDT). Here, we used targeted optogenetic activation alongside DA detection with fast-scan cyclic voltammetry to test whether ChIs alone and/or brainstem afferents to the striatum can account for how ACh drives and modulates DA release in rat striatum. We demonstrate that targeted transient light activation of rat striatal ChIs drives striatal DA release, corroborating and extending previous observations in mouse to rat. However, the same light stimulation targeted to cholinergic brainstem afferents did not drive DA release, and nor did it modulate DA release activated subsequently by electrical stimulation, whereas targeted activation of ChIs did so. We were unable to obtain any evidence for DA modulation by PPN/LDT stimulation. By contrast, we could readily identify that striatal ChIs alone are sufficient to provide a source of ACh that powerfully regulates DA via nAChRs.