RESUMO
Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Autofagia Mediada por Chaperonas/fisiologia , Neurônios/metabolismo , Proteostase , Envelhecimento/patologia , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Caseína Quinase I/genética , Autofagia Mediada por Chaperonas/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Neurônios/patologia , ProteomaRESUMO
α-Synuclein is expressed at high levels at presynaptic terminals, but defining its role in the regulation of neurotransmission under physiologically relevant conditions has proven elusive. We report that, in vivo, α-synuclein is responsible for the facilitation of dopamine release triggered by action potential bursts separated by short intervals (seconds) and a depression of release with longer intervals between bursts (minutes). These forms of presynaptic plasticity appear to be independent of the presence of ß- and γ-synucleins or effects on presynaptic calcium and are consistent with a role for synucleins in the enhancement of synaptic vesicle fusion and turnover. These results indicate that the presynaptic effects of α-synuclein depend on specific patterns of neuronal activity.
Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismo , Anestésicos Inalatórios/farmacologia , Animais , Sinalização do Cálcio , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Feminino , Isoflurano/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurotransmissores/metabolismo , Substância Negra/citologia , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/genética , gama-Sinucleína/metabolismoRESUMO
Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. The impact of these highly lipophilic sensors has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a nongenetic molecular platform for cell- and molecule-specific targeting of synthetic VSDs in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of VSDs by dynamic encapsulation and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.
Assuntos
Encéfalo , Cocaína/análogos & derivados , Dopamina/análise , Corantes Fluorescentes/química , Norepinefrina/análise , Animais , Encéfalo/citologia , Cocaína/síntese química , Cocaína/química , Corantes Fluorescentes/síntese química , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Estrutura Molecular , Imagem ÓpticaRESUMO
Altered prefrontal cortex function is implicated in schizophrenia (SCZ) pathophysiology and could arise from imbalance between excitation and inhibition (E/I) in local circuits. It remains unclear whether and how such imbalances relate to genetic etiologies. We used a mouse model of the SCZ-predisposing 22q11.2 deletion (Df(16)A+/- mice) to evaluate how this genetic lesion affects the excitability of layer V prefrontal pyramidal neurons and its modulation by dopamine (DA). Df(16)A+/- mice have normal balance between E/I at baseline but are unable to maintain it upon dopaminergic challenge. Specifically, in wild-type mice, D1 receptor (D1R) activation enhances excitability of layer V prefrontal pyramidal neurons and D2 receptor (D2R) activation reduces it. Whereas the excitatory effect upon D1R activation is enhanced in Df(16)A+/- mice, the inhibitory effect upon D2R activation is reduced. The latter is partly due to the inability of mutant mice to activate GABAergic parvalbumin (PV)+ interneurons through D2Rs. We further demonstrate that reduced KCNQ2 channel function in PV+ interneurons in Df(16)A+/- mice renders them less capable of inhibiting pyramidal neurons upon D2 modulation. Thus, DA modulation of PV+ interneurons and control of E/I are altered in Df(16)A+/- mice with a higher excitation and lower inhibition during dopaminergic modulation.
Assuntos
Dopamina/metabolismo , Interneurônios/fisiologia , Canal de Potássio KCNQ2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Córtex Pré-Frontal/fisiopatologia , Esquizofrenia/genética , Esquizofrenia/fisiopatologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Córtex Pré-Frontal/metabolismo , Células Piramidais/fisiologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Esquizofrenia/metabolismoRESUMO
Dopamine neurotransmission in the dorsal hippocampus is critical for a range of functions from spatial learning and synaptic plasticity to the deficits underlying psychiatric disorders such as attention-deficit hyperactivity disorder. The ventral tegmental area (VTA) is the presumed source of dopamine in the dorsal hippocampus. However, there is a surprising scarcity of VTA dopamine axons in the dorsal hippocampus despite the dense network of dopamine receptors. We have explored this apparent paradox using optogenetic, biochemical, and behavioral approaches and found that dopaminergic axons and subsequent dopamine release in the dorsal hippocampus originate from neurons of the locus coeruleus (LC). Photostimulation of LC axons produced an increase in dopamine release in the dorsal hippocampus as revealed by high-performance liquid chromatography. Furthermore, optogenetically induced release of dopamine from the LC into the dorsal hippocampus enhanced selective attention and spatial object recognition via the dopamine D1/D5 receptor. These results suggest that spatial learning and memory are energized by the release of dopamine in the dorsal hippocampus from noradrenergic neurons of the LC. The present findings are critical for identifying the neural circuits that enable proper attention selection and successful learning and memory.
Assuntos
Dopamina/fisiologia , Hipocampo/fisiologia , Memória , Aprendizagem Espacial , Animais , Axônios/fisiologia , Comportamento Animal , Locus Cerúleo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Optogenética , Transmissão Sináptica/fisiologiaRESUMO
1-Methyl-4-phenylpyridinium (MPP(+)), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP(+) exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DAcyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP(+) exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP(+) concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP(+) depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DAcyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP(+)-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DAcyt levels in MPP(+)-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in â¼30% reduction of MPP(+)-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP(+) on neuronal DA homeostasis and neurotoxicity.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurotoxinas/toxicidade , Animais , Células Cultivadas , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Homeostase/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Understanding the impact of long-term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency. However, this has been limited by human brain inaccessibility and cross-species differences in animal models. Here, a human midbrain model is established that uses hiPSC-derived midbrain organoids to assess cell-type-specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single-cell mRNA sequencing of 25,510 cells from organoids in different treatment groups reveals that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single-cell level.
Assuntos
Analgésicos Opioides , Mesencéfalo , Organoides , Humanos , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Analgésicos Opioides/farmacologia , Fentanila/farmacologia , Neurogênese/efeitos dos fármacosRESUMO
In Parkinson's disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA-/- neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.
RESUMO
Ethanol is a wildly abused substance that causes various problems and damage in our society. We examined the connection between the action of ethanol and the endocannabinoid system in corticostriatal synaptic transmission by recording excitatory post-synaptic currents (EPSCs). Acute treatment of ethanol (100 mM) inhibited corticostriatal EPSCs. In the presence of AM 251 (5 µM), a cannabinoid 1 (CB(1))-receptor antagonist, or AM 404 (5 µM), a cannabinoid transporter inhibitor, the inhibition of corticostriatal EPSCs caused by ethanol was significantly reduced. This result suggests the possibility that the endocannabinoid system is involved in the action of ethanol. To support this result, brain slices were pre-treated with WIN 55,212-2 (1 µM), a CB(1)-receptor agonist, following treatment of ethanol or treated with WIN 55,212-2 alone. There was no significant difference between each other, indicating that when CB(1) receptors are previously activated, the effect of ethanol is blunted. These results suggest that the activation of the endocannabinoid system is one of the possible mechanisms involved in ethanol-induced corticostriatal synaptic depression.
Assuntos
Encéfalo/efeitos dos fármacos , Etanol/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Receptor CB1 de Canabinoide/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Benzoxazinas/farmacologia , Encéfalo/fisiologia , Canabinoides/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Morfolinas/farmacologia , Naftalenos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidoresRESUMO
Optical imaging of changes in the membrane potential of living cells can be achieved by means of fluorescent voltage-sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer-supported probes for chemical, nongenetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring into the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with nongenetically targeted VSDs in native brain tissue.
Assuntos
Neurônios Dopaminérgicos , Corantes Fluorescentes , Animais , Corantes Fluorescentes/química , Potenciais da Membrana/fisiologia , Camundongos , Polímeros , XantonasRESUMO
Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell-type specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find that most proteins encoded by DA neuron-enriched genes are localized within striatal dopaminergic axons, including ion channels with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.
Assuntos
Encéfalo/citologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Neurônios Dopaminérgicos/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Proteômica , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Feminino , Masculino , Camundongos , Enzimas Multifuncionais/genética , Sinaptossomos/metabolismoRESUMO
P2X4 receptors are calcium-permeable cation channels gated by extracellular ATP. They are found close to subsynaptic sites on hippocampal CA1 neurons. We compared features of synaptic strengthening between wild-type and P2X4 knockout mice (21-26 days old). Potentiation evoked by a tetanic presynaptic stimulus (100 Hz, 1 s) paired with postsynaptic depolarization was less in P2X4(-/-) mice than in wild-type mice (230 vs. 50% potentiation). Paired-pulse ratios and the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) were not different between wild-type and knockout mice. Prior hyperpolarization (ten 3 s pulses to -120 mV at 0.17 Hz) potentiated the amplitude of spontaneous EPSCs in wild-type mice, but not in P2X4(-/-) mice; this potentiation was not affected by nifedipine, but was abolished by 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) in the recording pipette. The amplitude of N-methyl-d-aspartate EPSCs (in 6-cyano-7-nitroquinoxaline-2,3-dione, 10 or 30 µm, at -100 mV) facilitated during 20 min recording in magnesium-free solution. In wild-type mice, this facilitation of the N-methyl-d-aspartate EPSC was reduced by about 50% by intracellular BAPTA (10 mM), ifenprodil (3 µm) or 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 µm). In P2X4(-/-) mice, the facilitation was much less, and was unaffected by intracellular BAPTA, ifenprodil (3 µm) or mitogen-activated protein (MAP) kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 µm). This suggests that the absence of P2X4 receptors limits the incorporation of NR2B subunits into synaptic N-methyl-d-aspartate receptors.
Assuntos
Região CA1 Hipocampal/citologia , Neurônios/fisiologia , Receptores Purinérgicos P2X4/metabolismo , Sinapses/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos P2X4/genética , Bloqueadores dos Canais de Sódio/farmacologia , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tetrodotoxina/farmacologiaRESUMO
Proanthocyanidin is a phenolic compound present in plants, that has antioxidant, antinociceptive, anti-emetic, and neuroprotective properties. We investigated the actions of proanthocyanidin from grape seeds on 5-hydroxytryptamine (5-HT)(3) receptors in NCB-20 neuroblastoma cells using a whole-cell voltage clamp technique. Co-treatment of proanthocyanidin (0.3-100 µg/ml) and 3 µM 5-HT (near EC(50)) produced a slight inhibition of 5-HT-induced inward peak current (I(5-HT)) in NCB-20 cells, but pretreatment with proanthocyanidin for 30 s before application of 5-HT induced a much larger inhibition of I(5-HT) in an irreversible, concentration- and time-dependent manner (IC(50)=6.5±0.4 µg/ml, Hill coefficient=2.5±0.1). Proanthocyanidin also produced a concentration-dependent inhibition of currents induced by 30 µM 5-HT, near-maximal concentration (IC(50)=22.1±0.4 µg/ml, Hill coefficient=2.4±0.1). High concentrations (â§30 µg/ml) of proanthocyanidin caused a concentration-dependent inhibition of the activation and desensitization of currents induced by 30 µM 5-HT. Further studies showed that pretreatment of 20 µg/ml proanthocyanidin caused not only a rightward shift of the dose-response curve for 5-HT (EC(50) shift from 2.7±0.4 to 6.2±0.5 µM), but also a decreased E(max) (inhibition by 37.5±1.3%). The proanthocyanidin-induced inhibition of 5-HT(3) receptors did not show a significant difference within the testing holding potential ranges (-50-+30 mV). These results suggest that proanthocyanidin inhibits 5-HT(3) receptor function in NCB-20 cells in a noncompetitive mode, and that this inhibitory effect of proanthocyanidin probably contributes to the pharmacological actions of proanthocyanidin.
Assuntos
Proantocianidinas/farmacologia , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Sementes/química , Vitis/química , Linhagem Celular Tumoral , Humanos , Antagonistas da Serotonina/farmacologia , Vitis/embriologiaRESUMO
α-Synuclein (α-Syn) is a small cytosolic protein associated with a range of cellular compartments, including synaptic vesicles, the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. In addition to its physiological role in regulating presynaptic function, the protein plays a central role in both sporadic and familial Parkinson's disease (PD) via a gain-of-function mechanism. Because of this, several recent strategies propose to decrease α-Syn levels in PD patients. While these therapies may offer breakthroughs in PD management, the normal functions of α-Syn and potential side effects of its depletion require careful evaluation. Here, we review recent evidence on physiological and pathological roles of α-Syn in regulating activity-dependent signal transduction and gene expression pathways that play fundamental role in synaptic plasticity.
Assuntos
Expressão Gênica/fisiologia , alfa-Sinucleína/genética , Humanos , Plasticidade Neuronal/fisiologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Transdução de Sinais/fisiologiaRESUMO
Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.
Assuntos
Neurônios Dopaminérgicos , Células-Tronco Embrionárias Humanas , Animais , Diferenciação Celular , Mesencéfalo , Ratos , Via de Sinalização WntRESUMO
Changes in striatal cholinergic interneuron (ChI) activity are thought to contribute to Parkinson's disease pathophysiology and dyskinesia from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, but the physiological basis of these changes is unknown. We find that dopamine lesion decreases the spontaneous firing rate of ChIs, whereas chronic treatment with L-DOPA of lesioned mice increases baseline ChI firing rates to levels beyond normal activity. The effect of dopamine loss on ChIs was due to decreased currents of both hyperpolarization-activated cyclic nucleotide-gated (HCN) and small conductance calcium-activated potassium (SK) channels. L-DOPA reinstatement of dopamine normalized HCN activity, but SK current remained depressed. Pharmacological blockade of HCN and SK activities mimicked changes in firing, confirming that these channels are responsible for the molecular adaptation of ChIs to dopamine loss and chronic L-DOPA treatment. These findings suggest that targeting ChIs with channel-specific modulators may provide therapeutic approaches for alleviating L-DOPA-induced dyskinesia in PD patients.
Assuntos
Neurônios Colinérgicos/fisiologia , Corpo Estriado/fisiologia , Dopamina/administração & dosagem , Interneurônios/fisiologia , Levodopa/administração & dosagem , Animais , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
RATIONALE: GLT-1 is the major glutamate transporter in the brain and is expressed predominantly in astrocytes but is also present in excitatory axon terminals. To understand the functional significance of GLT-1 expressed in neurons, we generated a conditional GLT-1 knockout mouse and inactivated GLT-1 in neurons using Cre-recombinase expressed under the synapsin 1 promoter, (synGLT-1 KO). OBJECTIVES: Abnormalities of glutamate homeostasis have been shown to affect hippocampal-related behaviors including learning and memory as well as responses to drugs of abuse. Here, we asked whether deletion of GLT-1 specifically from neurons would affect behaviors that assessed locomotor activity, cognitive function, sensorimotor gating, social interaction, as well as amphetamine-stimulated locomotor activity. METHODS/RESULTS: We found that the neuronal GLT-1 KO mice performed similarly to littermate controls in the behavioral tests we studied. Although performance in open field testing was normal, the acute locomotor response to amphetamine was significantly blunted in the synGLT-1 KO (40% of control). We found no change in amphetamine-stimulated extracellular dopamine in the medial shell of the nucleus accumbens, no change in electrically stimulated or amphetamine-induced dopamine release, and no change in dopamine tissue content. CONCLUSIONS: These results support the view that GLT-1 expression in neurons is required for amphetamine-induced behavioral activation, and suggest that this phenotype is not produced through a change in dopamine uptake or release. Although GLT-1 is highly expressed in neurons in the CA3 region of the hippocampus, the tests used in this study were not able to detect a behavioral phenotype referable to hippocampal dysfunction.
Assuntos
Anfetamina/farmacologia , Dopamina/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Deleção de Genes , Locomoção/fisiologia , Neurônios/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/deficiência , Transportador 2 de Aminoácido Excitatório/genética , Medo/efeitos dos fármacos , Medo/fisiologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Relações Interpessoais , Locomoção/efeitos dos fármacos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , FenótipoRESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra (SN). Although mitochondrial dysfunction and dysregulated α-synuclein (aSyn) expression are postulated to play a role in PD pathogenesis, it is still debated why neurons of the SN are targeted while neighboring dopaminergic neurons of the ventral tegmental area (VTA) are spared. Using electrochemical and imaging approaches, we investigated metabolic changes in cultured primary mouse midbrain dopaminergic neurons exposed to a parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). We demonstrate that the higher level of neurotoxicity in SN than VTA neurons was due to SN neuron-specific toxin-induced increase in cytosolic dopamine (DA) and Ca2+, followed by an elevation of mitochondrial Ca2+, activation of nitric oxide synthase (NOS), and mitochondrial oxidation. The increase in cytosolic Ca2+ was not caused by MPP+-induced oxidative stress, but rather depended on the activity of both L-type calcium channels and aSyn expression, suggesting that these two established pathogenic factors in PD act in concert.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , alfa-Sinucleína/metabolismo , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , CamundongosRESUMO
The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.
Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/fisiologia , Animais , Animais Geneticamente Modificados , Dextroanfetamina/farmacologia , Drosophila , Proteínas de Drosophila/metabolismo , Concentração de Íons de Hidrogênio , Locomoção/efeitos dos fármacos , Mesencéfalo/metabolismo , Camundongos , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
The purpose of this study was to examine the effects of ethanol on synaptic transmission in the dorsal striatum in rat brain slices. The effects of ethanol on corticostriatal synaptic transmission were tested by whole-cell voltage-clamp recording. Ethanol significantly decreased corticostriatal excitatory postsynaptic currents (EPSCs) in a dose-dependent manner (10-200 mM). However, the paired-pulse ratio was not affected by the ethanol (100 mM) treatment. The amplitude of miniature EPSCs (mEPSCs) from these neurons, recorded without cortical stimulation, was decreased, but the frequency of the mEPSCs remained unchanged. Ethanol also decreased currents induced by the local pressure injection of glutamate into dorsal striatal neurons. These results suggest that ethanol inhibits glutamatergic synaptic transmission in the dorsal striatum, possibly through a postsynaptic mechanism.