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1.
Nat Commun ; 11(1): 5113, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037215

RESUMO

Striatal activity is dynamically modulated by acetylcholine and dopamine, both of which are essential for basal ganglia function. Synchronized pauses in the activity of striatal cholinergic interneurons (ChINs) are correlated with elevated activity of midbrain dopaminergic neurons, whereas synchronous firing of ChINs induces local release of dopamine. The mechanisms underlying ChIN synchronization and its interplay with dopamine release are not fully understood. Here we show that polysynaptic inhibition between ChINs is a robust network motif and instrumental in shaping the network activity of ChINs. Action potentials in ChINs evoke large inhibitory responses in multiple neighboring ChINs, strong enough to suppress their tonic activity. Using a combination of optogenetics and chemogenetics we show the involvement of striatal tyrosine hydroxylase-expressing interneurons in mediating this inhibition. Inhibition between ChINs is attenuated by dopaminergic midbrain afferents acting presynaptically on D2 receptors. Our results present a novel form of interaction between striatal dopamine and acetylcholine dynamics.


Assuntos
Neurônios Colinérgicos/metabolismo , Corpo Estriado/citologia , Interneurônios/metabolismo , Inibição Neural/fisiologia , Transmissão Sináptica/fisiologia , Acetilcolina/fisiologia , Animais , Condicionamento Clássico , Corpo Estriado/fisiologia , Dopamina , Feminino , Masculino , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Receptores de Dopamina D2/metabolismo , Recompensa
2.
J Vis Exp ; (163)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986023

RESUMO

Parkinson's disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Neurônios Dopaminérgicos/citologia , Mesencéfalo/citologia , Animais , Caspase 3/metabolismo , Células Cultivadas , Dependovirus/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Embrião de Mamíferos/citologia , Vetores Genéticos/metabolismo , Glutamatos/farmacologia , Processamento de Imagem Assistida por Computador , Camundongos , Quinoxalinas/farmacologia , Receptores de AMPA/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
3.
J Vis Exp ; (162)2020 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-32865527

RESUMO

The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson's disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Embrião de Mamíferos/citologia , Mesencéfalo/citologia , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animais , Citoesqueleto/metabolismo , Mesencéfalo/patologia , Camundongos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregados Proteicos
4.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831313

RESUMO

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6‒8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.


Assuntos
Técnicas de Cultura de Células/métodos , Córtex Cerebral/citologia , Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Automação , Dióxido de Carbono , Contagem de Células , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Mesencéfalo/citologia , Células-Tronco Neurais/citologia , Crescimento Neuronal , Interface Usuário-Computador
5.
PLoS One ; 15(7): e0223633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701951

RESUMO

BACKGROUND: Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. METHODS: We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. RESULTS: Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 µm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 µm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 µM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 µM, but not 100 µM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 µM 1-EBIO, and partially reduced by 100 µM 1-EBIO. CONCLUSION: The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/agonistas , Animais , Benzimidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/toxicidade
6.
Nat Commun ; 11(1): 3460, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651365

RESUMO

The learning of stimulus-outcome associations allows for predictions about the environment. Ventral striatum and dopaminergic midbrain neurons form a larger network for generating reward prediction signals from sensory cues. Yet, the network plasticity mechanisms to generate predictive signals in these distributed circuits have not been entirely clarified. Also, direct evidence of the underlying interregional assembly formation and information transfer is still missing. Here we show that phasic dopamine is sufficient to reinforce the distinctness of stimulus representations in the ventral striatum even in the absence of reward. Upon such reinforcement, striatal stimulus encoding gives rise to interregional assemblies that drive dopaminergic neurons during stimulus-outcome learning. These assemblies dynamically encode the predicted reward value of conditioned stimuli. Together, our data reveal that ventral striatal and midbrain reward networks form a reinforcing loop to generate reward prediction coding.


Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Tubérculo Olfatório/efeitos dos fármacos , Animais , Dopamina/farmacologia , Masculino , Mesencéfalo/citologia , Camundongos , Modelos Teóricos , Estriado Ventral/efeitos dos fármacos , Estriado Ventral/metabolismo
7.
Neuron ; 106(4): 637-648.e6, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32164875

RESUMO

Although the ventromedial hypothalamus ventrolateral area (VMHvl) is now well established as a critical locus for the generation of conspecific aggression, its role is complex, with neurons responding during multiple phases of social interactions with both males and females. It has been previously unclear how the brain uses this complex multidimensional signal and coordinates a discrete action: the attack. Here, we find a hypothalamic-midbrain circuit that represents hierarchically organized social signals during aggression. Optogenetic-assisted circuit mapping reveals a preferential projection from VMHvlvGlut2 to lPAGvGlut2 cells, and inactivation of downstream lPAGvGlut2 populations results in aggression-specific deficits. lPAG neurons are selective for attack action and exhibit short-latency, time-locked spiking relative to the activity of jaw muscles during biting. Last, we find that this projection conveys male-biased signals from the VMHvl to downstream lPAGvGlut2 neurons that are sensitive to features of ongoing activity, suggesting that action selectivity is generated by a combination of pre- and postsynaptic mechanisms.


Assuntos
Agressão/fisiologia , Mesencéfalo/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Núcleo Hipotalâmico Ventromedial/fisiologia , Animais , Feminino , Masculino , Mesencéfalo/citologia , Camundongos , Vias Neurais/citologia , Neurônios/citologia , Núcleo Hipotalâmico Ventromedial/citologia
8.
Biochem Biophys Res Commun ; 525(3): 570-575, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115149

RESUMO

Retrotransposon activation occurs in a variety of neurological disorders including multiple sclerosis and Alzheimer's Disease. While the origins of disease-related retrotransposon activation have remained mostly unidentified, this phenomenon may well contribute to disease progression by inducing inflammation, disrupting transcription and, potentially, genomic insertion. Here, we report that the inhibition of mitochondrial respiratory chain complex I by pharmacological agents widely used to model Parkinson's disease leads to a significant increase in expression of the ORF1 protein of the long interspersed nucleotide element 1 (LINE1) retrotransposon in human dopaminergic LUHMES cells. These findings were recapitulated in midbrain lysates from accordingly treated wild-type mice that mimic Parkinson's disease. Retrotransposon activation was paralleled by a loss of DNA cytosine methylation, providing a potential mechanism of retrotransposon mobilization. Loss of DNA methylation as well as retrotransposon activation were suppressed by the mitochondrial antioxidant phenothiazine, indicating that the well-established production of oxidants by inhibited complex I was causing these effects. Retrotransposon activation in some brain disorders may be less of a primary disease trigger rather than a consequence of mitochondrial distress, which is very common in neurodegenerative diseases.


Assuntos
Mitocôndrias/genética , Neurônios/metabolismo , Retroelementos/genética , Animais , Linhagem Celular , Metilação de DNA/genética , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Mesencéfalo/citologia , Camundongos Endogâmicos C57BL
9.
Nature ; 578(7795): 413-418, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051589

RESUMO

The mammalian claustrum, owing to its widespread connectivity with other forebrain structures, has been hypothesized to mediate functions that range from decision-making to consciousness1. Here we report that a homologue of the claustrum, identified by single-cell transcriptomics and viral tracing of connectivity, also exists in a reptile-the Australian bearded dragon Pogona vitticeps. In Pogona, the claustrum underlies the generation of sharp waves during slow-wave sleep. The sharp waves, together with superimposed high-frequency ripples2, propagate to the entire neighbouring pallial dorsal ventricular ridge (DVR). Unilateral or bilateral lesions of the claustrum suppress the production of sharp-wave ripples during slow-wave sleep in a unilateral or bilateral manner, respectively, but do not affect the regular and rapidly alternating sleep rhythm that is characteristic of sleep in this species3. The claustrum is thus not involved in the generation of the sleep rhythm itself. Tract tracing revealed that the reptilian claustrum projects widely to a variety of forebrain areas, including the cortex, and that it receives converging inputs from, among others, areas of the mid- and hindbrain that are known to be involved in wake-sleep control in mammals4-6. Periodically modulating the concentration of serotonin in the claustrum, for example, caused a matching modulation of sharp-wave production there and in the neighbouring DVR. Using transcriptomic approaches, we also identified a claustrum in the turtle Trachemys scripta, a distant reptilian relative of lizards. The claustrum is therefore an ancient structure that was probably already present in the brain of the common vertebrate ancestor of reptiles and mammals. It may have an important role in the control of brain states owing to the ascending input it receives from the mid- and hindbrain, its widespread projections to the forebrain and its role in sharp-wave generation during slow-wave sleep.


Assuntos
Claustrum/anatomia & histologia , Claustrum/fisiologia , Lagartos/anatomia & histologia , Lagartos/fisiologia , Sono/fisiologia , Animais , Claustrum/citologia , Claustrum/lesões , Masculino , Mamíferos/fisiologia , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Vias Neurais , RNA-Seq , Rombencéfalo/citologia , Rombencéfalo/fisiologia , Serotonina/metabolismo , Análise de Célula Única , Transcriptoma , Tartarugas/anatomia & histologia , Tartarugas/fisiologia
10.
Neuron ; 105(5): 822-836.e7, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31899072

RESUMO

Mutations in the GBA1 gene are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GBA1 encodes the lysosomal lipid hydrolase glucocerebrosidase (GCase), and its activity has been linked to accumulation of α-synuclein. The current study systematically examines the relationship between GCase activity and both pathogenic and non-pathogenic forms of α-synuclein in primary hippocampal, cortical, and midbrain neuron and astrocyte cultures, as well as in transgenic mice and a non-transgenic mouse model of PD. We find that reduced GCase activity does not result in aggregation of α-synuclein. However, in the context of extant misfolded α-synuclein, GCase activity modulates neuronal susceptibility to pathology. Furthermore, this modulation does not depend on neuron type but rather is driven by the level of pathological α-synuclein seeds. This study has implications for understanding how GBA1 mutations influence PD pathogenesis and provides a platform for testing novel therapeutics.


Assuntos
Astrócitos/metabolismo , Glucosilceramidase/genética , Neurônios/metabolismo , Transtornos Parkinsonianos/genética , Agregação Patológica de Proteínas/genética , alfa-Sinucleína/metabolismo , Animais , Astrócitos/citologia , Astrócitos/patologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Suscetibilidade a Doenças , Predisposição Genética para Doença , Glucosilceramidase/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Cultura Primária de Células , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Sinucleinopatias/genética , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia
11.
Acta Pharmacol Sin ; 41(1): 1-9, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31554960

RESUMO

Midbrain dopaminergic (DA) neurons are governed by an endogenous cholinergic system, originated in the mesopontine nuclei. Nicotine hijacks nicotinic acetylcholine receptors (nAChRs) and interferes with physiological function of the cholinergic system. In this review, we describe the anatomical organization of the cholinergic system and the key nAChR subtypes mediating cholinergic regulation of DA transmission and nicotine reward and dependence, in an effort to identify potential targets for smoking intervention. Cholinergic modulation of midbrain DA systems relies on topographic organization of mesopontine cholinergic projections, and activation of nAChRs in midbrain DA neurons. Previous studies have revealed that α4, α6, and ß2 subunit-containing nAChRs expressed in midbrain DA neurons and their terminals in the striatum regulate firings of midbrain DA neurons and activity-dependent dopamine release in the striatum. These nAChRs undergo modification upon chronic nicotine exposure. Clinical investigation has demonstrated that partial agonists of these receptors elevate the success rate of smoking cessation relative to placebo. However, further investigations are required to refine the drug targets to mitigate unpleasant side-effects.


Assuntos
Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Mesencéfalo/citologia , Vias Neurais/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Tabagismo/metabolismo , Animais , Neurônios Colinérgicos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Vias Neurais/metabolismo
12.
Neurobiol Aging ; 85: 22-37, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734438

RESUMO

The cause of midbrain dopaminergic (mDA) neuron loss in sporadic Parkinson's disease (PD) is multifactorial, involving cell autonomous factors, cell-cell interactions, and the effects of environmental toxins. Early loss of neurons in the locus coeruleus (LC), the main source of ascending noradrenergic (NA) projections, is an important feature of PD and other neurodegenerative disorders. We hypothesized that NA afferents provide trophic support for vulnerable mDA neurons. We demonstrate that depriving mDA neurons of NA input increases postnatal apoptosis and decreases cell survival in young adult rodents, with relative sparing of calbindin-positive subpopulations known to be resistant to degeneration in PD. As a mechanism, we propose that the neurotrophin brain-derived neurotrophic factor (BDNF) modulates anterograde survival effects of LC inputs to mDA neurons. We demonstrate that the LC is rich in BDNF mRNA in postnatal and young adult brains. Early postnatal NA denervation reduces both BDNF protein and activation of TrkB receptors in the ventral midbrain. Furthermore, overexpression of BDNF in NA afferents in transgenic mice increases mDA neuronal survival. Finally, increasing NA activity in primary cultures of mDA neurons improves survival, an effect that is additive or synergistic in the presence of different concentrations of BDNF. Taken together, our results point to a novel mechanism whereby LC afferents couple BDNF effects and NA activity to provide anterograde trophic support for vulnerable mDA neurons. Early loss of NA activity and anterograde neurotrophin support may contribute to degeneration of vulnerable neurons in PD and other neurodegenerative disorders.


Assuntos
Sobrevivência Celular , Neurônios Dopaminérgicos/patologia , Mesencéfalo/citologia , Norepinefrina/fisiologia , Doença de Parkinson/etiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Camundongos Transgênicos , Doença de Parkinson/patologia , Ratos Sprague-Dawley
13.
Exp Brain Res ; 237(12): 3333-3350, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31720812

RESUMO

The mesencephalic locomotor region (MLR) is an essential area for initiation of locomotion. Its functional roles and circuits underlying locomotion have been studied intensively in many species. Studies suggest that cuneiform nucleus and pedunculopontine nucleus (PPN) are two core regions in the MLR for locomotion. However, it remains unclear about cellular components and morphological and intrinsic membrane properties of the neurons in these regions, especially the serotonergic neurons. Using neonatal ePet-EYFP transgenic mice and immunofluorescent technique, we demonstrated existence of 5-HT neurons in the MLR and discovered that 5-HT neurons distributed mainly in the caudal PPN. 5-HT neurons were heterogeneous in MLR and had three types of firing pattern (single spike, phasic and tonic) and two subtypes of morphology (pyramidal and stellate). We measured parameters of 5-HT neurons (n = 35) including resting membrane potential (- 69.2 ± 4.2 mV), input resistance (1410.1 ± 616.9 MΩ), membrane capacitance (36.4 ± 14.9 pF), time constant (49.7 ± 19.4 ms), voltage threshold (- 32.1 ± 7.4 mV), rheobase (21.3 ± 12.4 pA), action potential amplitude (58.9 ± 12.8 mV) and half-width (4.7 ± 1.1 ms), afterhyperpolarization amplitude (23.6 ± 10.4 mV) and half-decay (331.6 ± 157.7 ms). 5-HT neurons were intrinsically different from adjacent non-5-HT neurons and less excitable than them. Hyperpolarization-activated inward currents and persistent inward currents were recorded in 5-HT neurons. NMDA increased excitability of 5-HT neurons, especially the tonic-firing neurons, accompanied with depolarization of membrane potential, hyperpolarization of voltage threshold, reduction of afterhyperpolarization half-decay, and left-shift of frequency-current relationship. This study provided insight into the distribution and properties of 5-HT neurons in the MLR and interaction between serotonergic and glutamatergic modulations.


Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Locomoção/fisiologia , Mesencéfalo/fisiologia , N-Metilaspartato/metabolismo , Neurônios Serotoninérgicos/fisiologia , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Potenciais da Membrana/fisiologia , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Formação Reticular Mesencefálica/citologia , Formação Reticular Mesencefálica/fisiologia , Técnicas de Patch-Clamp , Núcleo Tegmental Pedunculopontino/citologia , Núcleo Tegmental Pedunculopontino/fisiologia , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/metabolismo
14.
Regen Med ; 14(11): 1057-1069, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31718456

RESUMO

Current cell therapy product limitations include the need for in-depth product understanding to ensure product potency, safety and purity. New technologies require development and validation to address issues of production scale-up to meet clinical need; assays are required for process control, validation and release. Prior to clinical realization, an understanding of production processes is required to implement process changes that are essential for process control. Identification of key parameters forms the basis of process tolerances, allowing for validated, adaptive manufacturing processes. This enables greater process control and yield while withstanding regulatory scrutiny. This report summaries key milestones in specifically for ventral midbrain dopaminergic neuroprogenitor differentiation and key translational considerations and recommendations to enable successful, robust and reproducible current cell therapy product-manufacturing.


Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Mesencéfalo/citologia , Doença de Parkinson/terapia , Carcinogênese/patologia , Humanos , Pesquisa Médica Translacional
15.
Cell Reprogram ; 21(6): 285-295, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31651190

RESUMO

A simple, rapid, efficient, and specialized culture system was successfully developed in this study to induce human embryonic stem cells into dopaminergic neurons in vitro. It only took 5 days to generate quickly and directly a large number of homogeneous neural stem cell (NSC) spheres by the introduction of small molecules LDN (inhibitor of BMP [bone morphogenetic protein] pathway that inhibits BMP type I receptors ALK2 and ALK3), SB431542 (inhibitor of TGF-ß/Activin/Dodal pathway that inhibits ALK4, ALK5, and ALK7), CHIR99021 (inhibitors of GSK-3 [glycogen synthase kinase 3]), and basic fibroblast growth factor (bFGF). The dopaminergic neurons were successfully induced at day 25 (tyrosine hydroxylase [TH] expressed) and at day 32 (TH highly expressed) with high purity (TH/Tuj1: 84.14% and 93.15%, respectively) by the addition of FGF8 (fibroblast growth factor 8), sonic hedgehog (SHH), and Purmorphamine after the generation of NSC at day 5. And, the dopaminergic neurons induced by this system successfully survived and integrated into the striatum of cynomolgus monkey brain after transplantation, which verified the efficiency of the induction system developed in this study, suggesting the potential clinical application in cell therapy for neurological diseases.


Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias Humanas/metabolismo , Mesencéfalo/metabolismo , Animais , Neurônios Dopaminérgicos/citologia , Xenoenxertos , Células-Tronco Embrionárias Humanas/citologia , Humanos , Macaca fascicularis , Mesencéfalo/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Células-Tronco Neurais/transplante
16.
J Neurosci ; 39(48): 9521-9531, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31641054

RESUMO

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.


Assuntos
Proteínas com Homeodomínio LIM/metabolismo , Mesencéfalo/metabolismo , Transtornos Parkinsonianos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , Transplante de Células-Tronco/métodos , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Feminino , Previsões , Humanos , Masculino , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , Ratos , Ratos Nus
17.
J Neurosci ; 39(45): 8916-8928, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31541020

RESUMO

Our perceptual experience of sound depends on the integration of multiple sensory and cognitive domains, however the networks subserving this integration are unclear. Connections linking different cortical domains have been described, but we do not know the extent to which connections also exist between multiple cortical domains and subcortical structures. Retrograde tracing in adult male rats (Rattus norvegicus) revealed that the inferior colliculus, the auditory midbrain, receives dense descending projections not only, as previously established, from the auditory cortex, but also from the visual, somatosensory, motor, and prefrontal cortices. While all these descending connections are bilateral, those from sensory areas show a more pronounced ipsilateral dominance than those from motor and prefrontal cortices. Injections of anterograde tracers into the cortical areas identified by retrograde tracing confirmed those findings and revealed cortical fibers terminating in all three subdivisions of the inferior colliculus. Immunolabeling showed that cortical terminals target both GABAergic inhibitory, and putative glutamatergic excitatory neurons. These findings demonstrate that auditory perception and behavior are served by a network that includes extensive descending connections to the midbrain from sensory, behavioral, and executive cortices.SIGNIFICANCE STATEMENT Making sense of what we hear depends not only on the analysis of sound, but also on information from other senses together with the brain's predictions about the properties and significance of the sound. Previous work suggested that this interplay between the senses and the predictions from higher cognitive centers occurs within the cerebral cortex. By tracing neural connections in rat, we show that the inferior colliculus, the subcortical, midbrain center for hearing, receives extensive connections from areas of the cerebral cortex concerned with vision, touch, movement, and cognitive function, in addition to areas representing hearing. These findings demonstrate that wide-ranging cortical feedback operates at an earlier stage of the hearing pathway than previously recognized.


Assuntos
Vias Auditivas/citologia , Mesencéfalo/fisiologia , Córtex Sensório-Motor/fisiologia , Animais , Vias Auditivas/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico , Masculino , Mesencéfalo/citologia , Técnicas de Rastreamento Neuroanatômico , Neurônios/classificação , Neurônios/fisiologia , Ratos , Córtex Sensório-Motor/citologia
18.
Neurochem Int ; 131: 104549, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31539561

RESUMO

As a classic neurotransmitter in the brain, norepinephrine (NE) also is an important modulator to other neuronal systems. Using primary cultures from rat ventral mesencephalon (VM) and dopaminergic cell line MN9D, the present study examined the neuroprotective effects of NE and its effects on the expression of tyrosine hydroxylase (TH). The results showed that NE protected both VM cultures and MN9D cells against 6-hydroxydopamine-caused apoptosis, with possible involvement of adrenal receptors. In addition, treatment with NE upregulated TH protein levels in dose- and time-dependent manner. Further experiments to investigate the potential mechanisms underlying this NE-induced upregulation of TH demonstrated a marked increase in protein levels of the brain-derived neurotrophic factor (BDNF) and the phosphorylated extracellular signal-regulated protein kinase 1 and 2 (pERK1/2) in VM cultures treated with NE. In MN9D cells, a significantly increase of TH and pERK1/2 protein levels were observed after their transfection with BDNF cDNA or exposure to BDNF peptides. Treatment of VM cultures with K252a, an antagonist of the tropomyosin-related kinase B, blocked the upregulatory effects of NE on TH, BDNF and pERK1/2. Administration of MEK1 & MEK2 inhibitors also reversed NE-induced upregulation of TH and pERK1/2. Moreover, ChIP assay showed that treatment with NE or BDNF increased H4 acetylation in the TH promoter. These results suggest that the neuroprotection and modulation of NE on dopaminergic neurons are mediated via BDNF and MAPK/ERK pathways, as well as through epigenetic histone modification, which may have implications for the improvement of therapeutic strategies for Parkinson's disease.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Norepinefrina/farmacologia , Oxidopamina/toxicidade , Tirosina 3-Mono-Oxigenase/biossíntese , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carbazóis/farmacologia , Linhagem Celular , Epigênese Genética/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Alcaloides Indólicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Norepinefrina/antagonistas & inibidores , Gravidez , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos
19.
Folia Neuropathol ; 57(2): 196-204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31556578

RESUMO

INTRODUCTION: Exposure to acrylamide is increasing worldwide as a result of its heavy use in industry and formation in carbohydrate-rich food cooked at high temperature. Despite its neurotoxicity, no studies have shown its toxic effects on dopaminergic neurons yet. Therefore, the current study was carried out to show whether acrylamide adversely affects primary cultured dopaminergic neurons. MATERIAL AND METHODS: Acrylamide (0.001, 0.01, 0.1, 1, 2 mM) was added to two different groups of primary mesencephalic cell cultures on the 9th day in vitro for 24 and 48 h, respectively. Moreover, a group of cultures was treated with lower concentrations of acrylamide (0.01, 0.05, 0.1, 0.5 mM) on the 6th day in vitro for 5 consecutive days to investigate its long-term effects on dopaminergic neurons. Following each treatment, culture media were obtained for measuring lactate dehydrogenase, and cultured cells were stained immunocytochemically against tyrosine hydroxylase and neuronal nuclear antigens. RESULTS: Treatment of cultures with acrylamide for 48 h significantly reduced the number of dopaminergic neurons, adversely altered the morphology of the surviving neurons and increased levels of lactate dehydrogenase in the culture media. Similar treatment of cultures with acrylamide also resulted in lower numbers of total neuronal cells as shown by a reduced expression of the neuronal nuclear antigen. Prolonged treatment of cultures with lower concentrations of acrylamide slightly reduced the survival of dopaminergic neurons but increased the release of lactate dehydrogenase into the culture media as well. CONCLUSIONS: The current study shows, for the first time, neurotoxicity of acrylamide on dopaminergic neurons in the primary mesencephalic cell culture.


Assuntos
Acrilamida/toxicidade , Morte Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Animais , Células Cultivadas , Neurônios Dopaminérgicos/citologia , L-Lactato Desidrogenase/análise , Mesencéfalo/citologia , Camundongos
20.
Neurotoxicology ; 75: 148-157, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31545971

RESUMO

Manganese (Mn) is essential for neuronal health but neurotoxic in excess. Mn levels vary across brain regions and neurodevelopment. While Mn requirements during infanthood and childhood are significantly higher than in adulthood, the relative vulnerability to excess extracellular Mn across human neuronal developmental time and between distinct neural lineages is unknown. Neurological disease is associated with changes in brain Mn homeostasis and pathology associated with Mn neurotoxicity is not uniform across brain regions. For example, mutations associated with Huntington's disease (HD) decrease Mn bioavailability and increase resistance to Mn cytotoxicity in human and mouse striatal neuronal progenitors. Here, we sought to compare the differences in Mn cytotoxicity between control and HD human-induced pluripotent stem cells (hiPSCs)-derived neuroprogenitor cells (NPCs) and maturing neurons. We hypothesized that there would be differences in Mn sensitivity between lineages and developmental stages. However, we found that the different NPC lineage specific media substantially influenced Mn cytotoxicity in the hiPSC derived human NPCs and did so consistently even in a non-human cell line. This limited the ability to determine which human neuronal sub-types were more sensitive to Mn. Nonetheless, we compared within neuronal subtypes and developmental stage the sensitivity to Mn cytotoxicity between control and HD patient derived neuronal lineages. Consistent with studies in other striatal model systems the HD genotype was associated with resistance to Mn cytotoxicity in human striatal NPCs. In addition, we report an HD genotype-dependent resistance to Mn cytotoxicity in cortical NPCs and hiPSCs. Unexpectedly, the HD genotype conferred increased sensitivity to Mn in early post-mitotic midbrain neurons but had no effect on Mn sensitivity in midbrain NPCs or post-mitotic cortical neurons. Overall, our data suggest that sensitivity to Mn cytotoxicity is influenced by HD genotype in a human neuronal lineage type and stage of development dependent manner.


Assuntos
Encéfalo/efeitos dos fármacos , Doença de Huntington/metabolismo , Manganês/toxicidade , Neurônios/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Feminino , Humanos , Doença de Huntington/complicações , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/crescimento & desenvolvimento , Neurônios/metabolismo
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