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1.
Science ; 372(6546): 1068-1073, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083484

RESUMO

Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3→Ten3, Lphn2→Lphn2" rule instructed by reciprocal repulsions.


Assuntos
Orientação de Axônios , Axônios/fisiologia , Região CA1 Hipocampal/fisiologia , Hipocampo/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Região CA1 Hipocampal/citologia , Córtex Entorrinal/fisiologia , Feminino , Hipocampo/citologia , Ligantes , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Vias Neurais , Receptores de Peptídeos/genética , Transcriptoma
2.
Nat Commun ; 12(1): 3373, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099727

RESUMO

Theta rhythms temporally coordinate sequences of hippocampal place cell ensembles during active behaviors, while sharp wave-ripples coordinate place cell sequences during rest. We investigated whether such coordination of hippocampal place cell sequences is disrupted during error trials in a delayed match-to-place task. As a reward location was learned across trials, place cell sequences developed that represented temporally compressed paths to the reward location during the approach to the reward location. Less compressed paths were represented on error trials as an incorrect stop location was approached. During rest periods of correct but not error trials, place cell sequences developed a bias to replay representations of paths ending at the correct reward location. These results support the hypothesis that coordination of place cell sequences by theta rhythms and sharp wave-ripples develops as a reward location is learned and may be important for the successful performance of a spatial memory task.


Assuntos
Hipocampo/fisiologia , Células de Lugar/fisiologia , Desempenho Psicomotor/fisiologia , Memória Espacial/fisiologia , Ritmo Teta/fisiologia , Algoritmos , Animais , Teorema de Bayes , Hipocampo/citologia , Aprendizagem/fisiologia , Masculino , Ratos Long-Evans , Recompensa
3.
Nat Commun ; 12(1): 3892, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162876

RESUMO

The decline of brain function during aging is associated with epigenetic changes, including DNA methylation. Lifestyle interventions can improve brain function during aging, but their influence on age-related epigenetic changes is unknown. Using genome-wide DNA methylation sequencing, we here show that experiencing a stimulus-rich environment counteracts age-related DNA methylation changes in the hippocampal dentate gyrus of mice. Specifically, environmental enrichment prevented the aging-induced CpG hypomethylation at target sites of the methyl-CpG-binding protein Mecp2, which is critical to neuronal function. The genes at which environmental enrichment counteracted aging effects have described roles in neuronal plasticity, neuronal cell communication and adult hippocampal neurogenesis and are dysregulated with age-related cognitive decline in the human brain. Our results highlight the stimulating effects of environmental enrichment on hippocampal plasticity at the level of DNA methylation and give molecular insights into the specific aspects of brain aging that can be counteracted by lifestyle interventions.


Assuntos
Envelhecimento , Ilhas de CpG/genética , Metilação de DNA , Meio Ambiente , Hipocampo/metabolismo , Fatores Etários , Animais , Giro Denteado/metabolismo , Epigenômica/métodos , Feminino , Hipocampo/citologia , Humanos , Camundongos Endogâmicos C57BL , Neurogênese/genética , Plasticidade Neuronal/genética , Neurônios/metabolismo
4.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064454

RESUMO

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.


Assuntos
Potenciação de Longa Duração/genética , Neurônios/metabolismo , Receptor Cross-Talk , Receptores de Dopamina D5/genética , Receptores de GABA-A/genética , Transmissão Sináptica/genética , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peptídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D5/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/genética , Sinapses/metabolismo
5.
Phytomedicine ; 87: 153576, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33985879

RESUMO

BACKGROUND: Gastrodiae Rhizoma (Tianma), the dried tuber of Gastrodia elata Bl. (Orchidaceae), is listed as a top-grade herbal medicine in Shen-nong Ben-ts'ao Jing and has been used for treating headaches, dizziness, vertigo and convulsion. It has a neuroprotective effect and extends the lifespan in mouse models of Huntington's disease and Niemann-Pick type C disease. However, its effect on senescence remains unknown. PURPOSE: This study aimed to investigate the anti-aging effects and the underlying mechanism of Gastrodiae Rhizoma. METHODS: D-galactose (D-gal)- and BeSO4-induced cellular senescence and senescence-associated ß-galactosidase (SA-ß-gal) activity were evaluated in SH-SY5Y and PC12 cells. D-gal-induced aging mice were used as an in vivo model. Animal behaviors including nesting and burrowing and Morris water maze were conducted. Neurogenesis in the hippocampus was assessed by immunohistochemistry and confocal microscopy, and the aging-related proteins were assessed by Western blot analysis. The potential neuritogenesis activity of the partially purified fraction of Gastrodiae Rhizoma (TM-2) and its major ingredients were investigated in PC12 cells. RESULTS: TM-2 could improve D-gal-induced learning and memory impairement by inhibiting oxidative stress, increasing hippocampal neurogenesis and regulating the SH2B1-Akt pathway. Moreover, N6-(4-hydroxybenzyl)adenine riboside (T1-11) and parishins A and B, three constituents of TM-2, had anti-aging activity, as did T1-11 and parishin A induced neuritogenesis. CONCLUSION: Our data suggested that TM-2 slowed down D-gal-induced cellular and mouse brain aging. These results indicate that Gastrodiae Rhizoma has a beneficial effect on senescence. It may be used for neuroprotection and promoting neurogenesis.


Assuntos
Envelhecimento/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Gastrodia/química , Hipocampo/efeitos dos fármacos , Rizoma/química , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Galactose , Hipocampo/citologia , Hipocampo/fisiologia , Masculino , Camundongos , Neurogênese/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos
6.
Nat Commun ; 12(1): 3190, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045465

RESUMO

The hippocampus is essential for spatial and episodic memory but is damaged early in Alzheimer's disease and is very sensitive to hypoxia. Understanding how it regulates its oxygen supply is therefore key for designing interventions to preserve its function. However, studies of neurovascular function in the hippocampus in vivo have been limited by its relative inaccessibility. Here we compared hippocampal and visual cortical neurovascular function in awake mice, using two photon imaging of individual neurons and vessels and measures of regional blood flow and haemoglobin oxygenation. We show that blood flow, blood oxygenation and neurovascular coupling were decreased in the hippocampus compared to neocortex, because of differences in both the vascular network and pericyte and endothelial cell function. Modelling oxygen diffusion indicates that these features of the hippocampal vasculature may restrict oxygen availability and could explain its sensitivity to damage during neurological conditions, including Alzheimer's disease, where the brain's energy supply is decreased.


Assuntos
Hipocampo/irrigação sanguínea , Microcirculação/fisiologia , Neocórtex/irrigação sanguínea , Córtex Visual/irrigação sanguínea , Trifosfato de Adenosina/biossíntese , Doença de Alzheimer/fisiopatologia , Animais , Hipóxia Celular/fisiologia , Demência Vascular/fisiopatologia , Feminino , Hipocampo/citologia , Hipocampo/diagnóstico por imagem , Hipocampo/fisiopatologia , Humanos , Microscopia Intravital , Fluxometria por Laser-Doppler , Masculino , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Microvasos/diagnóstico por imagem , Microvasos/fisiologia , Modelos Animais , Neocórtex/citologia , Neocórtex/diagnóstico por imagem , Neocórtex/fisiopatologia , Neurônios/metabolismo , Acoplamento Neurovascular/fisiologia , Fosforilação Oxidativa , Oxigênio/análise , Oxigênio/metabolismo , Memória Espacial/fisiologia , Córtex Visual/citologia , Córtex Visual/fisiopatologia
7.
Nat Commun ; 12(1): 2849, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990590

RESUMO

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.


Assuntos
Proteína 4 Homóloga a Disks-Large/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Trifosfato de Adenosina/administração & dosagem , Animais , Autofagia/fisiologia , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/deficiência , Feminino , Hipocampo/citologia , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Modelos Neurológicos , N-Metilaspartato/administração & dosagem , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/fisiologia , Receptores Purinérgicos P2X/fisiologia
8.
Nat Commun ; 12(1): 2912, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006874

RESUMO

The hippocampal mossy fiber synapse is a key synapse of the trisynaptic circuit. Post-tetanic potentiation (PTP) is the most powerful form of plasticity at this synaptic connection. It is widely believed that mossy fiber PTP is an entirely presynaptic phenomenon, implying that PTP induction is input-specific, and requires neither activity of multiple inputs nor stimulation of postsynaptic neurons. To directly test cooperativity and associativity, we made paired recordings between single mossy fiber terminals and postsynaptic CA3 pyramidal neurons in rat brain slices. By stimulating non-overlapping mossy fiber inputs converging onto single CA3 neurons, we confirm that PTP is input-specific and non-cooperative. Unexpectedly, mossy fiber PTP exhibits anti-associative induction properties. EPSCs show only minimal PTP after combined pre- and postsynaptic high-frequency stimulation with intact postsynaptic Ca2+ signaling, but marked PTP in the absence of postsynaptic spiking and after suppression of postsynaptic Ca2+ signaling (10 mM EGTA). PTP is largely recovered by inhibitors of voltage-gated R- and L-type Ca2+ channels, group II mGluRs, and vacuolar-type H+-ATPase, suggesting the involvement of retrograde vesicular glutamate signaling. Transsynaptic regulation of PTP extends the repertoire of synaptic computations, implementing a brake on mossy fiber detonation and a "smart teacher" function of hippocampal mossy fiber synapses.


Assuntos
Fibras Musgosas Hipocampais/fisiologia , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia , Animais , Células Cultivadas , Estimulação Elétrica , Potenciais Evocados/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/fisiologia , Masculino , Técnicas de Patch-Clamp , Ratos , Potenciais Sinápticos/fisiologia
9.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923565

RESUMO

Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG.


Assuntos
Axônios/metabolismo , Magnetossomos/metabolismo , Crescimento Neuronal , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Transporte Biológico , Células Cultivadas , Feminino , Hipocampo/citologia , Magnetospirillum/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Mecânico
10.
Nat Commun ; 12(1): 2424, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893293

RESUMO

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Quinase 5 Dependente de Ciclina/genética , Endocitose/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Clatrina/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Dinamina I/genética , Dinamina I/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Células HeLa , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neurônios/metabolismo , Ligação Proteica , Interferência de RNA
11.
Nat Commun ; 12(1): 2112, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837214

RESUMO

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.


Assuntos
Potenciação de Longa Duração/fisiologia , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Potenciais Sinápticos/fisiologia , Regulação Alostérica , Animais , Células Cultivadas , Agonistas de Receptores de GABA-A/farmacologia , Células HEK293 , Hipocampo/citologia , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Cultura Primária de Células , Proteína Quinase C/metabolismo , Ratos , Receptores de GABA-A/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sinapses/efeitos dos fármacos , Potenciais Sinápticos/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo
12.
Neurosci Lett ; 751: 135777, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33647396

RESUMO

Previous research has implicated the ventral pole of the hippocampus in regulating anxiety. However, most rat studies examining the specific contribution of the ventral hippocampus have utilized techniques that have nonspecific effects and/or create nonreversible damage to the region. The present study sought to characterize the role of ventral hippocampal CaMKIIα-expressing neurons in modulating anxiety- and fear-like behavior during exposure to a variety of threatening stimuli. Five weeks prior to testing, adult male Long-Evans hooded rats received ventral hippocampal viral-vector infusions expressing either AAV8-CaMKIIα-hM4D-mCherry (DREADD) or AAV8-CaMKIIα-EGFP (GFP). DREADD transfection allowed for the specific, noninvasive and temporary inhibition of the ventral hippocampus (vHC) immediately before threat presentation. Rats were evaluated for behaviors congruent with anxiety- or fear-like defensive states during testing in the elevated plus-maze (EPM) and light-dark test (LDT), or post footshock freezing and footshock-induced contextual freezing, respectively. Analyses revealed a significant effect of vHC inhibition that was dependent on the type of threat exposure. Specifically, DREADD-induced silencing of vHC neurons reduced anxiety-like behavior in the EPM and LDT, without reliably affecting footshock-induced fear. These data add to a growing literature implicating the vHC as a key region involved in controlling the expression of anxiety in rodents, primates and humans.


Assuntos
Ansiedade/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Reação de Congelamento Cataléptica , Hipocampo/metabolismo , Animais , Ansiedade/fisiopatologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Drogas Desenhadas/farmacologia , Medo , Hipocampo/citologia , Hipocampo/fisiologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Long-Evans
13.
Neural Netw ; 139: 45-63, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33677378

RESUMO

The mammalian spatial navigation system is characterized by an initial divergence of internal representations, with disparate classes of neurons responding to distinct features including location, speed, borders and head direction; an ensuing convergence finally enables navigation and path integration. Here, we report the algorithmic and hardware implementation of biomimetic neural structures encompassing a feed-forward trimodular, multi-layer architecture representing grid-cell, place-cell and decoding modules for navigation. The grid-cell module comprised of neurons that fired in a grid-like pattern, and was built of distinct layers that constituted the dorsoventral span of the medial entorhinal cortex. Each layer was built as an independent continuous attractor network with distinct grid-field spatial scales. The place-cell module comprised of neurons that fired at one or few spatial locations, organized into different clusters based on convergent modular inputs from different grid-cell layers, replicating the gradient in place-field size along the hippocampal dorso-ventral axis. The decoding module, a two-layer neural network that constitutes the convergence of the divergent representations in preceding modules, received inputs from the place-cell module and provided specific coordinates of the navigating object. After vital design optimizations involving all modules, we implemented the tri-modular structure on Zynq Ultrascale+ field-programmable gate array silicon chip, and demonstrated its capacity in precisely estimating the navigational trajectory with minimal overall resource consumption involving a mere 2.92% Look Up Table utilization. Our implementation of a biomimetic, digital spatial navigation system is stable, reliable, reconfigurable, real-time with execution time of about 32 s for 100k input samples (in contrast to 40 minutes on Intel Core i7-7700 CPU with 8 cores clocking at 3.60 GHz) and thus can be deployed for autonomous-robotic navigation without requiring additional sensors.


Assuntos
Biomimética/métodos , Células de Grade/fisiologia , Redes Neurais de Computação , Células de Lugar/fisiologia , Navegação Espacial/fisiologia , Animais , Córtex Entorrinal/citologia , Córtex Entorrinal/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Ratos
14.
Bull Exp Biol Med ; 170(5): 649-653, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: covidwho-1159147

RESUMO

Ivermectin (IVM) belongs to the class of macrocyclic lactones, which is used as an antiparasitic agent. At present, the researchers focus on possibility to use IVM in treatment of certain forms of cancer and viral diseases such as COVID-19. The mechanisms of IVM action are not clear. It is assumed that IVM affects chloride channels and increases cytoplasmic concentration of chloride. This study examines the effect of IVM on chloride currents induced by glycine (IGly). Experiments were carried out on isolated pyramidal neurons of the rat hippocampus with whole-cell patch clamp. A short-term (600 msec) application of IVM in a concentration of 10 µM induced a slow inward current, which persisted after washing the neurons. The low concentrations (0.1-1000 nM) of IVM did not induce any novel current, but it rapidly and reversibly reduced the peak amplitude and accelerated desensitization of IGly in a dose-dependent manner. The threshold concentrations of IVM sufficient to reduce peak amplitude of IGly and to accelerate desensitization of IGly were 100 nM and 0.1 nM, respectively. The study revealed a high sensitivity of neuronal glycine receptors to IVM.


Assuntos
Canais de Cloreto/efeitos dos fármacos , Glicina/farmacologia , Ivermectina/farmacologia , Células Piramidais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antivirais/farmacologia , Células Cultivadas , Canais de Cloreto/metabolismo , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo
15.
Pharmacology ; 106(5-6): 254-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33691319

RESUMO

INTRODUCTION: Schisandrin which is derived from Schisandra chinensis has shown multiple pharmacological effects on various diseases including Alzheimer's disease (AD). It is demonstrated that mitochondrial dysfunction plays an essential role in the pathogenesis of neurodegenerative disorders. OBJECTIVE: Our study aims to investigate the effects of schisandrin on mitochondrial functions and metabolisms in primary hippocampal neurons. METHODS: In our study, rat primary hippocampal neurons were isolated and treated with indicated dose of amyloid ß1-42 (Aß1-42) oligomer to establish a cell model of AD in vitro. Schisandrin (2 µg/mL) was further subjected to test its effects on mitochondrial function, energy metabolism, mitochondrial biogenesis, and dynamics in the Aß1-42 oligomer-treated neurons. RESULTS AND CONCLUSIONS: Our findings indicated that schisandrin significantly alleviated the Aß1-42 oligomer-induced loss of mitochondrial membrane potential and impaired cytochrome c oxidase activity. Additionally, the opening of mitochondrial permeability transition pore and release of cytochrome c were highly restricted with schisandrin treatment. Alterations in cell viability, ATP production, citrate synthase activity, and the expressions of glycolysis-related enzymes demonstrated the relief of defective energy metabolism in Aß-treated neurons after the treatment of schisandrin. For mitochondrial biogenesis, elevated expression of peroxisome proliferator-activated receptor γ coactivator along with promoted mitochondrial mass was found in schisandrin-treated cells. The imbalance in the cycle of fusion and fission was also remarkably restored by schisandrin. In summary, this study provides novel mechanisms for the protective effect of schisandrin on mitochondria-related functions.


Assuntos
Ciclo-Octanos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Lignanas/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Policíclicos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/toxicidade , Animais , Animais Recém-Nascidos , Citocromos c/antagonistas & inibidores , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Biogênese de Organelas , Fragmentos de Peptídeos/toxicidade , Cultura Primária de Células , Ratos Sprague-Dawley
16.
J Vis Exp ; (168)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33645578

RESUMO

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.


Assuntos
Segmento Inicial do Axônio/metabolismo , Hipocampo/citologia , Neurônios/citologia , Animais , Anquirinas/metabolismo , Células Cultivadas , Edição de Genes , Hipocampo/metabolismo , Integrases/metabolismo , Camundongos , Neuroglia/citologia , Neurônios/metabolismo
17.
Food Funct ; 12(7): 2901-2913, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33710186

RESUMO

Cerebral stroke, a common clinical problem, is the predominant cause of disability and death worldwide. Its prevalence increases and infarctions exacerbate with age. A Tibetan plant, Brassica rapa L., possesses multiple medicinal effects, such as anti-altitude sickness, anti-hyperlipidemia and anti-fatigue, as mentioned in the noted ancient Tibet pharmacopeia "The Four Medical Tantras". Our preliminary studies also showed the anti-hypoxia protection mechanism of B. rapa L., implying its possible relationship with anti-ischemic neuroprotection. However, the potential molecular mechanism of the active constituent of turnip against cerebral ischemia/reperfusion remains unclear. In our study, oxidative stress markers, including LDH, ROS, SOD, GPx and CAT were assayed. In controlled in vitro assays, we found that the turnip's active constituent had remarkable anti-hypoxia capability. We further showed the profound effects of the active constituent of turnip on the levels of apoptosis-related proteins, including Bax, Bcl-2 and caspase-3, which contributed to its anti-inflammatory activity. Western blot analysis results also implied that active-constituent pretreatment reversed the diminished expression of the PI3K/Akt/mTOR pathway mediated by oxygen glucose deprivation/reperfusion (OGD/R); further experimental evidence showed that the protective role was limited in the PI3K inhibitor (LY294002) treatment group. Our results demonstrated that the functional monomer of B. rapa L. exerted a neuroprotective effect against OGD/R-induced HT22 cell injury, and its potential mechanism provides a scientific basis for future clinical applications and its use as a functional food.


Assuntos
Brassica rapa , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Animais , Isquemia Encefálica/tratamento farmacológico , Linhagem Celular/efeitos dos fármacos , Hipocampo/citologia , Humanos , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Traumatismo por Reperfusão/metabolismo , Tibet
18.
Biochem Biophys Res Commun ; 553: 99-106, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33765560

RESUMO

Spontaneous burst firing is a hallmark attributed to the neuronal network activity. It is known to be accompanied by intracellular calcium [Са2+]i oscillations within the bursting neurons. Studying mechanisms underlying regulation of burst firing is highly relevant, since impairment in neuronal bursting accompanies different neurological disorders. In the present study, the contribution of NMDA and GABA(A) receptors to the shape formation of spontaneous burst -was studied in cultured hippocampal neurons. A combination of inhibitory analysis with simultaneous registration of neuronal bursting by whole-cell patch clamp and calcium imaging was used to assess spontaneous burst firing and [Са2+]i level. Using bicuculline and D-AP5 we showed that GABA(A) and NMDA receptors effectively modulate burst plateau phase and [Са2+]i transient spike which can further affect action potential (AP) amplitudes and firing frequency within a burst. Bicuculline significantly elevated the amplitude and reduced the duration of both burst plateau phase and [Са2+]i spike resulting in an increase of AP firing frequency and shortening of AP amplitudes within a burst. D-AP5 significantly decreases the amplitude of both plateau phase and [Са2+]i spike along with a burst duration that correlated with an increase in AP amplitudes and reduced firing frequency within a burst. The effect of bicuculline was occluded by co-addition of D-AP5 revealing modulatory role of GABA(A) receptors to the NMDA receptor-mediated formation of the burst. Our results provide new evidence on importance of NMDA and GABA(A) receptors in shaping burst firing and Ca2+transient spikes in cultured hippocampal neurons.


Assuntos
Potenciais de Ação , Sinalização do Cálcio , Cálcio/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Astrócitos/citologia , Bicuculina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Feminino , Masculino , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
19.
Nat Commun ; 12(1): 1413, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658493

RESUMO

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).


Assuntos
Exocitose/fisiologia , Proteínas Luminescentes/metabolismo , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Mutação , Neurônios/citologia , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Sinápticas/fisiologia , Imagem com Lapso de Tempo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo
20.
Neurosci Lett ; 751: 135801, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33705932

RESUMO

Lidocaine hydrochloride (LC-HCl) and morphine hydrochloride (Mor-HCl) are two kinds of most prevalently used anesthetics. However, their influences on electrical excitability of hippocampal neuronal networks and hippocampal brain slices were rarely studied. Previously, our group have assessed the influence of acetylcholine, alcohol and temperature change on the excitability of neural networks with the so-called Voltage Threshold Measurement Method (VTMM) based on microelectrode array (MEA). In this paper, we will study the influence of LC-HCl and Mor-HCl on the electrical excitability of neural networks and the morphological features of neurons, and discuss the relations between the changes of electrical excitability of neural networks and the morphological changes of neurons. The results of VTMM showed: The voltage threshold (VTh) of hippocampal neuronal networks and hippocampal brain slices first increased and then decreased as the LC-HCl concentration increased. The VTh of hippocampal neuronal networks and hippocampal brain slices increased as the Mor-HCl concentration increased. The results of HCS experiments showed: The neurite length change of cultured hippocampal neuronal networks increased first and then decreased with increased LC-HCl concentration, but decreased as the Mor-HCl concentration increased. The combined analysis of VTMM and HCS experiments showed that under effects of the two drugs, the VTh and the hippocampal neurite length were strongly negatively correlated.


Assuntos
Hipocampo/efeitos dos fármacos , Lidocaína/farmacologia , Morfina/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Anestésicos Locais/farmacologia , Animais , Células Cultivadas , Interações Medicamentosas , Hipocampo/citologia , Hipocampo/fisiologia , Potenciais da Membrana , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley
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