RESUMO
We present a dry lift-off method using a chemically resistant spin-on plastic, polyimide, to pattern surfaces with high accuracy and resolution. Using well-known lithographic and reactive ion etching techniques, the spin-on polymer is patterned over a silicon dioxide surface. The plastic efficiently adheres to the silicon dioxide surface during the chemical modification and is readily lifted-off following the derivatization process, permitting highly reliable surface derivatization. The verticality of the reactive ion etch enables sub-micrometer features to be patterned, down to 0.8 µm. The technique is used to pattern neurons on silicon dioxide surfaces: efficient neuron placement over a 4 mm area is shown for patterns larger than 50 µm while process guidance is shown for 10 µm patterns.
Assuntos
Técnicas de Cultura de Células/métodos , Materiais Revestidos Biocompatíveis/química , Neurônios/fisiologia , Resinas Sintéticas/química , Animais , Células Cultivadas , Ratos , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Patch-clamp is an important method to monitor the electrophysiological activity of cells and the role of pharmacological compounds on specific ion channel proteins. In recent years, planar patch-clamp chips have been developed as a higher throughput approach to the established glass-pipette method. However, proper conditions to optimize the high resistance cell-to-probe seals required to measure the small currents resulting from ion channel activity are still the subject of conjecture. Here, we report on the design of multiple-aperture (sieve) chips to rapidly facilitate assessment of cell-to-aperture interactions in statistically significant numbers. We propose a method to pre-screen the quality of seals based on a dye loading protocol through apertures in the chip and subsequent evaluation with fluorescence confocal microscopy. We also show the first scanning electron micrograph of a focused ion beam section of a cell in a patch-clamp chip aperture.
Assuntos
Células/efeitos dos fármacos , Canais Iônicos/metabolismo , Técnicas de Patch-Clamp , Animais , Linhagem Celular , Ensaios de Triagem em Larga Escala/métodos , Camundongos , Microscopia Eletrônica de Varredura , Microscopia de FluorescênciaRESUMO
Choline is a micronutrient essential for the structural integrity of cellular membranes, and its presence at synapses follows either depolarization-induced pre-synaptic release or degradation of acetylcholine. Previous studies using whole-cell recording have shown that choline can modulate inhibitory input to hippocampal pyramidal neurons by acting upon nicotinic acetylcholine receptors (nAChRs) found on interneurons. However, little is known about how choline affects neuronal activity at the population level; therefore, we used extracellular recordings to assess its influence upon synaptic transmission in acutely prepared hippocampal slices. Choline caused a reversible depression of evoked field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner (10, 500, and 1000 µM). When applied after the induction of long-term potentiation, choline-mediated depression (CMD) was still observed, and potentiation returned on wash-out. Complete blockade of CMD could not be achieved with antagonists for the α7 nAChR, to which choline is a full agonist, but was possible with a general nAChR antagonist. The ability of choline to increase paired-pulse facilitation, and the inability of applied gamma-aminobutyric acid (GABA) to mediate further depression of fEPSPs, suggests that the principal mechanism of choline's action was on the facilitation of neurotransmitter release. Our study provides evidence that choline can depress population-level activity, quite likely by facilitating the release of GABA from interneurons, and may thereby influence hippocampal function.
Assuntos
Colina/farmacologia , Hipocampo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Nootrópicos/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip's polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)-polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.
Assuntos
Técnicas de Cultura de Células/instrumentação , Dispositivos Lab-On-A-Chip , Neurônios/citologia , Técnicas de Patch-Clamp/instrumentação , Polímeros/química , Animais , Dimetilpolisiloxanos/química , Condutividade Elétrica , Fenômenos Eletrofisiológicos , Imidas/química , Lymnaea/citologia , Microtecnologia , Neurônios/metabolismo , Sinapses/metabolismoRESUMO
Interface devices such as integrated planar patch-clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly-d-lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo-preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch-clamp interfaces on the chip.
Assuntos
Neurônios/citologia , Técnicas de Patch-Clamp/métodos , Potenciais de Ação , Animais , Encéfalo/citologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Dimetilpolisiloxanos/química , Rede Nervosa/citologia , Nylons/química , Polilisina/química , Ratos , Propriedades de SuperfícieRESUMO
We report on a simple and high-yield manufacturing process for silicon planar patch-clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high-quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high-impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high-fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole-cell current recordings obtained from a voltage-clamp stimulation protocol, and in current-clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch-clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high-information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity.
Assuntos
Biotecnologia/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Canais Iônicos/efeitos dos fármacos , Lymnaea , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Animais , Células Cultivadas , Capacitância Elétrica , Potenciais da Membrana/efeitos dos fármacos , SilícioRESUMO
Dravet syndrome is a developmental epileptic encephalopathy caused by pathogenic variation in SCN1A To characterize the pathogenic substitution (p.H939R) of a local individual with Dravet syndrome, fibroblast cells from the individual were reprogrammed to pluripotent stem cells and differentiated into neurons. Sodium currents of these neurons were compared with healthy control induced neurons. A novel Scn1aH939R/+ mouse model was generated with the p.H939R substitution. Immunohistochemistry and electrophysiological experiments were performed on hippocampal slices of Scn1aH939R/+ mice. We found that the sodium currents recorded in the proband-induced neurons were significantly smaller and slower compared to wild type (WT). The resting membrane potential and spike amplitude were significantly depolarized in the proband-induced neurons. Similar differences in resting membrane potential and spike amplitude were observed in the interneurons of the hippocampus of Scn1aH939R/+ mice. The Scn1aH939R/+ mice showed the characteristic features of a Dravet-like phenotype: increased mortality and both spontaneous and heat-induced seizures. Immunohistochemistry showed a reduction in amount of parvalbumin and vesicular acetylcholine transporter in the hippocampus of Scn1aH939R/+ compared to WT mice. Overall, these results underline hyper-excitability of the hippocampal CA1 circuit of this novel mouse model of Dravet syndrome which, under certain conditions, such as temperature, can trigger seizure activity. This hyper-excitability is due to the altered electrophysiological properties of pyramidal neurons and interneurons which are caused by the dysfunction of the sodium channel bearing the p.H939R substitution. This novel Dravet syndrome model also highlights the reduction in acetylcholine and the contribution of pyramidal cells, in addition to interneurons, to network hyper-excitability.
Assuntos
Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Epilepsias Mioclônicas/patologia , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Interneurônios/patologia , Células Piramidais/patologia , Animais , Região CA1 Hipocampal/metabolismo , Eletrofisiologia , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Interneurônios/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Células Piramidais/metabolismoRESUMO
Synthetic cannabinoids are marketed as legal alternatives to Δ9-THC, and are a growing worldwide concern as these drugs are associated with severe adverse effects. Unfortunately, insufficient information regarding the physiological and pharmacological effects of emerging synthetic cannabinoids (ESCs) makes their regulation by government authorities difficult. One strategy used to evade regulation is to distribute isomers of regulated synthetic cannabinoids. This study characterized the pharmacological properties of a panel of ESCs in comparison to Δ9-THC, as well as six JWH-122 isomers relative to its parent compound (JWH-122-4). Two cell-based assays were used to determine the potency and efficacy of ESCs and a panel of reference cannabinoids. HEK293T cells were transfected with human cannabinoid receptor 1 (CB1) and pGloSensor-22F, and the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live cells. All ESCs examined were classified as agonists, with the following rank order of potency: Win 55,212-2 > CP 55,940 > JWH-122-4 > Δ9-THC ≈ RCS-4 ≈ THJ-2201 > JWH-122-5 > JWH-122-7 > JWH-122-2 ≈ AB-CHMINACA > JWH-122-8 > JWH-122-6 > JWH-122-3. Evaluation of ESC-stimulated Ca2+ transients in cultured rat primary hippocampal neurons confirmed the efficacy of four of the most potent ESCs (JWH-122-4, JWH-122-5, JWH-122-7 and AB-CHMINACA). This work helps regulatory agencies make informed decisions concerning these poorly characterized recreational drugs.
Assuntos
Canabinoides/farmacologia , Hipocampo/citologia , Indazóis/farmacologia , Indóis/química , Naftalenos/química , Neurônios/efeitos dos fármacos , Valina/análogos & derivados , Canabinoides/química , Células HEK293 , Humanos , Indazóis/química , Isomerismo , Naftalenos/farmacologia , Valina/química , Valina/farmacologiaRESUMO
This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular catalytic antioxidants [superoxide dismutase (SOD) mimetics]. High SOD-active Mn(III)porphyrins meso-substituted with N,N'-dimethylimidazolium or N-alkylpyridinium groups did not protect neurons against oxygen-glucose deprivation (OGD), although lower SOD-active and -inactive para isomers protected against N-methyl-D-aspartate (NMDA) exposure. Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP), as well as SOD-inactive metalloTBAPs and other phenyl ring- or beta-substituted metalloporphyrins that contained redox-insensitive metals, protected cultures against OGD and NMDA neurotoxicity. Crucially, neuroprotective metalloporphyrins suppressed OGD- or NMDA-induced rises in intracellular Ca2+ concentration in the same general rank order as observed for neuroprotection. Results from paraquat toxicity, intracellular fluorescence quenching, electrophysiology, mitochondrial Ca2+, and spontaneous synaptic activity experiments suggest a model in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor-mediated Ca2+ rises, thereby indirectly preventing accumulation of neurotoxic mitochondrial Ca2+ levels. Though neuroprotective in a manner not originally intended, SOD-inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca2+ toxicity is implicated. Conventional syntheses aimed at improving the catalytic antioxidant capability and/or intracellular access of metalloporphyrins may not yield improved efficacy in some disease models.
Assuntos
Cálcio/metabolismo , Metaloporfirinas/química , Fármacos Neuroprotetores/farmacologia , Superóxido Dismutase/química , Animais , Antioxidantes/metabolismo , Isquemia Encefálica/patologia , Cálcio/química , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Catálise , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Eletrofisiologia , Sequestradores de Radicais Livres/farmacologia , Glucose/metabolismo , Imidazóis/química , Metaloporfirinas/farmacologia , Mitocôndrias/metabolismo , Modelos Químicos , N-Metilaspartato/química , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxirredução , Oxigênio/metabolismo , Paraquat/farmacologia , Permeabilidade , Ratos , Superóxido Dismutase/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Fatores de TempoRESUMO
There has been a worldwide proliferation of synthetic cannabinoids that have become marketed as legal alternatives to cannabis (marijuana). Unfortunately, there is a dearth of information about the pharmacological effects of many of these emerging synthetic cannabinoids (ESCs), which presents a challenge for regulatory authorities that need to take such scientific evidence into consideration in order to regulate ECSs as controlled substances. We aimed to characterize the pharmacological properties of ten ESCs using two cell based assays that enabled the determination of potency and efficacy relative to a panel of well-characterized cannabinoids. Agonist-mediated inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live HEK293T cells transfected with human cannabinoid receptor 1 gene (CNR1) and pGloSensor-22F. Pharmacological analysis of this data indicated that all of the ESCs tested were full agonists, with the following rank order of potency: Win 55212-2≈5F-PB-22≈AB-PINACA≈EAM-2201≈MAM-2201>JWH-250≈ PB-22>AKB48 N-(5FP)>AKB-48≈STS-135>XLR-11. Assessment of agonist-stimulated depression of Ca(2+) transients was also used to confirm the efficacy of five ESCs (XLR-11, JWH-250, AB-PINACA, 5F-PB-22, and MAM-2201) in cultured primary hippocampal neurons. This work aims to help inform decisions made by regulatory agencies concerned with the profusion of these poorly characterized recreational drugs.
Assuntos
Canabinoides/síntese química , Canabinoides/farmacologia , Hipocampo/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Activation of cannabinoid receptor 1 (CB1) inhibits synaptic transmission in hippocampal neurons. The goal of this study was to evaluate the ability of benchmark and emerging synthetic cannabinoids to suppress neuronal activity in vitro using two complementary techniques, Ca(2+) spiking and multi-electrode arrays (MEAs). Neuron culture and fluorescence imaging conditions were extensively optimized to provide maximum sensitivity for detection of suppression of neural activity by cannabinoids. The neuronal Ca(2+) spiking frequency was significantly suppressed within 10min by the prototypic aminoalkylindole cannabinoid, WIN 55,212-2 (10µM). Suppression by WIN 55,212-2 was not improved by pharmacological intervention with signaling pathways known to interfere with CB1 signaling. The naphthoylindole CB1 agonist, JWH-018 suppressed Ca(2+) spiking at a lower concentration (2.5µM), and the CB1 antagonist rimonabant (5µM), reversed this suppression. In the MEA assay, the ability of synthetic CB1 agonists to suppress spontaneous electrical activity of hippocampal neurons was evaluated over 80min sessions. All benchmark (WIN 55,212-2, HU-210, CP 55,940 and JWH-018) and emerging synthetic cannabinoids (XLR-11, JWH-250, 5F-PB-22, AB-PINACA and MAM-2201) suppressed neural activity at a concentration of 10µM; furthermore, several of these compounds also significantly suppressed activity at 1µM concentrations. Rimonabant partially reversed spiking suppression of 5F-PB-22 and, to a lesser extent, of MAM-2201, supporting CB1-mediated involvement, although the inactive WIN 55,212-3 also partially suppressed activity. Taken together, synthetic cannabinoid CB1-mediated suppression of neuronal activity was detected using Ca(2+) spiking and MEAs.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Canabinoides/farmacologia , Eletrofisiologia/instrumentação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Canabinoides/síntese química , Eletrodos , Feminino , Hipocampo/citologia , Gravidez , RatosRESUMO
The growth-arrest-specific protein gas7 is required for morphological differentiation of cultured mouse cerebellar neurons and PC12 cells. Moreover, its overexpression in various cell types induces neurite-like outgrowth. The role of gas7 in neuronal differentiation was further characterized by adenovirus-mediated overexpression in PC12 cells and quantification of the expression of various neuronal markers, in the absence and presence of different concentrations of nerve growth factor (NGF). The potential neuroprotective activity of gas7 against various neurotoxic insults was also assessed. In addition to promoting the formation of neurite-like extensions, overexpression of gas7 potentiated NGF-mediated neuronal differentiation of PC12 cells, as shown by the enhanced expression of the neuronal proteins betaIII-tubulin, synaptotagmin, alpha7 subunit of the acetylcholine receptor, and dihydropyrimidinase related protein-3. This effect was exerted independently of cell cycle progression, as gas7 did not affect proliferation of PC12 cells. While some differentiation enhancers protect PC12 cells against lethal insults, gas7 overexpression in PC12 cells did not protect against oxygen-glucose deprivation, the calcium ionophore A23187, or the nitric oxide donor sodium nitroprusside, suggesting that gas7 is not neuroprotective. The ability of gas7 to potentiate neuronal differentiation makes it a potential therapeutic target to promote re-establishment of neuronal connections in the injured or diseased brain, such as following stroke.
Assuntos
Diferenciação Celular/fisiologia , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Biomarcadores/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Vetores Genéticos , Ionóforos/farmacologia , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/metabolismo , Doadores de Óxido Nítrico/farmacologia , Células PC12 , Ratos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , TransfecçãoRESUMO
Although organotypic hippocampal slice cultures (OHSCs) are used to study function within the hippocampus, the effect of maintenance in vitro upon protein expression is not fully understood. Therefore, we examined developmental changes in cultures prepared from P8 rats and maintained on porous membranes between medium and atmosphere. Between 7 and 28 days following explantation, altered hippocampal morphology could not be detected despite a significant decrease in both MAP-2c and a mid-range tau isoform by 21 DIV. During the same period, lower GFAP expression was observed, and GFAP labeling suggested a migration of astrocytes to the slice-atmosphere interface. In contrast, levels of the synaptic proteins synaptophysin and PSD-95 were significantly increased, but GAP-43 was not. The preservation of myelinated axons and synapses, along with glial and endothelial cells, was confirmed by ultrastructural analysis. Furthermore, intranuclear inclusion bodies, which are associated with normal aging in vivo, were detected in the CA1 pyramidal layer in cultures older than 14 DIV. When OHSCs were maintained for approximately 3, 4, and 10 weeks, a rise and then fall in the expression of synaptophysin and, especially, PSD-95 were found, and the biphasic trend paralleled by significant changes in Schaffer collateral-evoked excitatory post-synaptic potentials from CA1 neurons. Our data not only describe changes in cytoskeletal, synaptic, and nuclear proteins related to the maintenance of interface OHSCs, but also emphasize the potential of the model for the study of age-related phenomena within the hippocampus.
Assuntos
Citoesqueleto/metabolismo , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Proteínas Nucleares/metabolismo , Sinapses/metabolismo , Animais , Western Blotting/métodos , Proteína 4 Homóloga a Disks-Large , Proteína GAP-43/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/ultraestrutura , Imuno-Histoquímica/métodos , Indóis , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestrutura , Sinaptofisina/metabolismo , Fatores de Tempo , Proteínas tau/metabolismoRESUMO
The Cl(-) channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) dose-dependently protected against oxygen-glucose deprivation in cultured rat cortical neurons. DIDS or SITS attenuated oxygen-glucose deprivation-induced increases in extracellular glutamate concentrations and intracellular Ca(2+). DIDS or SITS provided moderate protection against N-methyl-D-aspartate (NMDA) toxicity and decreased NMDA receptor-mediated increases in intracellular Ca(2+). Whole-cell NMDA receptor currents were attenuated 39+/-2% and 21+/-3% by 1 mM DIDS and SITS, respectively. Other Cl(-) channel blockers as equipotent as DIDS and SITS did not decrease oxygen-glucose deprivation- or NMDA-mediated neuronal Ca(2+) influx or toxicity. Neurotoxicity by exogenous glutamate was not prevented by SITS and was exacerbated by DIDS. Reductions in oxygen-glucose deprivation-induced increases in intracellular Ca(2+) levels underlie neuroprotection by DIDS and SITS. This was a reflection of lower extracellular [glutamate], direct inhibition of Ca(2+) influx through postsynaptic NMDA receptors, and possibly through other protective properties associated with DIDS and SITS.
Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Córtex Cerebral/efeitos dos fármacos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Anaerobiose , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Relação Dose-Resposta a Droga , Feminino , Glucose/farmacologia , Ácido Glutâmico/farmacologia , Ácido Glutâmico/toxicidade , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/toxicidade , Neurônios/citologia , Neurônios/fisiologia , Oxigênio/farmacologia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
The hippocampus is a complex network tightly regulated by interactions between excitatory and inhibitory neurons. In neurodegenerative disorders where cognitive functions such as learning and memory are impaired this excitation-inhibition balance may be altered. Interestingly, the uncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist memantine, currently in clinical use for the treatment of Alzheimer's disease, may alter the excitation-inhibition balance in the hippocampus. However, the specific mechanism by which memantine exerts this action is not clear. To better elucidate the effect of memantine on hippocampal circuitry, we studied its pharmacology on NMDAR currents in both pyramidal cells (PCs) and interneurons (Ints) in the CA1 region of the hippocampus. Applying whole-cell patch-clamp methodology to acute rat hippocampal slices, we report that memantine antagonism is more robust in PCs than in Ints. Using specific NMDAR subunit antagonists, we determined that this selective antagonism of memantine is attributable to specific differences in the molecular make-up of the NMDARs in excitatory and inhibitory neurons. These findings offer new insight into the mechanism of action and therapeutic potential of NMDA receptor pharmacology in modulating hippocampal excitability.
RESUMO
Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons; recently, we have also reported progress towards interrogating mammalian neurons. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel .The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
Assuntos
Análise em Microsséries/instrumentação , Neurônios/fisiologia , Técnicas de Patch-Clamp/instrumentação , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Técnicas de Cultura de Células/métodos , Fenômenos Eletrofisiológicos , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Lymnaea , Neurônios/citologia , Técnicas de Patch-Clamp/métodosRESUMO
Planar patch-clamp chip technology has been developed to enhance the assessment of novel compounds for therapeutic efficacy and safety. However, this technology has been limited to recording ion channels expressed in isolated suspended cells, making the study of ion channel function in synaptic transmission impractical. Recently, we developed single- and dual-recording site planar patch-clamp chips and demonstrated their capacity to record ion channel activity from neurons established in culture. Such capacity provides the opportunity to record from synaptically connected neurons cultured on-chip. In this study we reconstructed, on-chip, a simple synaptic circuit between cultured pre-synaptic visceral dorsal 4 neurons and post-synaptic left pedal dorsal 1 neurons isolated from the mollusk Lymnaea stagnalis. Here we report the first planar patch-clamp chip recordings of synaptic phenomena from these paired neurons and pharmacologically demonstrate the cholinergic nature of this synapse. We also report simultaneous dual-site recordings from paired neurons, and demonstrate dedicated cytoplasmic perfusion of individual neurons via on-chip subterranean microfluidics. This is the first application of planar patch-clamp technology to examine synaptic communication.
Assuntos
Potenciais de Ação/fisiologia , Análise em Microsséries/instrumentação , Microeletrodos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , CaramujosRESUMO
All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions - including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.
RESUMO
Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.
Assuntos
Isquemia Encefálica , Precondicionamento Isquêmico , Neurônios/metabolismo , Animais , Encéfalo/embriologia , Cálcio/metabolismo , Técnicas In Vitro , Microscopia de Fluorescência/métodos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Fenótipo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Tetrodotoxina/farmacologiaRESUMO
The importance of the hippocampus to learning and memory has attracted significant attention to how the structure responds to damage. Although many studies have used either the acute hippocampal slice preparation or organotypic hippocampal slice cultures, little work has been done to determine if the choice of model is an important variable. The present study examined whether differences exist in how each model responds to a commonly studied ischemic-like paradigm, oxygen-glucose deprivation. Following the insult, synaptic activity was examined by recording orthodromically evoked CA1 subfield responses, while mitochondrial activity was assessed by spectrophotometric measurement of formazan produced by metabolism of 2,3,5-triphenyltetrazolium chloride. The insult significantly decreased both synaptic and mitochondrial activity within acutely prepared slices, but a disparity existed between these measures in cultured slices. While evoked activity was greatly reduced by an insult of moderate duration, a much longer period was required to cause a comparable decrease in formazan production. Quantitative immunoblotting revealed that one possible explanation for the discrepancy was an elevated expression of astrocytes, which display resistance to hypoxia-aglycemia. Our data indicate that acutely prepared and cultured slices respond differently to ischemic-like challenge; therefore, assays examining viability in these models must consider their innate differences.