culturing-and-electrophysiology-of-cells-on-nrcc-patch-clamp-chips

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

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 ...

The connections of limbic structures are remarkably constant across species. However, different species express fear in distinct ways. Because the central amygdaloid nucleus is considered an important mediator of fear responses, we compared the physiological properties of central amygdala neurons in guinea pigs, rats and cats using whole-cell recordings in coronal slices in vitro. In guinea pigs, most central medial and lateral neurons displayed an outward rectification that delayed firing onset in response to depolarizing current pulses (hence the designation late-firing neurons). Late-firing neurons were also prevalent in the lateral sector of the cat central nucleus. In contrast, late-firing neurons were rare in the rat central nucleus and the cat central medial nucleus. There, most neurons generated Ni2+-sensitive low-threshold bursts of Na+ spikes and/or displayed pronounced inward rectification in the hyperpolarizing direction. Species differences in passive properties were also observed, but they were mainly related to the relative prevalence of late-firing cells. Indeed, late-firing cells had a significantly lower input resistance and more negative membrane potential than other types of central neurons. Thus, there are marked species differences in the physiological properties of central neurons. Because the synaptic responsiveness of neurons is constrained by their physiological properties, our findings raise the possibility that the contrasting behavioural responsiveness observed across species is partly dependent on the species-specific physiological properties of central neurons.

Physiological properties of central amygdala neurons: species differences.

Physiologische Eigenschaften von zentralen Neuronen der Amygdala: Arten Unterschiede.

The organization of the neuronal hippocampal network depends on the tightly regulated interaction between pyramidal cells (PCs) and interneurons (Ints). NMDA receptor (NMDAR) activation requires the binding of glutamate and co-activation of the 'glycine site'. It has been reported that D-serine is a more potent endogenous agonist than glycine for that site. While many studies have focused on NMDAR function in PCs, little is known regarding the modulation of NMDARs in Ints. We studied the modulatory effect of D-serine on NMDAR EPSCs in PCs and in stratum radiatum Ints using whole-cell patch-clamp recording in rat acute hippocampal slices. We found that D-serine enhances NMDAR function and differently modulates NMDAR currents in both cell types. The augmentation of NMDAR currents by D-serine was significantly larger in PCs compared with Ints. Moreover, we found differences in the kinetics of NMDAR currents in PCs and Ints. Our findings indicate that regulation of NMDAR through the 'glycine site' depends on the cell types. We speculate that the observed differences arise from assemblies of diverse NMDAR subunits. Overall, our data suggest that D-serine may be involved in regulation of the excitation-inhibition balance in the CA1 hippocampal region.

D-Serine differently modulates NMDA receptor function in rat CA1 hippocampal pyramidal cells and interneurons.

D-Serin unterschiedlich moduliert NMDA-Rezeptor-Funktion bei Ratten in CA1 Pyramidenzellen des Hippocampus und Interneuronen.

Long-term potentiation (LTP) in the hippocampal CA1 region requires the activation of NMDA receptors (NMDARs). NMDAR activation in turn requires membrane depolarization as well as the binding of glutamate and its coagonist glycine. Previous pharmacological studies suggest that the glycine transporter type 1 (GlyT1) maintains subsaturating concentrations of glycine at synaptic NMDARs. Antagonists of GlyT1 increase levels of glycine in the synaptic cleft and, like direct glycine site agonists, can augment NMDAR currents and NMDAR-mediated functions such as LTP. In addition, stimulation of the glycine site initiates signalling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis. We have used a new potent GlyT1 antagonist, CP-802,079, with whole-cell patch-clamp recordings in acute rat hippocampal slices to determine the effect of GlyT1 blockade on LTP. Reverse microdialysis experiments in the hippocampus of awake, freely moving rats, showed that this drug elevated only the extracellular concentration of glycine. We found that CP-802,079, sarcosine and glycine significantly increased the amplitude of the NMDAR currents and LTP. In contrast, application of higher concentrations of CP-802,079 and glycine slightly reduced NMDAR currents and did not increase LTP. Overall, these data suggest that the level of glycine present in the synaptic cleft tightly regulates the NMDAR activity. This level is kept below the 'set point' of the NMDAR internalization priming mechanism by the presence of GlyT1-dependent uptake.

Glycine transporter type 1 blockade changes NMDA receptor-mediated responses and LTP in hippocampal CA1 pyramidal cells by altering extracellular glycine levels.

Glycin-Transporter Typ-1-Blockade &#xC4;nderungen NMDA-Rezeptor-vermittelten Reaktionen und LTP in hippocampalen CA1 Pyramidenzellen durch Ver&#xE4;nderung der extrazellul&#xE4;ren Glycin-Spiegel.

N-methyl-d-aspartate receptor (NMDAR) activation requires both the binding of glutamate to its recognition site and occupancy of the strychnine insensitive glycine modulatory site (GMS). Pharmacological studies suggest that the glycine transporter, GlyT1, maintains subsaturating concentrations of glycine at synaptic NMDARs. To characterize further the role of GlyT1, we generated mice in which the gene encoding GlyT1 was inactivated by homologous recombination through insertion of a PGK-Neo cassette in place of exons 2 and 3. Real-time quantitative PCR revealed no transcripts in newborn homozygous [GlyT1(-/-)] mice and a 50% reduction in heterozygous (HZ) [GlyT1(+/-)] mice as compared with WT littermates. The activity of Na(+)-dependent glycine transport in forebrain homogenates was similarly affected. Homozygous mice died within 12 h of birth. In acute hippocampal slices, exogenous glycine or d-serine (10 microM) enhanced NMDAR currents with Schaffer collateral stimulation in WT mice but not HZ mice, suggesting that the GMS was more occupied in the latter. The NMDAR/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ratio of the excitatory postsynaptic currents was significantly increased in the HZ mice. In the water maze, the HZ mice exhibited better spatial retention. Furthermore, HZ mice were less sensitive to an amphetamine disruption of prepulse inhibition than WT mice but were more sensitive to the effects of MK-801. Thus, reduced expression of GlyT1 enhances hippocampal NMDAR function and memory retention and protects against an amphetamine disruption of sensory gating, suggesting that drugs which inhibit GlyT1 might have both cognitive enhancing and antipsychotic effects.

Gene knockout of glycine transporter 1: characterization of the behavioral phenotype.

Gen-Knockout von Glycin-Transporter-1: Charakterisierung des Verhaltens-Ph&#xE4;notyp.

To investigate the effects of persistent elevation of synaptic glycine at Schaffer collateral-CA1 synapses of the hippocampus, we studied the glutamatergic synaptic transmission in acute brain slices from mice with reduced expression of glycine transporter type 1 (GlyT1+/-) as compared to wild type (WT) littermates using whole-cell patch-clamp recordings of CA1 pyramidal cells. We observed faster decay kinetics, reduced ifenprodil sensitivity and increased zinc-induced antagonism in N-methyl-d-aspartate receptor (NMDAR) currents of GlyT1+/- mice. Moreover, the ratio alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)/NMDAR was decreased in mutants compared to WT. Surprisingly, this change was associated with a reduction in the number of AMPARs expressed at the CA1 synapses in the mutants compared to WT. Overall, these findings highlight the importance of GlyT1 in regulating glutamatergic neurotransmission.

Reduced glycine transporter type 1 expression leads to major changes in glutamatergic neurotransmission of CA1 hippocampal neurones in mice.

Reduzierte Glycin-Transporter Typ-1-Expression f&#xFC;hrt zu erheblichen Ver&#xE4;nderungen in glutamatergen Neurotransmission von CA1 Neuronen des Hippocampus bei M&#xE4;usen.

The sigma receptor (sigmaR), once considered a subtype of the opioid receptor, is now described as a distinct pharmacological entity. Modulation of N-methyl-D-aspartate receptor (NMDAR) functions by sigmaR-1 ligands is well documented; however, its mechanism is not fully understood. Using patch-clamp whole-cell recordings in CA1 pyramidal cells of rat hippocampus and (+)pentazocine, a high-affinity sigmaR-1 agonist, we found that sigmaR-1 activation potentiates NMDAR responses and long-term potentiation (LTP) by preventing a small conductance Ca2+-activated K+ current (SK channels), known to shunt NMDAR responses, to open. Therefore, the block of SK channels and the resulting increased Ca2+ influx through the NMDAR enhances NMDAR responses and LTP. These results emphasize the importance of the sigmaR-1 as postsynaptic regulator of synaptic transmission.

The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus.

Der Sigma-Rezeptor (SigmaR), einst als Untertyp des opioid-Rezeptor, wird jetzt als eine eigenst&auml;ndige pharmakologische Entit&auml;t beschrieben. Modulation des N-Methyl-D-Aspartat-Rezeptor (NMDAR) Funktionen von SigmaR-1 Liganden ist gut dokumentiert; jedoch ist der Mechanismus nicht vollst&auml;ndig verstanden. Mit Patch-Clamp-ganz-Zell-Aufnahmen in CA1 Pyramidenzellen der Ratte Hippocampus und (+) Pentazocin, ein hoher Affinit&auml;t SigmaR-1 Agonist, wir fanden, dass SigmaR-1 Aktivierung NMDAR Antworten und langfristige Erm&ouml;glichung (LTP) verst&auml;rkt durch die Verhinderung einer kleinen Leitwert Ca2 +-aktivierte K + Strom (SK-Kan&auml;le), bekannt, dass die shunt-NMDAR Antworten, zu &ouml;ffnen. Daher steigert der Block der SK-Kan&auml;le und die daraus resultierende erh&ouml;hte Ca2 + Zustrom durch die NMDAR NMDAR Antworten und LTP. Diese Ergebnisse unterstreichen die Bedeutung des SigmaR-1 als postsynaptisches Regulator der synaptischen &Uuml;bertragung.

Der Sigma-1-Rezeptor moduliert synaptische &Uuml;bertragung von NMDA-Rezeptor und Plastizit&auml;t &uuml;ber SK-Kan&auml;le Ratte Hippocampus.

Dopamine (DA) receptor and NMDA receptor (NMDAR) activation in the lateral (LA) nucleus of the amygdala plays a critical role in emotional processing. Several distinct mechanisms regulate the molecular cross-talk between DA receptors and NMDARs in different brain regions; however, the cellular mechanism through which DA modulates NMDARs in LA projection neurons has not been studied. Here, we investigated the effect of DA receptor activation on NMDAR currents in LA projection neurons recorded in amygdala slices obtained from young rats. We found that DA reduces NMDAR current amplitudes in an additive manner through the activation of both D1-like and D2-like receptors. The reduction of NMDAR current amplitudes by D1-like receptor activation is mediated by a protein-protein interaction between the D1R and the NMDAR, while the regulation of NMDAR activity by D2-like receptors is elicited through a G protein-dependent pathway controlled by D4R. The results of our investigation show for the first time a functional interplay between D1R and D4R that mediates coincident G protein-independent and dependent regulation of NMDARs.

D1 and D4 dopaminergic receptor interplay mediates coincident G protein-independent and dependent regulation of glutamate NMDA receptors in the lateral amygdala.

Dopamin (DA)-Rezeptors und NMDA-Rezeptor (NMDAR) Aktivierung im seitlichen (LA) Kern der Amygdala spielt eine wichtige Rolle in der emotionalen Verarbeitung. Mehrere verschiedene Mechanismen regulieren die molekulare Nebensprechen zwischen DA-Rezeptoren und NMDARs in verschiedenen Hirnregionen; jedoch ist der zellulare Mechanismus, DA durch den NMDARs in LA Projektion Neuronen moduliert, nicht gepr&uuml;ft worden. Hier untersuchen wir die Wirkung der DA-Rezeptor-Aktivierung auf NMDAR Str&ouml;mungen in LA Projektion Neuronen aufgenommen in Amygdala Scheiben aus jungen Ratten gewonnen. Wir fanden, dass DA NMDAR aktuelle Amplituden in additiver Weise durch die Aktivierung wie D1 und D2-artigen Rezeptoren reduziert. Verringerung der aktuellen NMDAR-Amplituden von D1-Like-Rezeptor-Aktivierung wird durch eine Protein-Protein-Interaktion zwischen den D1R und die NMDAR, vermittelt, w&auml;hrend die Regulierung der NMDAR Aktivit&auml;t von D2-artigen Rezeptoren durch ein G-Protein-abh&auml;ngige-Pathway von D4R kontrolliert hervorgerufen wird. Die Ergebnisse unserer Untersuchung zeigen zum ersten Mal eine funktionale Zusammenspiel zwischen D1R und D4R, die zusammenfallenden G-Protein-unabh&auml;ngige und abh&auml;ngige Regelung der NMDARs vermittelt.

D1 und D4 Zusammenspiel der dopaminergen-Rezeptor vermittelt die zusammenfallenden G-Protein-unabh&auml;ngige und abh&auml;ngige Regelung der Glutamat-NMDA-Rezeptoren in der lateralen Amygdala.

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.

A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neurons.

Wir berichten &uuml;ber eine einfache und hochverzinsliche Herstellungsprozess f&uuml;r Silizium planar Patch-Clamp-Chips, die niederohmigen Kapazit&auml;ts- und Serie von individuell bestimmte kultivierten Neuronen erm&ouml;glichen. &Ouml;ffnungen sind in einem qualitativ hochwertige Silizium-Nitrid-Film auf einem Silizium-Wafer ge&auml;tzt; Brunnen sind auf der R&uuml;ckseite des Wafers durch Nass&auml;tzen ge&ouml;ffnet und passiviert ein dickes hinterlegten Siliciumdioxid-Film, die Kapazit&auml;t des Chips zu reduzieren und die Bildung einer Zelle hochohmige auf Blende Siegel zu erleichtern. Die Chip-Oberfl&auml;che eignet sich f&uuml;r die Kultur der Neuronen &uuml;ber eine kleine &Ouml;ffnung in das Substrat mit aktuellen minimal Leck. Diese Features werden kollektiv, High-Fidelity-elektrophysiologische Aufnahme Transmembran Str&ouml;mungen aus Ion Channel-Aktivit&auml;ten in kultivierten Neuronen aktivieren. Mit kultivierte Lymnaea Neuronen zeigen wir ganze Zellen aktuelle Aufnahmen ein Klemm-Spannung Anregung Protokoll entnommen und im Klemm-Strom-Modus berichten wir Aktionspotentiale durch Membrane Depolarisierung Schritte angeregt. Trotz der relativ gro&szlig;en Gr&ouml;&szlig;e dieser Neuronen zeigte gute zeitliche und r&auml;umliche Kontrolle der Zellmembran Spannung. Unseres Wissens ist dies der erste Bericht der Aufnahme von Ion Channel-Aktivit&auml;ten und Aktionspotentiale von Neuronen, die direkt auf eine planare Patch-Clamp-Chip kultiviert. Dieses Verh&ouml;r-Plattform hat enormes Potential als neuartige Werkzeug leicht hoher Informationsgehalt w&auml;hrend pharmazeutische Proben zur Untersuchung von in-vitro-Modelle von Krankheit, sowie neuronale Physiologie und synaptische Plastizit&auml;t bereitstellen.

Eine neuartige Patch-Clamp-Siliziumchip erlaubt originalgetreue Aufnahme von Ion Channel-Aktivit&auml;ten von funktional definierte Neuronen.

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.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip.

Wir pr&auml;sentieren einen Polymer-Mikrochip &Uuml;berwachung neuronaler Aktivit&auml;t mit einer Treue f&auml;hig, noch nie auf eine planare Patch-Clamp-Ger&auml;t erh&auml;ltlich. Kardio-respiratorische Neuronen linke Pedal Dorsal 1 (LPeD1) von Weichtieren Lymnaea wurden auf dem Mikrochip-Polyimid-Oberfl&auml;che f&uuml;r 2 bis 4 Stunden kultiviert. Kultivierte Neuronen gebildet Hochwiderstandsf&auml;hige Dichtungen (Gigaseals) zwischen der Zellmembran und die Oberfl&auml;che rund um &Ouml;ffnungen in die Polyimid ge&auml;tzt. Gigaseal Bildung wurde beobachtet, ohne &auml;u&szlig;ere Kraft, wie z. B. Absaugung, auf Neuronen zu &uuml;bernehmen. Die Bildung von Gigaseals, sowie die niedrigen Zugang Widerstand und Shunt Kapazit&auml;tswerte von Polymer-Mikrochip f&uuml;hrten zu High-Fidelity-Aufnahmen. Auf dem Chip Kultur von Neuronen erlaubt, zum ersten Mal auf einem Polymeren Patch-Clamp-Ger&auml;t, die Aufzeichnung von High Fidelity physiologische Aktionspotentiale. Microfabrication des Hybrid Poly (Dimethylsiloxane)-Polyimid (PDMS-PI) Mikrochip wird diskutiert, einschlie&szlig;lich eine zweischichtige PDMS Verarbeitung-Technik, was zu schrumpfen Variationen minimiert.

High-Fidelity-Patch-Clamp-Aufnahmen von Neuronen, die auf einem Mikrochip Polymer kultiviert.

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.

Cell to aperture interaction in patch-clamp chips visualized by fluorescence microscopy and focused-ion beam sections.

Patch-Clamp ist ein wichtiges Verfahren zur &Uuml;berwachung der elektrophysiologischen Aktivit&auml;t von Zellen und die Rolle der pharmakologischen Verbindungen auf bestimmte Ionen-Kanal-Proteine. In den letzten Jahren wurden planare Patch-Clamp-Chips als einen h&ouml;heren Durchsatz-Ansatz f&uuml;r die etablierten Glas-Pipette-Methode entwickelt. Ordnungsgem&auml;&szlig;en Zustand, die hohe Best&auml;ndigkeit-Zelle-zu-Probe-Dichtungen ben&ouml;tigt, um die kleine Str&ouml;me aus Ion Channel-Aktivit&auml;ten zu messen zu optimieren sind allerdings noch Gegenstand der Vermutung. Hier berichten wir &uuml;ber die Gestaltung des Multiple-Blende (Sieb) Chips schnell Bewertung der Zelle-zu-Blende Interaktionen in statistisch signifikanten Zahlen zu erleichtern. Wir schlagen eine Methode, um die Qualit&auml;t der Dichtungen, die auf der Grundlage eines Farbstoffes laden Protokoll durch &Ouml;ffnungen in den Chip und die anschlie&szlig;ende Auswertung mit konfokale Fluoreszenzmikroskopie Vorpr&uuml;fung. Wir zeigen auch die ersten Scan elektronenmikroskopische Aufnahme eines focused Ion Beam Abschnitts einer Zelle in einem Patch-Clamp-Chip-Blende.

Handy-Blende Interaktion in Patch-Clamp-Chips von Fluoreszenzmikroskopie und konzentriert-Ionen Balkenquerschnitte visualisiert.

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.

Recordings of cultured neurons and synaptic activity using patch-clamp chips.

Planare Patch-Clamp-Chip-Technologie wurde entwickelt, um die Beurteilung der neuartigen Compounds f&uuml;r die therapeutische Wirksamkeit und Sicherheit zu verbessern. Jedoch ist diese Technologie beschr&auml;nkt sich auf die Aufnahme von Ionenkan&auml;len in lokalisierten gesperrten Zellen ausgedr&uuml;ckt, machen das Studium der Ionen-Kanal-Funktion in der synaptischen &Uuml;bertragung unpraktisch gewesen. K&uuml;rzlich wir Single - und Dual-Recording-Website-planare Patch-Clamp-Chips entwickelt und demonstriert ihre Kapazit&auml;t aufzeichnen Ion Channel-Aktivit&auml;ten von Neuronen, die Kultur gegr&uuml;ndet. Dieser Kapazit&auml;t bietet die M&ouml;glichkeit, die Aufzeichnung von synaptisch verbundenen Neuronen kultiviert auf dem Chip. In dieser Studie, die wir rekonstruiert, isoliert auf dem Chip, eine einfache synaptische Schaltung zwischen kultivierten pre-synaptic viszerale dorsalen 4 Neuronen und post-synaptic linken Pedal dorsal 1 Neuronen aus dem Mollusk Spitzschlammschnecke. Hier berichten wir die ersten planare Patch-Clamp-Chip-Aufnahmen der synaptischen Ph&auml;nomene aus diesen Neuronen gekoppelt und pharmakologisch demonstrieren die cholinerge Natur dieser Synapse. Wir auch simultane Dual-Site-Aufnahmen von gepaarten Neuronen zu melden und engagierten zytoplasmatischen Perfusion einzelner Neuronen &uuml;ber unterirdische Mikrofluidik-Chip zu demonstrieren. Dies ist die erste Anwendung der planare Patch-Clamp-Technik, synaptische Kommunikation zu pr&uuml;fen.

Aufnahmen von kultivierten Neuronen und synaptische Aktivit&auml;t mit Patch-Clamp-Chips.

Glycine serves a dual role in neurotransmission. It is the primary inhibitory neurotransmitter in the spinal cord and brain stem and is also an obligatory coagonist at the excitatory glutamate, N-methyl-D-aspartate receptor (NMDAR). Therefore, the postsynaptic action of glycine should be strongly regulated to maintain a balance between its inhibitory and excitatory inputs. The glycine concentration at the synapse is tightly regulated by two types of glycine transporters, GlyT1 and GlyT2, located on nerve terminals or astrocytes. Genetic studies demonstrated that homozygous (GlyT1-/-) newborn mice display severe sensorimotor deficits characterized by lethargy, hypotonia, and hyporesponsivity to tactile stimuli and ultimately die in their first postnatal day. These symptoms are similar to those associated with the human disease glycine encephalopathy in which there is a high level of glycine in cerebrospinal fluid of affected individuals. The purpose of this investigation is to determine the impact of chronically high concentrations of endogenous glycine on glutamatergic neurotransmission during postnatal development using an in vivo mouse model (GlyT1+/-). The results of our study indicate the following; that compared with wild-type mice, CA1 pyramidal neurons from mutants display significant disruptions in hippocampal glutamatergic neurotransmission, as suggested by a faster kinetic of NMDAR excitatory postsynaptic currents, a lower reduction of the amplitude of NMDAR excitatory postsynaptic currents by ifenprodil, no difference in protein expression for NR2A and NR2B but a higher protein expression for PSD-95, an increase in their number of synapses and finally, enhanced neuronal excitability.

Chronically saturating levels of endogenous glycine disrupt glutamatergic neurotransmission and enhance synaptogenesis in the CA1 region of mouse hippocampus.

Glycin dient eine Doppelrolle in Neurotransmission. Es ist die prim&auml;re inhibitorischer Neurotransmitter im R&uuml;ckenmark und Hirnstamm und auch eine obligatorische Coagonist an der exzitatorischen Glutamat, N-Methyl-D-Aspartat-Rezeptor (NMDAR). Daher sollte die postsynaptisches Aktion von Glycin stark geregelt werden, um die Aufrechterhaltung eines Gleichgewichts zwischen seiner inhibitorischen und exzitatorischen Eing&auml;nge. Die Glycin-Konzentration an der Synapse wird durch zwei Arten von Glycin-Transporter, GlyT1 und GlyT2, gelegen auf Nervenenden oder Astrozyten eng geregelt. Genetische Studien zeigten, dass homozygote (GlyT1-/-) Neugeborene M&auml;use zeigen schwere sensomotorischen Defizite gekennzeichnet durch Lethargie, Muskelhypotonie und Hyporesponsivity auf taktile Reize und letztlich zu ihrer ersten postnatalen Zeit sterben. Diese Symptome &auml;hneln denen der menschlichen Krankheit Glycin Enzephalopathie in denen besteht ein hohes Ma&szlig; an Glycin im Liquor von betroffenen Personen zugeordnet. Diese Untersuchung ist f&uuml;r die Bestimmung die Auswirkungen der chronisch hohe Konzentrationen von endogenen Glycin auf Glutamat Neurotransmission w&auml;hrend der postnatalen Entwicklung mit einem in-vivo Mausmodell (GlyT1 +/-). Die Ergebnisse unserer Studie zeigen Folgendes: das im Vergleich mit Wildtyp M&auml;usen, CA1 pyramidaler Neurons von Mutanten Anzeige erhebliche St&ouml;rungen in hippocampal Glutamat Neurotransmission, wie von einem schneller kinetische NMDAR exzitatorischen postsynaptisches Str&ouml;mungen, eine geringere Reduktion der Amplitude der NMDAR exzitatorischen postsynaptisches Str&ouml;me von Ifenprodil, keinen Unterschied in der Protein-Ausdruck f&uuml;r NR2A und NR2B, sondern einen h&ouml;heren Proteinausdruck f&uuml;r PSD-95, ihre Zunahme der Synapsen und schlie&szlig;lich, verbesserte neuronale Erregbarkeit.

Chronisch S&auml;ttigung Niveaus von endogenen Glycin st&ouml;ren Glutamat Neurotransmission und Verbesserung der Synaptogenese in der Region CA1 des Hippocampus Maus.

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.

From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology.

Alle erregbar Zelle Funktionen st&uuml;tzen sich auf Ionenkan&auml;le, die in ihrer Plasmamembran eingebettet sind. St&ouml;rungen von Ionen-Kanal-Struktur oder Funktion f&uuml;hren Pathologien von kardialen Dysfunktion bis hin zu neurodegenerativen Erkrankungen. Um die Funktionen von excitable Zellen zu verstehen und ihre Pathophysiologie zu beseitigen, ist es daher wichtig zu verstehen, die Ion-Kanalfunktionen unter verschiedenen experimentellen Bedingungen - einschlie&szlig;lich der Gef&auml;hrdung durch neuartige Droge-Ziele. Glas-Pipette Patch-Clamp ist die modernste Technik, die intrinsischen und synaptischen Eigenschaften der Neuronen zu &uuml;berwachen. Aber diese Technik ist arbeitsintensiv und hat geringen Datendurchsatz. Planare Patch-Clamp-Chips, Automationen, integriert bieten hohe Durchs&auml;tze aber begrenzt an isolierten Zellen von Suspensionen, ihre Verwendung bei der Modellierung physiologische Funktion eingeschr&auml;nkt sind. Diese Chips sind daher nicht f&uuml;r Studien mit neuronalen Kommunikation am geeignetsten. Multielectrode Arrays (MEAs), im Gegensatz dazu haben die Netzwerk-Aktivit&auml;t &uuml;berwachen durch Messung lokales Feld Potenziale mehrerer extrazellul&auml;re Standorte, aber spezifische Ion Channel-Aktivit&auml;ten ist eine Herausforderung, um aus dieser Multiplex-Signale zu extrahieren. Hier beschreiben wir eine neuartige planare Patch-Clamp-Chip-Technologie, die es erm&ouml;glicht die gleichzeitige hochaufl&ouml;sende elektrophysiologische Befragung des einzelnen Neuronen an mehreren Standorten in synaptisch neuronale Netzwerke, damit kombinieren die Vorteile von MEA und Patch-Clamp-Technik. Jedes Neuron kann durch eine Blende sondiert werden, die einen dedizierten unterirdischen Microfluidic-Kanal verbindet. Neuronen wachsen in Netzwerken werden die &Ouml;ffnungen von Physisorbed oder chemisorbed chemische Signale ausgerichtet. In diesem Bericht beschreiben wir den Prozess-Design und Fertigung dieser Chips, Ans&auml;tze zur chemischen Musterung f&uuml;r Zelle Platzierung und physiologische Daten von kultivierten neuronalen Zellen.

Article	Year
Physiological properties of central amygdala neurons: species differences. The European journal of neuroscience\| PubMed ID: 11876782	2002
D-Serine differently modulates NMDA receptor function in rat CA1 hippocampal pyramidal cells and interneurons. The Journal of physiology\| PubMed ID: 12611916	2003
Glycine transporter type 1 blockade changes NMDA receptor-mediated responses and LTP in hippocampal CA1 pyramidal cells by altering extracellular glycine levels. The Journal of physiology\| PubMed ID: 15064326	2004
Gene knockout of glycine transporter 1: characterization of the behavioral phenotype. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15159536	2004
Reduced glycine transporter type 1 expression leads to major changes in glutamatergic neurotransmission of CA1 hippocampal neurones in mice. The Journal of physiology\| PubMed ID: 15661817	2005
The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus. The Journal of physiology\| PubMed ID: 17068104	2007
D1 and D4 dopaminergic receptor interplay mediates coincident G protein-independent and dependent regulation of glutamate NMDA receptors in the lateral amygdala. Journal of neurochemistry\| PubMed ID: 18662324	2008
A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neurons. Biotechnology and bioengineering\| PubMed ID: 20648547	2010
High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip. Biomedical microdevices\| PubMed ID: 20694518	2010
Cell to aperture interaction in patch-clamp chips visualized by fluorescence microscopy and focused-ion beam sections. Biotechnology and bioengineering\| PubMed ID: 21391207	2011
Recordings of cultured neurons and synaptic activity using patch-clamp chips. Journal of neural engineering\| PubMed ID: 21540486	2011
Chronically saturating levels of endogenous glycine disrupt glutamatergic neurotransmission and enhance synaptogenesis in the CA1 region of mouse hippocampus. Synapse (New York, N.Y.)\| PubMed ID: 21633974	2011
From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology. Frontiers in pharmacology\| PubMed ID: 22007170	2011

Marzia Martina

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