Name: culturing and electrophysiology of cells on nrcc patch-clamp chips
Uploaded: 2012-02-07T12:00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips at JoVE.com

Forfatterne ønsker at anerkende Alexei Bogdanov til fremstilling af patch-clamp-chips på CPFC og Hue Tran, Ping Zhao og Matthew Shiu for hjælp til montering. Naweed Syed blev understøttet af en canadisk Institute of Health Research (CIHR) tilskud. Collin Luk er modtager af NSERC og Alberta Heritage Foundation for medicinsk forskning (AHFMR) stipendier.

1. Chip fabrikation Fremgangsmåden beskrevet i 6 resulterer i en 3 um tyk selvstændig film, en lav 1 Mohm adgang modstand og en glat overflade tragtformet åbning, der letter en intim forsegling med cellen 9, se fig 2. De chips er singulated og limet i plexiglas pakker med blænden over et hul forbinder chip til en underjordisk kanal. Limning dispenseres på en måde, der minimerer shunten kapacitansen til en nominel 17 pF. Pakker er forsynet med to 1,5 mm i diameter og 6 mm lange rør, som strømningstekniske porte (figur 3). <img alt="Figur 2" src="/files/ftp_upload/3288/3288fig2.jpg" /> Figur 2. Scanningselektronmikrofotografi af en fokuseret ionstråle sektion af en NRcC patch-clamp chip viser en glat siliciumdioxid overflade og tragtform, der begunstiger intime celle-kontakter, og en flad dyse, der udgør en lav adgang resistance. <img alt="Figur 2" src="/files/ftp_upload/3288/3288fig2.jpg" /> Figur 3. En chip er limet på bunden af kulturen hætteglasset i en plexiglas pakke forsynet med underjordiske fluidik og glasrør. 2. Sterilisation, grunding og test Følgende trin skal udføres i en biologi sikkerhedskabinet for at undgå forurening eller tilstopning af hullet. <ul type="disc"><li> Steriliser chips i en Harrick grundlæggende plasma renere (www.harrickplasma.com) med 0,1-0,3 mbar resterende lufttryk i 15 minutter ved maksimal effekt på 18 W. Andre luft plasma systemerne kan anvendes, med sammenlignelig effekttæthed (24 mW / cm 3) og holde produktet af effekttæthed og tidskonstant. Plasmabehandlingen gør også chips hydrofile, hvilket letter priming. </li><li> Passer glasrør med sterilt Silastic Laboratory siliconerør, 1 mm ID x 2 mm OD (Cole ParmerKat. # 96.115-08), 3inch på den ene side en inch på den anden side. </li><li> Fylde fluidik gennem rørene med sterilfiltreret standard phosphatpufferopløsning (PBS), at sørge for ikke boble er fanget. </li><li> Klippe den lange silikoneslange medens tryk PBS fra den korte side ved 1 atm. En pulje af PBS siver gennem åbningen kan være synlige i hætteglasset. Klip tryk levering af PBS og tag den korte silikoneslange fra forsyningen. </li><li> Fylde hætteglasset med filtreret PBS under anvendelse af en sprøjte. </li><li> Nedsænkes en Ag / Ag: Cl elektrode i kort rør, og en modelektrode i hætteglasset. En impedans meter anvendes til at bekræfte, at adgang til modstanden mellem 300kohm og 3Mohm og shunt kapacitans mellem 10pF og 25Pf. En chip med en lavere modstand sandsynligvis har en lækage, og en højere kapacitet anses uhensigtsmæssig til anvendelse som det ikke kan indfange de hurtigste dynamik cellens elektrofysiologi 6. En chip med en lavere kapacitanseller højere modstand anses sluttet, selvom det kunne være simpelthen at en boble er fanget i åbningen. Nogle chips testes igen efter 1H og fundet at have den korrekte elektrokemiske impedans. </li><li> Skylle strømningstekniske kanal med sterilt deioniseret vand, tømme det øverste hætteglasset og skylles med samme to gange. </li><li> Nedsænkes emballerede chip med rør i frisk sterilt deioniseret vand i 30 minutter og derefter indlæse med andre chips i en steril beholder fyldt med sterilt deioniseret vand. Dette sikrer, at chips forbliver hydrofil, og beskyttet mod forurening. </li></ul> 3. Chips forberedelse i cellebiologi Lab De følgende trin udføres i et HEPA-filtreret laminar strømningshætte anvendelse af aseptisk teknik, skal alle opløsninger, der anvendes i denne procedure filtreres steriliseres før brug under anvendelse af en 0,22 um filter. <ul type="disc"><li> Åbn en chip beholder under et HEPA filtreret laminar flow hætte og fjerne chips fra container nedad for at undgå øse mulige flydende forureninger i top hætteglasset. </li><li> Skylning toppen af ​​hætteglasset chip 2x med frisk filtreret sterilt deioniseret vand for at fjerne eventuel resterende snavs fra chip overfladen. </li><li> Aspirer sterilt deioniseret vand fra toppen hætteglasset. </li><li> Forbinde den ene ende af silastic-rør fastgjort til den fluide kanal til en tryksat sprøjte indeholdende sterilt filtreret deioniseret vand. I vores system er sprøjter fyldt med opløsning tryk ved forbindelse til en tryklufttank indstillet til 20 psi. For at sikre sterilitet, bliver luften filtreret (0,22 um filter) forud for indtræden i sprøjten, og vores system har også en on / off ventil, der giver os mulighed for at stoppe eller lægge pres når det er nødvendigt. </li><li> Ved hjælp af en hæmostat, udgangsenden af ​​røret klemme. </li><li> Åbne on / off ventiler at presse opløsningen. </li><li> At skylle subterranen strømningstekniske af chippen med frisk sterilt deioniseret vand, løsne klemmerne udgangsenden af ​​røret. </li><li> Til force vand op gennem åbningen, klemme udgangsenden af ​​røret med en hæmostat. </li><li> For at undgå vandafledning, klemme indgangs-og udgangsenderne af slangen med en hemostats og lukke on / off ventiler. </li><li> Løsnes indgangsenden af ​​røret fra sprøjten. </li><li> Indsætte glas propper i begge ender af røret og fjerne hemostats. </li><li> Fyld top hætteglas med filter steriled deioniseret vand. </li><li> Låget af en 35 mm steril skål ind i bunden af ​​en 100 mm steril skål </li><li> Sted chip oven på 35 mm låget og dækslet 100 mm skål låg. </li><li> Billeddata chips for at sikre, at membranen og åbningen af spånerne er fri for snavs og / eller luftbobler. I vores laboratorium har vi anvender en opretstående mikroskop og et 20x lang arbejdsafstand mål. </li><li> Hvis snavs og / eller luftbobler er til stede, skyl fluidisk kanal og top hætteglasset. Hvis snavs og / eller luftbobler ikke kan fjernes chippen kasseres. </li><li> Når de chips, erafbildes, tilbage til laminært flow og trække begge ender af røret. </li><li> Vedhæfte den ene ende af røret til en tryksat sprøjte indeholdende fysiologiske medier. </li><li> Klemme udgangsenden af ​​røret med en hæmostat </li><li> Åbne overtryksventil, fjerne hæmostat fra udgangsenden af ​​fluidik og skyl strømningstekniske kanal med fysiologiske medier </li><li> At skylle underjordiske strømningstekniske med fysiologiske medier, åbne on / off ventiler og fjerne hæmostat fra udgangsenden af ​​røret. </li><li> At tvinge de fysiologiske medier op gennem åbningen, klemme udgangsenden af ​​røret med en hæmostat. </li><li> Clamp input-ende af røret med en hæmostat og lukke on / off ventiler. </li><li> Fjerne indgang ende af røret fra sprøjten og proppen begge ender af røret med glas propper. </li><li> Fjern hemostats. </li><li> Fjerne vandet ovenfra hætteglasset. </li><li> Fyld top hætteglasset med filtersteriliseres fysiologiske medier. </li><li>Placere chip tilbage til 100 mm petriskål. </li><li> Placere bunden af ​​en 35 mm skål i 100 mm skål og fylde den med filtersteriliseret deioniseret vand for at berige fugtighed. </li><li> Dæk fadet, indtil det er tid til plettering </li></ul> 4. Celleudpladning af snegl neuroner De patch-clamp chips kan være egnet til en række præparater. Vi tester i øjeblikket vores chips med pattedyr primære kortikale neuroner og har fået de foreløbige resultater med celler dyrket i 14 dage 7, hvilket indikerer, at vores sterilisation protokollen er tilstrækkelig, og at de chips ikke er cytotoksisk i længerevarende kulturer. Med henblik på denne protokol blev sneglen neuroner valgt, fordi de repræsenterer en enkel, men veletableret model til at studere neuronal elektrofysiologi 11, og det er med de celler, vi har fået de væsentligste resultater til dato 10. Detaljeret celleisolering og kultur procedurer harblevet beskrevet tidligere 12, 13. <ul type="disc"><li> Fjerne den ydre skal 2-3 måneder gamle Lymnaea stagnalis med stumpe pincet og bedøve dyr i Lymnaea saltvand indeholdende 10% Listerine. Alle efterfølgende trin udføres aseptisk i en laminar luftstrøm hætte med steriliseret dissektion udstyr og opløsninger ved stuetemperatur. </li><li> Erstatte fysiologiske medier i fluidik med det passende optagelse opløsning under anvendelse af en Eppendorf-pipette. I tilfælde af Lymnaea neuroner opløsningen indeholder (i mM): 50 KCI, 5 MgCl2, 5 ethylenbis (oxyethylennitrilo) tetraeddikesyre (EGTA) og 5 4 - (2-hydroxyethyl)-1-piperazinethansulfonsyre (HEPES; pH 7,4 ; 130 mOsm) 14 </li><li> Pin sneglene i en Sylgard skål fyldt med Lymnaea saltvand og fjerne hele hjernen som tidligere beskrevet 13 </li><li> Behandle isolated hjerner i definerede medier (DM) med trypsin enzym (0,2% opløsning, Sigma, katalog nr. T-9201) i 18 minutter efterfulgt af behandling i DM og trypsin-inhibitor (Sigma, sojabønne, Catalog # T-9003) i 15 minutter. </li><li> Pin hjernerne i en lille Sylgard skål fyldt med høj osmolaritet definerede medier (HODM - DM med glucose tilsættes 750 pi 1 M glucoseopløsning til 20 ml DM). </li><li> Ved hjælp af fine tænger og dissektion sakse, det ydre og indre lag af ganglierne af interesse fjernes, og udsætter neuronerne. </li><li> Under anvendelse af en flammepoleret glaspipette fyldt med HODM og fastgjort til en mikrosprøjte, forsigtigt sugning nær cellen af ​​interesse (venstre pedal dorsal 1), indtil neuron frigøres fra hjernen og suspenderes i pipetten. </li><li> Den glaspipette bevæges derefter og dyppes i individuelle neurochips hvor blid udsendelse fra mikrosprøjten giver de neuroner, der skal forsigtigt skubbet ud af pipetten og anbringes på toppen af ​​de enkelte patch hullerne på chips.</li><li> Tillader celler at sidde uforstyrret i mindst 2 timer ved stuetemperatur for at fremme deres fastgørelse til spånfladen omgiver åbningen. </li></ul> 5. Elektrofysiologiske optagelser For at forbinde chips til forstærkeren (i vores tilfælde en Multiclamp 700B forstærker, Molecular Devices, Foster City, CA, USA) <ul type="disc"><li> Erstatte opløsningen i den underjordiske strømningstekniske og chippen hætteglasset med de passende optagelse løsninger. I tilfælde af Lymnaea neuroner, er de øvre kultur kamre fyldes med Lymnaea saltvand (i mM: 51,3 NaCl, 1,7 KCI, 4 CaCl2, og 1,5 MgCl2) pufret til pH 7,9 med HEPES 14. </li><li> For stabilitet. Lim chippen på et objektglas, og læg det under et mikroskop </li><li> For at tilslutte chip til forstærkeren (i vores tilfælde en Multiclamp 700B forstærker, Molecular Devices, Foster City, CA, USA), skæres den ene ende af slangen og sæt en Silver tråd, der er forbundet til hoved-fase. </li><li> Anbring referenceelektroden i toppen hætteglasset af chippen. </li><li> Chippen er nu klar til optagelse. </li><li> Anvende en 5 mV trin med forstærkeren til måling af total modstand (R t) og bestemme konfigurationen af neuroner: celle-tilknyttet (Rt&gt; 1 GQ) helcelle-(Rt = 50-100 MQ plus kapacitiv transienter, vejledende af membranbrud over åbningen), eller nogen tætning (Rt &lt;5 MQ). I vores sidste sæt af eksperimenter 10, havde 58% procent af cellerne høj modstand sæler, og af dem, 80% af cellerne viste irritable reaktioner. </li></ul> 6. Repræsentative resultater <ul type="disc"><li> Anvend depolariserende strømimpulser (20 pA tilvækst) til neuron (i dette tilfælde LPeD1). </li><li> Hold neuron ved Vm tæt på sin hvilende potentiale (~ - 60 mV). </li><li> Overhold svarene på disse depolariserende impulser. Overskridelse aktions potentialer børskal overholdes, hvis neuron er levedygtig. </li><li> Figur 4 viser et repræsentativt resultat, der er diskuteret i detaljer i 14, 15. </li></ul><img alt="Figur 4" src="/files/ftp_upload/3288/3288fig4.jpg" /> Figur 4. Voltage reaktioner (øverst) i en LPeD1 neuron til trindelt række intracellulære strømimpulser (nedenfor). De nuværende pulser blev anvendt ved Vm = - 60mV. Fejlfinding <ul type="disc"><li> Før plating, at billedet chips afgøre, om membranen og blænde er fri for snavs og egnet til plettering. Vi bruger en oprejst mikroskop og en 20x lang arbejdsafstand mål. I vores sidste sæt af forsøg 1, blev 67% procent af chips succes grundes på denne måde. </li><li> Hvis snavs og / eller luftbobler er til stede kassere chip og prøv en anden. </li><li> Forskellige overfladebehandlinger (plasma) eller overtræk (PDL, PEI etc.) kan prøvet, men succes kan være meget afhængig af celletypenanvendes. </li><li> Sammensætningen af ​​strømningstekniske (&quot;pipette&quot;)-opløsning i kritisk for dannelsen af ​​Giga-tætning og hel-celle. Juster løsning, at være opmærksom på osmolaritet, pH og ion-makeup. </li><li> Ved længere tids kultur, begynder med medier i mikrofluidkanal, derefter skifte til pipettering løsning forud for optagelse via blid tyngdekraften fodret perfusion (~ 0,5 ml / min). </li></ul>

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Ingen interessekonflikter erklæret.

NRcC patch-clamp chip afhøring platform er et potentielt effektivt redskab til høje informationsindhold farmaceutiske assays og til at undersøge in vitro modeller af sygdom. Dens fordele sammenlignet med glaspipetter er en lav adgang modstand, hvilket er en fordel at probe store celler, og på trods af en noget større kapacitans vil resultere i sammenlignelige dynamik for mindre celler. Spontan celle til blænde sæler er rutinemæssigt blevet indhentet, og hele celleindtastning er blevet observeret at være spontan 14. En klar forskel mellem chips og glaspipette metode er, at proben er en del af celledyrkningsskål og ikke manuelt bringes i kontakt med cellemembranen. Dyrkning af celler, eventuelt en del af funktionelle net, resulterer i mere biologisk relevante modeller som sygdomsmodeller og en anden mekanisme til fastgørelse høj celle til at probe forseglinger 16. Imidlertid, i modsætning til cellesuspensioner, canno aspirationt anvendes til at positionere en celle på proben. Sneglen neuroner, som andre store celler, er egnede til manuel placering på toppen af ​​sonden. For mindre celler kræver længere kultur gange, har vi undgås behovet for en manipulation og holde en høj sandsynlighed for at opnå en forsegling ved mønstring vedhæftning polypeptider oven af ​​proberne til at placere celler oven på prober, og viste placering af celler på sonde 17,18. NRcC udvikler også en polyimid mikrofluid patch-clamp chip 19 med en kapacitans sammenlignelig med glaspipette. Det endelige mål for dette projekt er en multiple-sonder patch-clamp-chip, som gør det muligt samtidig monitorering af den elektrofysiologiske aktivitet i flere neuroner involveret i netværket adfærd på løsningen af de enkelte ionkanaler 14. Denne metode er en høj opløsning supplerende metode til multi-elektrode-arrays 20.

culturing and electrophysiology of cells on nrcc patch-clamp chips

På grund af sin udsøgte følsomhed og evnen til at overvåge og kontrollere de enkelte celler på niveauet af ionkanaler, patch-fastspænding er guld standard for elektrofysiologi anvendes på sygdomsmodeller og farmaceutiske skærme både 1. Fremgangsmåden traditionelt involverer forsigtigt at kontakte en celle med en glaspipette fyldt med en fysiologisk opløsning med henblik på at isolere en patch af membranen under dens spids 2. En elektrode indsættes i pipetten indfanger ion-kanal-aktivitet inde i membranen plaster eller, når brydes for hele celler. I det sidste årti har patch-clamp chips blevet foreslået som et alternativ 3, 4: en ophængt film adskiller fysiologisk medium fra dyrkningsmediet, og en åbning mikrofabrikerede i filmen erstatter toppen af pipetten. Patch-clamp chips er blevet integreret i automatiserede systemer og kommercialiseret til high-throughput screening 5. Til Incrlette gennemløb, omfatter de strømningstekniske levering af celler fra suspensionen, deres placering på åbningen ved sugning, og automatiserede rutiner for at opdage celle-til-probe forseglinger og træde ind i helcelle tilstand. Vi har rapporteret om fremstilling af en silicium patch-clamp chip med optimeret impedans og åbning form, der tillader høj kvalitet optagelse af action potentialer i dyrkede sneglen neuroner 6, for nylig, har vi også rapporteret om fremskridt i retning af forhør af pattedyr neuroner 7. Vores patch-clamp-chips er fremstillet på den canadiske Photonics Fabrication Centre 8, en kommerciel støberi, og fås i store serier. Vi er ivrige efter at engagere sig i samarbejde med electrophysiologists at validere brugen af ​​NRcC teknologi i forskellige modeller. De chips, der anvendes ifølge den almindelige opbygning vist i figur 1: silicium chip er i bunden af ​​en plexiglas kultur hætteglas og bagsiden af ​​åbningen er forbundet med en underjordisk channEl forsynet med rør ved hver ende af emballagen. Cellerne dyrkes i hætteglasset, og cellen på enden af ​​sonden overvåges ved en måleelektrode indsat i channel.The to uden strømningstekniske porte lette opløsning udveksling med minimal forstyrrelse af cellen, hvilket er en fordel i forhold til glaspipetter til intracellulær perfusion. <img alt="Figur 1" src="/files/ftp_upload/3288/3288fig1.jpg" /> Figur 1. Princip for måling ved hjælp af NRcC patch-clamp chip Vi detaljer her de protokoller, der sterilisere og primes chips, indlæse dem med medium, plade dem med celler, og til sidst bruge dem til elektrofysiologiske optagelser.

Watch this Scientific Journal Video about Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips at JoVE.com

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Vi viser, hvordan plane patch-clamp-chips fremstillet på National Research Council of Canada er steriliseret, grundes, fyldt med medium, belagt med celler, og anvendes til elektrofysiologiske optagelser.

Dyrkning og Elektrofysiologi af celler på 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 ...

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

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch clamp optagelser i indre øre-hårceller isoleret fra zebrafisk

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

Hele Cell Patch Clamp for Undersøgelse af mekanismerne i Infrarød Neural Stimulation

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolering af sensoriske neuroner i<em&gt; Aplysia californica</em&gt; For patchclamp Optagelser af glutamaterge strømforhold

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patchclamp Optagelser fra embryonale zebrafisk Mauthner Cells

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

En computer-assisteret Multi-elektrode Patch-clamp System

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Helcelle-patch-clamp Optagelser fra Morphologically- og Neurochemically identificerede Hippocampale interneuroner

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-celle Patch-clamp Optagelser i Brain Skiver

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcellulære Patch-clamp Optagelser fra Somatodendritisk Domain of nigrale dopamin neuroner

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch Clamp Registrering af Starburst amacrine Celler i en væg-mount Udarbejdelse af Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.