Forberedelse af akut rygmarven skiver til hele-celle Patch-klemme optagelse i Substantia Gelatinosa neuroner
Summary
Her beskriver vi de væsentlige skridt for hele-celle patch-klemme optagelser foretaget fra substantia gelatinosa (SG) neuroner i rygmarven skive i vitro . Denne metode giver den iboende membran egenskaber, synaptisk transmission og morfologiske karakterisering af SG neuroner som skal undersøges.
Abstract
Nylige hele-celle patch-klemme undersøgelser fra substantia gelatinosa (SG) neuroner har givet et stort antal oplysninger om de underliggende sensoriske transmission, nociceptive regulering og kronisk smerte eller kløe udvikling spinal mekanismer. Implementeringer af elektrofysiologiske optagelser med morfologiske undersøgelser baseret på nytte af akut rygmarven skiver er yderligere forbedret vores forståelse af neuronal egenskaber og sammensætning af lokale kredsløb i SG. Vi præsenterer her, en detaljeret og praktisk vejledning til forberedelse af rygmarven skiver og Vis repræsentative hele-celle optagelse og morfologiske resultater. Denne protokol tillader ideelle neuronal bevarelse og kan efterligne i vivo betingelser til en vis grad. I Resumé, evnen til at opnå en in vitro- forberedelse af rygmarven skiver giver stabil strøm – og spænding-klemme optagelser og kunne dermed lette detaljerede undersøgelser af de iboende membran egenskaber, lokale kredsløb og neuronal struktur ved hjælp af forskellige eksperimentelle metoder.
Introduction
Substantia gelatinosa (SG, lamina II af spinal dorsal Hornet) er et ubestrideligt vigtigt relæ center for fremsendelse og regulering af sensorisk information. Det er sammensat af excitatoriske og hæmmende interneurons, der modtager input fra de primære afferente fibre, lokale interneurons og den endogene faldende hæmmende system1. I de seneste årtier, har udvikling af akut rygmarven skive forberedelse og fremkomsten af hele-celle patch-klemme optagelse aktiveret forskellige undersøgelser på de iboende elektrofysiologiske og morfologiske egenskaber af SG neuroner2, 3 , 4 samt undersøgelser af lokale kredsløb i SG5,6. Desuden ved hjælp af in vitro- rygmarven skive forberedelse, forskere kan fortolke ændringer i neuronal excitabilities7,8, funktionen af ion-kanaler9,10, og Synaptic aktiviteter11,12 forskellige patologiske betingelser. Disse undersøgelser har uddybet vores forståelse af den rolle, som generalsekretæren neuroner spille i udviklingen og vedligeholdelse af kroniske smerter og neuropatiske klør.
Det væsentlige, den vigtigste forudsætning for at opnå en klar visualisering af neuronal soma og ideelle hele-celle lappe ved hjælp af akut rygmarven skiver er at sikre den fremragende kvalitet af skiver så sunde og patchable neuroner kan opnås. Forberede rygmarven skiver indebærer imidlertid flere trin, som udfører en ventrale laminektomi og fjerne pia-arachnoid membran, der kan være forhindringer i at opnå sund skiver. Selv om det ikke er let at tilberede rygmarven skiver, har udfører optagelser i vitro på rygmarven skiver flere fordele. I forhold til celle kultur præparater, kan rygmarven skiver delvis bevare iboende synaptiske forbindelser, der er i en fysiologisk relevante tilstand. Derudover kunne hele-celle patch-klemme optagelse ved hjælp af rygmarven skiver kombineres med andre teknikker, såsom dobbelt patch klemme13,14, morfologiske undersøgelser15,16 og encellede RT-PCR 17. derfor, denne teknik giver flere oplysninger om kendetegner de anatomiske og genetiske forskelle inden for et bestemt område og giver mulighed for undersøgelse af sammensætningen af lokale kredsløb.
Her give vi et grundlæggende og detaljeret beskrivelse af vores metode til at forberede akut rygmarven skiver og erhverve hele-celle patch-klemme optagelser fra SG neuroner.
Protocol
Representative Results
Discussion
Denne protokol detaljer trin for at forberede skiver, rygmarv, som vi har brugt med succes, når du udfører hele-celle patch-klemme eksperimenter på SG neuroner18,19,20,21. Ved at implementere denne metode, vi for nylig rapporteret, at minocycline, en anden generation af tetracyklin, kan markant forbedre hæmmende synaptisk transmission via en præsynaptiske mekanisme i SG neuroner<sup class=…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af tilskud fra den National Natural Science Foundation of China (nr. 81560198, 31660289).
Materials
| NaCl | Sigma | S7653 | Used for the preparation of ACSF and PBS |
| KCl | Sigma | 60130 | Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution |
| NaH2PO4·2H2O | Sigma | 71500 | Used for the preparation of ACSF, sucrose-ACSF and PBS |
| CaCl2·2H2O | Sigma | C5080 | Used for the preparation of ACSF and sucrose-ACSF |
| MgCl2·6H2O | Sigma | M2670 | Used for the preparation of ACSF and sucrose-ACSF |
| NaHCO3 | Sigma | S5761 | Used for the preparation of ACSF and sucrose-ACSF |
| D-Glucose | Sigma | G7021 | Used for the preparation of ACSF |
| Ascorbic acid | Sigma | P5280 | Used for the preparation of ACSF and sucrose-ACSF |
| Sodium pyruvate | Sigma | A7631 | Used for the preparation of ACSF and sucrose-ACSF |
| Sucrose | Sigma | S7903 | Used for the preparation of sucrose-ACSF |
| K-gluconate | Wako | 169-11835 | Used for the preparation of K+-based intracellular solution |
| Na2-Phosphocreatine | Sigma | P1937 | Used for the preparation of intracellular solution |
| EGTA | Sigma | E3889 | Used for the preparation of intracellular solution |
| HEPES | Sigma | H4034 | Used for the preparation of intracellular solution |
| Mg-ATP | Sigma | A9187 | Used for the preparation of intracellular solution |
| Li-GTP | Sigma | G5884 | Used for the preparation of intracellular solution |
| CsMeSO4 | Sigma | C1426 | Used for the preparation of Cs+-based intracellular solution |
| CsCl | Sigma | C3011 | Used for the preparation of Cs+-based intracellular solution |
| TEA-Cl | Sigma | T2265 | Used for the preparation of Cs+-based intracellular solution |
| Neurobiotin 488 | Vector | SP-1145 | 0.05% neurobiotin 488 could be used for morphological studies |
| Agar | Sigma | A7002 | 3% agar block was used in our protocol |
| Paraformaldehyde | Sigma | P6148 | 4% paraformaldehyde was used for immunohistochemical processing |
| Na2HPO4 | Hengxing Chemical Reagents | Used for the preparation of PBS | |
| Mount Coverslipping Medium | Polyscience | 18606 | |
| Urethan | National Institute for Food and Drug Control | 30191228 | 1.5 g/kg, i.p. |
| Borosilicate glass capillaries | World Precision Instruments | TW150F-4 | 1.5 mm OD, 1.12 mm ID |
| Micropipette puller | Sutter Instrument | P-97 | Used for the preparation of micropipettes |
| Vibratome | Leica | VT1000S | |
| Vibration isolation table | Technical Manufacturing Corporation | 63544 | |
| Infrared CCD camera | Dage-MIT | IR-1000 | |
| Patch-clamp amplifier | HEKA | EPC-10 | |
| Micromanipulator | Sutter Instrument | MP-285 | |
| X-Y stage | Burleigh | GIBRALTAR X-Y | |
| Upright microscope | Olympus | BX51WI | |
| Osmometer | Advanced | FISKE 210 | |
| PH meter | Mettler Toledo | FE20 | |
| Confocol microscope | Zeiss | LSM 700 |
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