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Abstract

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Neuroscience

In vivo Calciumbilleddannelse af neuronale ensembler i netværk af primære sensoriske neuroner i intakte dorsale rodganglier

Published: February 10th, 2023

DOI:

10.3791/64826

1Department of Oral & Maxillofacial Surgery, School of Dentistry, University of Texas Health Science Center at San Antonio, 2Programs in Integrated Biomedical Sciences, Translational Sciences, Biomedical Engineering, Radiological Sciences, University of Texas Health Science Center at San Antonio

Denne protokol beskriver den kirurgiske eksponering af dorsalrodsganglion (DRG) efterfulgt af GCaMP3 (genetisk kodet Ca2+ indikator; Grønt fluorescerende protein-calmodulin-M13-protein 3) Ca2+ billeddannelse af neuronale ensembler ved hjælp af Pirt-GCaMP3-mus, mens der påføres en række stimuli på den ipsilaterale bagpote.

Ca 2+ billeddannelse kan bruges som en proxy for cellulær aktivitet, herunder handlingspotentialer og forskellige signalmekanismer, der involverer Ca 2+ indgang i cytoplasmaet eller frigivelse af intracellulære Ca2+ lagre. Pirt-GCaMP3-baseret Ca2+ billeddannelse af primære sensoriske neuroner i dorsalrodsganglion (DRG) hos mus giver fordelen ved samtidig måling af et stort antal celler. Op til 1.800 neuroner kan overvåges, hvilket gør det muligt at studere neuronale netværk og somatosensoriske processer som et ensemble i deres normale fysiologiske kontekst på populationsniveau in vivo. Det store antal neuroner, der overvåges, gør det muligt at detektere aktivitetsmønstre, der ville være udfordrende at opdage ved hjælp af andre metoder. Stimuli kan påføres musebagpoten, hvilket gør det muligt at undersøge de direkte virkninger af stimuli på DRG-neuronensemblet. Antallet af neuroner, der producerer Ca 2+ transienter samt amplituden af Ca2+ transienter indikerer følsomhed over for specifikke sensoriske modaliteter. Diameteren af neuroner giver bevis for aktiverede fibertyper (ikke-skadelige mechano vs. skadelige smertefibre, Aβ-, Aδ- og C-fibre). Neuroner, der udtrykker specifikke receptorer, kan genetisk mærkes med td-Tomat og specifikke Cre-rekombinaser sammen med Pirt-GCaMP. Derfor giver Pirt-GCaMP3 Ca2+ billeddannelse af DRG et kraftfuldt værktøj og en model til analyse af specifikke sensoriske modaliteter og neuronundertyper, der fungerer som et ensemble på befolkningsniveau for at studere smerte, kløe, berøring og andre somatosensoriske signaler.

Primære sensoriske neuroner innerverer huden direkte og bærer somatosensorisk information tilbage til centralnervesystemet 1,2. Dorsalrodsganglier (DRG'er) er cellekropsklynger på 10.000-15.000 primære sensoriske neuroner 3,4. DRG-neuroner præsenterer forskellig størrelse, myelineringsniveauer og gen- og receptorekspressionsmønstre. Neuroner med mindre diameter inkluderer smertefølende neuroner, og neuroner med større diameter reagerer typisk på ikke-smertefulde mekaniske stimuli 5,6. Forstyrre....

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Alle procedurer, der er beskrevet her, blev udført i overensstemmelse med en protokol, der er godkendt af Institutional Animal Care and Use Committee ved University of Texas Health Science Center i San Antonio.

BEMÆRK: Når dyrekirurgi (trin 1) og billeddannelse (trin 2) er påbegyndt, skal de gennemføres løbende. Dataanalyse (trin 3) kan udføres senere.

1. Kirurgi og sikring af dyret til højre side L5 DRG-billeddannelse

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Figure 4
Figur 4: Repræsentative billeder af L5 dorsale rodganglier af Pirt-GCaMP3-mus. (A,D) Single frame højopløsningsscanninger af L5 dorsale rodganglier hos Pirt-GCaMP3-mus vises. (B,E) . Gennemsnitlige intensitetsfremskrivninger af 15 billeder af Pirt-GCaMP3 L5 DRG-ganglier fra henholdsvis panel A og.......

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Vedvarende smerter er til stede i en lang række lidelser, invaliderende og / eller reducerer livskvaliteten for omkring 8% af mennesker29. Primære sensoriske neuroner registrerer skadelige stimuli på huden, og deres plasticitet bidrager til vedvarende smerte8. Mens neuroner kan studeres i cellekultur og eksplanter, fjerner det dem fra deres normale fysiologiske kontekst. Kirurgisk eksponering af DRG efterfulgt af Pirt-GCaMP3 Ca2+ billeddannelse tillader unders.......

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Dette arbejde blev støttet af National Institutes of Health Grants R01DE026677 og R01DE031477 (til Y.S.K.), UTHSCSA startup fund (Y.S.K.) og en Rising Star Award fra University of Texas system (Y.S.K.).

....

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NameCompanyCatalog NumberComments
Anased Injection (Xylazine)Covetrus, Akorn33197
C Epiplan-Apochromat 10x/0.4 DICCal Zeiss422642-9900-000
Cotton Tipped ApplicatorsMcKesson24-106-1S
Curved HemostatFine Science Tools13007-12
DC Temperature ControllerFHC40-90-8D
DC Temperature Controller Heating PadFHC40-90-2-05
Dumont Ceramic Coated ForcepsFine Science Tools11252-50
FHC DC Temperature ControllerFHC40-90-8D
Fluriso (Isoflurane)MWI Animal Health, Piramal Group501017
Friedman-Pearson RongeursFine Science Tools16221-14
GelFoamPfizer09-0353-01
Ketaset (Ketamine)ZoetisKET-00002R2
Luminescent Green Stage TapeJSITON/ AmazonB803YW8ZWL
Matrx VIP 3000 Isoflurane VaporizerMidmark91305430
Micro dissecting scissorsRobozRS-5882
Micro dissecting spring scissorsFine Science Tools15023-10
Micro dissecting spring scissorsRobozRS-5677
Mini Rectal Thermistor ProbeFHC40-90-5D-02
Operating scissorsRobozRS-6812
Pirt-GCaMP3 C57BL/6J miceJohns Hopkins UniversityN/AEither sex can be imaged equally well. Mice should be at least 8 weeks old due to weak or intermittent Pirt promoter expression in younger mice.
SMALGO small animal algometerBioseb In vivo Research InstrumentsBIO-SMALGO
Stereotaxic frameKopf Model 923-B923-B
td-Tomato C57BL/6J miceJackson Laboratory7909
Top Plate, 6 in x 10 inNewport290-TP
TrpV1-Cre C57BL/6J miceJackson Laboratory17769
Zeiss LSM 800 confocal microscopeCal ZeissLSM800
Zeiss Zen 2.6 Blue Edition SoftwareCal ZeissZen (Blue Edition) 2.6

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