Juxtacellular Overvågning og lokalisering af Single neuroner inden subcorticale hjernestrukturer af indberetning, Head-behersket rotter
Summary
This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.
Abstract
There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal’s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.
Introduction
Overvågning neuronal aktivitet i en advarsel dyr aktivt engageret i en adfærdsmæssig opgave er afgørende for at forstå funktion og organisering af nervesystemet. Ekstracellulær registrering af den elektriske aktivitet fra enkelte neuronale enheder har længe været en hæfteklamme værktøj systemer neurovidenskab og er stadig i vid udstrækning anvendes på nuværende tidspunkt. En række af elektrode typer og konfigurationer, afhængigt af de videnskabelige og tekniske krav, som en bestemt eksperiment. Kronisk implanteret Microdrives eller elektrode arrays anvendes ofte i frit bevægelige dyr, herunder fugle, gnavere og ikke-menneskelige primater 1-4. Alternativt er akutte gennemføringer med metal eller glas mikroelektroder via en ekstern mikromanipulator ofte til at optage fra bedøvede eller head-behersket dyr. Glasmikropipette elektroder har den fordel, at de kan anvendes i juxtacellular eller "celle vedlagte" konfiguration utvetydigt isolereaktivitet af enkelte neuroner uden komplikationer af post-hoc spike sortering 5. Disse elektroder endvidere muliggøre optagelse fra anatomisk identificerede celler eller steder, da de kan anvendes til at injicere små aflejringer af farvestof eller neuroanatomiske sporstoffer, eller endda til at fylde den enkelte optaget celle. Denne konfiguration er blevet anvendt med succes i rotter, mus og fugle 6-10. Det for tiden beskrevne teknik fokuserer på juxtacellular overvågning og ekstracellulære farvestof indskud i alarm, head-behersket rotter. Bemærk, at i modsætning til enkelt celle juxtacellular fylder, disse farvestof indskud ikke give oplysninger om cellemorfologi eller axonale fremspring 11, men de gør det muligt nøjagtigt anatomisk lokalisering til ca. 50 um og kritisk, har en betydeligt højere udbytte i Alert dyr. Oplysninger om encellede juxtacellular fylder ikke desto mindre fastsat som en alternativ strategi for anatomiske mærkning.
Kort sagt, denprotokol består af tre hovedfaser. I den første fase, er rotten akklimatiseret til kroppen tilbageholdenhed i en klud sok (Figur 1) over en periode på 6 dage. I den anden fase, er en nakkestøtte apparat (figur 2) og optagelse kammer indopereret sådan, at rotten kan opretholdes i stereotaktisk fly under flere efterfølgende indspilningerne (Figur 3); denne procedure giver forsøgslederen at målrette bestemte subcorticale områder af hjernen for elektrofysiologiske undersøgelse baseret på standard henvisning koordinater 12. Den tredje fase indebærer at placere rotten i et passende jig for udførelse af adfærdsmæssige og elektrofysiologiske forsøg (figur 4), konstruere elektroden fra en kvarts kapillarrør (figur 5), hvilket gør juxtacellular neuronale optagelser, entydigt isolere enkelte enheder 6-9, og markerer anatomiske locatipå af optagelsen site med Chicago Sky blå farvestof (figur 6 og 7). Optagelserne udføres med samtidig adfærdsmæssige overvågning; dog vil de tekniske detaljer i den adfærd afhænge af de videnskabelige mål for hvert forsøg, og er således uden for rammerne af en enkelt protokol. Efter afslutning af den eksperimentelle procedure, der kan gentages på flere dage, dyret aflivet. Hjernen udvindes og forarbejdes i overensstemmelse med standard neuroanatomiske teknikker, der anvender enten lyse felt eller fluorescens mikroskopi.
Protocol
Representative Results
Discussion
Konstruktion af den eksperimentelle jig
Beskrivelsen af de mekaniske dele, der anvendes til at bygge den eksperimentelle jig (figur 4) er udeladt fra protokol, da det kan konstrueres i en række forskellige måder. I denne demonstration standard opto-mekaniske dele og support klemmer bruges til at montere nakkestøtten bar og kroppen tilbageholdenhed rør (se Materialer afsnit). Lignende opto-mekanisk dele kan bruges til at mon…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to the Canadian Institutes of Health Research (grant MT-5877), the National Institutes of Health (grants NS058668 and NS066664), and the US-Israeli Binational Foundation (grant 2003222) for funding these studies.
Materials
| Name of the Reagent | Company | Catalogue Number | Comments |
| Ketaset (Ketamine HCl) | Fort Dodge | N/A | |
| Anased (Xylazine solution) | Lloyd Laboratories | N/A | |
| Betadyne (Povidone-Iodine) | CVS Pharmacy | 269281 | |
| Loctite 495 | Grainger Industrial Supply | 4KL86 | 20-40 cp cyanoacrylate |
| Vetbond | 3M | 1469SB | |
| Grip cement powder | Dentsply Intl | 675571 | For the base of the recording chamber |
| Grip cement liquid | Dentsply Intl | 675572 | For the base of the recording chamber |
| Silicone Gel | Dow Corning | Mar-80 | |
| Jet denture repair acrylic powder | Lang Dental Manufacturing Co. | N/A | For securing the head restraint apparatus to the cranium |
| Ortho-Jet Fast curing orthodontic acrylic resin liquid | Lang Dental Manufacturing Co. | N/A | For securing the head restraint apparatus to the cranium |
| Chicago sky blue | Sigma | C8679 | |
| Paraformaldehyde | Sigma | 158127 | For perfusion and tissue fixation |
| Phosphate-buffered saline | Sigma | P3813 | For perfusion and tissue fixation |
| Cytochrome C | Sigma | C2506 | For cytochrome-oxidase staining (Figure 7) |
| Diaminobenzidine | Sigma | D5905 | For cytochrome-oxidase staining (Figure 7) |
| Material Name | Company | Catalogue Number | Comments |
| Rat sock | Sew Elegant (San Diego, CA) | N/A | Custom made, Figures 1, 4 |
| PVC tube 2 ½” | U.S. Plastic Co. | 34108 | Figure 4 |
| Subminiature D pins & sockets | TE Connectivity | 205089-1 | Figure 3 |
| Stainless steel music wire 0.010” diameter | Precision Brand Products, Inc. | 21010 | Figure 3 |
| Stereotaxic holding frame | Kopf Instruments | Model 900 | Figure 3 |
| Stereotaxic ear bars | Kopf Instruments | Model 957 | Figure 3 |
| Stereotaxic manipulator | Kopf Instruments | Model 960 | Figure 3 |
| ½ mm drill burr | Henry Schein | 100-3995 | |
| Quiet-Air dental drill | Midwest Dental | 393-1600 | |
| Stainless steel 0-80 1/8” screw | Fastener superstore | 247438 | Figure 3 |
| 0.2mL centrifuge tube | Fisher Scientific | 05-407-8A | Figure 3 |
| Custom head-holding bar | UCSD SIO Machine Shop | N/A | Custom made, Figures 2, 3, 4 |
| Custom head-holding plate | UCSD SIO Machine Shop | N/A | Custom made, Figure 2, 3, 4 |
| Right angle post-clamp | Newport | MCA-1 | Figure 3,4; standard opto-mechanical parts for the experimental jig (Figure 4) are also from Newport Corp. |
| 8-32 3/4” screw | Fastener Superstore | 240181 | For head-restraint, Figure 3 |
| 4-40 ¼” screw | Fastener Superstore | 239958 | For head restraint, Figures 3, 4 |
| Quartz capillary tubing | Sutter Instruments | QF-100-60-10 | Figure 5 |
| Carbon dioxide laser puller | Sutter instruments | P-2000 | |
| Motorized micromanipulator | Sutter Instruments | MP-285 | |
| Microelectrode amplifier | Molecular Devices | Multiclamp 700B | Alternate part: Molecular Devices Axoclamp 900A |
| Microelectrode amplifier head stage | Molecular Devices | CV-7B | Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A |
| Isolated pulse stimulator | A-M Systems | Model 2100 | Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A |
| Audio monitor | Radio Shack | 32-2040 | |
| Pipette holder | Warner Instruments | #MEW-F10T | Alternate parts: see Discussion |
| Figure 6A | |||
| Electrode lead wire | Cooner wire | NEF34-1646 | (optional), Figure 6D |
| Relay for amplifier head-stage | COTO Technology | #2342-05-000 | (optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), Figure 6A-C |
| Digital video camera | Basler | A602fm | (optional) For behavioral monitoring, Figure 7 |
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