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Template in use: EO Publishing App for SR-RD-EC Eng 2018-12-19_v1 (new disclaimer).dotm © Her Majesty the Queen in Right of Canada (Department of National Defence), 2019

© Sa Majesté la Reine en droit du Canada (Ministère de la Défense nationale), 2019

CAN UNCLASSIFIED

IMPORTANT INFORMATIVE STATEMENTS

This document was reviewed for Controlled Goods by Defence Research and Development Canada (DRDC) using the Schedule to the Defence Production Act.

Disclaimer: This publication was prepared by Defence Research and Development Canada an agency of the Department of National Defence. The information contained in this publication has been derived and determined through best practice and adherence to the highest standards of responsible conduct of scientific research. This information is intended for the use of the Department of National Defence, the Canadian Armed Forces (“Canada”) and Public Safety partners and, as permitted, may be shared with academia, industry, Canada’s allies, and the public (“Third Parties”). Any use by, or any reliance on or decisions made based on this publication by Third Parties, are done at their own risk and responsibility. Canada does not assume any liability for any damages or losses which may arise from any use of, or reliance on, the publication.

In conducting the research described in this report, the investigators adhered to the “Guide to the Care and Use of Experimental Animals, Vol. I, 2nd Ed.” published by the Canadian Council on Animal Care.

Endorsement statement: This publication has been published by the Editorial Office of Defence Research and Development Canada, an agency of the Department of National Defence of Canada. Inquiries can be sent to: [email protected].

DRDC-RDDC-2019-D111 i

Abstract

Electrophysiology is the study of electrical properties of biological cells and tissues. It studies the flow of ions (ion current) in biological systems, including the brain. It involves measurements of changes in voltage or electric current on a wide variety of scales from single ion channel proteins to whole organs. Electrophysiological techniques are widely used to investigate mechanisms of physiological or pathological processes in a variety of organs, including the brain. To assist studies at DRDC – Suffield Research Centre in traumatic brain injury research, we have recently acquired an electrophysiology / patch clamp system. This system is being used to study the molecular mechanisms of traumatic brain injury, either caused by primary blast waves or concussion by weight drop in small laboratory animals.

This Standard Operating Procedure (SOP) is design to guide the operation of this system to perform field recordings to study changes in Long Term Potentiation (LTP) and Long Term Depression (LTD). Both LTP and LTD are considered the molecular basis of learning and memory.

ii DRDC-RDDC-2019-D111

Résumé

L’électrophysiologie est l’étude des propriétés électriques des cellules et des tissus biologiques. Son principal champ d’étude est le flux (courant) ionique dans les systèmes biologiques, comme le cerveau. Il s’agit de mesurer les variations de tension ou de courant électrique à diverses échelles allant des canaux ioniques (protéines) uniques jusqu’aux organes entiers. Les techniques électrophysiologiques sont largement utilisées dans l’analyse des mécanismes en jeu dans les processus physiologiques ou pathologiques dans divers organes, notamment le cerveau. Afin de contribuer aux recherches au sein de RDDC – Centre de recherches de Suffield sur les traumatismes cérébraux, nous avons récemment fait l'acquisition d’un système d’enregistrement patch-clamp. Un tel système sert à l’étude moléculaire des mécanismes impliqués dans les traumatismes cérébraux causés par une onde de souffle primaire ou une commotion consécutive à une chute de poids sur de petits animaux de laboratoire.

La présente instruction permanente d’opération (IPO) est destinée à faciliter l’utilisation de ce système pour effectuer des enregistrements sur le terrain afin d’étudier les changements dans la potentialisation et la dépression à long terme (PLT/DLT). On considère que la PLT et la DLT constituent le fondement moléculaire de l’apprentissage et de la mémoire.

DRDC-RDDC-2019-D111 iii

Table of contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Résumé . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

Table of contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

1 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.1 Materials used for acute brain slice preparation . . . . . . . . . . . . . . . . . 2 2.2 Materials and equipment used for electrophysiology recordings . . . . . . . . . . . 2

3 Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Annex A Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

iv DRDC-RDDC-2019-D111

List of figures

Figure 1: A snapshot of materials used for brain slice preparation. . . . . . . . . . . . . . 3

Figure 2: Field recording electrode placement. . . . . . . . . . . . . . . . . . . . . . 7

Figure 3: Patch clamp electrode placement. . . . . . . . . . . . . . . . . . . . . . . 8

Figure 4: Example test pulses during whole-cell approach. . . . . . . . . . . . . . . . . 9

DRDC-RDDC-2019-D111 v

List of tables

Table A.1: Composition of brain slice solution (1X). . . . . . . . . . . . . . . . . . . . 10

Table A.2: Composition of perfusion solution (ACSF, 10X). . . . . . . . . . . . . . . . . 10

Table A.3: Intracellular solution for GABAa current recording. . . . . . . . . . . . . . . . 11

Table A.4: Intracellular solution for voltage clamp for young rats. . . . . . . . . . . . . . . 11

Table A.5: Intracellular solution for voltage clamp for old rats. . . . . . . . . . . . . . . . 12

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DRDC-RDDC-2019-D111 1

1 Purpose

To describe the procedures for electrophysiology using acute brain slices including:

1. Preparation of materials

2. Preparation of acute brain slices

3. Setup for electrophysiology recording

4. Electrophysiology recording methods

a. Extracellular field recording for excitatory postsynaptic potential (fEPSP)

b. Input-Output field recording

c. Long-Term Potentiation (LTP) field recording

d. Blind whole-cell patch clamp recording

5. Data analysis

2 DRDC-RDDC-2019-D111

2 Materials

2.1 Materials used for acute brain slice preparation

200 mL and 1 L glass beaker Dissection tray (plastic)

400 mL stainless steel beaker Bone cutters

100 mm petri dish Large scissors

Vibrating-blade microtome : Leica VT1000 S Spatula

Slice solution Fine dissecting scissors

Perfusion Solution (ACSF) Loctite 495™ instant adhesive glue

Inhalation chamber with Isoflurane Filter paper

Guillotine 95% O2 / 5% CO2 Tank

Blue absorbent pad Perfusion Apparatus and mesh holder unit

Biohazard bag in disposal container Air stone

Plastic transfer pipettes with end cut off Heat Bath

Paint brush Intracellular solution (for patch clamp)

2.2 Materials and equipment used for electrophysiology recordings

Vibration isolation table Video system

Stimulator Perfusion control system

Isolator Bipolar tungsten stimulation electrode

Amplifier Silver wire (chlorided by immersion in bleach for at least half hour)

Digidata (AD/DA interface) Glass capillaries (ends fire-polished with flame)

Sharps Container Pipette puller

Microscope pClamp software

Micromanipulator Clampex software

Vacuum System (Pump or H20)

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k. Preload the inhalation chamber with isoflurane for 5 minutes.

l. Remove the slice solution from the freezer and mix until it forms a slurry with a slush-like consistency.

m. Fill the dissecting tray and petri dish with the semi-frozen slice solution.

2. Preparation of acute brain slices

a. Place the rat into the charged induction chamber for several minutes, until the rat no longer responds to a toe-pinch.

b. Euthanize the rat using a guillotine by pressing swiftly and firmly on the handle of the guillotine and separating the head from the body.

c. Wrap the body in the absorbent pad and place in the biohazard bag.

d. Immediately transfer the head to the dissecting tray filled with slice solution slurry and immerse in the solution. Keep the head as fully immersed as possible throughout the following steps.

e. Trim excess muscle from the posterior end of the head using scissors.

f. Remove the skin from the head by cutting a line from the back end of the skull towards the eyes.

g. Peel the skin away from the skull towards the ears before cutting it completely away from the skull.

h. Flip the occipital bones sideways to remove them so that the cerebellum is exposed. Cut along the central line of the skull up to the coronal suture using the large scissors or bone cutters.

i. Flip the parietal bones open to the temporal suture and cut them off to expose the entire brain.

j. Gently remove the meninges. Scoop the brain out by running a spatula gently underneath the brain, severing the nerves.

k. Cut off the cerebellum as well as most of the frontal cortex to present a flat surface.

l. Place a drop of glue on the stage disc and gently dab the moisture off the brain using the filter paper. The stage is a flat disc with a cut away ½ circle on one side to allow it to attach into the buffer tray with a countersunk screw.

m. Place the brain onto the glue on the stage disc with front down. The glue will set in 5 seconds.


n. Attach the stage disc manipulator onto the stage and screw into the vibratome buffer tray using the stage Allen key. See p. 20 of the Leica manual for detailed instructions.

o. Pour ice-cold slice solution into the vibratome chamber to cover the brain.

p. Place tubing with needle tip into the solution to ensure the brain is bubbled with the carbogen throughout the procedure.

q. Insert a new vibratome blade into the knife holder, secure with clamping screw and attach to the vibratome.

r. Raise the vibratome chamber until the top surface of the brain is just above the level of the blade using the up direction of the UP/DOWN toggle switch.

s. Move the blade forward until it has almost reached the top of the brain using the forward direction on the REV/FORW toggle switch.

t. Press the button for setting the limit stops of the sectioning window to set the first limit of the sectioning window.

u. Advance the blade through the first section of tissue by holding forward on the toggle switch until it has cut through the entire brain. Use the paint brush to gently support the brain as the blade advances.

v. Press the button for setting the limit stops of the sectioning window once more to set the second sectioning window limit.

w. Ensure the vibratome is set to 400µm slices and press the start button to continue to slice until the desired level of brain is reached.

x. Use the paint brush to gently coax the slices of interest over the vibratome blade until they have detached into the buffer tray and the blade has retracted.

y. Separate the two hemispheres of the brain using fine dissecting scissors.

z. Transfer the brain slices to the mesh holder in the beaker with ACSF solution using a cut off plastic transfer pipette and bubble continuously with carbogen.

aa. Remove the blade, dispose of blade in sharps container and empty the buffer tray and cooling bath when finished cutting.

bb. Allow the slices to recover and stabilize for at least an hour at room temperature prior to further manipulation (for patch clamp, incubate the beaker containing brain slices in a heat bath at 32C for 1 to 1.5 hours to return to physiological temperature).

cc. Leave beaker with brain slices at room temperature following the recovery period.


3. Setup for electrophysiology recording

a. Thaw 1mL of intracellular solution if performing patch clamp and keep on ice.

b. Turn on all the instruments and open the pClamp and Clampex software.

c. Prepare the perfusion system by filling the perfusion tube with 40–50 ml of perfusion solution while constantly bubbling the perfusion tube with carbogen. Place the in-flow tubing from the pump in the remaining perfusion solution and turn on the pump to allow continuous flow of solution into the perfusion tube. Open the valve on the bottom of the tube to begin gravity-fed perfusion of perfusion solution into the chamber.

d. Turn on the water flow to create vacuum suction to drain excess perfusion buffer from the chamber and allow constant refresh of the perfusion solution in the chamber.

e. Pull fire-polished glass capillary pipettes on the pipette puller with a two line program designed to give the right resistance, tip size and taper length tip. See Pipette Cookbook for instructions.

f. Transfer a brain slice into the recording chamber using a cut off transfer pipette.

g. Adjust the slice position with the region of interest in the microscope field using a paintbrush.

h. Place the paperclip weight on top of the slice to hold it in position.

4. Electrophysiology methods

a. Extracellular field recording for excitatory postsynaptic potential (fEPSP):

i. Lower the bipolar tungsten stimulation electrode into the Schaffer collateral-commissural pathway of the CA1 area (see stimulating electrode in Figure 1).

ii. Mount the glass micropipette recording electrode (2–4 M) filled with perfusion solution onto the electrode holder.

iii. Lower the recording electrode into the stratum radiatum, 60–80 µm from the cell body layer of the CA1 (see recording electrode in Figure 1).

iv. Stimulate synaptic responses with 0.05 ms duration in constant current 0.1–1 mA in the presence of 10 µM bicuculline (to block GABAa receptor-mediated inhibitory synaptic currents).

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Annex A Solutions

Slice solution (1X) (older animals):

Table A.1: Composition of brain slice solution (1X).

Component [Conc.] mM MW Amount for 0.5L(g)

KCl 2.5 74.56 0.093

CaCl2 1 147 0.074

MgCl2 7 202 0.707

NaH2PO4 1.36 138 0.094

NaHCO3 25 84 1.05

N-Methy-D –glucamine 195.21 195.21 11.7

Sodium L-ascorbate 1.35 198 0.134

Sodium Pyruvate 2.43 110 0.134

Glucose 20 180 1.8

pH with HCl 7.35 Osmolarity 300–310

Perfusion solution (ACSF) 10X (for brain slices):

Note: Make the perfusion solution as a 10X stock solution without NaHCO3 and glucose and keep refrigerated. Dilute the stock solution, add the final ingredients then check and adjust pH and osmolality on the day of the experiment.

Table A.2: Composition of perfusion solution (ACSF, 10X).

Component [Conc.] mM MW Amount for 1 L(g)

NaCl 125 58.44 73.05

KCl 2.5 74.56 1.86

CaCl2 2 147 2.94

MgCl2 1 or 2 or 0 202 2.02 or 4.04 or 0

NaH2PO4 1.25 138 1.73

NaHCO3 26 84 2.18 for 1X*

Glucose 25 180 4.5 for 1X*


* add when making 1X only.

To block GABAa inhibitory currents add bicuculline.


Intracellular solutions:

Note: make the intracellular solutions in advance, check and adjust pH and osmolarity, filter and freeze aliquots in microcentrefuge tubes.

For voltage clamp, particularly for GABAa current:

Table A.3: Intracellular solution for GABAa current recording.

Component [Conc.] mM MW Amount for 20ml(g)

CsCl 140 168.4 0.4715

CaCl 0.1or 0 147 0.00029 or 2 μl/1M

MgCl2 2 202 0.0081 or 40 μl/1M

HEPES 10 238.2 0.0476

BAPTA 10 628.8 0.126

or EGTA*

0.5 or 10

380.4 0.0038 or 0.0761

10μl or 200 μl/1M ATP (K) 4 583.4 0.046


*Soluble in 1M CsOH.

To block Na+ channel add 5 mM QX-314.

For voltage clamp:

Table A.4: Intracellular solution for voltage clamp for young rats.


Cs-gluconate* 122.5 328.05 0.8037 or 2.45 ml/1M

CsCl 17.5 168.4 0.0589

MgCl2 2 202 0.0081 or 40 μl/1M

HEPES 10 238.2 0.0476

BAPTA 10 628.8 0.126

or EGTA

0.5 or 10

380.4

0.0038 or 0.0761 10μl or 200 μl/1M

ATP (K) 4 583.4 0.046


*Add 1.68 g CsOH-H2O to 3.92g 50% C6H12O7 to make 10 ml C6H11O7Cs.



For voltage clamp:

Table A.5: Intracellular solution for voltage clamp for old rats.


CsMeSo3 130 228 0.592

NaCl 8 58.44 0.0094

MgCl2 2 202 0.0081 or 40 μl/1M

HEPES 10 238.2 0.0476

BAPTA 10 628.8 0.126

or EGTA

0.5 or 10

380.4 0.0038 or 0.0761 10 μl or 200 μl/1M

ATP (K) 4 583.4 0.046



DOCUMENT CONTROL DATA *Security markings for the title, authors, abstract and keywords must be entered when the document is sensitive

1. ORIGINATOR (Name and address of the organization preparing the document. A DRDC Centre sponsoring a contractor's report, or tasking agency, is entered in Section 8.)

DRDC – Suffield Research Centre Defence Research and Development Canada P.O. Box 4000, Station Main Medicine Hat, Alberta T1A 8K6 Canada

2a. SECURITY MARKING (Overall security marking of the document including special supplemental markings if applicable.)

CAN UNCLASSIFIED

2b. CONTROLLED GOODS

NON-CONTROLLED GOODS DMC A

3. TITLE (The document title and sub-title as indicated on the title page.)

Electrophysiology and field recording in rat brain slices for traumatic brain injury research: Standard operating procedure

4. AUTHORS (Last name, followed by initials – ranks, titles, etc., not to be used)

Barnes, J.; Weiss, T.; Wang, Y.

5. DATE OF PUBLICATION (Month and year of publication of document.)

August 2019

6a. NO. OF PAGES (Total pages, including Annexes, excluding DCD, covering and verso pages.)

18

6b. NO. OF REFS (Total references cited.)

0

7. DOCUMENT CATEGORY (e.g., Scientific Report, Contract Report, Scientific Letter.)

Reference Document

8. SPONSORING CENTRE (The name and address of the department project office or laboratory sponsoring the research and development.)

DRDC – Suffield Research Centre Defence Research and Development Canada P.O. Box 4000, Station Main Medicine Hat, Alberta T1A 8K6 Canada

9a. PROJECT OR GRANT NO. (If appropriate, the applicable research and development project or grant number under which the document was written. Please specify whether project or grant.)

04j

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10a. DRDC PUBLICATION NUMBER (The official document number by which the document is identified by the originating activity. This number must be unique to this document.)

DRDC-RDDC-2019-D111

10b. OTHER DOCUMENT NO(s). (Any other numbers which may be assigned this document either by the originator or by the sponsor.)

11a. FUTURE DISTRIBUTION WITHIN CANADA (Approval for further dissemination of the document. Security classification must also be considered.)

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11b. FUTURE DISTRIBUTION OUTSIDE CANADA (Approval for further dissemination of the document. Security classification must also be considered.)

12. KEYWORDS, DESCRIPTORS or IDENTIFIERS (Use semi-colon as a delimiter.)

Traumatic Brain Injury (TBI); primary blast; electrophysiology

13. ABSTRACT (When available in the document, the French version of the abstract must be included here.)

Electrophysiology is the study of electrical properties of biological cells and tissues. It studiesthe flow of ions (ion current) in biological systems, including the brain. It involves measurements of changes in voltage or electric current on a wide variety of scales from single ion channelproteins to whole organs. Electrophysiological techniques are widely used to investigatemechanisms of physiological or pathological processes in a variety of organs, including the brain. To assist studies at DRDC – Suffield Research Centre in traumatic brain injury research,we have recently acquired an electrophysiology / patch clamp system. This system is being used to study the molecular mechanisms of traumatic brain injury, either caused by primary blastwaves or concussion by weight drop in small laboratory animals.

This Standard Operating Procedure (SOP) is design to guide the operation of this system toperform field recordings to study changes in Long Term Potentiation (LTP) and Long TermDepression (LTD). Both LTP and LTD are considered the molecular basis of learning andmemory.

L’électrophysiologie est l’étude des propriétés électriques des cellules et des tissus biologiques.Son principal champ d’étude est le flux (courant) ionique dans les systèmes biologiques,comme le cerveau. Il s’agit de mesurer les variations de tension ou de courant électrique àdiverses échelles allant des canaux ioniques (protéines) uniques jusqu’aux organes entiers. Lestechniques électrophysiologiques sont largement utilisées dans l’analyse des mécanismes enjeu dans les processus physiologiques ou pathologiques dans divers organes, notamment lecerveau. Afin de contribuer aux recherches au sein de RDDC – Centre de recherches deSuffield sur les traumatismes cérébraux, nous avons récemment fait l'acquisition d’un systèmed’enregistrement patch-clamp. Un tel système sert à l’étude moléculaire des mécanismesimpliqués dans les traumatismes cérébraux causés par une onde de souffle primaire ou une commotion consécutive à une chute de poids sur de petits animaux de laboratoire.

La présente instruction permanente d’opération (IPO) est destinée à faciliter l’utilisation de cesystème pour effectuer des enregistrements sur le terrain afin d’étudier les changements dansla potentialisation et la dépression à long terme (PLT/DLT). On considère que la PLT et la DLTconstituent le fondement moléculaire de l’apprentissage et de la mémoire.

standard operating procedure · vibrating-blade microtome : leica vt1000 s spatula slice solution...

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