Neuropak demo

NeuroPak™ PracticalWORKSHOP SUPPORT

Prepared by H. Gharbi | © Mauna Kea Technologies

NeuroPak™ Description• NeuroPak is a complete solution for Neuroscientists

• Developped with the Institut Pasteur (Paris)

• Enables deep brain access in freely-moving animals

‣ Calcium transients

‣ Neuron activity

‣ Perfusion studies

‣ Animal model validation

‣ ...

2

Behavioral Studies Calcium flux


Microprobe with beveled tip Stereotaxic holder Implants Fixing kit User’s Booklet

CerboFlex or CerboFlex J Compatible with most stereotaxic frames Small weight < 0,3 g Screws Description

Length 4 m Allows accurate positioning of the CerboFlex and Implants

unoxidablemade of Titanium

Skull screws (smaller)

CerboFlex and Stereotaxic holder

(larger screws)

Implantation protocol

Diameter 350 µm or 470 µm (J)

Guide the CerboFlex into deep brain structures Screwdriver Well Illustrated

Resolution 3,3 µmYet adapted to mice

(max penetration depth: 6,5 mm)

NeuroPak™ Description

3


NeuroPak™ Description

4

x6

Installation disk


Requirements• Protocol preparation

• Animal model & neuron labelling

‣ Mouse or rat

‣ WT, transgenic (GFP, Thy1 YFP), transfected (AAV-GCaMP3)

‣ Chemical calcium indicator: Oregon Green BAPTA 1 AM

• Surgery

‣ Needs to be performed at least one week prior to imaging

‣ Detailed implantation procedure inside the Booklet

‣ Surgical skills

‣ Stereotaxic frame control

5

Protocol for CNS deep brain imagery using Cellvizio with anaesthetizedwildtype mouse (protocol used and developped by A. Cressant)

Requirements:

Mouse model: adult wildtype mouse

Fluorophore/Marker oregon green BAPTA 1

Material:

Cellvizio 488 nm

Calibration kit

Microprobe: S300/b

Laboratory material:

Surgery equipment: syringe, hair clipper, scalpel, compresses, scissors, forceps,anesthetic (Ketamine/xylazine solution for example), drilling tool (ex: F.S.T, withsmall drills 0.5 mm of diameter), lead pencil, ear bars, dissecting microscope andstereotactic frame (with mouse adaptator if needed) with electrode/syringe holder

Solutions: 70% ethanol solution, ice, PBS solution, Oregon Green BAPTA 1 solution

Mouse Brain Atlas

Injection system: glass injection needle, precision pump (Harvard, PHD 2000),Hamilton syringe (10 microliter), tubing 0.11”x. 0.24”x.0065” (A-M System, Inc), extrastrong glue

I – Protocol (described for Oregon Green BAPTA 1 AM fluorescent dye use)

Oregon Green BAPTA-1 AM (OGB1) solution preparation: solution is prepared as describedin Stosiek C et coll. (2003). The AM ester (Molecular Probes Invitrogen Ltd, Paisley, UK) isdissolved in DMSO plus 20% Pluronic F-127 (e.g., 2 g of Pluronic F-127 in 10 mlof DMSO).The final concentration is 10 mM. Prior cell loading the OGB1 AM is diluted 1:10, in a solutioncontaining in mM: 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4. The final solution concentration is 1mM.

If brightness is too strong, OGB1 1mM solution can be successfully diluted 1:5 in PBS beforeinjection into the brain.

Mauna Kea Technologies S.A.S.9, rue d’Enghien75010 Paris, FranceTel +33 1 48 24 03 45Fax +33 1 48 24 12 18

www.maunakeatech.com

Mauna Kea Technologies, Inc.660 Newtown-Yardley Rd., Suite 107

Newtown, PA 18940, USATel +1 215 478 6288Fax +1 215 279 8463

Société par Actions Simplifiée au capital de EUR 351 924,03 • RCS PARIS B 431 268 028 (2000B18081) • NAF 7219Z • SIRET: 431 268 028 00021

Protocol

Calcium imaging using Cellvizio Lab system and GCaMP3 (Genetically encoded calcium indicators)


Material Checklist

6

• Material checklist• Anaesthetic (Ketamine/Xylazine or Isoflurane)

• Mouse/Rat brain stereotaxic coordinates Atlas (Paxinos)

• Syringe, hair clipper, scalpel, compresses, scissors, forceps, 30G needles, Q-tips, lead pencil.

• Drilling tool (ex: F.S.T, with small drills 0.5 mm of diameter).

• Dissecting microscope.

• Stereotactic frame (with mouse adaptator if needed) with electrode/syringe holder, ear bars.

• Solutions: 70% ethanol solution, ice, PBS solution, povidine/iodine.

• Dental acrylic solution (cement).

• Injection system


System and Microprobe Warm-Up• Turn on the Laser Scanning Unit and the Computer

• Open the NeuroPak™ case and take the CerboFlex microprobe

• Unfold it carefully, clean its proximal part using the Cletop-S®

• Plug the probe into the connector

• Launch ImageCell™ (Cellvizio) or Cellvizio Dual Software (Cellvizio Dual Band)

• If the probe is not recognized, use the CD to install the probe

• Click on Start Laser and let the microprobe warm-up for at least 30 min. This will contribute to best imaging performances

• Once the microprobe ready, proceed with calibration using the Quantikit® vials by following the onscreen guidelines

7

Note 1 : NeuroPak contents & How to pack the Cerboflex

30 min warm-up

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Animal Preparation• Anaesthetize the animal and shave off fur:

• From the ears to just in-between the eyes

• Mount it onto the stereotaxic apparatus • Use the right mouth adapter (mouse or rat)

• Secure the animal with the ear bars

8

Dissecting microscope

*

*

*

*


Stereotaxic Holder• The stereotaxic holder will replace the standard

vertical manipulator of the stereotaxic frame.• Unmount the original arm using appropriate tools• Place the stereotaxic holder as shown below

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Toutes les cotes sont en mm/All dimensions in mm Tol. generales :

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Accessoire Stéréo

Inox 316L (1,4404)

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ISO 2768mK

This document is the property of Mauna Kea Technologies. Reproduction without authorizationof company MKT is strictly prohibited.

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Mount the CerboFlex on the Holder

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Toutes les cotes sont en mm/

All dimensions in mm

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Ce document est la propriété de Mauna Kea Technologies. Toute reproduction totale ou partielle sous quelque forme que ce

soit de ce document sans une autorisation écrite préalable de la société MKT est strictement interdite.

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Bouchon connecteur jaune

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PTFE extruded special, ID 0,551mm OD 0,8mm

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Colle epotek 301 USP Class VI

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• Place the CerboFlex tip body into the holder’s cavity• Make sure the U shape of the CerboFlex tip is correctly orientated• Secure the microprobe with the locking screws

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Wrong orientation


Skull Surgery: Mouse or Rat• Secure the mouth with the anterior

mount of the stereotaxic frame• The incision is then made down the

midline using a scalpel or surgical scissors

• The top of the skull is exposed

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11 2 3

2

3

• Find bregma on the skull, and position the CerboFlex tip right over this location.

• Write down the anterior/posterior and lateral coordinates.

• From bregma, find the correct coordinates needed for the placement of your probe with the aid of the stereotaxic atlas.

• This is where you will be drilling.• Better results are obtained if the drill is

fixed to the stereotaxic frame.

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4


Skull Surgery: Mouse or Rat

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• Next, using a hand drill, make three holes for skull screws.

• Screws are inserted into frontal, parietal, and occipital bones on the opposite side to the imaging target structure/hole.

• Sterilize the screws and place them onto the skull until they are tightly anchored on.

• Remove the CerboFlex and sterilize your drill bit. • Carefully drill a hole at the pencil mark until you

get through the width of the skull. • Check with a sterile 30G needle to see if it would

clear the hole without touching the sides. • Once the hole is made, use a sterile needle to

gently punch the meninges in order to allow unobstructed insertion of the microprobe.

Drilled hole Skull screws


Implant Placing

13

Plug and lock the implant onto the holder Align the implant cannula with the hole

Lower the implant into the hole

Leave a space between the holder and the implant

Apply dental cement around the implant base and the skull screws

Once the dental cement is dry: release the implant from the holder

Prepared by H. Gharbi | © Mauna Kea Technologies14

Implant Placing into the holder



Incision Suture• The incision is sutured around the

implant• The animal is then allowed to recover for

one week in its cage• Make sure the cement is completely dry• Plug a protective cap into the implant’s

cannula

16


Microprobe Insertion• One week after implantation,

anaesthetize the animal• Then place it on the stereotaxic frame• Attach the CerboFlex to the holder

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0 . 00 . 02

211-01230

5/01/2009

Jerry (sans lame)

NIB

ISO 2768 mK

1 : 1


PN

Description

Rév.

Qté

211-0009

Connecteur LAB

1 . 4

1

215-0002

Valise Proflex

1 . 0

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313-0024

Guide 10000 Ø0,35mm - D15 - 4m

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321-0117

Vis CS M1 x 2 mm A2

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Tol. generales :

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Modifications

A3

Approuvé

Matière/Material :

Dessiné

Validé

Nom

Date

Obs :

Feuille : 1 / 1

Echelle/Scale :

PN : Rév. :


0 . 00 . 02

211-01230

5/01/2009

Jerry (sans lame)

NIB

ISO 2768 mK

1 : 1


PN

Description

Rév.

Qté

211-0009

Connecteur LAB

1 . 4

1

215-0002

Valise Proflex

1 . 0

1

313-0024

Guide 10000 Ø0,35mm - D15 - 4m

---

1

321-0117

Vis CS M1 x 2 mm A2

---

1

322-0048


1 . 3

1

322-0276

Guide sonde J

0 . 00 . 04

1

322-0277

Ferrule J

0 . 00 . 05

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325-0030


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1

325-0031


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1

327-0034


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327-0037


WHITE L=4500 Etched

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361-0028


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• Set the 0 value at the entry of the probe into the cannula

• Orientate the probe with respect to the implant placement

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4 Toutes les cotes sont en mm/


Tol. generales :

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A

1

A

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F

G

H

234

B

C

D

E

F

G

H

234



Modifications

A3

Approuvé

Matière/Material :

Dessiné

Validé

Nom

Date

Obs :

Feuille : 1 / 1

Echelle/Scale :

PN : Rév. :


0 . 00 . 02

211-01230

5/01/2009

Jerry (sans lame)

NIB

ISO 2768 mK

1 : 1


PN

Description

Rév.

Qté

211-0009

Connecteur LAB

1 . 4

1

215-0002

Valise Proflex

1 . 0

1

313-0024

Guide 10000 Ø0,35mm - D15 - 4m

---

1

321-0117

Vis CS M1 x 2 mm A2

---

1

322-0048


1 . 3

1

322-0276

Guide sonde J

0 . 00 . 04

1

322-0277

Ferrule J

0 . 00 . 05

1

325-0030


---

1

325-0031


---

1

327-0034


---

1

327-0037


WHITE L=4500 Etched

1 . 4

1

353-0003


1 . 0

1

353-0004


1 . 0

1

361-0028


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Microprobe Insertion and locking

19

• The microprobe is slightly lowered into the cannula

• Reach the Z coordinates• Then lock the CerboFlex on the

implant’s guide with the lateral screw

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Release the CerboFlex Microprobe

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Tol. generales :

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Modifications

A3

Approuvé

Matière/Material :

Dessiné

Validé

Nom

Date

Obs :

Feuille : 1 / 1

Echelle/Scale :

PN : Rév. :


0 . 00 . 02

211-01230

5/01/2009

Jerry (sans lame)

NIB

ISO 2768 mK

1 : 1


PN

Description

Rév.

Qté

211-0009

Connecteur LAB

1 . 4

1

215-0002

Valise Proflex

1 . 0

1

313-0024

Guide 10000 Ø0,35mm - D15 - 4m

---

1

321-0117

Vis CS M1 x 2 mm A2

---

1

322-0048


1 . 3

1

322-0276

Guide sonde J

0 . 00 . 04

1

322-0277

Ferrule J

0 . 00 . 05

1

325-0030


---

1

325-0031


---

1

327-0034


---

1

327-0037


WHITE L=4500 Etched

1 . 4

1

353-0003


1 . 0

1

353-0004


1 . 0

1

361-0028


---

1



Tol. generales :

1

A

1

A

B

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D

E

F

G

H

234

B

C

D

E

F

G

H

234



Modifications

A3

Approuvé

Matière/Material :

Dessiné

Validé

Nom

Date

Obs :

Feuille : 1 / 1

Echelle/Scale :

PN : Rév. :


0 . 00 . 02

211-01230

5/01/2009

Jerry (sans lame)

NIB

ISO 2768 mK

1 : 1


PN

Description

Rév.

Qté

211-0009

Connecteur LAB

1 . 4

1

215-0002

Valise Proflex

1 . 0

1

313-0024

Guide 10000 Ø0,35mm - D15 - 4m

---

1

321-0117

Vis CS M1 x 2 mm A2

---

1

322-0048


1 . 3

1

322-0276

Guide sonde J

0 . 00 . 04

1

322-0277

Ferrule J

0 . 00 . 05

1

325-0030


---

1

325-0031


---

1

327-0034


---

1

327-0037


WHITE L=4500 Etched

1 . 4

1

353-0003


1 . 0

1

353-0004


1 . 0

1

361-0028


---

1

Unlock the CerboFlex by unscrewing the holder’s screws Release the CerboFlex from the holder


Before Starting Imaging• Calibration must be done before inserting the CerboFlex

microprobe into the implant.• Always control the tip cleanliness on the screen.• In case objects remain stuck on the field of view, clean the probe• Clean the probe’s tip by soaking it into the Quantikit™ cleaning vial

(pink), then rinse in the blue vial.• If the tip is still dirty, use the Coton Q-tips provided with Cellvizio,

impregnated with the blue vial solution.• Always clean from the base to the top of the probe’s bevel

21


After the procedure• Catch the animal and anesthetize it• Place it under the stereotaxic frame• Attach the CerboFlex body to the stereotaxic holder• Carefully unlock the CerboFlex’s screws from the implant’s guide• Slightly remove the CerboFlex from the implant• Clean the CerboFlex tip by keeping the Laser ON for onscreen check up• Put the protection cap into the implant’s cannula• Carefully remove the CerboFlex microprobe from the holder• Fold the probe as shown

22


Disconnect the microprobe by turning the Cellvizio’s connector clockwise until the click. Then, release and remove the CerboFlex.

1


Put the distal and proximal protection back over the tip of the CerboFlex.


2

Note 1 : N

euroPak contents & How

to pack the Cerboflex

Curve the fiber into a circle as shown. The diameter should fit the CerboFlex print in the storage box.

3

CAUTION: Do NOT twist or fold the optical fiber bundle of the CerboFlex


References

23

• Additional information can be found in the NeuroPak USER BOOKLET

• For any inquiry:

[email protected]

mailto:[email protected]

mailto:[email protected]