NeuroPak

www.cellviziolab.com

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80%

+10°C

+35°C

NeuroPak™

NeuroPak™

Deep Brain Imaging in Freely-Moving Mice

Deep Brain Imaging in Freely-Moving Mice

In Vivo Molecular Imaging at Cellular Resolution

www.maunakeatech.com

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Image neurons at work, anywhere in the brainwith

EUROPEMauna Kea Technologies SA

9 rue d’Enghien,75010 Paris, France

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USAMauna Kea Technologies, Inc.3240 South White Road, PMB#122San Jose, CA 95148Ph : 408-729-3155Fax : 408-729-1889

STRIATUMS-300, NeuroPak™CaMK–eGFP transgenicmice somata of medium spiny neurons

HIPPOCAMPUSS-300, S-650, NeuroPak™AAV transfected neurons

CORTEXS-1500, Mini-Z,FITC injection,Microcirculation

SUBSTANTIA NIGRAS-650, Mini-Z, Oregon Green BAPTA-1, ParkinsonNeuroPak™

ACCUBENSNeuroPak™, S-300,S-650, Addiction

VTAS-300, S-650, NeuroPak™

Lentivirus-transfectedneurons and individual

dendrites

VISUAL CORTEXS-650, Mini-Z, Oregon Green,

BAPTA-1, Neuron activation

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flyer-Neuro corrections (2.indd 1 22/08/12 16:50



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USER BOOKLETIn Vivo Molecular Imaging at Cellular Resolution


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NeuroPak™ User Booklet 2



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“Cellvizio®Lab is the only integrated platform currently on the market for endomicroscopic confocal imaging with cellular resolution appropriate for in vivo imaging of the brain in behaving mice.The dedicated CerboFlex system permits secure and firm installation of the microprobes onto the head of an animal and the unprecedented small weight of the device -300 milligrams- combined with the extreme flexibility of the probe itself now makes the animal totally free to move and behave while imaging.”

Eisenstein, Nature Methods 2009




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NeuroPak™

http://www.nature.com/nmeth/journal/v6/n10/abs/nmeth1009-773.html

http://www.nature.com/nmeth/journal/v6/n10/abs/nmeth1009-773.html

NEUROPAK™ CONTENTS




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CerboFlex™ Screwdriver

Sterotaxic holder

Locking Screws

Implants + caps

Installation disk

User Booklet

Protection case

TECHNICALSPECIFICATIONS




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

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Accessoire Stéréo

Inox 316L (1,4404)

nicolasb

ISO 2768mK

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

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

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N° doc Description Rév. Qté

322-0275 Guide implant J 0 . 00 . 04 1

322-0282 Socle implant J 0 . 00 . 06 1

329-0005 Goupille Ø2 x 8 A2 --- 1

CerboFlex™ Microprobe• Diameter 350 µm or 470 µm (Hard tip)• 45° beveled tip• 6,5 mm penetration capability• Adapted to mouse brain• Guide Length 4 m• Resolution 3,3 µm• Flexible, reusable

Stereotaxic holder • Fits to any stereotaxic frame• Acurate positionning of the implants

and the CerboFlex™

Implants (x6)• Weight < 300 mg• Made of Titanium• Guide post and cannula• Caps provided to

protect the brain after imaging

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

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CELLVIZIO®PREPARATION

The over goal of this procedure is to implant the CerboFlex microprobe for chronic neuron activity recordings with Cellvizio Lab.

1. Turn on the Laser Scanning Unit and the Computer.

2. Open the NeuroPak™ case and take the CerboFlex microprobe.

3. Unfold it carefully and plug it to the connector.

4. Launch ImageCell™ (Cellvizio) or Cellvizio Dual Software (Cellvizio Dual Band).

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

6. Once the microprobe ready, proceed with calibration using the Quantikit vials by following the onscreen guidelines.

Important Note: • In case of failure during calibration, please proceed again to Step 5• If the system won’t calibrate, please contact [email protected]




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MATERIALCHECKLIST

๏ Surgery equipment: syringe, hair clipper, scalpel, compresses, scissors, forceps, 30G needles, Q-tips, lead pencil.

๏ Anesthetic (Ketamine/xylazine solution for example).

๏ 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 arylic solution (cement).

๏ Mouse Brain Atlas (Paxinos).

๏ 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), extra strong glue.

๏ Cellvizio® or Cellvizio® Dual Band

๏ NeuroPak™ or NeuroPak™-J

๏ Quantikit.

๏ ImageCell or Cellvizio Dual Software.




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IMPLANTATIONPROTOCOL

1. Set up the stereotaxic frame and all the materials needed. Make sure the area and instruments are cleaned and sterilized.

2. Shave off fur from the ears to just in-between the eyes with an electric hair clipper. Once done, apply povidine/iodine to shaved area but protect the eyes from it.

3. Mount the animal onto the stereotaxic apparatus by placing the ear bars into the ear canal. Secure the mouth with the anterior mount of the stereotaxic [A].

4. The incision is then made down the midline using a scalpel or surgical scissors and the top of the skull is exposed [B, C].

5. Replace the stereotaxic holder from the frame by the NeuroPak™ stereotaxic holder 6. Secure the CerboFlex™ head with the screws from the holder and tape the fiber-optic guide

to the stereotaxic frame arm.

7. Then 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. A mark can be made with a pencil at this location. This is where you will be drilling.

8. 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 [D]. 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 micriprobe.

9. 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 [D].

10. Pick up an implant, remove its cap and clean it with ethanol and saline. Then plug it into the NeuroPak™ stereotaxic holder, secure with the two screws, and lower it slowly to the proper ventral coordinate. The protruding part of the implant cannula should penetrate the skull straight through the hole [E].

11. Now mix a thin batch of liquid dental cement and cover the implant base, screws, and the rest of the skull with a sterile spatula. Make sure to cover the base of the implant and not the stereotaxic holder. Make another batch, thicker this time, and completely cover the area and the implant’s base and anchor screw enough to secure it [F].

12. Allow the dental cement to completely dry before removing the stereotaxic holder from the implant. To do this, just unscrew it.

13. The incision is sutured around the implant and the animal is allowed to recover for one week prior to imaging [G].

14. One week after implantation [H], position again the animal on the stereotaxic frame, then place the CerboFlex on the holder as described step 6.

15. The microprobe is slightly lowered into the cannula. After the Z coordinates have been reached, secure the CerboFlex on the implant’s guide with the lateral screw [I].

16. Unmount the CerboFlex from the holder by unscrewing the two lateral screws. Cellvizio imaging session can now be started.




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IMPLANTPOSITIONNING




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[A] [B] [C]

[D] [E] [F]

[G] [H] [I]

CERBOFLEXPACKING

After use, the CerboFlex distal tip must be cleaned thoroughly using Q-tips impregnated with peroxide (pink vial from the Quantikit). Keep laser on during this step and check the tip’s cleanliness on Cellvizio’s screen. When done, rinse in the blue vial (demineralized water).




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Note 1 : NeuroPak contents & How to pack the Cerboflex

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.

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

3


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

1

USEFULREFERENCES

1. Michael Eisenstein (2009), Getting inside their minds Nature Methods; 6, 773-781

2. Chang JY et al (2008), Studies of the neural mechanisms of deep brain stimulation in rodent models of Parkinson’s disease. Neurosci. Biobehav. Rev., 32, 352–366.

3. Enwere, E. et al. (2004), Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination.J. Neurosci. 24, 8354–8365

4. Gheusi, G. et al. (2000), Importance of newly generated neurons in the adult olfactory bulb for odor discrimination. PNAS 97, 1823-1828

5. Heim et al. (2007), Nature Methods, 4 (2) : 127-129

6. Holschneider DP and Maarek JM (2008)

7. Paxinos and Franklin, The Mouse brain in stereotaxic coordinates - Academic Press

8. Rochefort, C.et al. (2002) Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory.J. Neurosci. 22, 2679–2689

9. Sawamoto et al. (2001), Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters, J. Neurosci. 65 (3): 220-7.

10. Stosiek C et al. (2003), in vivo two-photon calcium imaging of neuronal network, PNAS 10.1073

11. Vincent et al. (2006) Live imaging of neural structure and function by fibred fluorescence microscopy. EMBO Rep. 7(11): 1154–1161.

12. Zhao, M. et al. (2003), Evidence of neurogenesis in the adult mammalian substantia Nigra, PNAS U S A. 100 (13): 7925–30.




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In Vivo Imaging at Cellular Resolution

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http://www.cellviziollab.com

http://www.cellviziollab.com

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