ICV Cannulation

For other stereotaxic coordinates, see also Amygdala, JA Lateral Hypothalamus Lesions.

Lateral Ventricular Cannula

1.2 mm caudal to bregma

1.5 mm lateral to the midline

4 mm below the skull surface

Under isoflurane anesthesia, each rat was stereotaxically implanted with a 22-gauge, stainless steel, guide cannula (model C313G, Plastics One, Roanoke, VA) aimed towards the lateral cerebral ventricle (1.2 mm caudal to bregma, 1.5 mm lateral to the midline, and 4 mm below the skull surface; Houpt 1998 PMID 9437750). Guide cannulas were held in place with dental acrylic bonded to stainless steel screws anchored to the skull. A 28-gauge obdurator was inserted into each guide cannula and remained in place except during injections when it was removed and replaced with an 28-gauge injector that extended 1.0 mm beyond the tip of the guide cannula.

To test the patency and placement of the cannula in the lateral ventricle, water-replete rats were injected with 100 ng human angiotensin II (Ang II; Sigma Chemical Co, St Louis, MO) dissolved in 5 µl volume of saline (0.15 M). The volume of all icv injections was 5 µl, delivered over 30s with a handheld 50 µl syringe (Hamilton Co, Reno, NV). Immediately after the ICV injection, rats were returned to their home cage with access to a water bottle. The latency to drink was recorded; rats that failed to drink within 2 minutes of the angiotensin II injection were dropped from the study. Cannula placements were also verified postmortem by sectioning through the brain. ICV injections of 5 µl volume were given by hand to lightly restrained rats using a 50 µl Hamilton microsyringe.

Fourth Ventricular Cannula

3.2 mm caudal to lambda

0 mm lateral to the midline

7.2 mm below the skull surface

Under isoflurane anesthesia, each rat was stereotaxically implanted with a single 22-gauge stainless steel guide cannula (model C315G; Plastics One, Inc., Roanoke, VA) aimed towards the fourth ventricle (4V). The tip of the guide cannula was positioned on the midline 3.2 mm caudal to lambda and 7.2 mm below the skull surface (stereotaxic coordinates taken from (Baird et al. 2009 PMID 19008313)). Guide cannulas were held in place with dental acrylic bonded to stainless steel screws anchored to the skull. A 28-gauge obdurator extending 1.0 mm beyond the guide cannula was inserted into each guide cannula and remained in place except during injections when it was removed and replaced with a 28-gauge injector that extended 1.0 mm beyond the tip of the guide cannula.

To test the patency and placement of the cannula in the lateral ventricle, water-replete rats were injected with XXX ng bombesin (California Peptides) dissolved in 5 µl volume of saline (0.15 M). The volume of all icv injections was 5 µl, delivered by hand over 30s to lightly restrained rats using a 50 µl Hamilton microsyringe (Hamilton Co, Reno, NV). Immediately after the ICV injection, rats were returned to their home cage with access to a water bottle. The latency to initiate and duration of grooming was recorded; rats that failed to groom within 2 minutes of the bombesin injection or groomed for less than XXX minutes were dropped from the study. Cannula placements were also verified postmortem by sectioning through the brain.

Supplies

Plastics One Cannulas and Screws

P1 Technologies (née Plastics One)

6591 Merriman Rd

Roanoke VA 24018

Tel: 540-772-7950

Fax: 540-777-5900

Check prices on annual price list.

22 gauge guide cannulas for ventricular injections

Use C313 components, which provide 28 gauge dummy and injectors. e.g., for guide cannula(G) projecting 5 mm below skull surface, with large-cap dummy cannulas (DC) and injectors (I) projecting 1 mm beyond tip of the guide cannula:

C313G/SPC 5 mm

C313DC/SPC(LG) for 5 mm C313G with 1 mm projection

C313I/SPC for 5 mm C313G with 1 mm projection

26 gauge guide cannulas for site-specific injections

Use C315 components, which provide 33 gauge dummy and injectors. e.g., for guide cannula(G) projecting 8 mm below skull surface, with small-cap dummy cannulas (DC) and injectors (I) projecting 0.5 mm beyond tip of the guide cannula:

C315G/SPC 8 mm

C315DC/SPC(SM) for 8 mm C315G with 0.5 mm projection

C315I/SPC for 8 mm C315G with 0.5 mm projection

Screws for skull

We use screws of 0-80 size with shaft length 3/32 inches (2.4 mm). These are a little long, but they are stronger and easier to work with than 1/16 inch (1.6 mm) long screws. Also need the small drill bit.

0-80 X 3/32 (packages of 100)

D#56 1.18 mm Drill Bit for 0-80 screws

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Other supplies needed - more detail to come

Pin Vise for hand drilling the screws

Most pin vises are nickel plated, and so cannot be autoclaved but must be sterilized e.g. with 70% EtOH.

VWR 63041-530 Pin Vise - Double-Ended, stainless steel

Little tubing for washer around top of guide cannula?

Superglue

Size of screwdriver? source of SS screw drivers?

Trephin drill bits

Hamilton microsyringe and needle

PE-50 tubing to connect with injector

Syringe Pump

New Truliner Denture Lining Standard Kit, Pink

From either Henry Schein Dental, #2502527EZ, $105.29, or, Bosworth #0921970 $45.00

Harry J. Bosworth Company

7227 North Hamlin Avenue

Skokie, Illinois 60076 USA

Toll-Free: 800-323-4352

Phone: 847-679-3400

Fax: 847-679-2080

E-Mail: HJBinfo@bosworth.com