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added 4V cannulation method
===Method===
===Method===
Each rat was stereotaxically implanted with a 22 gauge, stainless steel, guide
Each rat was stereotaxically implanted with a 22-gauge, stainless steel, guide
cannula (Plastics One, Roanoke, VA) aimed towards the
cannula (Plastics One, Roanoke, VA) aimed towards the lateral cerebral ventricle (1.2 mm caudal to bregma, 1.5 mm
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
lateral to the midline, and 4 mm below the skull surface).
stainless steel screws anchored to the skull. An obdurator was inserted into each guide cannula and remained in place
Guide cannulas were held in place with dental acrylic
except during injections when it was removed and replaced with an injector that extended 1.0 mm beyond the tip of the guide cannula.
bonded to stainless steel screws anchored to the skull. An
obdurator was inserted into each guide cannula and remained
in place except during injections when it was removed
and replaced with an injector that extended 1.0 mm
beyond the tip of the guide cannula.
===Test===
===Test===
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.
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
Plastics One Cannula:
===Method===
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. An obdurator (model C315DC; Plastics One, Inc.) 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 XXX-gauge injector that extended 1.0 mm beyond the tip of the guide cannula.
===Test===
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.