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thclear cbd 8 1

Thclear cbd 8 1

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Thclear cbd 8 1

Rat brainstem tissues were homogenized in ice-cold Choi lysis buffer (Tris–HCl 20 mM, sucrose 0.32 M, EDTA 0.2 mM, EGTA 0.5 mM, pH 7.5) containing Roche © protease inhibitor cocktail (1:40 v/v; Roche Diagnostics, Mannheim, Germany) and phenylmethylsulphonyl fluoride (1 mM). The homogenate was centrifuged at 13 500×g for 15 min, and the resulting pellet was kept in −80°C for at least 2 h. The pellet was then resuspended in TME buffer (50 mM Tris–HCl; EDTA 1.0 mM; MgCl2 3.0 mM; pH 7.4), homogenized and stored at −80°C.

When administered intracranially into the DRN, WAY100635 was prepared in sterile saline at a concentration of 21 ng in 0.5 µL (Herges and Taylor, 1999) and intracranially microinfused at 0.5 µL·min −1 for 1 min. Intracranial CBD was prepared in 45% 2-hydroxypropyl-β-cyclodextrin at 10 µg·µL −1 and intracranially microinfused into the DRN at 1 µL·min −1 for 1 min (based on Murillo-Rodriguez et al., 2008).

Effect of systemic injections of 5-HT1A receptor antagonists on CBD-induced suppression of LiCl-induced conditioned gaping in rats

Verification of cannula placement into the DRN was determined by histological evaluation of tissue. Rats were injected with 85 mg·kg −1 sodium pentobarbital (Euthansol, Intervet Canada Corp., Kirkland, QC, Canada) and were transcardially perfused with PBS (0.1 M) and 4% formalin. The brains were removed and stored at 4°C in 4% formalin solution for 24–48 h, after which they were placed in a 20% sucrose solution overnight at room temperature. The brains were then sliced in 60 µm sections using a CM1850 Leica cryostat (Leica Microsystems Inc., Concord, ON, Canada), and relevant sections were mounted on glass microscope slides. The tissue was later stained with cresyl violet and examined for accurate injector tip placement using a Leica MZ6 Stereomicroscope with a Leica DFC420 Digital Camera and Leica Application Suite software.

The accurate injector tip placements (circles) are presented in Figure 4A . The tips of the injectors were located in the DRN between 2.04 and 0.84 mm anterior to the inter-aural line for a total of 29 rats. A total of 20 rats had placements outside the DRN. The rats in group WAY-CBD with inaccurate placements (triangles) were included in the analysis as a separate group (WAY-CBD-OUT; n= 5) for comparison with those receiving the antagonist in the DRN. Figure 4 B presents a representative photomicrograph of the tip/track of the injector in the DRN.


(A) Traces of infusion sites in the DRN for all groups (circles). Numbers indicate sections relative to inter-aural zero. (B) A representative photomicrograph of the tip/track of the injector in the DRN.