The CMS Model of Depression

We have now set up the CMS model to evaluate the presence of CB2 cannabi-noid receptors in the brain and whether or not they play a role in depression. This was achieved as follows:

1. Male and female BALBc mice were exposed to mild stressors every day for 4 wk to simulate the symptoms of anhedonia, a major feature in depression.

2. The experimental animals were subjected to a weekly CMS regime consisting of three 10-h periods of 45° cage tilt; three periods of overnight stroboscopic illumination; two 10-h periods of empty water bottle; two periods of overnight food or water deprivation; two 10-h periods of damp bedding.

3. The control animals were housed in a separate room. Anhedonia and weight changes were measured weekly in both the CMS and control animals.

4. Behavioral and rewarding effects of abused substances were also determined in the CMS and control animals.

5. The effect of depression in reward showed that the data obtained are consistent with anhedonia, a lack of pleasure in sucrose consumption, as demonstrated by the CMS animals.

6. We were surprised that the CMS animals also consumed less alcohol compared to control animals (Fig. 1).

7. The CMS animals demonstrated anhedonia because of their lack of interest in reward, whereas the control animals that were not exposed to CMS consumed more sucrose solution, as shown in Fig 1.

3.2. Elevated Plus-Maze Test of Anxiety

1. The influence of CMS with or without challenge with WIN55,212-2, a nonspecific cannabinoid agonist in male and female mice, was evaluated in the plus-maze test.

2. CMS induced gender-specific aversions, which were blocked by WIN55,212-2,1.0 mg/kg (data not shown).

3. The intracerebroventricular (ICV) administration of CB2 antisense oligos, 20 |g, on the performance of mice was assessed before and after 3 d of twice-daily microinjection and compared to mice injected with sense and mismatched oligos (Fig. 2).

4. Direct CB2 antisense oligonucleotide microinjection into the mouse brain induced anxiolysis, indicating that CB2 or CB2-like receptors may influence behavior (Fig. 2). The CB2 antisense oligonucleotide used is 5'-TGTCTCCCGGATCCTC-3', CB2 sense is 5'-GAGGGATGCCGGGAGACA-3', and CB2 mismatch is 5'-TCTATCCGGTCTTGCGTC-3'.

3.3. Western Blotting

1. Equal amounts of protein 20 |g obtained from the brains of stressed and control mice were loaded and separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVD membrane.

Fig. 3. Western blots of CB2 cannabinoid receptor immunoreactivities with CB2 receptor antibody (A) and postincubation with CB2 blocking peptide (B) in mouse brain from CMS and control mice. M is the marker, lane 1 is control brain, and lane 2 is CMS brain. No immunoractivities were detected when primary antibodies were preincubated with the CB2- blocking peptide.

Fig. 3. Western blots of CB2 cannabinoid receptor immunoreactivities with CB2 receptor antibody (A) and postincubation with CB2 blocking peptide (B) in mouse brain from CMS and control mice. M is the marker, lane 1 is control brain, and lane 2 is CMS brain. No immunoractivities were detected when primary antibodies were preincubated with the CB2- blocking peptide.

2. The membrane was washed and blocked in Tris buffered saline containing 5% bovine serum albumin and incubated with the CB2 antibody overnight. The membranes were washed and incubated with a conjugated goat anti-rabbit secondary antibody and processed for immunoreactivities with and without preincubation of the primary antibodies with CB2 peptide.

3. The CB2 antibody used in these studies has been raised against a sequence between the N-terminus and the rst transmembrane domain of the protein of the human CB2 receptor (Cayman Chemicals, MI), which is claimed to be used for Western blotting and immunohistochemical applications as utilized in these studies.

4. There were CB2 immunoreactivities with enhanced expression in the whole brains of CMS animals in comparison to controls (Fig. 3A) and no detectable immunoreactivities postincubation with CB2 blocking peptide (Fig. 3B).

3.4. Mouse CB2 Gene Expression and Rat CB2 Brain Immunohistochemistry

1. CMS and control mice in-groups of 5-10 were sacri ced and brains removed immediately to extract RNA. The expression of CB2 RNA was compared by real-time PCR system (TaqMan, ABI) using the CB2 cannabinoid receptor gene-speci c probe and primers.

str mid hip

Fig. 4. C57B1/6 mice were subjected to acute and chronic mild stress and assessed for anhedonia (CMS animals) by weekly sucrose consumption. Mice were sacri ced at the end of wk 4 or following acute stress, and the brains were quickly dissected to extract RNA for CB2 cannabinoid receptor gene expression by real-time PCR system using the gene-speci c probe and primers (T aqMan real-time PCR system, ABI 7900). CB2 gene expression was detected in the acute and chronic mild stress mice in the striatum (str), midbrain (mid), and the hippocampus (hip).

2. CB2 cannabinoid gene transcripts are expressed in the striatum (str), midbrain (mid), and hippocampus (hip) in naïve and stressed mice (Fig. 4).

3. For immunohistochemical studies, animals were anesthetized and perfused through the left ventricle with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB) pH 7.4. The brains were removed, post xed in the same xative solution for 2 h, and cryoprotected in 30% sucrose in PB at 4°C overnight, then frozen on dry ice. Serial 30-|jm sagittal sections were cut on a cryostat. Standard immunohistochemical protocol, as described previously (10-13), was used. Brie y, sections were prepared, and after incubation overnight with the CB2 polyclonal antibody, the sections were washed and incubated with biotinylated goat anti-rabbit antibody, followed by avidin-biotin complex. Treating the sections with diaminobenzidine and hydrogen peroxide produced visible reaction. Mounted sections were observed and photographed. Dense CB2 cannabinoid receptors are abundantly expressed in the rat hippocampus, striatum and cerebral cortex (data not shown).

3.5. Future Directions

The preliminary results obtained and presented here will help us to lay the foundation to continue these exciting studies. The preliminary result indicating that CB2 or some form of CB2 cannabinoid receptor subtype may be present in the

Fig. 4. C57B1/6 mice were subjected to acute and chronic mild stress and assessed for anhedonia (CMS animals) by weekly sucrose consumption. Mice were sacri ced at the end of wk 4 or following acute stress, and the brains were quickly dissected to extract RNA for CB2 cannabinoid receptor gene expression by real-time PCR system using the gene-speci c probe and primers (T aqMan real-time PCR system, ABI 7900). CB2 gene expression was detected in the acute and chronic mild stress mice in the striatum (str), midbrain (mid), and the hippocampus (hip).

mouse brain with an enhanced expression in CMS model of depression may open new areas and approaches in our understanding of depression and addictive disorders, which may be associated with neuroinflammation, since CB2 cannabinoid receptors have traditionally been known to be present in immune cells.

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