Behavioural Pharmacology, Substance Abuse, and CB2-R Function
Source: Onaivi et al., PLoS ONE 3(2): e1640
Difficulty: Advanced | Prerequisites: Part 1 of these study notes (CB2 expression and genetics), basic behavioural pharmacology, rodent behavioural models (elevated plus-maze, locomotor activity, sucrose preference test). Some familiarity with antisense oligonucleotide technology is helpful.
Part 1 established that CB2 receptors exist in brain neurons. This second set of notes covers the functional consequences: what happens when you activate or block those receptors, how drugs of abuse change CB2 gene expression, and what antisense gene targeting reveals about CB2's role in behaviour. This is where the receptor biology meets pharmacology and addiction science. The data here build the case that CB2-Rs are not merely present in the brain but actively modulate behaviour, including locomotion, anxiety, hedonic responses, and alcohol consumption.
Drugs of abuse (heroin, cocaine, alcohol) alter CB2 gene expression in the brain. Activating CB2 receptors with an agonist changes locomotor activity, increases anxiety-like behaviour, and enhances alcohol consumption in stressed mice. Directly knocking down CB2 gene expression in the brain with antisense oligonucleotides changes mouse behaviour in the plus-maze, confirming these receptors are functional in the CNS.
Elevated plus-maze
A standard rodent test for anxiety-like behaviour. It consists of four arms arranged in a plus shape: two open (no walls) and two closed (with walls). Anxious animals spend more time in the closed arms and avoid the open arms. Reduced aversion to the open arms indicates an anxiolytic (anti-anxiety) effect.
Think of it as a platform where mice choose between safe, enclosed spaces and exposed, open ones. More time on the open arms means less anxiety.
Locomotor activity
Spontaneous movement measured in activity monitors (beam-break or infrared sensors). Changes in locomotor activity reflect sedation, stimulation, or motor impairment caused by a drug.
Stereotypy
Repetitive, purposeless behaviours (e.g. grooming, head-bobbing, sniffing) observed in rodents, often as a measure of dopaminergic or stimulant drug effects. Measured alongside locomotor activity in this study.
Two-compartment black and white box
An anxiety test where mice can move between a brightly lit white chamber and a dark black chamber. Anxious animals prefer the dark side. Time spent in each chamber is the primary measure.
In simple terms, this tests whether a drug makes mice more or less fearful of being in a bright, exposed space.
Antisense oligonucleotide (ASO)
A short synthetic strand of nucleic acid designed to be complementary to a target mRNA. When delivered to cells, it binds the mRNA and blocks translation or triggers its degradation, effectively silencing the gene. Here, CB2 antisense oligos were injected directly into the brain (ICV) to knock down CB2-R expression.
Think of it as a molecular tool that specifically shuts off a single gene's protein production in living tissue.
Intracerebroventricular (ICV) injection
Direct injection of a substance into the brain's lateral ventricles. This bypasses the blood-brain barrier and delivers the compound directly to the cerebrospinal fluid surrounding the brain.
Sucrose consumption test
A measure of hedonic (pleasure) responses in rodents. Reduced consumption of a dilute sucrose solution (compared to water) indicates anhedonia. Used as the primary readout in the CMS model of depression.
Self-medication hypothesis
The idea that individuals use drugs of abuse to alleviate pre-existing psychiatric symptoms (e.g. depression, anxiety). Relevant here because cannabis use and depression are commonly comorbid.
Heroin (10 mg/kg, chronic): Increased CB2 gene expression in whole mouse brain, though the increase did not reach statistical significance (p > 0.05)
Cocaine (40 mg/kg, chronic): Significantly increased CB2 gene expression in whole mouse brain (p < 0.05)
Alcohol (chronic varying intake paradigm):
Mice that developed alcohol preference showed significantly reduced CB2 gene expression in the striatum (p = 0.05) and ventral midbrain (p = 0.04)
Mice with little preference for alcohol showed no changes in CB2 gene expression in these regions
This is a key finding: the direction of CB2 expression change with alcohol preference is opposite to that seen with heroin and cocaine
The pattern suggests that CB2 gene expression is differentially modulated by different classes of abused substances
CB2-R agonist JWH015 enhanced alcohol consumption in stressed mice but not in unstressed controls
CB2-R antagonist AM630 reduced alcohol intake in stressed mice (p = 0.08) but had no effect on unstressed mice
These pharmacological results are consistent with the gene expression data: reduced CB2 expression in alcohol-preferring mice, and exogenous CB2 activation increasing alcohol intake in stressed animals
CB2-R agonist JWH015 (1 to 20 mg/kg, acute):
Reduced spontaneous locomotor activity in a dose-dependent fashion across three mouse strains (C57BL/6, BALBc, DBA/2)
Effects were strain- and gender-dependent, with females generally more sensitive than males
In the two-compartment black and white box, JWH015 produced an anxiogenic (anxiety-increasing) profile in C57BL/6 mice:
Decreased time spent in the white (aversive) chamber
Increased time spent in the black (safe) chamber
Females of the C57BL/6 strain were more sensitive to this effect
These locomotor and anxiogenic effects are consistent with reports for another CB2-R agonist, GW405833
At higher doses, CB2-R agonists induce sedation and catalepsy
CB2-R antagonist SR144528 (1 to 20 mg/kg, acute):
Enhanced locomotor activity and stereotypy in DBA/2 mice (p < 0.05) compared to vehicle controls
Males were more susceptible to locomotor activation than females
Had little or no effect on time spent in the black and white box chambers, except at the highest dose (20 mg/kg), where males spent less time in the white compartment
CB2-R ligand effects on food consumption:
Both enhancement and suppression of feeding were observed, depending on strain and timing
Strain and gender differences were prominent throughout
Key pattern: CB2-R activation (agonist) tends to suppress locomotor activity and increase anxiety-like behaviour; CB2-R blockade (antagonist) tends to increase locomotor activity. Effects are strongly modulated by strain and sex.
Purpose: to test whether reducing CB2-R expression specifically in the brain alters behaviour, providing direct evidence that brain CB2-Rs are functional
Method: direct intracerebroventricular (ICV) microinjection of CB2 antisense oligonucleotide (20 micrograms in 5 microlitres) bilaterally into the mouse lateral ventricles, twice daily for 3 days
Controls: sense oligonucleotide and mismatched oligonucleotide injections
Result in the elevated plus-maze:
CB2 antisense oligo significantly reduced mouse aversions to the open arms (p < 0.05), meaning mice were less anxious
Sense and mismatched oligo-injected mice performed no differently from untreated controls
Interpretation: knocking down CB2-R expression in the brain reduces anxiety-like behaviour, which is the inverse of what the CB2-R agonist JWH015 does (JWH015 increases anxiety-like behaviour). This pharmacological consistency strengthens the case that CB2-Rs in the brain functionally modulate emotionality.
Separately, CMS-exposed or prenatally capsaicin-exposed mice were tested on the plus-maze after acute JWH015 (20 mg/kg):
Stress (CMS or capsaicin) induced gender-specific aversions in the plus-maze
JWH015 significantly reduced these stress-induced aversions (p < 0.05)
The antisense data are particularly important because they rule out the possibility that behavioural effects of systemically administered CB2-R ligands are mediated entirely through peripheral receptors
CMS regime was applied for 4 weeks to produce anhedonia (validated by significant reduction in sucrose consumption, p < 0.05 to p < 0.01, compared to unstressed controls)
Water consumption was not affected by CMS, confirming that reduced sucrose intake reflects loss of hedonic response rather than general fluid aversion
CB2-R agonist JWH015 (20 mg/kg, daily for 4 weeks):
In CMS mice: variable sucrose consumption; stressed mice treated with JWH015 did not differ from untreated CMS mice (no reversal of anhedonia)
In control (unstressed) mice: JWH015 enhanced sucrose consumption by weeks 2 and 4 (p < 0.05)
Important context: JWH015 dramatically enhanced alcohol intake in stressed mice in a separate paradigm. So while it did not reverse sucrose anhedonia, it did enhance consumption of another rewarding substance (alcohol) under stress
CB2-R antagonist AM630 (1 and 3 mg/kg, daily for 4 weeks):
Did not significantly modify sucrose intake in either CMS or control mice
However, in the separate alcohol paradigm, AM630 blocked the stress-enhanced alcohol consumption (p > 0.05)
Summary of reward-related findings:
CB2-R agonist JWH015 did not reverse anhedonia for sucrose but enhanced alcohol consumption in stressed mice
CB2-R antagonist AM630 blocked stress-enhanced alcohol consumption but did not affect sucrose intake
This dissociation suggests that CB2-R modulation of reward may be substance-specific or that alcohol and sucrose engage different motivational circuits under stress
The observation that CB2-R agonism enhances alcohol consumption in stressed animals, and that CB2-R antagonism blocks this effect, has direct implications for understanding alcohol use disorder. If CB2-R blockade can reduce stress-driven alcohol consumption without the psychiatric side effects of CB1-R antagonists, it could represent a safer pharmacological strategy for treating alcoholism, particularly in patients with comorbid depression.
Students often assume that if a CB2-R agonist does not reverse anhedonia in the sucrose test, CB2 receptors have no role in depression. This is too simplistic. The agonist did modulate alcohol intake under stress, and the Q63R polymorphism data (Part 1) link CB2 to depression genetically. Reward is not one-dimensional.
A frequent error is to assume that behavioural effects of systemically administered CB2-R ligands must be mediated peripherally (since CB2 was thought to be a peripheral receptor). The antisense oligonucleotide data, where CB2 was knocked down directly in the brain and behaviour changed, rule this out.
Students sometimes conflate anxiolytic and antidepressant effects. The antisense data show reduced anxiety (plus-maze), not necessarily antidepressant action. These are distinct behavioural domains tested by different assays.
It is incorrect to think that CB2-R effects are uniform across strains and sexes. This study found pronounced strain and gender differences in every behavioural measure, which is itself an important finding.
⚠️ Be able to explain the logic of the antisense oligonucleotide experiment: why injecting CB2 antisense directly into the brain ventricles provides stronger evidence for central CB2-R function than systemic drug administration alone.
⚠️ Know the dissociation between sucrose anhedonia and alcohol consumption results. JWH015 did not reverse sucrose anhedonia but enhanced stress-driven alcohol intake. This is a nuanced result that examiners may test.
⚠️ Understand strain and sex differences in CB2-R pharmacology. C57BL/6, BALBc, and DBA/2 strains responded differently to the same drugs. Females were generally more sensitive to the CB2-R agonist, males to the antagonist.
⚠️ The direction of CB2 gene expression change differs by substance: cocaine and heroin increase it; alcohol preference decreases it. Be ready to explain why this might matter for understanding the neurobiology of different addictions.
True or false: Chronic cocaine treatment decreased CB2 gene expression in the mouse brain. _____ (False, it significantly increased it)
CB2-R agonist JWH015 enhanced alcohol consumption in _______ mice but not in _______ mice. (stressed; unstressed/control)
True or false: ICV injection of CB2 antisense oligonucleotide increased anxiety-like behaviour in the plus-maze. _____ (False, it reduced aversions to the open arms, indicating reduced anxiety)
The CB2-R antagonist AM630 _______ (blocked / enhanced) stress-induced alcohol consumption. (blocked)
True or false: CB2-R agonist JWH015 successfully reversed anhedonia in the CMS sucrose consumption test. _____ (False, it did not reverse anhedonia for sucrose in CMS mice)
Q: How do the effects of chronic heroin, cocaine, and alcohol on CB2 gene expression differ, and what does this suggest about CB2-R involvement in substance abuse?
A: Chronic heroin treatment increased CB2 gene expression (non-significantly), chronic cocaine significantly increased it (p < 0.05), and alcohol preference was associated with significantly decreased CB2 expression in the striatum and midbrain. This differential pattern suggests that CB2-Rs are modulated by drugs of abuse in a substance-specific manner, rather than through a single common mechanism, and that CB2 may play distinct regulatory roles in the rewarding or reinforcing effects of different drug classes.
Q: Explain how the CB2 antisense oligonucleotide experiment demonstrates that brain CB2 receptors, rather than peripheral CB2 receptors, influence behaviour.
A: The antisense oligonucleotide was injected directly into the brain ventricles (ICV), bypassing peripheral tissues entirely. The antisense oligo specifically reduces CB2 mRNA in the brain, leading to decreased CB2-R protein production. The resulting behavioural change (reduced aversion to open arms of the plus-maze) occurred only with the antisense oligo, not with sense or mismatched controls. This demonstrates that the behavioural effect is mediated by reducing CB2-R expression specifically in the CNS.
Q: Why might the CB2-R agonist JWH015 enhance alcohol consumption in stressed mice while failing to reverse sucrose anhedonia in the CMS model?
A: Several explanations are possible. Alcohol and sucrose may engage different motivational or reward circuits that are differentially sensitive to CB2-R modulation. Stress may alter the CB2-R signalling context in a way that specifically enhances the reinforcing properties of alcohol (which has pharmacological effects on multiple neurotransmitter systems) but not the hedonic response to a simple sweet taste. The finding highlights that CB2-R involvement in reward is not uniform across all rewarding stimuli.
Q: What is the significance of strain and sex differences in the behavioural pharmacology of CB2-R ligands?
A: The strain differences (C57BL/6, BALBc, DBA/2) and sex differences observed in this study indicate that genetic background and hormonal factors modulate CB2-R-mediated behavioural responses. This has translational relevance: human populations are genetically diverse (as shown by the Q63R polymorphism data), and sex differences in depression and addiction prevalence are well documented. These findings caution against assuming uniform CB2-R pharmacology and support the need for pharmacogenomic approaches in developing CB2-R-targeted therapies.
The behavioural pharmacology here connects to courses on addiction biology, particularly the neurocircuitry of reward (mesolimbic dopamine pathway, striatum, ventral tegmental area) and how stress interacts with reward systems. The antisense oligonucleotide work ties into molecular pharmacology and gene therapy concepts. The strain and sex differences connect to pharmacogenomics and personalised medicine. The CMS model links to clinical psychiatry and the ongoing search for better animal models of depression beyond the monoamine hypothesis.
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