1. Cannabinoid CB1 receptor
Role in extinction and sensitization

2. Say it with me
Kah nab a noid
Ka banna boy

3. outline Receptor and receptor subtypes Endogenous ligand
Location, function and distribution
Endogenous ligand
Kamprath paper
Pamplona et al paper
Results
Clinical applications

4. CB receptor subtypes CB1-found in brain
CB1A-very little info, but does exist
CB2-found in immune cells (not in brain)
CB1 is of most interest to us today
acts via G-protein which, when activated, inhibits adenylate cyclase as well as voltage gated CA++ channels, stimulates K+ channels

5. Distribution of CB1 Cerebellum- Cortex- Basal Ganglia Limbic system
motor function
Cortex-
association
Basal Ganglia
Limbic system
Hippocampus-learning
Amygdala-fear
NO CB1 in thalamus,medulla or brainstem

6. Limbic CB1 receptors
Exclusively localized to GABA and Cholecystokinin (CCK) containing presynaptic terminals
Receptor activates G-protein that suppresses GABA release
Less GABA=more anxiety?
In hippocampus- release of ACh is inhibited

7. CB1 in a RAT BRAIN

8. Relative density of CB1R in select brain regions

9. Anandamide Endogenous cannabinoid
Synthesis not known
When bound in high doses, behaviors include: hypothermia, analgesia, hypomobility, catalepsy
LIPID NT, can pass membrane, thought to be synthesized on demand
Acts on membrane bound and intracellular locations

10. Anandamide Brain levels rival that of DA (felder et al 96)
Binds to both CB1 and CB2
Acts as agonist
Highest conc in hippo, striatum, ctx and cerebellum
THC acts as CB1 agonist
Some diet drugs act as CB1 antagonist
Rimonabant (aka SR141716)

11. Kamprath et al Neuroscience 06
Sensitization: nonassociative learning
General increase in reponse to potentially harmful stimuli after aversive experience
E.g. inescapable footshock=alter behav& cort
Fear conditioning: tone paired with shock
Re-exposure to tone activates memory of the tone-punishment association and induces response
E.g. rats freeze upon re-exposure to tone

12. Extinction vs Habituation
Extinction: new association btwn tone and the nonappearance of predicted punisment (safety learning)
Suppresses the expression of the memory of the tone-shock pairing
Habituation: repeated non-reinforced tone presentation will lead to a decrease in response to the tone

13. Role of CB1
CB1 role in extinction is limited to aversive testing conditions
Genetic ablation or pharmacological blockade of CB1 impairs the extinction of fear memories
CB1 plays no role in conditioning to tasks involving positive reinforcement

14. Goals of study
Examine the role of CB1 in extinction and habituation of acquired fear responses
Utilize genetic mutant CB -/- and ‘wild type’ CB+/+
Utilize SR selective CB1 antagonist

15. Cond
Sens
CB-/- showed same freezing
Behavior with or without shock
CB-/-
No difference
in acquisition
sensitized
conditioned
Conditioning: 80 dB tone with .7 mA footshock
next day tone only in novel envir
CB-/- show prolonged and stronger freezing to tone
CB+/+
sensitized
conditioned
Sensitization: .7 mA footshock only NO TONE
next day tone only in novel envir
CB-/- show prolonged and longer freezing to tone

16. Can CB-/- associate memories?
Because no difference in freezing was seen between tone-shock pair and tone alone in CB-/- animals, authors wanted to make sure that CB-/- animals could in fact form an associative memory i.e. associate the tone with the impending shock
Utilize electrophysiology to record auditory evoked potentials in CA1 region of hippo

17. No significant differences between CB-/- and CB+/+ in the potentiation of auditory-evoked potentials
Both groups have similar activation in CA1 region when exposed to loud tone

18. Within session vs long term
Next, authors wanted to see what changes in extinction are seen over time between the two groups
Testing occurred as before, with the addition of another testing round 5 days after the first test

19. SR CB1 controls both within session and long term fear adaptation
sensitized
conditioned
CB+/+
sensitized
conditioned
CB-/- again froze longer
And stronger both 1 day
and 6 days after conditioning SR
CB+/+ animals treated with
SR show same trend as the
Mutant strain, even 5 days
later
CB1 controls both
within session and
long term
fear adaptation

20. Pamplona et al Psychopharmacology 2006
The cannabinoid receptor agonist
WIN 55,212-2 facilitates the extinction of contextual fear memory and spatial memory in RATS

21. Main Objectives
Examine whether admin of CB1 agonist WIN could faciliate the extinction of recent and/or remote contextual fear memory in rats
Investigate the role of CB1 antagonist SR in the extinction process
Determine if not only fear memory but also spatial memory was affected by these drugs

22. Methods Animals Male Wistar rats, group housed
Behavorial testing occurred during light phase of the day/night cycle….. Why?
N of 7-10 animals per group

23. Animals injected IP 0.2mL per 100 g BW
Drugs
Win and SR dissolved in 0.9% saline with 10% DMSO and 0.1% Tween 80
Controls received vehicle only
Animals injected IP 0.2mL per 100 g BW
Win given 30 min before testing
SR given 20 minutes before testing

24. Behavioral Procedures
Fear Conditioning
Contextual fear: 3 min in cond chamber and then received 1 sec 1.5 mA shock, 60 seconds after shock they were removed
Tone fear: 3 min in cond chamber and then 10 sec of 80 dB tone that co-terminated with 1 sec 1.5 mA shock, 60 seconds after shock they were removed
Freezing was the behavior measured during subsequent testing

25. Effects of CB1R on extinction of recent contextual fear memory
Successive exposures to conditioning chamber were used to assess short term (within exposure) and long term (between exposure) extinction of cond fear
24, 48 and 72 hours after contextual conditioning animals were placed back in chamber and freezing recorded
WIN or SR treatment before each session

26. Dose Dependant Effects!
Control animals froze less on each
successive re-exposure
1.0 mg/Kg dose of SR disrupted this
extinction (they froze more than
control group)
Low dose of WIN facilitated
extinction (they froze less than
controls); high dose of WIN
disrupted extinction
Mid dose of WIN mimicked control
group
Dose Dependant Effects!

27. In the first 3 minutes (retrieval) of the
9 minute test block SR had no effect
But WIN at the mid dose showed
Decreased freezing time, this means
That there is no effect of WIN on
Memory retrieval

28. Effects of WIN on extinction of remote contextual fear memory

29. 5 days of exposure to context
shows that WIN treated rats
freeze less, extinction is
facilitated in short term fear
memory by WIN
Drug free rats 48 hours after
the 5 day extinction protocol
WIN treated, contextual cond
rats froze less than control and
SR treated rats
WIN facilitates extinction of
remote fear memory

30. Unconditioned freezing behavior
Day 1 rats placed in cond chamber for 3 minutes, shocked (1 sec 1.5mA), 60 seconds later they were removed
Day 2 rats treated with WIN or SR, placed in novel environment and freezing behavior was measured
Also, separate group of rats treated with WIN and SR and placed in open field to measure effects on locomotion

31. No effect of WIN or SR on unconditioned freezing or
on locomotion in open field test

32. Day 1 and 2 were training,
6 trials each day
Drug admin on day 3, platform
moved to opposite side, and
6 trials followed
WIN treated animals had
decreased latency to find
platform on first trial, no diff
from controls after that
SR treated animals showed
increased latency on trial 2
Compared to control, no diff

33. Discussion
Disruption of CB1 cannabinoid signaling decreases the ability of rodents to extinguish fear memories
WIN acting as a CB1 agonist facilitates fear extinction
WIN also facilitates spatial memory
Not by disrupting acquisition, memory retrieval or affecting sensory-motor ability

34. CB1 antagonists
Administration of selective CB1 antagonists (SR or rimonabant) or genetic ablation of CB1 gene leads to a pronounced deficit in the extinction of conditioned fear
Also leads to deficits in previously learned spatial information
In Rats!

35. Administration of CB1 agonist WIN facilitates the extinction of contextual fear and may have long term implications
In humans, pharmacotherapies directed at the endocannabinoid system may help treat psychiatric disorders such as retrival of fear memories, panic, phobias (like being afraid of russians) and PTSD

36. You may cease learning…….
………………….now!
The End

37. Pat Ronan
Rules!