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CANNABINOID AUGMENTATION OF EXTINCTION LEARNING

R. Andrew Sewell MD, Mohini Ranganathan MD, Harlan Fichtenholtz PhD, Patrick Skosnik PhD, Ashley Schnakenberg BS, Lawrence Rispoli BA, Brynn Huguenel BA, Sahani Jayatilaka BS, Christina Luddy BS, and D. Cyril D’Souza MD Yale University School of Medicine, New Haven, CT and VA Healthcare System, West Haven, CT

BACKGROUND
600,000 SOLDIERS, OR 40% OF RETURNING VETERANS, WILL EXPERIENCE POSTTRAUMATIC STRESS DISORDER (PTSD) AND OTHER MENTAL HEALTH PROBLEMS WHEN THEY RETURN HOME1 Problem: existing medications are not disease-modifying, are only partly effective, and are associated with treatment-limiting side effects THE INCREASE IN NEW CASES, AND SHORTCOMINGS IN CURRENT PHARMACOTHERAPY GENERATE A NEED FOR NEW TREATMENTS BASED ON NOVEL HYPOTHESES

Drugs that enhance CB1 receptor function in humans should also enhance extinction learning

Each session (i.e drug condition) consists of three study days (Figure 4) Day 1: Habituation: subject is familiarized with testing apparatus, CS+U, CS+E, and CS15 trials of CS+U, CS+E, and CS- presented unpaired with the US. Each trial will consist of four presentations of each CS+ and four presentations of the CS-, in a random counterbalanced order, for 4 seconds with a 12-second inter-trial interval (ITI). Eight trials of two startle probes, which consist of a 40 ms burst of 102 dB white noise, will also be presented, in order to reduce initial startle reactivity.

Acquisition:

both CS+U and CS+E are paired with the US

Eight trials of CS+E, CS+U, and CS-, both CS+s paired with the US, which will consist of a 500ms pulse delivered immediately at the end of each CS+ without delay in a 2/3 reinforcement ratio.

Δ9-THC Δ9-THC stimulates CB1 receptors. Intravenous administration standardizes drug delivery and minimizes inter- and intra-individual variability in plasma levels.

PF-04457845 PF-04457845 inhibits fatty acid amide hydrolase (FAAH). FAAH degrades the endocannabinoid anandamide. Therefore, oral PF-04457845 boosts naturally occurring anandamide in the brain.6

Day 2: Drug administration: Δ9-THC, FAAH inhibitor, or placebo Extinction learning: at peak effect, the association learned in the acquisition phase is eliminated
Ten trials of CS+E and CS- unpaired with the US. Startle probes will be delivered during the CS+E, CS-, and inter-trial interval.

EXTINCTION IS A LEARNING PROCESS BY WHICH A PREVIOUSLY ACQUIRED FEAR RESPONSE IS INHIBITED

Extinction recall:

Day 3: the strength of association remaining is measured in the drug-free state

Ten trials of CS+U, CS+E, and CS-, following the same methodology as in the extinction-learning phase, except that neither Δ9-THC nor placebo will be administered, and startle probes will be delivered during the CS+, CS-, and ITI.

OUTCOME MEASURES
Heart rate, blood pressure
robust, non-invasive measures of autonomic activity.

CONDITIONING
US: 500 ms electrical pulse: “painful but not intolerable” CS+U: fear-relevant cue (shark, spider etc.) paired with a shock in the acquisition phase) CS+E: fear-relevant cue paired with a shock in acquisition and extinguished in extinction phase CS-: fear-relevant cue (never paired with a shock)

HYPOTHESIS:
Conditioned fear (based on Figure 1): •  pairs neutral stimulus (CS) with aversive stimulus (US) •  is used to understand how aversive memories are formed FIGURE 1: CLASSICAL CONDITIONING •  models diseases such as PTSD and phobias Extinction of conditioned fear (based on Figure 2): •  pairs the original feared stimulus repeatedly without the aversive stimulus that generated the original fear memory •  is a form of learning parallel to and distinct from the process that created the original fear memory (acquisition)3 •  defects have been implicated in the pathophysiology of PTSD and other anxiety disorders. Extinction learning deficits: •  contribute to inappropriate fear persistence •  predict the development of PTSD after trauma
FIGURE 2: EXTINCTION LEARNING2

acoustic startle electromyography (EMG)
a reflexive blinking to a loud noise (startle probe) potentiated by fear; measures anticipatory anxiety;7 used in many fear conditioning studies.

skin conductance response (SCR)

Administration of cannabinoid receptor agonists, either exogenous or endogenous (anandamide) by way of a FAAH inhibitor, will enhance extinction learning in both healthy subjects and veterans with PTSD.

(Δ9-THC)

the fastest-responding measure of stress response and the primary outcome measure in many studies of extinction learning.8

salivary β-amylase serum cortisol

indicator of sympathetic nervous system response. Peak levels occur immediately after stress and return to baseline after 10-20 minutes. an index of hypothalamic-pituitary-adrenal (HPA) axis activity; correlated with emotional distress and stress response.

METHODS:
•  60 healthy normal subjects (20 per group: placebo, Δ9-THC, FAAH inhibitor) and 60 veterans with PTSD (20 per group: placebo, Δ9-THC, FAAH inhibitor) •  randomized double-blind design •  three test days (acquisition, extinction learning, extinction recall) •  0.015 mg/kg dose of Δ9-THC (1.05 mg in a 70-kg individual), 4 mg PF-04457845 or saline placebo on the first test day

subjective effects
“high”, “anxiety”, etc. assessed with a Visual Analog Scale (VAS) as well as other measures of perceptual and cognitive alterations.

PRELIMINARY RESULTS
µS1/2
1.20# 1.00# 0.80# 0.60# 0.40# CS+# CS/#

GSR$(Hab)$ Habituation

µS1/2 1.20#

GSR$(Acq)$ Acquisition

1.00# 0.80# 0.60# 0.40# 0.20# 0.00#
Hab1# Hab2#

CS+# CS0#

THE CANNABINOID SYSTEM IS CRITICAL FOR THE EXTINCTION OF AVERSIVE MEMORIES

0.20# 0.00#

Amygdalar nuclei (center) are roughly divided into two subsystems (Figure 3):
•  The basolateral complex (lateral, basolateral, and basomedial nuclei; center) integrates auditory, gustatory, somatosensory, spatial, and olfactory information, and is likely to be where the CS is associated with the US. •  This association is then conveyed to the central nucleus, which projects to brain areas responsible for freezing, acoustic startle response, increases in heart rate, blood pressure and breathing, and glucocorticoid release.
This proposal is supported by the Brain and Behavior Research Foundation (NARSAD Young Investigator Award) and the National Center for PTSD.

a
medial&& geniculate& posterior& intralaminar& THALAMUS& auditory& cortex& perirhinal& cortex& insula& piriform& cortex& bed&nucleus& of&the&stria& terminalis&

b

Acq1#

Acq2#

Acq3#

THC,!PF=04457845,!or!placebo!
! Habitua(on!

µS1/2
0.40# 0.35# 0.30# 0.25# 0.20# 0.15# 0.10# 0.05# 0.00#

GSR$ Extinction learning

µS1/2
0.40# 0.35# 0.30# 0.25#

GSR$ Extinction recall

Skin Conductance Response (SCR) in a healthy subject given PF-04457845. a: Habituation to CS+ and CS-. b: When paired with a mild electric shock, aversive conditioning is robustly acquired to the CS +. c: When the CS+ is unpaired with the shock again, the aversive response is extinguished on the second day. d: The extinction learning persists on the third day (extinction retention). REFERENCES
1. Manderscheid R (2008). Our returning veterans: Status report and update on care for PTSD. American Public Health Association 136th Annual Meeting, San Diego, CA. 2. Milad MR, Rauch SL, Pitman RK & Quirk GJ (2006). “Fear extinction in rats: Implications for human brain imaging and anxiety disorders”. Biological Psychology 73:61–71 3. Myers KM & Davis M (2002). “Behavioral and neural analysis of extinction”. Neuron 36(4): 567-84. 4. Glass M, Dragunow M & Faull RLM (1997). “Cannabinoid receptors in the human brain: A detailed anatomical and quantitative autoradiographic study in the fetal, neonatal and adult human brain”. Neuroscience, 77(2), 299-318. 5. Maren S (2001). “Neurobiology of Pavlovian fear conditioning”. Annual Review of Neuroscience. 24: 897-931. 6. Li GL, Winter H, Arends R, Jay GW, Le V, Young T & Huggins JP (2012). “Assessment of the pharmacology and tolerability of PF-04457845, an irreversible inhibitor of fatty acid amide hydrolase-1, in healthy subjects”. British Journal of Clinical Pharmacology, 73(5), 706–16. 7. Orr SP, Metzger LJ et al. (2000). "De novo conditioning in traumaexposed individuals with and without posttraumatic stress disorder." J Abnorm Psychol 109(2): 290-8. 8. Milad MR, Orr SP et al. (2008). "Presence and acquired origin of reduced recall for fear extinction in PTSD: results of a twin study." J Psychiatr Res 42(7): 515-20.

NEOCORTEX& lateral& nucleus& central& nucleus& ventromedial& nucleus& HYPOTHALAMUS&

CA1& ventral& subiculum& entorhinal& cortex& HIPPOCAMPAL& FORMATION&

lateral& nucleus&

DAY!1!

! Acquisi(on!

! Delay!

DAY!2!

! Ext.!learning!

DAY!3! !
Ext.!recall!

CS+# CS/#

0.20# 0.15# 0.10# 0.05#

CS+# CS/#

basolateral&&& basomedial& AMYGDALA& nuclei&

c

Ext1#

Ext2#

0.00#

d

Ext1#

Ext2#

•  Six subjects have been tested so far (2 placebo, 2 THC, 2 FAAH inhibitor) FIGURE 3: ANATOMY OF FEAR CONDITIONING CIRCUITS IN THE FIGURE 4: SCHEDULE OF STUDY DAYS BRAIN. D CB Figure 9: Study design. Habituation and acquisition occur on day #1. Drug administration and extinction learning occur on • day #2. .   Study is ongoing Extinction retention is tested two weeks later on day #3. •  Methodology is safe and tolerable
periaqueductal& grey& parabrachial& nucleus& MIDBRAIN& nucleus&reFcularis& ponFs&caudalis& MEDULLA&
EPTH OF SHADING IS CORRELATED WITH DENSITY OF RECEPTORS 4,5
1

vagal& motor& nucleus&