Endogenous Cannabinoid System In Basolateral Amygdala X X Endogenous Cannabinoid System In Basolateral Amygdala memory consolidation Timothy M. Hanna, MS Advanced Behavioral Neuroendocrinology Endocannabinoids Fall 2015 University of South Dakota

Objectives When relevant Maslow-ian information is encountered a slow response is no response. Therefore, it is imperative that the arousal response be mediated by rapid endogenous modulators. Need for a non-genomic explanation for rapid GC- mediated cascade action during emotionally aversive learning and memory has lead researchers to the eCB system. This slide lets the audience know that everyone is at “some stage” of retirement planning.

Endocannabinoid System Review Endocannabinoid System “Neuromodulatory system capable of transiently or persistently suppressing transmitter release from both excitatory or inhibitory synapses throughout the CNS” (Younts & Castillo 2014, 42). Insofar as BLA concerned GABAergic interneuron suppression plays an important role

Amygdaloid Complex Divisions

AB, accessory basal; B, basal nucleus; Ce, central nucleus; ic intercalated cells; La, lateral nucleus; M, medial nucleus; CO, cortical nucleus; (non-amygdala areas): AST, amygdalo-striatal transition area; Cpu, caudate putamen; CTX, cortex Amygdala (Rat Brain) 1=Nissl Stain 2=Acetylcholinesterase Stain 3=Silver Fiber Stain Joseph E. LeDoux (2008) Amygdala. Scholarpedia, 3(4):2698.

Pharmacological Agents Encountered In Today’s Papers

WIN55, 212-2 Aminoalkylindole (AAI) Family Full agonist THC-like effects  affinity than THC Ki=1.9nM vs. Ki=41nM Implicated in meadiating neuropathic pain and may decrease/prevent inflammation caused by amyloid beta proteins in AD

SR141716A (Rimonabant) SR141716A (Rimonabant) Selective CB1R antagonist Inverse agonist-reverses adenylyl cyclase inhibition by WIN55, 212-2

AM251 Antagonist/Inverse agonist at CB1R Similar to SR141716A (Rimonabant) AM251=Better affinity (Ki =7.5nM vs Ki =11.5nM) 2x selectivity CB1R

RU 28362 Corticoid Type II Receptor Agonist (GR Only) High Affinity Do not confuse with the MR antagonist RU 28318 http://en.chembase.cn/substance-346948.html

RU 38486 (Mifepristone) Very Effective Antiglucocorticoid Cortisol antagonist (competitive at receptor) Anti-progesterone Well known due to abortion controversy

Cort:BSA BSA Conjugated Cortisol Corticosterone conjugated to bovine serum albumin Membrane-impermeable glucocorticoid ligand

CRF6-33 Corticotropin-releasing factor binding protein inhibitor peptide Displaces CRF from CRFBP &  Free CRF C141H231N41O43S CRFBP  affinity than CRFR—binds CRF and regulates cascade

Clenbuterol β-adrenoceptor agonist Mimics NE Not just for COPD…

Propranolol β-adrenoceptor antagonist Inhibits the action of NE Nonselective Beta-blocker β1 & β2 Indirect α1 agonist ? Partial 5HT1B agonist ?

1st Background Paper

Endocannabinoids in the rat basolateral amygdala enhance memory consolidation and enable glucocorticoid modulation of memory Campolongo, P., Roozendaal, B., Trezza, V., Hauer, D., Schelling, G., McGaugh, J.L., & Cuomo, V.

Memory & Learning Preface

Memory & Learning 10 Second Tom Learning w/o memory would leave us reflex driven What if 10s-Tom encountered Bear-Man1, Bear, or Bear-Man2?

eCB Memory & Learning CB1R expression: Hippocampus, PFC, & Amygdala Suggests (regulatory) role in memory & learning Amygdaloid Complex eCB facilitate memory consolidation & fear learning + extinction of aversive memories BLA implicated in fear memory

Synaptic Transmission Neuronal firing Modulate mood and emotion BLA Complex (La, B, & AB) mediates stress hormone effects on memory formation of emotionally arousing experiences CB1R Highly Expressed GABA Interneurons Modulate Synaptic Transmission Neuronal firing Modulate mood and emotion

BLA Complex (La, B, & AB) mediates stress hormone effects on memory formation of emotionally arousing experiences …but is the BLA critical for mediating eCB effects on memory consolidation or extinction???

Glucocorticoids, HPA axis, and the BLA Complex…oh my eCB may mediate central action of GC GC admin  rapid eCB production and release (t= ≤10 min) And we all know, GC enhance long-term consolidation of emotionally arousing experiences

Campolongo et al. Study

Purpose “[Investigates] whether the cannabinoid system in the BLA influences memory consolidation of emotionally arousing experiences and whether CB1 activity plays an important role in mediating glucocorticoid effects on memory enhancement.”

Experiment 1: Will WIN55, 212-2 Infused into BLA Modulate Memory Consolidation? 5, 10, & 50 (ng/ 0.2 μl) of WIN55, 212-2 to BLA immediately after aversively motivated inhibitory avoidance training. Retention 48 h after training trial

Effects of WIN55,212-2 on retention of an inhibitory avoidance response. Effects of WIN55,212-2 on retention of an inhibitory avoidance response. Step-through latencies (mean and SEM) on a 48-h retention test. (A) Immediate posttraining intra-BLA infusions of the cannabinoid agonist WIN55,212-2 (WIN; 5, 10, 50 ng per 0.2 μL) enhanced memory consolidation. *, P < 0.05 vs. vehicle (Veh; n = 10–11 per group). (B) Delayed infusions of WIN55,212-2 (WIN; 50 ng per 0.2 μL) administered into the BLA 3 h after training did not enhance memory consolidation (n = 11–12 rats per group). (C) Immediate posttraining infusions of WIN55,212-2 (WIN 50 ng per 0.2 μL) into the CeA did not enhance memory consolidation (n = 7 rats per group). Patrizia Campolongo et al. PNAS 2009;106:4888-4893 ©2009 by National Academy of Sciences

Experiment 2: Will Blockade of CB1R (AM251) in BLA Impair Memory Consolidation? Effect of immediate post-training intra-BLA infusion of CB1R antagonist AM251 to determine whether or not eCB play role in memory consolidation Other groups received AM251 or WIN55, 212-2 3h after training infused into either the BLA or Ce

Effects of AM251 on retention of an inhibitory avoidance response. Effects of AM251 on retention of an inhibitory avoidance response. Step-through latencies (mean and SEM) on a 48-h retention test. (A) Immediate posttraining intra-BLA infusions of the cannabinoid antagonist AM251 (0.07, 0.14, 0.28 ng per 0.2 μL) impaired memory consolidation. *, P < 0.05 vs. vehicle (Veh; n = 9–11 per group). (B) Delayed infusions of AM251 (0.28 ng per 0.2 μl) administered into the BLA 3 h after training did not impair memory consolidation (n = 10–11 rats per group). (C) Immediate posttraining infusions of AM251 (0.28 ng per 0.2 μL) into the CeA did not impair memory consolidation (n = 5–6 rats per group). Patrizia Campolongo et al. PNAS 2009;106:4888-4893 ©2009 by National Academy of Sciences

Experiment 3: Are Memory Enhancing Effects of WIN55, 212-2 Due To Activation of CB1R Is WIN55, 212-2 activating the eCB system and providing a causal link in enhanced memory consolidation? Test by blocking CB1R (AM251; 0.14ng)

Effects of intra-BLA infusions of WIN55,212-2, either alone or together with AM251, on an inhibitory avoidance response. Effects of intra-BLA infusions of WIN55,212-2, either alone or together with AM251, on an inhibitory avoidance response. Step-through latencies (mean and SEM) on a 48-h retention test. Immediate posttraining infusions of AM251 (0.14 ng per 0.2 μL) blocked the memory-enhancing effects of concurrently administered WIN55,212-2 (WIN; 50 ng) into the BLA. **, P < 0.01 compared with the corresponding vehicle (Veh) group; ♦, P < 0.05 compared with the corresponding AM251 group (n = 14–15 per group). Patrizia Campolongo et al. PNAS 2009;106:4888-4893 ©2009 by National Academy of Sciences

Experiment 4: Is CB1R Activity in BLA Required for Enabling the Memory-Enhancing Effects Induced by Systemically Administered Corticosterone? If the GC system alone enhances memory Corticosterone (3mg/kg; sc) X AM251

Effects of intra-BLA administered AM251 on systemic corticosterone-induced enhancement of an inhibitory avoidance response. Effects of intra-BLA administered AM251 on systemic corticosterone-induced enhancement of an inhibitory avoidance response. Step-through latencies (mean and SEM) on a 48-h retention test. Immediate posttraining infusions of AM251 (0.14 ng per 0.2 μL) into the BLA blocked the memory enhancement induced by s.c. injections of corticosterone (CORT; 3.0 mg/kg; n = 10–12 per group). *, P < 0.05 compared with the corresponding vehicle (Veh) group; ♦, P < 0.05 compared with the corresponding CORT group. Patrizia Campolongo et al. PNAS 2009;106:4888-4893 ©2009 by National Academy of Sciences

Histology

(A) Diagram of the rat BLA, CeA, and adjacent structures (62). (A) Diagram of the rat BLA, CeA, and adjacent structures (62). (B) Representative photomicrograph (Microscope Nikon 801, original magnification ×20) of a needle track terminating in the BLA. (C) Diagrams of the rat brain sections (62) showing 40 infusion needle termination sites randomly selected from rats included in the final analyses. Only data from animals that had needle tracks terminating in the BLA and had no lesions in the surrounding BLA tissue were included in the analyses. Patrizia Campolongo et al. PNAS 2009;106:4888-4893 ©2009 by National Academy of Sciences

Beginning our Journey

Corticosterone (CORT) binds to a yet-uncharacterized membrane-bound glucocorticoid receptor (mbGR) that activates the Gs–cAMP/PKA pathway to induce endocannabinoid (eCB) synthesis. Corticosterone (CORT) binds to a yet-uncharacterized membrane-bound glucocorticoid receptor (mbGR) that activates the Gs–cAMP/PKA pathway to induce endocannabinoid (eCB) synthesis. Endocannabinoids are released into the synapse where they bind to CB1 receptors on GABAergic terminals inhibiting GABA release. This inhibition of GABA release disinhibits norepinephrine (NE) release and increases NE activation of postsynaptic β-adrenoreceptors, increasing the consolidation of emotionally-aversive memories. Matthew N. Hill, and Bruce S. McEwen PNAS 2009;106:4579-4580 ©2009 by National Academy of Sciences

Direction eCB and GC relationship is bidirectional  eCB X GABA release  stimulates noradrenergic activity  memory consolidation for adverse events

Endogenous cannabinoid signaling at inhibitory interneurons Younts, T.J. & Castillo, P.E. 2014 Review Article

Unde

Take-home = A lot is yet to be discovered about the role of GABAergic—eCB interactions

The Main Event

Endocannabinoid Signaling within the Basolateral Amygdala Integrates Multiple Stress Hormone Effects on Memory Consolidation Atsak, P., Hauer, D., Campolongo, P., Schelling, G., Fornari, R. V., and Roozendaal, B.

Experiment 1 …to what extent is eCB signaling implicated in regulating GC effects on memory consolidation? One-trial inhibitory avoidance task Immediately after GR agonist RU 28362 alone RU 28362 with CB1R antagonist AM251 Retention tested 48h later

Figure 1 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

Experiment 2 Do GC effects on the eCB system in regulating memory consolidation involve the activation of a corticosteroid receptor on the cell surface? Post-training admin to BLA Cort:BSA Cort:BSA + AM251

Figure 1 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

Experiment 3 How significant is the eCB pathway to memory-enhancement Direct eCB activation via WIN55, 212-2 (intra-BLA) GC path blocked via RU 38486 Significant results in favor of eCB (fig 1c)

Figure 1 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

SR141716A (Rimonabant) SR141716A (Rimonabant) Selective CB1R antagonist Inverse agonist-reverses adenylyl cyclase inhibition by WIN55, 212-2

Experiment 4-pCREB & CAMKII Is eCB activity required for mediating GC effects on CREB activation? SR141716 + Corticosterone administered immediately after inhibitory avoidance training pCREB assessed 1h post admin

Experiment 4-Retention 48h post training/corticosterone/Rimonabant admin memory assessment Corticosterone alone enhanced retention & co-admin of rimonabant removed effect

Figure 2 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

Propranolol β-adrenoceptor antagonist Inhibits the action of NE Nonselective Beta-blocker β1 & β2 Indirect α1 agonist ? Partial 5HT1B agonist ?

Experiment 5 Do the memory enhancing effects of eCB depend on noradrenergic activity in BLA? Inhibitory avoidance task Post-training infusion propranolol (.5 μg) + WIN55, 212-2 (10, 30, 100 ng) 48h retention test

Figure 3 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

Clenbuterol β-adrenoceptor agonist Mimics NE Not just for COPD…

RU 38486 (Mifepristone) Very Effective Antiglucocorticoid Cortisol antagonist (competitive at receptor) Anti-progesterone Well known due to abortion controversy

Experiment 6 & 7 Is eCB system primary route for GC alteration of NE sensitivity? 6 GR antagonist RU 38486 Clenbuterol WIN55, 212-2 (non-enhancing dose) 7 AM251 admin reproduces shift

Figure 3 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

CRF6-33 Corticotropin-releasing factor binding protein inhibitor peptide Displaces CRF from CRFBP &  Free CRF C141H231N41O43S CRFBP  affinity than CRFR—binds CRF and regulates cascade

Experiment 8 Does eCB serve a more general role GR blockade RU 38486 WIN55, 212-2 (non-enhancing dose) CRF6-33

Figure 4 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

Figure 5 Neuropsychopharmacology (2015) 40, 1485-1494; doi:10.1038/npp.2014.334

Take-Home & References

Take-Home CB1R signaling in BLA required for enabling memory-enhancing effect of GC cascade eCB primary route GC alter memory-enhancing effects of noradrenergic stimulation Antagonism of CB1R or GR= complete & comparable X of CRF-induced memory enhancement eCB system of BLA involved in neural plasticity mechanisms related to emotional learning and memory

Take-Home Cont. eCB system mediates BLA sensitivity to noradrenergic stimulation Downstream eCB recruitment essential eCB system mediates GC effects onto CRF

Putting it all together GC signaling  rapid  eCB mobilization  excitability of BLA pyramidal neurons  more sensitive to NE & CRF = Memory- enhancement of emotionally aversive learning BLA CB1R on GABA Interneurons. CB1R signaling (AEA) X GABA Interneurons…LTD of GABA Interneurons allows for  excitability of BLA neurons  enhanced memory consolidation

Suggested Articles & References Want to know more? Knapska, E., Radwanska, K., Werka, T. & Kaczmarek, L. (2007). “Functional Internal Complexity of Amygdala: Focus on Gene Activity Mapping After Behavioral Training and Drugs of Abuse.” Physiological Review. 87: 1113-73 McIntyre, C.K., Power, A.E., Roozendaal, B. & McGaugh, J.L. (2003). Role of the Basolateral Amygdala in Memory Consolidation. Annual New York Academy of Sciences. 985: 273-93 Paré, D. (2003). “Role of the basolateral amygdala in memory consolidation.” Progress in Neurobiology. 70: 409-20 Roozendaal, B., de Quervain, D.J., Ferry, B., Setlow, B. & McGaugh, J.L. (2001). Basolateral Amygdala—Nucleus Accumbens Interactions in Mediating Glucocorticoid Enhancement of Memory Consolidation. The Journal of Neuroscience. 21(7): 2518-25 Sah, P., Faber, E.S.L., Lopez De Armentia, M. & Power, J. (2003). “Amygdaloid Complex: Anatomy and Physiology.” Physiological Review. 83: 803-34