Nanxin Li, et al. Science 329, 959 (2010) R1 黃泰翰 V.S. 洪成志2010.09.21 mTOR-Dependent Synapse Formation Underlies the Rapid Antidepressant Effects of NMDA Antagonists Nanxin Li, et al. Science 329, 959 (2010) R1 黃泰翰 V.S. 洪成志2010.09.21

Ketamine Antagonist of Glutamate NMDA receptor analgesia, anesthesia, sedation Psychological effects similar to phencyclidine (PCP) Dissociative state Hallucination Schizophrenia like symptoms

Behavior model of depression Despair Forced Swim Test (FST) Learned hopelessness(LH) Anxiety Novelty-Suppressed Feeding Test (NSFT)

Forced Swim Test (FST) 1st swim for 15 mins - 24 hrs later drug treatment - 24hrs later 2nd swim for 5 mins Duration of Immobility

Learned-Helplessness (LH) Inescapable footshock (IES) 60 footshocks, duration: 15 s, intershock interval: 60 s -24hrs- drug treatment -24hrs- Active avoidance testing 30 trials of escapable footshock duration: 35s Number of escape failure recorded

Novelty-Suppressed Feeding Test (NSFT) food-deprived for 24hrs placed in an open field 76.5 cm X 76.5 cm X 40 cm food in the center latency to feed

A Randomized Trial of an NMDA Antagonist in Treatment-Resistant Major Depression Placebo controlled, double-blinded trial 18 subjects with treatment refractory MDD A single low dose of Ketamine (0.5mg/Kg) C. A. Zarate Jr. et al., Arch. Gen. Psychiatry 63, 856 (2006).

Ketamine Signaling Pathway? Anti-depression effects

mTOR Signaling Pathway Mammalian target of rapamycin Rapamycin = Sirolimus Immunosuppresant Ubiquitous Protein kinase translation regulation S6K 4E-BP

Translation Regulation Protein Synthesis Translation from mRNA Initiation, elongation, termination Regulation eIF (eukaryotic initiation factor) eEF Ribosome

S6K 4E-BP S6K phosphorylates ribosomal protein S6, component of the small, 40S ribosomal subunit. 4E-BP eIF4E binding protein

Methods Ketamine Measurement Synaptoneurosome in Prefrontal Cortex intraperitoneal injection Measurement Synaptoneurosome in Prefrontal Cortex

Ketamine (ip) induce transient, dose-dependent mTOR signaling in synaptoneurosome of PFC 10 mg/Kg 1 hr

ECS Electroconvulsive seizure, imipramine, or fluoxetine did not significantly influence mTOR signaling Chronic (21D) Acute (1hr)

Ketamine 4E-BP mTOR S6K

mTOR and growth factor signaling pathway MAPK/ERK cascade Extracellular signal–regulated kinase Mitogen-activated protein kinase MAP3K -> MAP2K -> MAPK Akt/PKB pathway PI3K -> PDK -> Akt C. A. Hoeffer, E. Klann, Trends Neurosci. 33, 67 (2010)

Ketamine transiently and dose-dependently increases pERK & pAkt 1 hr 10 mg/Kg Ketamine transiently and dose-dependently increases pERK & pAkt

Pretreatment (30 mins before Ketamine): U0126 (20 nmol, ICV): inhibitors of ERK LY294002 (20 nmol, ICV): inhibitor of PI-3k/Akt

Ketamine ERK, Akt mTOR 4E-BP S6K

Glutamate receptor Ionotropic Glutamate receptor NMDA receptor AMPA receptor Kainate receptor Metabotropic Glutamate receptor

Antidepressant actions of ketamine & AMPA receptor Glutamate AMPA receptor NBQX a selective AMPA receptor inhibitor it attenuate the reduction in immobility time induced by ketamine Pretreatment (10 min before Ketamine) with NBQX (10 mg/kg, ip) S. Maeng et al., Biol. Psychiatry 63, 349 (2008).

NBQX blocked ketamine activation of mTOR signaling and upstream ERK & Akt

Ketamine AMPA ERK, Akt mTOR 4E-BP S6K

mTOR and synaptic protein synthesis Presynaptic protein: Synapsin I Postsynaptic proteins: PSD95, GluR1 Arc: activity-regulated cytoskeletal-associated protein C. A. Hoeffer, E. Klann, Trends Neurosci. 33, 67 (2010).

Ketamine induces intermediate (1-2 hr) but transient increase of Arc Ketamine induces delayed (2-72 hr) increase of synaptic proteins Pretreatment (30 min) with a selective mTOR inhibitor, rapamycin (0.2 nmol, ICV) block the effect

Synape Formation ?

Dendritic spine formation Y. Yoshihara, M. De Roo, D. Muller, Curr. Opin. Neurobiol. 19, 146 (2009).

Spine density analysis Layer V Pyramidal Cell in PFC Tips of tuft branches approaching the pial membrane Proximal tuft dendrites just distal to the bifurcation Density Head diameter Length

ketamine increased spine density in distal and proximal segments of the apical tuft

Head diameter Spine length

Excitatory postsynaptic current (EPSC) Apical dendrites of Layer V pyramidal cells in mPFC 5-HT and Hypocretin Increase EPSCs Restraint stress 5-HT and hypocretin-induced EPSCs decreased Apical tuft dendritic branch length and spine density decreased R. J. Liu, G. K. Aghajanian, Proc. Natl. Acad. Sci. U.S.A. 105, 359 (2008).

EPSC measurement

Rapamycin was infused (0 Rapamycin was infused (0.2 nmol, ICV) 30 min before ketamine (10 mg/kg, ip) Rapid behavioral actions of ketamine require mTOR signaling

Infusion of rapamycin (0 Infusion of rapamycin (0.01 nmol) into the mPFC blocked the antidepressant actions of ketamine (10 mg/kg, ip) in the FST and NSFT

Pretreatment with inhibitors of ERK (U0126, 20 nmol, ICV) or PI3 kinase / Akt (LY294002, 20 nmol, ICV) block of ketamine effects in FST and NSFT

Learned helpless with Inescapable Shock (IES) Synapsin I, PSD95 and GluR1 Ketamine given 24hr after IES Tissue collected 24 hr after ketamine Single dose of ketamine reverse this effect Pretreatment with rapamycin (ICV, 30 min before) block ketamine effect

Dose-dependent ketamine antidepressant action FST Low dose 10 mg/kg high anesthetic dose 80 mg/kg Similar with dose of ERK, Akt, and mTOR induction

Ketamine for Depression ? Risk of abuse and adverse effects Ro 25-6981 selective NMDA receptor subunit 2B (NR2B) antagonists NMDA Receptor = 2 NR1 + 2 NR2 NR1: 8 subtype NR2: 4 subtype NR2A, NR2B, NR2C, NR2D

Ro 25-6981 produced rapid (24 hr before), dose-dependent antidepressant action in the FST

Pretreatment with rapamycin (0 Pretreatment with rapamycin (0.2 nmol, ICV) abolished the actions of Ro 25-6981 (10 mg/kg, ip) in FST and NSFT

mTOR signaling ERK and Akt signaling synaptic proteins 4E-BP1, p70S6K, mTOR ERK and Akt signaling synaptic proteins Arc, PSD95, GluR1, synapsin I

Conclusion Rapid antidepressant actions of ketamine Fast activation of mTOR signaling in PFC Rapid and sustained elevation of synapse associated proteins and spine number Elevated 5-HT neurotransmission

Discussion Rapid antidepressant therapy mTOR signal pathway

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