Role of Mu-Opioid Receptors in the Behavioral Effects of the Antidepressant Tianeptine Jaena Han Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2021 © 2021 Jaena Han All Rights Reserved Abstract Role of Mu-Opioid Receptors in the Behavioral Effects of the Antidepressant Tianeptine Jaena Han For over half a century, the monoamine hypothesis has been the dominant theoretical framework guiding depression research and drug development. This hypothesis posits that depression arises from a deficiency in the monoaminergic neurotransmitters serotonin, norepinephrine, and possibly dopamine, and that antidepressants function by increasing extracellular availability of these monoamines in the brain, especially at the synaptic level. It is clear however, that the monoamine hypothesis cannot fully explain either the pathophysiology of depression nor the mechanisms of antidepressant action. Tianeptine is an atypical antidepressant used in Europe to treat patients who respond poorly to selective serotonin reuptake inhibitors (SSRIs). The recent discovery that tianeptine is a mu opioid receptor (MOR) and delta opioid receptor (DOR) agonist has provided a potential avenue for expanding our understanding of antidepressant treatment beyond the monoamine hypothesis. This dissertation aims to understand the neural circuits underlying tianeptine’s antidepressant effects. We first characterized the acute and chronic effects of tianeptine on depressive-like and other opioid-related behaviors in mice, and used genetic and pharmacological models to test whether these behavioral effects are mediated by MOR and/or DOR. We found that acute tianeptine administration produced an antidepressant-like reduction in immobility time in the forced swim test, as well as classic opioid-like effects including analgesia, hypophagia, hyperactivity, and conditioned place preference. These behavioral responses to tianeptine are abolished in MOR knockout (KO) mice and in mice that have been pretreated with an MOR antagonist. By contrast, all responses to tianeptine remained intact in DOR KO mice. Remarkably, unlike other classic opiates such as morphine, chronic tianeptine treatment did not produce tolerance to tianeptine’s analgesic effect, nor naloxone-precipitated withdrawal. The acute behavioral effects of tianeptine (excluding analgesic effects, which were present at 15 minutes, but not 1 hour) were established to occur at 1 hour post-injection and to be largely absent by 3 hours post-injection. Chronically, tianeptine produced an antidepressant effect in corticosterone-treated mice, and prevented the development of restraint-stress-induced depression-like behavior, both in an MOR-dependent manner. Interestingly, tianeptine’s chronic antidepressant-like effects were evident in mice after as little as one week of treatment, rather than several weeks as might be expected for SSRIs. Using tissue-specific MOR knockouts, we further showed that MOR expression on GABAergic cells, specifically somatostatin-positive neurons, is necessary for the acute and chronic antidepressant-like responses to tianeptine. By contrast, tianeptine’s behavioral effects did not require the expression MORs on D1- and parvalbumin-expressing cells, nor the expression of ß-arrestin 2. These experiments also revealed a dissociation between the antidepressant-like phenotype and other opioid-like phenotypes resulting from acute tianeptine administration such as analgesia, conditioned place preference, and hyperlocomotion. Critically, we found that tianeptine’s mechanism of action is distinct from fluoxetine in three important aspects: 1) tianeptine requires MORs but not DORs for its chronic antidepressant-like effect, while fluoxetine is the opposite, 2) unlike fluoxetine, tianeptine does not promote hippocampal neurogenesis, and 3) tianeptine’s effects appear to persist even after serotonin depletion. Taken together, these results suggest a novel entry point for understanding what circuit dysregulations may occur in depression, as well as possible targets for the development of new classes of antidepressant drugs. Table of Contents List of Figures ................................................................................................................................ v Acknowledgments ...................................................................................................................... viii Dedication ..................................................................................................................................... xi Chapter 1: Introduction ............................................................................................................... 1 1.1 The Monoamine Hypothesis ..................................................................................................... 1 1.2 The Opioid System and Depression .......................................................................................... 2 1.2.1 History........................................................................................................................ 2 1.2.2 Endogenous Opioid System ....................................................................................... 4 1.2.3 Preclinical Evidence of Opioid Involvement in Depression ...................................... 7 1.2.4 Opioid Regulation of Mood in Humans..................................................................... 9 1.2.5 Clinical Use of Opioids as Antidepressants ............................................................. 12 1.3 Tianeptine ............................................................................................................................... 12 1.4 Cell Type and Brain Region Specificity ................................................................................. 16 1.4.1 GABA and Depression ............................................................................................ 16 1.4.2 Brain Regions of Interest ......................................................................................... 20 1.4.2.1 Hippocampus ............................................................................................ 20 1.4.2.2 Amygdala .................................................................................................. 23 1.4.2.3 Habenula ................................................................................................... 25 1.4.2.4 Ventral Tegmental Area ............................................................................ 26 1.4.2.5 Striatum ..................................................................................................... 27 1.4.2.6 Ventral Pallidum ....................................................................................... 28 i 1.4.2.7 Anterior Cingulate Cortex......................................................................... 29 Chapter 2: Materials and Methods ........................................................................................... 32 2.1 Mice ........................................................................................................................................ 32 2.2 Drugs ....................................................................................................................................... 33 2.3 BrdU and DCX Immunohistochemistry ................................................................................. 34 2.4 Chronic Varied Odor Restraint Stress..................................................................................... 35 2.5 Behavioral Testing .................................................................................................................. 36 2.5.1 Open Field Test ........................................................................................................ 36 2.5.2 Home Cage Feeding Test ........................................................................................ 36 2.5.3 Novelty Suppressed Feeding Test ............................................................................ 37 2.5.4 Forced Swim Test .................................................................................................... 37 2.5.5 Conditioned Place Preference Test .......................................................................... 38 2.5.6 Hot Plate Test ........................................................................................................... 38 2.5.7 Withdrawal Test ....................................................................................................... 39 2.5.8 Sucrose Preference Test ........................................................................................... 39 2.6 RNAscope ............................................................................................................................... 40 2.7 [3H]DAMGO Autoradiography .............................................................................................. 41 2.8 Cannulations ........................................................................................................................... 41 Chapter 3: Results....................................................................................................................... 43 3.1 Opioid Mechanisms for Tianeptine’s Behavioral Effects ....................................................... 43 3.1.1 The Acute Behavioral Effects of Tianeptine Require MORs, but not DORs