Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 1-1-2019 The Medial Prefrontal Cortex To Dorsal Raphe Circuit In The Antidepressant Action Of Ketamine Alexandra Thomas Follow this and additional works at: https://elischolar.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Thomas, Alexandra, "The Medial Prefrontal Cortex To Dorsal Raphe Circuit In The Antidepressant Action Of Ketamine" (2019). Yale Medicine Thesis Digital Library.edu/ymtdl/3538 This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact elischolar@yale.
The Medial Prefrontal Cortex to Dorsal Raphe Circuit in the Antidepressant Action of Ketamine A Thesis Submitted to the Yale University School of Medicine in Partial Fulfilment of the Requirements for the Degree of Doctor of Medicine By Alexandra Moran Thomas Dissertation Director: Ronald S. May 2019 ABSTRACT Major depressive disorder is a common and debilitating illness for which there is a notable lack of efficient, effective treatment. While currently available pharmacotherapies typically take eight weeks to take effect and fail to do so at all for about a third of patients, the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has shown a much more favorable effectiveness profile, including improvements in symptoms within hours of administration, even for many patients who do not respond to typical antidepressants. Ketamine, as a modulator of glutamate signaling in the brain, has a distinct mechanism of action from the serotonin and norepinephrine modulators that are currently the mainstay of depression treatment.
This dissertation seeks to contribute to the understanding of this unique mechanism, and particularly the brain circuits affected. Rodent studies have shown that ketamine induces a burst of glutamatergic activity in the medial prefrontal cortex (mPFC), which is necessary to produce its antidepressant effect. The downstream targets of this glutamatergic activity that are relevant to the ketamine antidepressant effect are unclear, but recent research has suggested a role for the dorsal raphe nucleus (DRN), which contains most of the brain’s serotonin-producing cells. In this thesis, I first provide a synthesis of the literature on the mechanism of ketamine’s antidepressant effect and the neural circuits that might underlie it.
I then investigate the projection from the mPFC to the DRN using optogenetic stimulation of mPFC-originating axon terminals in the DRN, finding that activation of this pathway produces an antidepressant effect on the forced-swim test (FST), which measures “behavioral despair” induced by a stressful environment, but not on other measures of depression-like behavior. I also perform immunohistochemical studies of the DRN, which indicate that both serotonergic and non-serotonergic cells are ii activated by this stimulation. I then find additional support for this behavioral selectivity using a pharmacological approach: by inhibiting serotonin release during ketamine administration, I find that DRN activity is needed for the antidepressant effect of ketamine on the FST but not on other behavioral tests. Finally, I interrogate the projection from the mPFC to the nucleus accumbens using the same optogenetic approach as before.
These experiments show that activation of the mPFC-to-DRN pathway produces an antidepressant effect on a particular subset of depression-like behavior and supports a role for serotonin signaling in the behavior measured by the FST. iii © Alexandra Moran Thomas All rights reserved. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS. vi LIST OF FIGURES.
viii LIST OF ABBREVIATIONS. ix CHAPTER 1: The neural and molecular mechanisms of the antidepressant effect of ketamine. Brain pathology in depression. Mechanism of action of currently available antidepressants.
Mechanism of action of ketamine. Neural circuits involved in the function of rapid-acting antidepressants. 17 CHAPTER 2: Optogenetic stimulation of mPFC-originating axon terminals in the dorsal raphe nucleus produces an antidepressant effect. 36 CHAPTER 3: Inhibition of DRN serotonin release inhibits the antidepressant effect of ketamine.
52 CHAPTER 4: Optogenetic stimulation of infralimbic-originating terminals in the nucleus accumbens does not produce an antidepressant effect. 62 CHAPTER 5: Conclusions and future directions. 70 v ACKNOWLEDGEMENTS I have been fortunate to have many mentors, close friends, and family members who have supported me on my journey through graduate school. First among them is my advisor, Ron Duman, who has helped me develop and execute this dissertation at every step, and whose immense patience and kindness along the way has modeled for me how a good mentor should be.
Yale as a whole has provided a wonderful environment in which to develop as a scientist and physician, and particularly the psychiatry department. I have greatly benefited from the input and expertise of my thesis committee, Ralph DiLeone, Marina Picciotto, and Alex Kwan; and from the depth and breadth of knowledge of my oral exam readers, John Krystal, Jane Taylor, and Angelique Bordey. The leadership and staff of the MD/PhD program has provided indispensable guidance on this long road, most notably Barbara Kazmierczak, Jim Jamieson, Cheryl Defilippo, and Sue Sansone; and the leadership of the MD program and Interdepartmental Neuroscience Program have been patient and helpful in navigating the transition from medical school to grad school and back again, especially Nancy Angoff, Michael O’Brien, Charlie Greer, Carol Russo, and Donna Carranzo. I am also grateful to the National Institute of Mental Health for the F30 grant that financed a portion of this work.
My development as a scientist has been influenced by many collaborators and colleagues. George Aghajanian and Rong-Jian Liu, as well as Ben Land and Rich Trinko of the DiLeone lab, were wonderful vi collaborators when I started my project. Manabu Fuchikami taught me nearly every technique I used in this project with care and diligence. I have learned from and gotten vital assistance from many members of the Duman lab, which made it a great place to go to work everyday: particular thanks to Kenichi Fukumoto, Brendan Hare, and Taro Kato, who directly contributed to some of the experiments in this dissertation; as well as Mouna Banasr, Astrid Becker, Cathy Duman, Jason Dwyer, Tina Franklin, Danielle Gerhard, Matthew Girgenti, Sri Ghosal, Ashley Lepack, Xiao-Yuan Li, Georgia Miller, Rose Terwiliger, Manmeet Virdee, and Eric Wohleb.
I have been blessed with an immensely supportive family, who have always trusted that I would make it to the finish line, even when I doubted it myself. I remember especially those who passed away during these years and whose love and encouragement I still carry with me: my uncle Monte Sliger, stepmom Sandy Thomas, grandmother Bertine Sliger, and especially my dad, George Thomas. I continue to be uplifted by my mother Janice Sliger, brother Luke Thomas and his wife Joanie, and the very best family-in- law: Joan Russo, Donald Burset, Stephanie Burset, and Charlie King. Finally, the best decision I made during grad school was to marry Christian Burset, who has picked me up and pulled me through even the toughest parts of the last five years with his love and patience.
I am especially thankful that our most ambitious collaborative project, our son Dominic, was completed in perfect form, needing not a single revision, almost simultaneously with this thesis. vii LIST OF FIGURES Figure 1.1 Mechanisms of synapse loss in depression ………………………….2 Signaling pathways involved in the response to rapid-acting antidepressants ……………………………………………………….1 Distribution of GFP-labeled ChR2 throughout the brain…………26 Figure 2.2 DRN axon-terminal stimulation produces an antidepressant effect on the FST ………………………………………………………………28 Figure 2.3 DRN axon-terminal stimulation had no effect on the NSFT, FUST, or 7-day post-stimulation FST ……………………………………….4 Cannula placement and viral expression in the mPFC and DRN………………………………………………………………………33 Figure 2.5 c-Fos activation is increased in the DRN but not in the ilPFC in response to DRN axon-terminal stimulation ………………………35 Figure 2.6 Stimulation induces c-Fos expression in non-TPH2-expressing cells ……………………………………………………………………….1 8OH-DPAT blocks the antidepressant effect of ketamine on the FST ……………………………………………………………………….2 Ketamine increases swimming, not climbing, on the FST ……….3 8OH-DPAT does not interfere with the effect of ketamine on the NSFT …………………………………………………………………….4 Depression-like behavior is higher in control groups when drugs are administered by a male experimenter than by a female experimenter ……………………………………………………………52 Figure 4.1 ChR2 expression in the nucleus accumbens after viral injection into the mPFC ………………………………………………………….2 Stimulation of mPFC-originating NAC axon terminals does not produce an antidepressant effect ……………………………………62 viii LIST OF ABBREVIATIONS 8OH-DPAT, 8-hydroxy-n,n-dipropylaminotetralin AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BDNF, brain-derived neurotrophic factor DBS, deep brain stimulation DRN, dorsal raphe nucleus DSM, Diagnostic and Statistical Manual of Mental Disorders eEf2K, eukaryotic elongation factor-2 kinase GABAR, l-aminobutyric acid receptor GSK, glycogen synthase kinase HNK, hydroxynorketamine; mAchR, muscarinic acetylcholine receptor LHB, lateral habenula MDD, Major Depressive Disorder mGluR, metabotropic glutamate receptor mPFC, medial prefrontal cortex MSN, medium spiny neuron mTORC1, mammalian target of rapamycin complex 1 NAC, nucleus accumbens NMDAR, N-methyl-D-aspartate receptor SNRI, selective norepinephrine-reuptake inhibitors SSRI, selective serotonin-reuptake inhibitors TrkB, tropomysin receptor kinase B VDCC, voltage-gated calcium channel ix CHAPTER 1: The neural and molecular mechanisms of the antidepressant effect of ketamine This chapter contains a modified version of material that appeared in the author’s publication: Alexandra Thomas & Ronald Duman. Novel rapid-acting antidepressants: molecular and cellular signaling mechanisms. Brain pathology in depression Major Depressive Disorder (MDD) affects an estimated 5% of the global population at any given time, and it is the leading cause of disability worldwide (Ferrari et al.
In addition to the high toll of personal suffering it exacts, depression drains over $50 billion per year from the US economy alone in lost work productivity and medical costs (P. Wang, Simon, & Kessler, 2003). Despite the widespread need for effective treatment, currently available antidepressants often take 6-8 weeks to take effect, and only one-third of patients respond to their first trial on any given drug. One-third of depressed patients never get relief from typical antidepressants, even after multiple trials (Gaynes et al.
Perhaps the biggest obstacle to the development of better medications has been the lack of understanding of the molecular mechanisms that underlie antidepressant 1 effects. But several innovations in the past two decades have begun to reveal answers to this puzzle. First, the drug ketamine, which had long been used in high doses as an anesthetic, was found to have a rapid antidepressant effect in low, sub- anesthetic doses (Berman et al. It relieves symptoms within hours, even in many patients who have not responded to typical antidepressants.
Notably, it acts primarily through a different neurotransmitter, glutamate, than do all currently available antidepressants, which primarily affect the transmission of serotonin and/or norepinephrine. The discovery of the rapid antidepressant action of ketamine and a handful of other drugs has spurred a rethinking of fundamental questions about how antidepressants work, and especially about the role of glutamatergic signaling in antidepressant mechanisms. To aid in this reassessment, new tools in neuroscience have shed light on the intracellular signals and neuronal networks that underlie the effects of rapid-acting agents. In order to understand how antidepressants relieve the symptoms of depression, it is helpful to understand how the brains of depressed people differ from those who are not depressed.
This question has been difficult to study due to the wide diversity of clinical presentations that meet criteria for MDD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM) (American Psychiatric Association, 2013). Derangements in a variety of biological processes have been imputed to lead to depression, including inflammation (Iwata, Ota, & Duman, 2012), metabolism (Abdallah et al., 2 2014), and stress-response pathways (Duman, 2014), and it is possible that these mechanisms interact in different ways in different subgroups of patients with MDD. But despite the probable heterogeneity of MDD mechanisms, there seem to be several common features of the depressed state that serve as hallmarks of the depressed brain. Human neuroimaging studies have consistently demonstrated reduced brain volume in key areas associated with mood regulation, including the frontal cortex, cingulate cortex, and hippocampus (Arnone, McIntosh, Ebmeier, Munafò, & Anderson, 2012).