Natalie Hesselgrave Education University of Maryland School Of

Psilocybin as a Rapid Acting Antidepressant: Are Hallucinations Necessary?

Item Type dissertation

Authors Hesselgrave, Natalie

Publication Date 2020

Abstract Major depressive disorder (MDD) is a leading cause of mental illness world-wide. Economists estimate MDD to be one of the leading causes of disability worldwide. The cost is not just due to treatment, but comorbidities, lost work and suicide, too. Ho...

Keywords antidepressants; depression; psychedelics; Anhedonia; Antidepressive Agents; Depressive Disorder, Major; Hallucinogens; Psilocybin; Reward

Download date 02/10/2021 13:04:27

Link to Item http://hdl.handle.net/10713/14474 Natalie Hesselgrave [email protected] Degree and date to be conferred: Ph.D., 2020 University of Maryland School of Medicine Program in Neuroscience, Graduate Program in Life Sciences

Education University of Maryland School of Medicine MSTP Baltimore, MD Doctorate of Philosophy December 2020 Doctor of Medicine expected May 2022

Postbaccalaureate Premedical Program, School of General Studies, Columbia University New York, NY Certificate in Premedical Sciences; GPA: 3.94 October 2012

Teachers College, Columbia University New York, NY Master of Education, Psychological Counseling May 2008 Master of Arts, Psychological Counseling; GPA: 3.92 February 2008

Cornell University, College of Human Ecology Ithaca, NY Bachelor of Science, Human Development; GPA: 3.35 May 2003 Research Experience

Graduate Research Assistant; Program in Neuroscience 2014 – current University of Maryland School of Medicine; Baltimore, MD Rotation Projects ◼ Dissertation Project: Characterizing Psilocybin as a rapid acting antidepressant in rodents: Are hallucinations necessary? ◼ MRI imaging in the rodent model of chronic pain: structural and functional connectivity. 2015, PI: Dr. David Seminowicz ◼ L-655,708 induced electrophysiological changes in the hippocampus and nucleus accumbens of the rat brain. 2014, PI: Dr. Scott Thompson

Research Assistant, Technician-B 2008-2012 Molecular Imaging and Neuropathology Division, Columbia University; New York, NY ◼ Worked under principal investigator Ramin Parsey, M.D., Ph.D. on several NIH funded grants investigating neuroreceptors in Major Depressive Disorder using brain imaging techniques. ◼ Compiled and clean clinical and imaging data for data analysis and presentation purposes using Microsoft Excel and Matlab. ◼ Contributed to manuscript development, writing and preparation. ◼ Conducted literature searches and reviews utilizing various Internet databases. ◼ Communicated with researchers and principal investigators to create, update and maintain study specific spreadsheets.

◼ Performed initial statistical analyses using SPSS. ◼ Implemented image processing stream, review and approval of each step for every MRI and PET scan collected.

Student Research Assistant 2006-2008 Social-Cognitive-Affective-Neuroscience Lab, Columbia University; New York, NY ◼ Worked with Drs. Kevin Ochsner and Jamil Zaki to study the basis of Empathic Accuracy. ◼ Designed an emotional word association task using E-prime. ◼ Participated in the data collection process by scheduling and running volunteers according to experimental protocol. ◼ Reviewed and contributed to the development of future project designs. ◼ Prepared protocols for IRB using RASCAL interface.

Professional Experience Student Rehabilitation Counselor, Mental Health Counseling Internship 2007-2008 Personality Disorders Unit, New York Presbyterian Hospital; White Plains, NY ◼ Conducted psychosocial assessments with new patients within the first 72 hours of admission date. ◼ Worked with a multidisciplinary team to develop treatment plans for and track progress of patients. ◼ Lead 3-4 psychoeducational groups per week addressing the current needs of the diverse patient population. ◼ Met with patients when indicated for bi-weekly individual counseling sessions.

Mental Health Counselor Fall 2007 Center for Educational and Psychological Services, Teachers College, Columbia University; New York, NY ◼ Held individual counseling sessions with client focusing on residual emotional problems related to vocational choice ◼ Involved client in the counseling process to establish rapport and develop a therapeutic relationship, counseling goals and appropriate intervention strategies. ◼ Completed progress notes, intake and termination reports to assist subsequent counselor. ◼ Engaged in supervision and peer feedback to increase insight and improve counseling skills and process.

Community Service and Training Specialist 2005-2006 Young Adult Institute (YAI)/National Institute for People with Disabilities; New York, NY ◼ Implemented therapeutic services for 4-5 adults with developmental disabilities using behavioral therapy techniques and completed service notes daily. ◼ Completed Applied Behavioral Analysis training. ◼ Coordinated and wrote service plans for participants in conjunction with Medicaid service providers and residential counselors.

◼ Represented the Manhattan Day Habilitation program for YAI/NIPD Network’s Central Park Challenge and successfully raised over $4500. Publications

N.Hesselgrave, T.A. Troppoli, A.Wulff, A. Cole, S.M. Thompson. Harnessing Psilocybin: antidepressant behavioral and synaptic actions of psilocybin are independent of 5-HT2R activation in mice. Nature neuroscience. Submitted, 2020.

S.R. Krimmel, H. Qadir, N. Hesselgrave, M.G. White, D.H. Reser, B.N. Mathur, D.A. Seminowicz. Resting state functional connectivity of the rat claustrum. Frontiers in Neuroanatomy. 2019.

M.S. Milak, S. Pantazatos, F. Zanderigo, C. DeLorenzo, N. Hesselgrave, R.T. Ogden, M.A. Oquendo, S.T. Mulhern, A.K. Burke, J.M. Miller, R.V. Parsey, J.J. Mann. Higher 5-HT1A Autoreceptor Binding as an Endophenotype for Major Depressive Disorder Identified in High Risk Offspring. A Pilot Study. Psychiatry Research: Neuroimaging. 2018, 276, 15-23.

J.M. Miller, N. Hesselgrave, R.T. Ogden, M.A. Oquendo, J.J. Mann, R.V. Parsey. Brain Serotonin 1A Receptor Binding as a Predictor of Treatment Outcome in Major Depressive Disorder. Biological Psychiatry; 2013, 74(10), 760-767.

M. Lan, N. Hesselgrave, A. Ciarleglio, R.T. Ogden, G. Sullivan, J.J. Mann, R.V. Parsey. Highter 5-HT1A Receptor Binding Potential in Bipolar Disorder Depression is Associated with Treatment Remission: A Naturalistic Treatment Pilot PET Study. Synapse, 2013; 67(11), 773-778.

J.M. Miller, N. Hesselgrave, R.T. Ogden, G. Sullivan, M.A. Oquendo, J.J. Mann, R.V. Parsey. Positron Emission Tomography Quantification of Serotonin Transporter in Suicide Attempters with Major Depressive Disorder. Biological Psychiatry; 2013, 74(4), 287-295.

N. Hesselgrave, & R.V. Parsey. Imaging the serotonin 1A receptor using [11C]WAY100635 in healthy controls and major depression. Philosophical Transactions of the Royal Society B: Biological Sciences, 2013; 368(1615).

R.V. Parsey, R.T. Ogden, J.M. Miller, A. Tin, N. Hesselgrave, E. Goldstein, et al. Higher Serotonin 1A Binding in a Second Major Depression Cohort: Modeling and Reference Region. Biological Psychiatry, 2010; 68(2), 170-178. Abstracts and Presentations

Psilocybin as a rapid acting antidepressant: are hallucinations necessary? Winter conference on brain research. January 2020, Big Sky, MT. Advancements on psychedelic neuroscience panelist, Chair: David Martin.

Uncovering the role of hippocampal projections to nucleus accumbens in depression and the antidepressant response. Student Research Seminar Series; University of Maryland, Baltimore. November 2018.

L-655,708 induced electrophysiological changes in the hippocampus and nucleus accumbens of the rat brain. Medical student research day. University of Maryland, School of Medicine. September 2014.

N. Hesselgrave & S. Thompson. Hippocampal, nucleus accumbens and prefrontal cortical activity in the awake behaving rodent. Program in Neuroscience Retreat; University of Maryland, Baltimore. June 2018. (Abs.)

D.A. Seminowicz, S. Krimmel, A. Ramsey, J. Teixeira Da Silva, N. Hesselgrave, M.G. White, M. Panicker, D.H. Reser, B.N. Mathur. Functional connectivity of the claustrum in humans and rats in 7T MRI. Society for Neuroscience, November 2016. (Abs)

N. Hesselgrave and S. Thompson. Oscillatory changes in the Hippocampus and Nucleus Accumbens following partial inverse agonism of α5-GABA receptor. Program in Neuroscience Retreat; University of Maryland, Baltimore. June 2016. (Abs)

S. Thompson, M. Kvarta, J. Fischell, N. Hesselgrave, T. LeGates, A. Van Dyke. Breaking sad: Stress induced dysfunction of excitatory synapses in the genesis and treatment of anhedonia. The Neurobiology of stress workshop, April 2016. (Abs)

M. Kvarta, J. Fischell, N. Hesselgrave, T. LeGates, A. Van Dyke, S. Thompson. From synapses to depression and back: Plasticity of excitatory drive in cortico-mesolimbic synapses. Society for Neuroscience, October 2015. (Abs).

N. Hesselgrave & S. Thompson. L-655,708 induced electrophysiological changes in the hippocampus and nucleus accumbens of the Rat Brain. MSTP Summer research symposium. August 2014. (Abs.)

J.M. Miller, N. Hesselgrave, R.T. Ogden, M.A. Oquendo, J.J. Mann, R.V. Parsey. Elevated Serotonin 1A Receptor Binding in Raphe Nuclei is Associated with Remission to the Antidepressant Escitalopram. Society of Biological Psychiatry, 67th Annual Meeting; May 3-5, 2012. Poster 616/Session 055

J. Miller, N. Hesselgrave, R.T. Ogden, M.A. Oquendo, J.J. Mann, R.V. Parsey. Serotonin 1A binding as Assessed by PET and Prediction of Antidepressant Treatment Outcome. Society of Biological Psychiatry, 66th Annual Meeting; May 12-14, 2011. 54/1000. (abs).

M. Milak, N. Berg-Hesselgrave, C. Delorenzo, R.T. Ogden, J.J. Mann, R.V. Parsey. A Neurochemical Phenotype for Major Depressive Disorder. Society of Biological Psychiatry, 66th Annual Meeting; May 12-14, 2011. 54/1010. (abs).

G. Sullivan, M.A. Oquendo, N. Berg-Hesselgrave, J.J. Mann, R.V. Parsey. Quantitative [C-11]DASB PET Study of Serotonin Transporter (5-HTT) Binding in Healthy Volunteers: Effects of Sex, Age, and 5-HTTLPR Genotype. Society of Biological Psychiatry, 65th Annual Meeting; May 20-22, 2010. 125/776. (abs). Honors and Awards The Glaser Prize in Imaging 2019

Medical Student Research Day – First place oral presentation September 2014 L-655,708 induced electrophysiological changes in the Hippocampus and Nucleus Accumbens of the rat brain

Structure and Development Honors Project Summer 2014 MR Imaging of Cranial Nerves Mentor: Dr. Theresa Kouo

Dean’s List, Columbia University Fall 2011

Postbaccalaureate Premedical Program Scholarship 2011-2012 Extracurriculars and Committees Medical Student Interviewer 2013-2014

Co-President of the Diagnostic, Therapeutic and Interventional Radiology Student Interest Group 2013-2014

MSTP Admissions Committee 2014-2016 Student member of the admissions committee required attendance at all committee meetings, review policy changes for the MSTP program, interview, present and vote for candidates.

MSTP Director Search Committee 2016 Student member of the search committee required attendance at all meetings and interviewing of prospective candidates.

MSTP Stephen R. Max Memorial Lectureship Committee 2017; 2019 Student member of the search committee responsible for nominating and selecting the speaker for the Stephen R. Max memorial lecture.

Abstract

Major depressive disorder (MDD) is a leading cause of mental illness world-wide.

Economists estimate MDD to be one of the leading causes of disability worldwide. The cost is not just due to treatment, but comorbidities, lost work and suicide, too. However, treatments are available and range from psychotherapy, pharmacotherapy or more invasive treatments like electroconvulsive therapy or deep brain stimulation. For decades, the standard of pharmacotherapy care has been selective serotonin reuptake inhibitors

(SSRIs). Yet, SSRIs are only effective in 60% of MDD patients and require weeks of treatment before reaching therapeutic efficacy. Thus, a large proportion of individuals experiencing MDD endure symptoms for weeks without guaranteed relief.

In 2000, low doses of ketamine, a dissociative anesthetic, was found to have rapid acting antidepressant effects. Prior to this, electroconvulsive therapy was the only treatment known to induce rapid antidepressant effects. The use of ketamine as an antidepressant has been met with resistance since it is a known drug of abuse, has an addictive potential and psychotomimetic side effects.

Findings from recent human studies suggest a single oral administration of psilocybin has antidepressant effects within 7 days. However, the hallucinatory effects of psilocybin, like ketamine, severely limit its clinical use. Currently, psilocybin is administered in a controlled environment with psychological support, which greatly increases the cost of care and patient burden. Administration of ketanserin, a serotonin 2 receptor (5HT2R) antagonist, prior to psilocybin attenuates the psychedelic side effects.

But whether ketanserin would also block the antidepressant effect is unknown.

I sought to test the hypothesis that psilocybin has an antidepressant-like effect in rodents independent of the 5HT2R. To this end, I have demonstrated that psilocybin has a

rapid antidepressant effect in anhedonic mice and increases synaptic strength in hippocampus following chronic stress. Prior administration of ketanserin blocks neither psilocybin’s antidepressant-like effect nor the increase in synaptic strength. In vitro electrophysiology experiments failed to show acute effects of psilocin, possibly due to psilocin’s rapid oxidation. While the antidepressant mechanism of action of psilocybin remains unknown, my data suggest the 5HT2R may not be necessary for the antidepressant effects.

Psilocybin as a Rapid Acting Antidepressant: Are Hallucinations Necessary?

by Natalie Hesselgrave

Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2020 © Copyright 2020 by Natalie Hesselgrave

To Wesley, Evyn Mae and Hayden,

My world would not turn without you. May you always nurture your wildest ideas and have the courage to follow your dreams. Without your smiles, hugs and laughs this work

would have never come to fruition.

iii Acknowledgements

As was recently pointed out to me by an astute observer, my memoir would be very short if it weren’t for my perseverance, or my stubbornness as those close to me might call it. However, I cannot attribute my successes to persistence alone. Along the way I have had the privilege of working with so many brilliant scientists who have encouraged me to pursue my dreams and supported me in my failures. I would like to thank Drs. Ramin Parsey and Christine DeLorenzo, with whom I worked during my time as a research assistant at Columbia University. They helped me discover my love of science and planted the seeds of confidence I needed to pursue medical school and then an MD-PhD. It was under their mentorship that I learned I was a scientist.

I would also like to thank my mentor, Dr. Thompson, whom, along with my committee have helped me achieve this goal, which at times seemed quite unattainable.

Without their patience and guidance this work would have never come to light. I have learned so much from them and it is with much gratitude that I move forward with the next phase of my career.

iv Table of Contents

1. Introduction ...... 1 1.1 Evolution of treatments for major depression: from slow to fast ...... 1 1.1.a. History of Major Depressive Disorder ...... 1 1.1.b. Treatments for MDD ...... 4 1.1.c. Rapid acting antidepressants ...... 7 1.2. Therapeutic psychedelics ...... 11 1.2.a. History and development of psychedelics ...... 11 1.2.b. The Chemistry and Mechanisms of Classical Psychedelics ...... 17 1.2.c. Therapeutic psychedelics ...... 28 1.2.d. Summary ...... 32

2. Hypothesis...... 36 2.1 Aim 1...... 36 2.2 Aim 2...... 36 2.3 Aim 3...... 36

3. General Methods ...... 37 3.1 Animals ...... 37 3.2 Chronic Multimodal Stress...... 37 3.3 Hedonic behavior ...... 38 3.4. Head twitch response ...... 39 3.5 Locomotion ...... 39 3.6 Electrophysiology: synaptic strength ...... 40 3.7 Drug treatment...... 42 3.8 Statistics ...... 42 3.9 Electrophysiology: in vitro potentiation ...... 43 3.10 Drugs ...... 44

4. Results ...... 45 4.1. Aim 1. Psilocybin will have an anti-anhedonic effect in the rodent chronic stress model of depression ...... 45 4.1.a. Prediction 1. Psilocybin will restore hedonic behaviors, assessed by sucrose preference and female urine preference, in male C57 mice following chronic multimodal stress...... 45

4.1.b. Prediction 2. Psilocybin will restore hedonic behaviors in other strains of mice following chronic multimodal stress ...... 51 4.1.c. Prediction 3. Psilocybin will restore synaptic strength in stress-weakened TA- CA1 synapses following chronic multimodal stress...... 53 4.2 Aim 2. Psilocybin will have anti-anhedonic and synaptic strengthening effects even when 5HT2Rs are blocked...... 55 4.2.a. Prediction 1. Pretreatment with ketanserin, a 5-HT2A/C antagonist, will not block the anti-anhedonic effects of psilocybin in rodents following chronic multimodal stress...... 55 4.2.b. Prediction 2. Pretreatment with ketanserin will not block psilocybin induced restoration of synaptic strength following chronic multimodal stress...... 64 4.3 Aim 3...... 68 4.3.a. Prediction 1. Bath application of psilocin in hippocampal brain slices will potentiate the field EPSP response...... 68 4.3.b. Prediction 2. Ketanserin will not block psilocin’s ability to potentiate the field EPSP response at the TA-CA1 synapse...... 71

5. Discussion ...... 73 5.1 Psilocybin has an anti-anhedonic like effect in the rodent chronic stress model of depression...... 73 5.2. Psilocybin has an anti-anhedonic effect even when 5HT2Rs are blocked...... 75 5.3 Psilocybin increases TA-CA1 synaptic strength following CMMS ...... 77 5.4. Psilocin failed to acutely potentiate the TA-CA1 synapse...... 80 5.5. Summary ...... 81

6. Conclusion ...... 86

7. Appendix ...... 87 7.1. Psilocin concentrations measured via mass spectrometry by Dr. John Hamlyn. ... 87 7.2. G-protein coupled receptor activation assay from Dr. Samuel Slocum (Roth Laboratory, University of North Carolina) ...... 90

8. References ...... 91

List of Tables

Table 1. Diagnostic criteria for Major Depressive Disorder from the DSM-V...... 2

Table 2. Binding affinities (Ki) of Serotonin and Classic Psychedelics...... 18

Table 3. No differences in Sucrose Preference Test between cohorts at baseline or post- CMMS...... 49

vii List of Figures Figure 1. Chemical structures of classical psychedelic...... 15

Figure 2. Experimental timeline ...... 46

Figure 3. Psilocybin restores sucrose preference following exposure to chronic stress. .. 47

Figure 4. Psilocybin increases sucrose preference following CMMS; a replication...... 49

Figure 5. Psilocybin increases preference for female urine scent following chronic

multimodal stress...... 50

Figure 6. Psilocybin does not have an effect on sucrose preference in B6129/F1 following

chronic multimodal stress...... 52

Figure 7. Psilocybin increases TA-CA1 synapses...... 54

Figure 8. Ketanserin does not block psilocybin's antidepressant-like effect...... 56

Figure 9. Pre-treatment with ketanserin does not prevent psilocybin induced recovery of

Female Urine Preference...... 58

Figure 10. Psilocybin treatment increases hedonic behavior compared to vehicle treated

rodents...... 59

Figure 11. Head twitches in mice do not effect Sucrose or Female Urine Preference

following chronic stress...... 61

Figure 12. Ketanserin pretreatment promotes hypolocomotion compared to Psilocybin

and Vehicle control conditions...... 63

Figure 13. Ketanserin pretreatment does not block psilocybin from increasing synaptic

strength...... 65

Figure 14. Psilocybin increases the AMPA/FV component of fEPSP...... 66

Figure 15. Synaptic strength is correlated with changes in Sucrose Preference...... 67

viii

Figure 16. Physiologically relevant doses of psilocin did not potentiate the TA-CA1

synapse...... 69

Figure 17. Ketanserin does not alter the effect of psilocin on TA-CA1 fEPSP...... 72

Figure 18. Summary: Hypothetical mechanisms beyond the hippocampus...... 83

Figure A1. Psilocin is present in abundance in freshly made and frozen aliquots...... 87

Figure A2. Psilocin concentration is greatly reduced after 60 minutes in artificial cerebrospinal fluid...... 88

Figure A3. Different psilocin concentrations between reservoir and eluate 60 minutes after addition to artificial cerebrospinal fluid...... 89

Figure A4. Psilocin activates a similar constellation of receptors as serotonin...... 90

List of Abbreviations

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

CMMS Chronic multimodal stress

DBS Deep brain stimulation

ECT Electroconvulsive therapy

FST Forced swim test

FU Female urine

FUST Female urine sniff test

Hipp Hippocampus

MAOI Monoamine oxidase inhibitor

MDD Major Depressive Disorder

NMDA N-methyl-D-aspartate receptor

PFC Prefrontal cortex

5-HT Serotonin

5-HT2R Serotonin Receptor – 2A subtype

SP Sucrose preference

SPT Sucrose Preference Test

SSRI Selective Serotonin Reuptake Inhibitor

TA-CA1 Temporoammonic-CA1 pathway

TCA Tricyclic antidepressant

1. Introduction

1.1 Evolution of treatments for major depression: from slow to fast

1.1.a. History of Major Depressive Disorder

Major depressive disorder (MDD) is one of the leading causes of mental illness world-wide. It is estimated that over 300 million individuals suffer from MDD1. Within the United States, recent estimates place the prevalence of MDD at 7.1% of adults, which equates to approximately 17 million people2. Clinically, MDD is defined by a constellation of heterogeneous symptoms. According to the Diagnostic and Statistical

Manual of Mental Disorders3, to receive a diagnosis of MDD and individual must experience either depressed mood AND/OR anhedonia for at least two weeks along with four other symptoms ranging from cognitive disturbances to physical symptoms impacting appetite or energy (Table 1). With such diversity of classification, it’s not uncommon that two patients with major depression can present with only few overlapping symptoms. While these symptoms can range from mild to severe, most adults experiencing a depressive episode report severe impairments2.

At least 1 of 2 core symptoms: Depressed mood (sadness, hopelessness) almost all day Anhedonia: loss of interest of pleasure in activities Additional symptoms (must total at least Appetite disturbance: Weight loss or 5 including core symptom): weight gain Sleep disturbance: Hypo- or hyper-somnia Psychomotor- agitation or -retardation Loss of energy Feelings of worthlessness or guilt Inability to focus, indecisiveness Thoughts of death or suicide Table 1. Diagnostic criteria for Major Depressive Disorder from the DSM-V. *modified from DSM-V, American Psychiatric Association

Symptoms of MDD have been described throughout history. Depression, or depressive like symptoms are documented as far back as the times of Ancient Greeks, when Hippocrates described pathological melancholy4. Mental illness in general has been documented across centuries. However, scholars attribute the first formal clinical description to Robert Burton’s The Anatomy of Melancholy5. Despite this early description, systematic study of depression remained scant. Various attempts were made to collect mental health information through the United States census in the 1800’s through categorizations of “idiocy/insanity” and, later, more affective descriptors6.

However, it was not until after the second world war when the World Health

Organization included a mental health section in the ICD-6 and the American Psychiatric

Association published the Diagnostic and Statistical Manual of Mental Disorders (1952) that true classification and systematic study of mental health began6. Categorical descriptions of psychopathology allowed scientists and clinicians to group and study patients based on shared behavioral characteristics.

While it remains difficult to estimate rates of MDD prior to the mid-twentieth century, studies have demonstrated the rates of MDD, especially during recent decades, are rising7. Along with these epidemiological studies, economic studies have revealed that MDD is extremely costly. The World Health Organization ranks MDD as the fourth leading cause of disability world-wide and predicts only increasing costs1. It is not only cost of treatment that drives up the expense of major depression, but also lost wages, comorbidities and suicide. In fact, costs associated with productivity losses may contribute as much as 40% of disability costs8. But to those suffering from MDD or know

3 someone suffering from MDD, their concern maybe more sinister. Suicide is highly associated with MDD9 and deaths due to suicide have increased over the past 2 decades10.

Although treating MDD reduces the costs and burden of the disease, costs associated with

MDD continue to increase despite increasing rates of treatment8,11.

1.1.b. Treatments for MDD

Treatments for MDD exist and range in levels of invasiveness, from completely non-invasive techniques like psychotherapy to surgical interventions like deep brain stimulation. There are also myriad psychopharmacological options for treating MDD.

Despite these options, 2017 estimates suggest that 35% of U.S. adults experiencing a major depressive episode receive no treatment2. Current clinical practice guidelines rely heavily on antidepressant drugs for first line treatment for MDD12. However, consistent with previous reports, a recent review found that approximately 50% of patients receiving psychopharmacological treatment for MDD fail to respond to first line treatment13,14.

Traditional psychopharmacological treatments for MDD also take weeks before clinical effects are reported15,16. Yet, in spite of the failure rate and lengthy treatment period before response, the prescription rates of antidepressants continue to rise17.

Antidepressants as a medication class were first discovered and named in the

1950s18,19. Physicians treating tuberculosis patients noted the psychostimulant-euphoric side effects of iproniazid, a hydrazine drug known to inhibit monoamine oxidase (MAO) and increase serotonin levels in the brains of experimental animals19–21. MAOs are enzymes found across species, responsible for breaking down endogenous and ingested monoamines22. They are expressed in both the periphery and central nervous system,23 and have two distinct types: MAO-A and MAO-B24. Human imaging data suggest an

4 over expression of MAOs in subjects with MDD25, which is consistent with findings from a rodent study reporting increased MAO-A activity following glucocorticoid administration26. Increased MAO activity could lead to decreased monoamine availability which is consistent with the monoamine hypothesis of MDD. The antidepressant effects of MAO inhibitors (MAOIs) are thought to be mediated by MAO-A as selective MAO-B inhibitors do not have antidepressant properties27. While acute administration of MAOIs decreased the firing rate of serotonergic projections from the dorsal raphe nucleus28, chronic administration induced downregulation of inhibitory autoreceptors allowing neuronal activity to normalize29. Presumably, the ultimate increases in extracellular monoamine levels underlie the antidepressant effects of MAOIs29.

Shortly after the initial reports from tubercular patients, psychiatrists published the first reports of the psychic effects of MAOIs in human subjects with depression30–32.

There was a quick but brief surge in treatment and development of hydrazine compounds with greater inhibitory affinities for MAO than the original compounds, but they were found to have hepatotoxic side effects19. Although the demand for antidepressant treatment remained high, these compounds were pulled from the marked19. Later non- hydrazine MAO inhibitors (MAOIs) were developed, but these were also plagued by side effects. Early studies linked the hypertensive crises reported in some patients to diet19, specifically diets rich in tyramine33,34. Although MAOIs can be effective antidepressants with proper guidance and dietary restriction, they are not considered first line treatments due to the potentially fatal side-effects and toxic drug interactions35.

Almost concurrent with the discovery of the antidepressant effects of MAOIs, tricyclic compounds developed from antihistamine research were found to have

5 antidepressant effects36. Imipramine, was the first tricyclic shown to have antidepressant properties developed specifically for psychiatric use37,38, but was quickly followed by others39. By the end of the 1950s, the development of pharmacological treatments for depression helped revolutionize the understanding of mental illness, taking the etiology from intrapsychic conflict or hysteria and providing evidence for biological dysregulation. That these drugs, despite different mechanisms, increased levels of endogenous neurotransmitters supported the catecholamine and monoamine hypotheses of depression in the 1960s which posits that decreased levels of serotonin, norepinephrine and dopamine underly depressive-like states39–42. These hypotheses now guided scientific research examining the neurobiological bases of MDD and drug development.

Evidence implicating the role of serotonin in depressive disorders began accruing.

In the late 1950s, it was noticed that a subset of patients undergoing reserpine therapy for hypertension experienced depressive episodes43. It was soon discovered that reserpine depleted levels of serotonin in the brain via inhibition of the vesicular monoamine transporter44,45. Decreased levels of serotonin were also found in a sample of brains from individuals completing suicide46. Thus, pharmaceutical companies began developing compounds that could specifically increase serotonin levels, hoping they would have antidepressant effects with fewer side effects than earlier MAOI and tricyclic antidepressants. Fluoxetine was one of the first selective serotonin reuptake inhibitors

(SSRIs) introduced in 197447 and SSRIs were approved as a novel class of antidepressants in the 1980s18.

Currently, antidepressants and SSRIs in particular, are some of the most widely prescribed medications in the United States48. However, despite their popularity, their

6 efficacy is limited. In one of the largest controlled trials to date, only 32% of patients remitted from their major depressive episode (MDE) following four weeks of treatment with an SSRI13.

SSRIs increase levels of available serotonin (5-HT) by blocking its presynaptic reuptake. As predicted, using a quantitative radioassay with [3H]citalopram and [3H]5-

HT, Owens et al reported high binding affinities of 13 SSRIs for the serotonin transporter

(SERT)49. The result of this inhibition was reduced 5-HT reuptake50 and increased 5-HT levels51. Interestingly, acute SSRI administration was found to rapidly increase 5-HT levels in rodents using in vivo microdialysis techniques52. A single dose of SSRI was shown to increase 5-HT levels for up to 24 hours53 to levels up to 5 times those measured at baseline53–56. When acutely applied to hippocampal tissue, SSRIs also rapidly increase the excitatory response, measured by local field potentials, in the CA1 region of the hippocampus57. A central paradox of understanding the role of serotonin and depression, however, is that despite this rapid increase in serotonin levels the therapeutic effects of

SSRIs and other 5-HT modulating antidepressants take weeks of chronic treatment before emerging15,16.

1.1.c. Rapid acting antidepressants

The antidepressant effect is not inherently slow. In fact, rapid antidepressant effects have been reported following electroconvulsive therapy58, deep brain stimulation59 and subanesthetic doses of ketamine60. Prior to the development of MAOIs, aside from psychotherapy, electroconvulsive therapy (ECT) was used to treat MDD and other mental illness. Many studies have demonstrated its efficacy throughout the years15 and a recent meta-analysis confirmed that ECT out-performed all other treatments58. When ECT is

7 used it can induce rapid relief of depressive symptoms with as few as one application61.

Yet, ECT remains a last resort treatment, which is understandable due to its invasive nature. Additionally, the reported side effects such as memory impairment62 and slowed cognitive processing63 can make ECT less palatable for patients. Preliminary data from preclinical and clinical studies suggest that deep brain stimulation (DBS) may exert rapid antidepressant effects59,64. Drawing conclusions and generalizing from DBS studies is complicated as effects vary by targeted brain region and stimulation intensity64. While

DBS has demonstrated its utility in treating movement disorders, it remains an experimental treatment when it comes to depression. Further studies demonstrating consistent antidepressant effects and long-term safety are needed. But similar to ECT,

DBS will always be more limited when compared to pharmacological treatments due to its invasive nature.

The initial report that ketamine has rapid acting antidepressant effects60 was ground-breaking. Pharmaceutical companies had developed newer variations of SSRIs through the 1990s attempting to increase efficacy and decrease side effects18, but these treatments remained similar in that they targeted the catecholaminergic system, consistent with the monoaminergic hypothesis of MDD. Simultaneously, evidence implicating the role of the N-methyl-d-aspartate receptors (NMDA) in MDD and the antidepressant response was growing. Mice chronically treated with SSRI, TCA or ECT had lower

NMDA expression65,66 and NMDA antagonists were found to have antidepressant effects in stress based animal models of depression67. Following these preclinical findings, ketamine, an NMDA antagonist, was shown to have antidepressant effects in humans, first within 3 days of infusion60 and then within hours68. However, there is something

8 unique about ketamine’s antidepressant actions. Clinical trials with other known NMDA antagonists do not show the same antidepressant effects69.

Significant efforts have been made to understand the mechanism of action underlying ketamine’s rapid antidepressant effect. Ketamine is a chiral molecule, similar to other anesthetic agents70. Racemic mixtures of (R,S)-ketamine have been used in most studies that report rapid acting antidepressant effects of subanesthetic doses of ketamine60,68,71. The S-ketamine enantiomer has higher affinity and potency for the

NMDA receptor than R-ketamine71–73. It also has a more positive clinical profile compared to R-ketamine when clearance rates, anesthetic potency and cognitive side effects are compared74. R-ketamine has yet to be tested for antidepressant efficacy. In animal models, the racemic mixture71 as well as the individual enantiomers75 were shown to have rapid antidepressant effects: increasing mobility time in the tail suspension and forced swim tests, and decreasing the reduction in sucrose preference following dexamethasone exposure75. However, only R-ketamine was reported to have sustained antidepressant effects, 7 days post-injection, in the tail suspension and forced swim tests75. Similar results were reported with R-ketamine following social defeat stress in mice and learned helplessness in rats76. Consistent with previous research77,78, pretreatment with the AMPA receptor antagonist NBQX 30 minutes prior blocked the antidepressant effects of both R- and S-ketamine following social defeat stress one-day after injection in forced swim test and tail suspension test, and seven days post injection in sucrose preference test76.

Ketamine is metabolized in the liver into many by-products79, of which only some have anesthetic properties80. Looking further into ketamine’s potential mechanism of

9 action, Zanos et al71 reported that NMDA antagonism alone is insufficient to produce antidepressant effects in stressed mice and that the hydroxynorketamine (HNK) metabolite is necessary for ketamine’s antidepressant response. Unlike ketamine, HNK does not antagonize NMDA but enhances AMPA signaling in the hippocampus, measured by local field potentials in the CA1 region, and prefrontal cortex, approximated by increased power in the gamma band of electroencephalography (EEG) in mice71.

Contrary to these findings, however, higher concentrations of HNK have been shown to decrease the amplitude of NMDA mediated post-synaptic responses in hippocampal pyramidal cells81. But it was shown that the lower concentration of HNK used by Zanos et al71 is more physiologically relevant and does, indeed, have no effect on NMDA mediated post synaptic responses in hippocampal tissue82. These findings are consistent with those previously described reporting no antidepressant effect of ketamine with

NBQX pretreatment.

Ketamine treatment, however, comes with great limitations. It has been safely used as an anesthetic in adults and children for decades83, but is known to cause dose- dependent respiratory depression84. Ketamine can also induce euphoric states, making it an appealing drug of abuse85. Preclinical studies have shown that ketamine can induce place preference on rodents86 and provide reinforcement in a fixed-ratio learning task in non-human primates87, suggesting that ketamine may have an addictive potential. Self- administration studies reported that rodents and nonhuman primates rapidly learn to lever press for ketamine, similar to other drugs of abuse like cocaine and heroin88,89. Ketamine is also known to have psychotomimetic effects even at subanesthetic doses90. Despite these side effects, ketamine is an invaluable clinical tool. In addition to its rapid

10 antidepressant effects, a meta-analysis reported that ketamine mitigated suicidal ideation within 4 hours of infusion91. The timing of the effects of ketamine also provided proof of concept that antidepressant effects can be induced rapidly pharmacologically.

Although the specific rapid antidepressant effects of ketamine were reported relatively recently, the mind-altering effects of ketamine have been known for decades92.

Ketamine has been classified as a psychedelic compound since its discovery in the 1960s because of its dissociative effects93. An infusion of S-ketamine, but not R-ketamine, produced increases in glucose metabolism in the frontal cortex which were highly correlated with psychotomimetic effects94. Alternatively, R-ketamine decreased glucose metabolism across the brain and did not induce hallucinations94. Clinical studies have reported positive correlations between the intensity of ketamine’s dissociative effects and antidepressant efficacy95,96. Similarly, a recent study with psilocybin, a psychedelic pan- serotonergic agonist, reported that the intensity of positive altered states of consciousness were correlated with decreases in depression rating scores97. Could a psychedelic pan- serotonergic agonist producing altered states of consciousness similar to ketamine also have antidepressant effects? Could the psychedelic experience account for rapid antidepressant properties?

1.2. Therapeutic psychedelics

1.2.a. History and development of psychedelics

Psychedelic compounds have been used by humankind for millennia. In fact, use of psychedelics dates so far back that it is thought by some “that the idea of the deity may have arisen as a result of their physiological effects.”98 Anthropologists have dated the use of psychoactive mushrooms to early Mayan and Aztec cultures, where these drugs

11 were used for sacred religious ceremonies and rituals.99 Modern science has been interested in identifying and classifying these compounds since the nineteenth century100 and many have struggled to describe and classify the unique effects of psychedelics. It wasn’t until the 1950s, well after the synthesis of lysergic acid diethylamide (LSD)101, that a unified definition of hallucinogens emerged which emphasized several unique aspects of this class of psychotropic drugs including: depersonalization, altered perception of space and time, and no major disruptions to the autonomic nervous system.98 But what are these compounds and how were they identified?

Many consider Albert Hofmann to be the founder of psychedelic science. After all, he was the first to synthesize LSD and psilocin, two powerful psychedelic compounds. Like most of modern-day pharmacology, his discoveries in the laboratory came from plants with known spiritual and medicinal uses. By the 1930s when Hofmann started in Sandoz laboratories, many therapeutic plant compounds had already been identified, like atropine102 and digitalis103. Hofmann continued purifying and comparing plant compounds under supervision from Dr. Arthur Stoll until he sought to focus on ergotamine, an alkaloid with therapeutic obstetric use that Stoll had purified in 1918101.

Claviceps purpurea, or Ergot, is a type of parasitic fungi that infects rye grasses. Over the course of history, outbreaks of ergotism have been recorded particularly in Eastern

European cultures consuming rye bread.98 Of the two types of ergotism identified, hallucinations are known to accompany one: ergotism convulsus.

Despite its known toxicity, ergot gained use in midwifery due to its ability to quicken labor, contract peripheral vessels and staunch post-partum bleeding101. Stoll’s purification of ergotamine enabled more widespread clinical use of the compound.101

Further purification of the ergot alkaloids and their components lead to the discovery of lysergic acid104 and ergobasine, the ergot alkaloid with parturient effects101. However, ergobasine was not one of the abundant ergot alkaloids. As such, Hofmann set about attempting to synthesize and improve ergobasine from the byproducts of alkaloid degradation: lysergic acid and amines. It was with the hope of creating a lysergic acid derivative with circulatory and respiratory stimulant effects that Hofmann developed

LSD-25,101 the incredibly potent psychoactive compound known today simply as LSD.

Between the documented symptoms of ergotism convulsus and the effects of mescaline, another psychoactive alkaloid used in Native American and Mexican cultures, the psychedelic effects of LSD were not surprising. However, the active compound in

Psilocybe mexicana, the mushrooms used in Mexican and South/Central American sacred ceremonies remained elusive. Hofmann’s initial attempts to isolate the active compound from Psilocybe mexicana were unsuccessful in eliciting behavioral responses in experimental animals.101 Only after careful extraction and self-experimentation, “since it is not appropriate for researchers to ask anyone else to perform self-experiments that they require for their own investigation,”101 Hofmann and his team of volunteers were able to identify 2 novel active compounds from the fractionation: psilocybin and psilocin105.

Months later, Hofmann and colleagues identified the structure of psilocybin and psilocin106.

Since they have similar psychic effects, it was not surprising that psilocybin and psilocin were found to be structurally similar in structure to LSD (Figure 1)107. Psilocybin is a naturally occurring indole compound93. While psilocin is not found in appreciable amounts in Psilocybe Mexicana101 or other psilocybin containing mushrooms, it is the

13 oxidized product of psilocybin and the psychoactive metabolite.108 Importantly, LSD, psilocybin and psilocin are both chemically and structurally similar to serotonin. This similarity to serotonin, along with early studies suggesting psychedelics can heighten self-awareness, peaked the interest of psychiatrists early on. In fact, by the 1970s there were over 1,000 papers on the clinical use of psychedelics109, although many did not employ the rigorous standards of current day methods, these initial studies set the foundation for today’s work.

Figure 1. Chemical structures of classical psychedelic. * adapted from National Center for Biotechnology Information. PubChem Database. Psilocybin, CID=10624; Psilocin, CID=4980; Serotonin, CID=5202; Lysergide, CID=5761; DOI, CID=1229; Mescaline, CID=4076 https://pubchem.ncbi.nlm.nih.gov/compound/Lysergide (accessed on May 21, 2020)

It quickly became clear that psychedelic substances, particularly psilocybin were safe for human consumption. Sandoz, after all, was marketing these compounds for therapeutic human use101. Early studies demonstrated that psilocybin activated the sympathetic nervous system less than LSD and had little effect on blood pressure, circulation, and other organ systems110. Original animal studies calculated the LD50 of intravenous psilocybin to be 250 mg/kg111, which is considerably higher than the effective doses in most human and rodent studies (.5-10 mg/kg)112,113. More recent work in rodents114 and primates115 has confirmed that psilocybin is safe and has relatively low abuse potential. Rigorous work regarding abuse potential in humans has not been completed, but preliminary studies suggest that the classic psychedelic compounds are safe and have low potential for abuse116,117.

However, the psychological effects of psychedelics have always been of concern and intrigue. Personality and environment can influence the psychedelic experience93. An early survey report found that LSD therapy increased the risk of adverse reaction, including psychosis and suicide109. Yet, a more recent double-blind, randomized, controlled trial of LSD therapy for anxiety reduction found positive effects of LSD treatment when administered in a safe, supervised therapeutic setting116. There is some evidence suggesting that the altered states of consciousness lead to heighted self- awareness and mediate the therapeutic effects of psychedelics97,118. Based on these findings, current psychiatric therapeutic use of psilocybin and ketamine occurs in carefully controlled environments with ample psychological support before, during and after the psychedelic experience119. This greatly increases the burden of treatment for the patient compared to SSRIs in terms of both time and cost. Understanding the molecular

16 mechanisms underlying these profound effects could aid in the development of compounds with targeted effects, while minimizing side-effects and potentially decreasing patient burden. But psychedelic research has been limited.

Unfortunately, clinical and scientific research with psychedelics came to a halt by the 1970s120. Despite the mounting evidence reporting positive outcomes of psychedelic therapeutics, psychedelics were increasingly associated with the counterculture of the

1960s. Recreational use of LSD and other psychedelics became popular and politicized119, resulting in the inclusion of these compounds as Schedule I substances under the Federal Comprehensive Substances Act of 1970121. Thus, when reviewing the literature elucidating the biological, molecular and clinical effects of psychedelics, a gap in continuity is expected and explained.

1.2.b. The Chemistry and Mechanisms of Classical Psychedelics

Concurrent with the progression of clinical psychedelic trials in 1960s, scientists gained better understanding of their mechanism of action. By the mid-1960s, psychedelic drugs were classified as “classical” psychedelics, those having serotonergic effects, or dissociative anesthetics, compounds like ketamine which modulate glutamate120.

Psilocybin and LSD are considered “classical” psychedelics, along with the phenethylamines like mescaline and 2,5-dimethoxy-4-iodo-amphetamine (DOI; Figure

1). As previously mentioned, the structural similarity of LSD and psilocybin to serotonin guided the initial direction of study. Although the classical psychedelics may all activate the 5HT system and share certain behavioral effects, they have different potencies, duration of action and affinities for known 5HT receptors (Table 2)122.

Serotonin Psilocin Mescaline Receptor LSD (nM) DOI (nM) (nM) (nM) (nM) 5-HT transporter 3801 5-HT1A 23.94 308.2 3.01 2355 5-HT1B 7.5 219.6 3.9 1261 5-HT1D 4.09 36.4 14 1241.4 5-HT1E 6.86 52.2 93 5-HT2A 123.07 107.2 5.6 1.65 150 5-HT2B 6.87 4.6 4.5 26.84 >10,000 5-HT2C 31.07 97.3 23.4 3.01 5-HT3 593 >10,000 5-HT5 83.7 6.9 5-HT6 79.47 57 2.3 5-HT7 7.66 3.5 6.6 Table 2. Binding affinities (Ki) of Serotonin and Classic Psychedelics.

*adapted from the pdsp database. https://pdsp.unc.edu/pdspweb/. Accessed July 11, 2020

Functional studies have confirmed that classical psychedelics have serotonergic effects. Initially, studies looked at the effects of these drugs on peripheral tissue. Like serotonin, both psilocin (0.005-0.030 µg/ml) and psilocybin (0.025-.25 µg/ml) were shown to induce dose-dependent increases in contractions on the rat uterus123. Moving into the central nervous system, using in vivo recording techniques in rodents, systemic

LSD administration was found to rapidly decrease the firing rate of serotonergic neurons in the raphe nucleus124. Direct application of psilocin or LSD to the raphe via iontophoresis also rapidly reduced firing, similar to the direct application of 5HT125,126.

Compared to the raphe, psilocin’s suppression of firing, measured by unit recordings, was not as great in the amygdala and ventral lateral geniculate, two areas receiving serotonergic input from the raphe125. This was true for N,N-dimethyltryptamine (DMT) and bufotenine, two other indolealkylamines tested125, suggesting that these psychedelics act on presynaptic 5HT projecting neurons in the raphe. Despite the decreased firing rates, downstream increases in 5HT levels have been observed. Looking at the effects in the rat forebrain, systemic LSD and DMT both increased 5HT levels127. Intraperitoneal injections of psilocin have also been observed to increase 5HT levels in rodents, but only at the highest dose of 10 mg/kg128. The LSD induced decrease in firing activity was blocked by either 5-HT2A or 5-HT1A antagonism129.

As illustrated in Table 2, psychedelics have varying degrees of affinities for the serotonin receptors. Generally, LSD has stronger affinities for the 5-HT receptors than psilocybin, even more so than 5-HT itself as in the case of 5-HT2AR. In contrast, DOI has strong affinities for the 5-HT2Rs but only weak attraction for 5-HT1Rs compared to either LSD or psilocybin. The role of the 5-HT2 receptor (5-HT2R) in the mechanism of

19 action of psychedelics has been of particular interest since radioligand binding assays correlated the potency of psychedelics with 5-HT2 binding130,131. The specific effects of

LSD and DOI on 5-HT2 receptors were first characterized using electrophysiological recording techniques on a population of 5-HT sensitive interneurons132 found in layer III of the rat cortex133. Both LSD and DOI produced a dose-dependent increase in the firing rate of these neurons which was blocked in brain slices pretreated with MDL-100,907, a

5-HT2A antagonist. The 5-HT stimulation of these layer III interneurons produce IPSCs in layer II pyramidal cells132. Using intracellular recording techniques to measure layer II

IPSCs, DOI was shown to induce IPSCs, but a lower frequency than 5HT133. The same group later reported an increase in frequency and amplitude of the spontaneous excitatory post synaptic potentials and currents (EPSP or EPSCs) of layer V pyramidal cells of rat cortex in response to 5-HT134, indicating that 5-HT may have excitatory properties. The

5-HT induced increases in EPSCs were attenuated pharmacologically by an AMPA selective glutamate antagonist, LY293558, and, separately, a 5-HT2A antagonist, MDL

100,907134. Application of DOI, a psychedelic compound with high 5-HT2A affinity, also increased the frequency and amplitude of EPSCs in layer V cortical cells135.

Microdialysis studies measuring glutamate levels further support the evidence implicating the 5-HT2AR in the actions of psychedelics. In independent studies, DOI136 and LSD137 have been found to increase glutamate levels in the rat cortex. In both studies, the glutamate increase was blocked by MDL-100,907, suggesting 5-HT2A activation mediates the increases in glutamatergic excitatory transmission.

To further explain and integrate the role of 5-HT2A in glutamatergic activity, it was hypothesized that 5-HT2A activation on cortical pyramidal cells lead to glutamate

20 release from thalamic fibers innervating the prefrontal cortex through retrograde signaling138–140. The idea of an alternate glutamatergic source was needed since 5-HT2A activation alone is not sufficient to induce depolarizations large enough to increase spiking or release probability140,141. However, experiments failed to identify putative retrograde messengers or that retrograde signaling was even occuring140. Instead, Beique et al identified a subset of pyramidal neurons in deep cortical layers in which 5-HT induced 5-HT2A mediated depolarizations strong enough to increase activity in these cells leading to increases in layer V EPSCs described above140. This finding suggests psychedelic effects may result from dysregulation of cortical networks within the prefrontal cortex.

The 5-HT2R has also been implicated in psychedelic induced synaptic plasticity.

Ly et al142 reported that LSD, DMT and DOI increased the number of dendritic spines and density of synapses in rat cortical cultures and that DMT increased the number of spines in adult rat prefrontal cortex 24 hours after injection. Electrophysiological studies following the in vivo DMT treatment showed increased frequency and amplitude of spontaneous excitatory post synaptic currents (EPSCs), confirming the functional impact of the anatomical changes142. Consistent with previous reports, treatment with ketanserin, a 5-HT2A and 5HT-2C antagonist, blocked the anatomic structural changes observed with LSD, DOI, DMT, and psilocin in cultured cortical neurons142. Behavioral studies have also confirmed the role of 5-HT2A in the effects of psychedelics.

1.2.c. Psychedelic behavior: Animal Models and Human experience

Almost 30 years after Hofmann’s initial synthesis of LSD, selective serotonin antagonists, drugs targeting and blocking specific serotonin receptors, were developed.

By combining these drugs with well characterized animal behaviors, the role of specific receptors or combinations of receptors on innate or induced behaviors have been elucidated. For example, rodents can be trained to discriminate between saline and certain classical psychedelics, like LSD, DMT or psilocybin. Stimulus generalization occurs when a rodent trained with one compound can accurately discriminate between saline and another psychedelic compound, indicating that the two substances, depending on which is used, are of the same or related pharmacological class143. Stimulus generalization varies based on the training compound. Ketanserin was shown to block stimulus generalization between LSD and other psychedelic compounds131, providing evidence for a behaviorally relevant role of 5-HT2A. Ketanserin has also been shown in humans to block the hallucinatory effects of psilocybin144 and LSD145. However, ketanserin in humans only partially blocks the hallucinatory effects of ayahuasca, a tea used in spiritual ceremonies containing a combination of psychedelic 5HT agonists146, notably DMT.

Due to the distinct human nature of the psychedelic experience, it has been difficult to quantify in a research setting. The altered states of consciousness scale (ASC) has become the preferred tool amongst human psychedelic researchers to quantify the psychedelic experience147,148. The scale can be scored in different ways which filters the type and intensity of the experience into 3-11 dimensions148. These dimensions allow researchers to compare the human psychedelic experience between and within individuals and across compounds in terms of: experience of unity, spiritual experience, blissful state, insightfulness, disembodiment, oceanic boundlessness, anxiety, imagery, synesthesia, etc.148 These dimensions describe experiences and at times behaviors that are

22 uniquely human. Yet, this does not mean that psychedelics do not affect behavior in animals.

Rodents demonstrate specific behaviors that can be reliably manipulated pharmacologically, observed and quantified. The forced swim test (FST), for example, is typically used to test the efficacy of antidepressants149, although its relationship to the symptoms of depression is unclear. In FST, a rodent is placed in a container of water for several minutes, during which time it exhibits periods of mobility (swimming) and immobility (floating)150. SSRIs induce an immediate response in the FST, decreasing immobility time in rodents, but take weeks of administration in phenotypically anhedonic rodents57 and humans with MDD13 before yielding an antidepressant response. Similarly, following demonstrations that injections of 5HT induce dose-dependent head twitch in mice151, the head twitch response (HTR) has been used to assess the behavioral or hallucinogenic effects of psychedelics in rodents139. In a follow-up to their original 5HT induced HTR finding, Corne and Pickering (1967) induced HTR with LSD and psilocybin in mice152. This finding has been replicated by multiple groups with multiple compounds known to produce hallucinations in humans139,153–156. Pharmacologically antagonizing the 5-HT2AR inhibits the psychedelic induced HTR with DOI157,158.

Similarly, in mice lacking the 5-HT2AR neither psilocin159 nor DOI155,156 nor LSD156 induced a HTR. However, not all 5-HT2A agonists induce HTR. Lisuride, a 5-HT2A agonist does not induce hallucinations in humans and fails to produce HTR in mice139,156.

Psychedelics may also induce changes in rodent locomotor behavior. In rats, LSD

(60 µg/kg) and DOI (.25 mg/kg) were observed to decrease overall activity, exploratory and organizational behavior, which effectively describes the locomotion pattern160. In this

23 same study, LSD induced decreases in activity were blocked with high doses of 5-

HT2AR antagonist but not the 5HT-2CR antagonist, while decreases in behavioral organization were inhibited by 5-HT2AR, 5HT-2CR and 5-HT1AR antagonism160. With low doses of DOI, pretreatment with either a 5-HT2AR antagonist or high dose of a 5HT-

2CR antagonist mitigated DOI induced decreases in activity160. However, in mice, lower doses of DOI increased activity while higher doses (5-20 mg/kg) reduced activity161. In 5-

HT2AR knock out mice, no change in locomotion was seen with low doses of DOI (1 mg/kg), but high doses of DOI (10 mg/kg) decreased locomotion. Prior administration of a 5HT-2C antagonist blocked the decreases in locomotion seen with high doses of

DOI161. Psilocin (4.8 mg/kg) decreased distance travelled in a novel arena between 30-50 minutes following injection in mice159. Interestingly, psilocin depressed locomotion in 5-

HT2AR knock out mice, as it did in wild type mice159. Prior injection of a 5-HT1AR antagonist but not a 5HT-2CR antagonist prevented psilocin from decreasing locomotor activity159. The dose dependent psilocin induced decreases in locomotion were replicated by a separate group in male and female rats162. However, at high doses of psilocin (4.0 mg/kg) they found that individual antagonism of 5-HT2AR, 5HT-2C and 5-HT1A reversed the decreases in locomotion162.

While it may appear contradictory, these data help elucidate the functional differences in the binding profiles of different psychedelic compounds and contribute to the general understanding of the shared and disparate mechanisms of action of psychedelic compounds. Looking back at Table 2, DOI has much stronger affinity for 5-

HT2Rs than other 5HT-Rs, specifically the 5-HT2A. Hence, it is not surprising that the effects of DOI on rodent behavior can be blocked via antagonism of 5-HT2Rs. The

24 psilocin data is not as clear-cut. However, this is consistent with the binding profile of psilocin. Unlike DOI which has 1,000 fold higher affinity for 5-HT2Rs than 5-HT1Rs, psilocin, like LSD, has comparable affinities for 5-HT2Rs and 5-HT1Rs (Table 2). In light of the finding that 5-HT2AR antagonism inhibited LSD decreases in activity and behavioral oganization160, it was surprising that psilocin depressed locomotion in the 5-

HT2AR knock out mouse159. This could be due to differences in study design, using pharmacological antagonism versus genetic knock out animals. That 5-HT1A antagonism inhibits similar activity patterns with both LSD and psilocin, supports the theory that study design may explain some discrepancies. Ultimately, these studies should be repeated using similar methods to distinguish the receptor similarities and differences underlying the locomotor effects of these two indolealkylamine compounds. But for now, based on the available data, psilocin and LSD exert their effects on locomotion and behavioral organization through different combinations of 5-HTRs163.

It is clear from the rodent behavior data and binding profiles of psychedelics that different collections of receptors may be agonized and downstream signaling proteins activated by psychedelic compounds. However, much of the human research on psychedelics has focused on the role of the 5-HT2AR. In a 1998 report, Vollenweider et al. demonstrated that a high dose of ketanserin (40 mg) completely blocked psilocybin induced hallucinations, assessed by the ASC (oceanic boundlessness, visionary restructuralization and dread of ego-dissolution), while 20 mg of ketanserin decreased psilocybin’s effects by 50-70%164. In contrast, prior administration of haloperidol, a D2 receptor antagonist, only reduced psilocybin’s effects on the oceanic boundlessness subscale164. A later study replicated the effects of ketanserin pretreatment on psilocybin

25 induced ACS, and extended these findings to inhibition processing using a pre-pulse inhibition and Stroop task165. Psilocybin decreased % pre-pulse inhibition and increased

Stroop error rates and reaction time, while pretreatment with ketanserin prevented these changes165. Kometer et al. extended the effects of psilocybin to affective state, reporting that psilocybin increased positive affect, decreased identification of negative facial expressions in a facial recognition task and increased reaction time for negative valence word cues166. As with the rodent behavioral data, pretreatment with ketanserin blocked these behavioral findings166.

In addition to behavioral effects, advances in brain imaging techniques have elucidated psychedelic biological signatures that are sensitive to pretreatment with ketanserin and, thus, thought to be mediated by 5HT2Rs. For example, pretreatment with ketanserin attenuated decreases in the P300 component of electroencephalogram (EEG) seen with psilocybin during an emotional go/no go task166. Similarly, the EEG measured decreases in cortical α oscillations and N170 potentials following psilocybin administration were found to be sensitive to ketanserin167. Results from a magnetoencephalography (MEG) study, which has greater structural resolution than

EEG, also demonstrated global activity decreases following psilocybin across a range of frequencies (1-100 Hz)168. Dynamic causal modeling of these data identified activity increases in the posterior cingulate, a region rich in 5-HT2ARs, as an underlying factor in the cortical desynchronization168. But these findings are not unique to psilocybin.

Decreases in EEG signal, mostly in the α power band, have been reported with LSD169, ayahuasca170 and DMT171. Like psilocybin, pretreatment with ketanserin blocked ayahuasca induced decrements in EEG power146.

Functional magnetic resonance imaging (fMRI) techniques have also reported psychedelic induced changes. Psilocybin decreased the cerebral blood flow and the blood-oxygen level-dependent (BOLD) signals172, with the greatest reductions found in the thalamus and cingulate cortices. The decreases in the cingulate and medial prefrontal cortex were correlated with the reported intensity of the psychedelic effects172. Increased variance in BOLD signal amplitude in the anterior cingulate cortices and hippocampi following psilocybin infusion have also been found173. In contrast, following psilocybin infusion resting state functional connectivity was increased in many, but not all, resting state networks examined174. The networks with decreased functional connectivity were between visual and sensorimotor cortices, which may be indicative of the visual alterations reported following psilocybin174. Psilocybin was also found to decrease amygdala activity in response to presentation of negative stimuli175. The decreased amygdala BOLD signals were correlated with psilocybin induced increases in positive mood175. Like psilocybin, using behavioral and fMRI measures, LSD was shown to increase positive biases in emotional processing tasks in a 5-HT2AR dependent manner145. Generally, these data are consistent with the notion that decreases in alpha and beta EEG power are correlated with increases in fMRI measures of functional connectivity176.

The imaging findings reviewed above are consistent with studies examining pathological states. Early human in vivo recordings reported large variations in oscillatory amplitudes in human subjects experiencing psychoses177,178. As with fMRI in healthy volunteers following psilocybin174, increased hippocampal activity has also been seen in fMRI of psychotic patients with schizophrenia179 but not in asymptomatic

27 schizophrenia180. Similarly, the alterations in consciousness, particularly along the derealization and depersonalization dimensions, that psilocybin induces were found to be similar to those seen in early stages of schizophrenia164. These consciousness alterations, however, were not sensitive to pretreatment with the typical antipsychotic haloperidol, a dopamine receptor type 2 antagonist164. Following doses of psilocybin, Vollenweider et al181 also reported decreases in attention and the human startle response, measured by prepulse inhibition, which were consistent with changes seen in schizophrenic subjects.

These findings suggest psilocybin may be of use as a model of psychosis. In addition to modelling mental illness, there is growing evidence that psilocybin and other psychedelics may have therapeutic effects for other mental health disorders.

1.2.c. Therapeutic psychedelics

We are only beginning to learn about the possible alternative effects of psychedelics. Since the early psychedelic assisted psychotherapy studies and the classification of psychedelics as a schedule 1 compound by the government, there has been little research published about the therapeutic effects of these compounds. And those studies which have been published often fail to meet rigorous scientific methodological standards. For example, a recent review on the subject found 151 reports between 1990-2015 pertaining to the antidepressant or anxiolytic effects of LSD, psilocybin or ayahuasca, but only six were in peer-reviewed journals182. However, a significant body of anecdotal reports together with the few controlled and reviewed studies suggest further research is warranted and potentially valuable to a vulnerable patient population.

Therapeutic psychedelic research began to pick up in the late 1990s following three seminal studies in healthy controls demonstrating the safety and practicality of psychedelic research183. Following these studies and others exploring psychedelic states in healthy volunteers with various psychedelic compounds, the first study exploring the effects of psilocybin in a psychiatric population was published in 2006184. Moreno et al. were the first to demonstrate that psilocybin (25-300 µg/kg) rapidly decreased symptoms of obsessive compulsive disorder within, and at times beyond, 24 hours in 9 patients184.

Higher doses of psilocybin (285-430 µg/kg) have also been shown to increase the efficacy of smoking cessation programs, increasing abstinence rates from ~35% to 80%, and alcohol abuse treatment programs with few reported side effects185,186. These findings, however, must be interpreted cautiously, because they utilized an open-label treatment study design, not a placebo-controlled double blinded design which has become the scientific gold standard.

In more carefully controlled studies, psilocybin and LSD have been shown to reduce anxiety and depressive symptoms co-occurring with terminal cancer diagnoses.

Grob et al reported decreases in depressive and anxiety measures, assessed by the Beck depression inventory (BDI) and State-trait anxiety inventory (STAI), respectively, up to six months after psilocybin (200 µg/kg) treatment187. Similar decreases in depression and anxiety in larger cohorts (n = 51 and n = 29) of terminally ill patients were found with higher doses of psilocybin (300-430 µg/kg) 1 day and up to 6 months after treatment188,189. Using a similar study design, Gasser et al reported similar long term anxiolytic effects of LSD in a small cohort (n=12) of terminally ill patients116. Together, these findings support the conclusion that psychedelics may have beneficial

29 antidepressive and anxiety reducing effects in terminally ill populations. But can they be effective in patients with depression and anxiety disorders not resulting from terminal illness?

In 2016 Carhart-Harris et al.190 reported the first findings that psilocybin has rapid acting antidepressant effects in patients with treatment resistant depression. In this cohort of n = 12, all patients were diagnosed with MDD, had depression ratings > 17 on the

Hamilton depression rating scale at baseline (moderate to severe range), and had failed at least 2 antidepressant trials190. A standard dose of 25 mg of psilocybin was used, 7 days after a test dose of 10 mg to ensure absence of negative reactions, and behavioral outcomes measured one week after the experimental dose. Remarkably, at the one week timepoint, self rated depression scores were significantly reduced from baseline and remained reduced in all subjects for three months190. A follow-up study in an expanded cohort (n = 20) replicated these results and extended the duration of the antidepressant findings out to the six month time point191. Functional imaging studies in these patients after treatment revealed decreased cerebral blood flow in key regions, such as the amygdala, which was correlated with decreases in depression rating scores192. Decreases in functional connectivity between the parahippocampus and prefrontal cortex were also correlated with decreases in depression symptoms192. While these findings are not entirely consistent with imaging studies in healthy volunteers following acute psychedelic experiences, they are consistent with reversals of elevations typically found in affective disorders. For example, increased blood flow has been reported in the amygdala of patients with MDD/anxiety193,194, as well as increased hippocampal-prefrontal cortex connectivity195. Although the authors also reported increases in the default mode network

30 in scans completed one day after psilocybin treatment, these increases were not correlated with any behavioral outcomes192, but are consistent with findings in patients following electroconvulsive therapy196. Lastly, post-hoc analyses of the data revealed that intensity of the mystical and spiritual experience during the acute phase of psilocybin treatment were predictive of changes in hippocampal resting state functional connectivity192.

As this last finding highlights, the acute effects of the psychedelic experience cannot be denied. Specifically examining the effects of the experience in this same cohort, Roseman et al97 found a significant correlation between the change in depressive rating score at 5-weeks post-psilocybin treatment and oceanic boundlessness, the ASC measure of mysticism and spirituality, and dread of ego dissolution, the ASC measure of anxiety97. In fact, great care is taken during psilocybin treatment sessions to ensure subjects have positive experiences: subjects undergo preparatory sessions with psychotherapists, therapists are present during the 6 hour treatment session which occurs in a quiet, calm and controlled environment, and post-treatment psychotherapy sessions offered to assist the subjects to “integrate” their experiences119. These costly protocols are in place to maximize the positive experiences, which may have beneficial effects on therapeutic outcomes, and minimize negative psychedelic experiences197. But are these experiences critical to the therapeutic outcome or an unnecessary side effect?

Although traditional SSRIs are not robustly efficacious in treating MDD, when they are successful the antidepressant response occurs without alterations of consciousness. Similar results occur with ECT, DBS and psychotherapy. Ketamine is the only other antidepressant known to induce altered states of consciousness. However, unlike ketamine, psilocybin has a better safety profile. Despite their reputation amongst

31 the general population as being dangerous, classical psychedelics do not lead to addiction, have no reported overdose deaths and have no known long term negative effects on mental health139. If the psychedelic effects of psilocybin can be safely inhibited with ketanserin, a 5-HT2A antagonist safe for human use, without attenuating the antidepressant effects, the cost and burden of treatment with psilocybin would be greatly reduced. As it stands, further studies are needed to elucidate the necessity of the psychedelic experience to the therapeutic potential of psilocybin as well as the underling mechanism of action.

1.2.d. Summary

Much of what we know about the signaling and molecular mechanisms of classical psychedelics come from electrophysiological and radiographic studies using

LSD and DOI. However, the recent human studies reporting therapeutic effects in MDD, end of life anxiety, obsessive compulsive disorder and nicotine cessation have used psilocybin. Psilocybin is more quickly metabolized than LSD and has a shorter

“psychedelic” experience, lending itself nicely to the clinical setting where patients can be treated and observed in a 6-8 hour session. Psilocin has its own unique binding profile

(Table 2) compared to LSD and DOI. Furthermore, rodent behavioral studies have attributed psilocin induced locomotor changes to different serotonergic receptors than

LSD and DOI. The behavioral pattern itself is also different.

Based on the human literature, it is most likely that the hallucinatory experiences of LSD, DOI and psilocybin are mediated by the 5-HT2A receptor. Thus far, findings from cell culture and animal research have supported the integral role of the 5-HT2A receptor in the functional changes psychedelics induce, from synaptogenesis to enhanced

32 neuronal excitability. However, the necessity of the 5-HT2A receptor to the therapeutic effects of psychedelics has yet to be elucidated. Over 15 types of 5-HT receptors have been identified and most psychedelics possess an appreciable affinity for and agonistic properties at more than one receptor or subtype. Many of these receptors are expressed in the hippocampus198.

Consistent with other groups, we have shown that exposure to chronic stress reduces hedonic behavior in rodents. We have also demonstrated that the strength of the temporoammonic-CA1 (TA-CA1) synapse in the hippocampus (Hipp) decreases following chronic stress due to decreases in AMPA expression199. In anhedonic rodents, chronic treatment with SSRIs, but not acute exposure57, restores synaptic strength and hedonic behavior199. The 5-HT1B receptor is necessary for fluoxetine mediated restoration of hedonic behavior in rodents following chronic stress and for fluoxetine and imipramine induced potentiation of the TA-CA1 synapse57. Lastly, we have shown that application of anpirtoline, a 5-HT1B agonist, is sufficient to potentiate the TA-CA1 pathway57.

Within the Hipp, the TA-CA1 pathway is unique in its sensitivity to stress and antidepressant treatment. Unlike the TA-CA1 pathway, the Schaffer collateral pathway is not stress sensitive199. Beyond the TA-CA1 pathway, the Hipp, as a whole has, been repeatedly implicated in the pathophysiology of depression in human and animal studies.

Changes in 5-HTR expression in the hippocampus have been reported patients with

MDD200 and in rodents following stress201. Animal studies have also reported alterations in 5-HTR expression following treatment with SSRIs202. Magnetic resonance imaging studies in humans showed that hippocampal volume is reduced in depression203,204.

Similarly, exposure to stress has been shown to decrease hippocampal volume in rats while treatment with an SSRI counteracts the stress induced reduction205. Presumably, these volumetric and molecular changes in the Hipp contribute to some of the symptoms reported in MDD, specifically memory related symptoms. For example, patients with depression had poorer performance on a spatial memory virtual reality task compared to controls206. Similarly, animals exposed to chronic stress performed more poorly on a water maze task testing spatial memory than controls199.

Stress as a precipitating factor of MDD is often discussed in both clinical and preclinical literature. In fact, many human studies have repeatedly linked actual or perceived stress to the development of depressive symptoms207–209. To better understand the effects of acute and chronic stress scientists have turned to animal models. There are several well characterized animal models of depression that are: known to induce depressive-like features, such as anhedonia, responsive to antidepressant treatment and replicate neurobiological features found in human MDD210. However, all stressors and animal models are not equal. Different types of stress have different molecular and behavioral consequences as described in Yang et al210. With respect to stress and anhedonia, the Thompson lab has repeatedly demonstrated that chronic stress in rodents leads to an anhedonic phenotype, as assayed by loss of preference for various rewards

(sucrose preference, social interaction, scent of female urine)57,199,211,212. The induction of anhedonia in rodents is accompanied by measurable changes in Hipp functioning, namely decreases in synaptic strength in the TA-CA1 synapse described above199,211. Directly connecting the effects of stress to rodent anhedonia, Kvarta et al demonstrated that

34 corticosterone, the rodent analog of the human stress hormone cortisol, is responsible for the Hipp synaptic weakening and loss of sucrose preference following chronic stress213.

Using well characterized animal models to examine the effects of stress and elucidate mechanisms of both known and unknown antidepressant treatments is key to developing effective therapies214. As previously described, Thompson lab has shown that chronic stress, through the effects of corticosterone, induces anhedonia and weakens the

TA-CA1 Hipp synapses. Increasing face validity of this animal model, both behavior and synaptic strength can be restored with chronic SSRI treatment in a manner dependent on the 5-HT1B receptor. In addition to the 5-HT1B receptor, the hippocampus is known to express many 5-HT receptors198. Psilocin is a pan-serotonergic agonist with known antidepressant effects in humans. While the psychedelic effects of psilocin are attenuated by ketanserin, it is unknown if ketanserin prevents the antidepressant effects. I have chosen to use chronic multimodal stress with hedonic behaviors and TA-CA1 synaptic strength as endpoints to test the potential antidepressant effects of psilocybin in rodents and its dependency on the 5-HT2 receptors.

2. Hypothesis Based on the literature presented, I hypothesize that psilocybin has rapid acting antidepressant effects and restores synaptic strength via mechanisms that are independent of 5-HT2 receptor activation.

2.1 Aim 1. Psilocybin will have an anti-anhedonic and synaptic strengthening effects in the rodent chronic stress model of depression.

Prediction 1. Psilocybin will restore hedonic behaviors, assessed by sucrose preference and female urine preference, in male C57 mice following chronic multimodal stress.

Prediction 2. Psilocybin will restore hedonic behaviors in other strains of mice following chronic multimodal stress

Prediction 3. Psilocybin will restore synaptic strength in stress-weakened TA-

CA1 synapses following chronic multimodal stress.

2.2 Aim 2. Psilocybin will have anti-anhedonic and synaptic strengthening effects even when 5HT2Rs are blocked.

Prediction 1. Pretreatment with ketanserin, a 5-HT2A/C antagonist, will not block the anti-anhedonic effects of psilocybin in rodents following chronic multimodal stress.

Prediction 2. Pretreatment with ketanserin will not block psilocybin induced restoration of in synaptic strength following chronic multimodal stress.

2.3 Aim 3. Psilocin will acutely potentiate the TA-CA1 synapse.

Prediction 1. Bath application of psilocin in hippocampal brain slices will potentiate the field EPSP response.

Prediction 2. Ketanserin will not block psilocin’s ability to potentiate the field

EPSP response at the TA-CA1 synapse.

3. General Methods 3.1 Animals All procedures were approved by the University of Maryland Baltimore Animal

Use and Care Committee and were conducted in full accordance with the National

Institutes of Health Guide for the Care and Use of Laboratory Animals.

The male C57Bl/6J mice were used in this study were bred in-house. The

B6129SF1/J male mice were purchased from Jackson laboratory. Mice were 8-weeks old at the start of the experiment, kept on a 12-hour light/dark cycle (lights on at 7am), and provided food and water ad libitum. Animals were group housed prior to the experiment but singly housed at the onset of the behavioral and stress protocols until the end of the study. Mice were assigned to balanced experimental and control groups based on hedonic behaviors assessed after stress.

Male rats used for in vitro electrophysiology experiments were acquired from

Envigo and ranged in age from 4-8 weeks. As with the mice, rats were kept on a 12-hour light/dark cycle and provided food and water ad libitum. Rats were group housed

(n=3/cage) in our in-house animal facility.

3.2 Chronic Multimodal Stress Chronic multimodal stress (CMMS) was used to induce an anhedonic-like phenotype in the animals215. The CMMS protocol consists of 4 hrs/day of restraint stress, in which mice were immobilized in appropriately sized plastic restraint tubes and exposed to strobe lighting and white noise to minimize habituation, for 10-14 consecutive days. Stress was initiated in the morning hours, between 9-10 am, near the onset of the animals’ light cycle. Following stress, rodents were returned to their home cages and singly housed.

3.3 Hedonic behavior Hedonic state was assessed using the sucrose preference test (SPT) and female urine sniff test (FUST) prior to stress (baseline), after 10-14 days of CMMS, and 24hrs after drug injection. For the SPT, mice were exposed to a 2% sucrose solution in their home cages prior to the baseline measurement. The baseline measurements began 1 day later. On each test day, one bottle containing tap water and another bottle containing a

1% sucrose solution were placed in the cages 1-2 hours prior to the onset of the animal’s dark cycle. Mice were free to consume liquid from either bottle for 14-16 hrs, after which bottles were weighed to measure consumption and replaced. The procedure was repeated for a second night with the position of the bottles reversed. Preference is expressed as a percentage and was calculated for each night as (the volume of 1% sucrose solution consumed/total liquid consumed)*100, and the preferences for the two nights were averaged.

For the FUST, mice were individually transferred to empty, freshly made cages and allowed to habituate for 15 mins. A fresh single cotton swab was then affixed to the rim of the cage, such that the tip was within reach of the mouse. 1 hour later, the swab was removed and replaced with 2 swabs spaced apart at the same end of the cage, one soaked in freshly collected urine from male mice and the other with urine from female mice in estrous. Video recording was started and animals were given 3 minutes to interact with the swabs. Videos were later scored by a trained experimenter blinded to the position of male and female urine swabs. Time spent sniffing each swab was recorded and percent preference scored as (time spent sniffing the female urine swab/total time spent sniffing both swabs)*100. The position of the female urine swab was switched between timepoints to account for potential side preference.

As a priori criteria for inclusion in the study, mice had to have a preference for sucrose of >65% at baseline. Mice which displayed a sucrose preference of <70% following CMMS, representing >3 standard deviations less than our historical mean baseline sucrose preference for C57 mice (89.2 ± 6.4% (SD), n=107 animals), were considered stress susceptible. Of those mice, only those displaying a female urine preference of >65% at baseline were included in the FUST arm of the study, accounting for the differences in the reported n’s. Mice having a sucrose preference >65% after 14 days of CMMS were classified as resilient.

3.4. Head twitch response

A distinct quantifiable head twitch response (HTR) or wet-dog shake is produced in rodents following administration of psychedelic compounds152,153. HTR was quantified in the second cohort of C57 mice exposed to CMMS and treated with vehicle-vehicle (n

= 7), ketanserin-vehicle (n = 7), vehicle-psilocybin (n = 7) and ketanserin-psilocybin (n =

8). Immediately following the second injection of either vehicle or psilocybin, mice were placed in their home cage and video recording was initiated with a camera positioned directly above the cage. 3 trained blinded experimenters scored the first 0-15 min of activity for HTR, noting the total number of and time stamp of each head twitch. Time stamps were compared across scorers and head twitches with agreement of at least 2/3 experimenters counted.

3.5 Locomotion A separate cohort of mice (n=12) were tested in a crossover design in an open field for effects of vehicle-vehicle, ketanserin-psilocybin, and vehicle-psilocybin injections on locomotor behavior. Control mice (n=3) received saline vehicle-vehicle

39 injections on both test days. Experimental mice received ketanserin (2 mg/kg)-psilocybin

(5 mg/kg; ket-psil; n = 5) or saline vehicle-psilocybin (5 mg/kg; veh-psil; n = 4) on day 1.

Conditions were switched for testing 3 days later. All animals were returned to their home cage for 60 minutes following the first injection. After the second injections, animals were immediately placed in the open field and video recording initiated. Animals were recorded for 90 minutes in the open field. Following recording on day 1 animals were returned to their home cage. Locomotor measures were scored in realtime by

Cleversys (Reston, Va). After day 2 recording sessions animals were immediately transported for perfusion.

3.6 Electrophysiology: synaptic strength Standard methods were used to prepare 400 µM-thick hippocampal slices.

Briefly, mice were euthanized via exposure to isoflurane followed by decapitation. Brains were excised and the hippocampus was quickly dissected from the brain and sectioned on a Leica VT1200 series vibratome in ice-cold artificial cerebrospinal fluid (ACSF) bubbled with 95% O2/5% CO2. The ACSF contained: 124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 1.5 mM MgSO4, 2.5 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose.

Slices were allowed to recover for a minimum of 60 mins at room temperature in ACSF in a humidified interface chamber before recording.

Use of extracellular recording, rather than whole-cell recording, was chosen for quantification of AMPA:NMDA ratios because of the complications of stress-induced changes in dendritic structure and their electrotonic influence on recordings of distal TA-

CA1 synapses. For quantification of AMPA:NMDA ratios, ACSF was prepared as stated, but without MgSO4, to leave NMDA channels unblocked. Picrotoxin (100µM) and

CGP54626 (2µM) were added to block GABAA and GABAB receptors, respectively. 40

Slices were placed in a recording chamber and perfused with this ACSF (1ml/min) for the duration of the experiment. Glass recording electrodes with resistance of 3-5MΩ were prepared and filled with recording ACSF. These electrodes were placed in stratum lacunosum moleculare (SLM) of area CA1. Concentric bipolar tungsten electrodes were positioned in SLM at least 500µM from the recording electrode to stimulate temporoammonic afferents (TA). Field excitatory postsynaptic potentials (fEPSPs) were acquired using Clampex software (pClamp 10 Series, Molecular Devices), amplified

(x1000, npi electronic), filtered (3kHz), and digitized (10kHz, Digidata 1440a, Molecular

Devices). Slices were stimulated (100µs) at 0.1 Hz at five different intensities ranging from 0.01-1.0 mA, in order to collect a range of responses around a fiber volley (FV) of

0.1 mV. DNQX (50µM) was then washed onto the slice for 15 minutes to block the

AMPA component of the fEPSP and reveal the NMDA component. Five fEPSPs were again collected at the same stimulation intensities recorded prior to DNQX. The NMDAR antagonist D-APV (80µM) was then washed onto the slice for 15 minutes to confirm that the fEPSP response remaining after DNQX was indeed NMDAR-mediated.

AMPA:NMDA ratios of the TA-CA1 fEPSPs were quantified as described previously15 in order to provide a measure of synaptic strength across slices from different mice. All traces at each intensity were first averaged and the amplitude of FVs quantified. The AMPA component of the fEPSP was quantified as the slope over 1.5ms at the earliest part of the linear portion of the response for each stimulation intensity, typically 0.1-2.0ms from its initiation. The NMDA component of the fEPSP slope was quantified over 4ms at the earliest point of the post-DNQX response fully eliminated by

APV. Both AMPA and NMDA slopes were normalized to their respective FVs. For

41 quantification, pairs of responses at the same stimulation intensities were chosen in which the response in the presence of DNQX was closest to 0.1mV in amplitude.

AMPA:NMDA ratios from each slice (1-6/mouse) were averaged to calculate each individual animal’s mean AMPA:NMDA. As an independent measure of synaptic strength, we also computed AMPA:FV and NMDA:FV ratios, calculated from the same pair of responses used for AMPA:NMDA ratios. Experimenters were blind to the treatment condition during quantification and values were confirmed by a second experimenter.

3.7 Drug treatment Psilocybin was obtained from Cayman Chemical (Ann Arbor, MI) and diluted to

1mg/ml in sterile 0.9% saline. Ketanserin (+)-tartrate salt was purchased from

MilliporeSigma (Burlington, MA) and also diluted to 1mg/ml. Ketanserin was administered 60 mins prior to injection with either vehicle control or psilocybin, consistent with previous studies of ketanserin’s ability to block hallucinogenic behavioral responses in humans144 and rodents114. Psilocybin injections were given at 1mg/kg and ketanserin at 2mg/kg, consistent with previous rodent studies113,114,216, or equivalent volumes of saline. I note that these doses are comparable to the oral doses used previously in human studies (psilocybin191 ca. 0.5mg/kg; ketanserin114 ca. 1mg/kg). Each experimental animal received two injections to control for any effects of injection or handling.

3.8 Statistics Statistical analysis consisted of linear regressions, Student’s t-tests, one -, two- and three-way repeated measures ANOVAs using GraphPad Prism 8. Multiple comparisons were corrected in Prism with Tukey’s or Sidak’s multiple comparisons test 42 for the 2-way ANOVA or with Holm-Sidak multiple comparisons corrections performed manually in Excel (Microsoft) for our comparisons of interest for the 3-way ANOVA.

Results from the two cohorts of animals were not statistically different and were therefore pooled. Statistical tests used are indicated within the figure legends. Where indicated, n = number of animals. For acute drug potentiation experiments n = number of slices.

3.9 Electrophysiology: in vitro potentiation

For drug wash-in experiments slices were prepared and electrodes placed as previously described. Once a response with a well differentiated fiber volley was observed, slices were left to acclimate to the recording chamber for 10 minutes. Slices were continuously perfused (1ml/min) with room temperature ACSF with picrotoxin

(100µM) and CGP54626 (2µM). After 10 minutes, stimulation intensity was set to 150% of threshold intensity and remained unchanged once recording was initiated. Slices were stimulated at 0.05 Hz with each stimuli lasting 100 µs. Baseline recordings were obtained for at least 20 minutes before the start of drug wash-in. Compounds washed onto the slices were added directly to the ACSF reservoir and continuously perfused (1ml/min) for the duration of the experiment. For ketanserin versus saline wash-ins, the experimenter was blinded to the drug condition through the experiment until analysis was complete.

All traces were collected and analyzed using Clampex software (pClamp 10

Series, Molecular Devices). The FV was measured and recorded for each trace over the duration of the recording. Similarly, the AMPA component of the response was measured for each trace as the linear slope over a 3ms window starting at the beginning of the response following the FV. Each AMPA measurement was normalized to its respective

FV. All AMPA/FV values were normalized to the mean baseline AMPA/FV and

43 averaged into 1 minute bins. Any slice showing >20% change from beginning to the end of the baseline period was excluded from analysis. For statistical analysis, means of the last 10 minutes of baseline were compared to the last 10 minutes of wash-in.

3.10 Drugs

Psilocin was obtained from Cayman Chemical (Ann Arbor, MI). Psilocin aliquots were prepared with diH20 at 0.5mM and frozen. Due to light sensitivity, psilocin was protected from light during dilution and stored in an opaque box. During electrophysiological recordings, room lights were off and reservoirs wrapped in aluminum foil.

4. Results

4.1. Aim 1. Psilocybin will have an anti-anhedonic effect in the rodent chronic stress model of depression

4.1.a. Prediction 1. Psilocybin will restore hedonic behaviors, assessed by sucrose preference and female urine preference, in male C57 mice following chronic multimodal stress.

As explained in depth in the previous methods section, mice were exposed to

CMMS for 10-14 days. Twenty-four hours after the last day of CMMS, mice were assessed for hedonic behavior via SPT and/or FUST. After the the 2-day SPT, mice had another day of CMMS to space our repeat behavioral assessments and ensure maintenance of the anhedonic phenotype. Four hours after the last CMMS, mice were injected with vehicle-vehicle or vehicle-psilocybin. Inclusion criteria included having a baseline sucrose preference (SP) ≥ 65%. To be categorized as anhedonic or “susceptible,” post-CMMS SP or FUST must have been ≤ 70%. For a graphical depiction of the experimental design, please see Figure 2.

In the first cohort of C57 mice tested, n = 11/26 mice met a priori criteria for anhedonia. Of the n = 26, there was a significant effect of stress (F2,46 = 19.66, p <

0.0001), treatment (F3,23 = 3.92, p = 0.021) and stress x treatment interaction (F6,46 = 6.19, p < 0.0001; Figure 3). Susceptible mice had lower sucrose preference (SP) following 14 days CMMS (p < 0.0001). Treatment with psilocybin increased SP to baseline levels

(baseline v. treatment p = ns) and had no effect on SP in resilient mice.

Figure 2. Experimental timeline

Figure 3. Psilocybin restores sucrose preference following exposure to chronic stress. Susceptible animals (n = 11) showed a significant decrease in sucrose preference (p < 0.0001) compared to resilient animals following CMMS exposure. Following treatment with psilocybin (n = 5), sucrose preference in susceptible animals increased to baseline levels (baseline v treatment, p = ns) while vehicle treated animals (n = 6) continued to demonstrate decreased sucrose preference (baseline v treatment, p = 0.022). Resilient animals had no change in sucrose preference from baseline regardless of treatment. The height of the bars represent group means and the error bars illustrate standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

To replicate our findings and expand our behavioral assessment of anhedonia, a second cohort of C57 mice (n=14) were obtained and exposed to a similar CMMS protocol. SPT and FUST were assessed at baseline, post stress and post treatment. Stress decreased SP (F2,24 = 34.79, p < 0.0001), but there was no effect of treatment (F1,12 =

0.11, p = 0.075) and no stress x treatment interaction (F2,24 = 1.57, p = 0.23). However, psilocybin significantly increased SP from post-stress (p = 0.008) while vehicle had no effect on post-stress SP (p = 0.069; Figure 4a). There were no differences at baseline or post-CMMS exposure in susceptible mice from cohort 1 and 2 (Table 3). As with cohort

2, when the cohorts were combined, there was a significant effect of stress (F2,46 = 48.27, p < 0.0001). Psilocybin increased SP to baseline levels (baseline v. psilocybin, p = ns; stress v. psilocybin, p < 0.0001), while vehicle treated animals maintained low SP following injection (baseline v. vehicle, p = 0.0002; stress v. vehicle, p = ns; Figure 4b).

Figure 4. Psilocybin increases sucrose preference following CMMS; a replication. A) In a second cohort of mice, psilocybin (n = 7) increased sucrose preference following CMMS (p = 0.0079), while vehicle treatment (n = 7) had no effect on sucrose preference following CMMS (p = 0.69). B) When susceptible animals from cohort 1 and 2 were combined, vehicle treated animals persistently demonstrated decreased sucrose preference (p = 0.0002) while psilocybin treated animal had sucrose preference levels no different from baseline (p = 0.11). The height of the bars represent group means and the error bars illustrate standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

Timepoint Comparison t-value df p-value Baseline 1.87 10 0.09 Vehicle-Vehicle Stress 1.05 10 0.32

Vehicle- Baseline 0.2 11 0.85 Psilocybin Stress 0.39 11 0.7 Table 3. No differences in Sucrose Preference Test between cohorts at baseline or post- CMMS.

As predicted, exposure to CMMS reduced preference for female urine (FU; F2,18 =

24.29, p < 0.0001). There was a significant stress x treatment interaction (F2,18 = 6.26, p

= 0.0086). Psilocybin (n = 5) increased preference for FU following stress (p < 0.0001), while vehicle (n = 6) had no effect (p = 0.30; Figure 5).

From these results, I conclude that psilocybin restores hedonic behavior to baseline levels following CMMS.

Figure 5. Psilocybin increases preference for female urine scent following chronic multimodal stress. Exposure to CMMS significantly decreased the preference mice had for female urine (p < 0.0001). Psilocybin treated animals (n = 5) had higher preference for female urine than after CMMS (p < 0.0001). Vehicle treated animals (n = 6) had no change in female urine preference between CMMS and treatment (p = ns). The height of the bars represent group means and the error bars illustrate standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

4.1.b. Prediction 2. Psilocybin will restore hedonic behaviors in other strains of mice following chronic multimodal stress

To see if psilocybin would have antidepressant-like effects in other mouse strains,

I ran a cohort of B6129SF1/J male mice through the previously described protocol

(Figure 2). As with the C57 mice, CMMS exposed hybrid mice demonstrated a significant decrease in SP (F2,26 = 27.01, p < 0.0001; Figure 6) but at a higher rate than

C57: n=15/20 met SP ≤ 70% criteria for anhedonia. However, both vehicle (n = 6, p =

0.013) and psilocybin injected mice (n = 9, p = 0.026) showed increased SP. These data suggest CMMS exposure in B6129SF1/J male mice does not produce lasting changes in hedonic behavior consistent with the well documented anhedonic phenotype seen in C57 following chronic stress exposure.

Figure 6. Psilocybin does not have an effect on sucrose preference in B6129/F1 following chronic multimodal stress. Exposure to CMMS decreases sucrose preference in B6129/F1 mice (p < 0.0001). Sucrose preference increased from CMMS following treatment with vehicle (n = 6, p = 0.013) or treatment with psilocybin (n = 9, p = 0.026). Sucrose preference for vehicle treated animals was not different from baseline (p = 0.12), but psilocybin treated animals had lower sucrose preference (p = 0.026). The height of the bars represent group means and the error bars illustrate standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

4.1.c. Prediction 3. Psilocybin will restore synaptic strength in stress-weakened TA-CA1 synapses following chronic multimodal stress.

Having investigated the behavioral effects of psilocybin following CMMS, I next asked if psilocybin would have a measurable electrophysiological effect. Following the final assessment of hedonic behavior, animals were sacrificed, and hippocampal brain tissue was prepared to measure local excitatory field potentials (fEPSPs) as a proxy for synaptic strength. As with SP, there were no differences between cohorts in matched treatment group in AMPA:NMDA (vehicle: t = 0.83, df = 10, p = 0.43; psilocybin: t =

1.35, df = 11, p = 0.20; Figure 7). Therefore, cohorts were combined. Anhedonic animals injected with psilocybin (1 mg/kg) had significantly higher TA-CA1 fEPSPs than vehicle controls (t = 4.15, df = 23, p = 0.0004; Figure 7). Thus, I conclude that along with hedonic behavior, psilocybin restores TA-CA1 synaptic strength following CMMS.

Figure 7. Psilocybin increases TA-CA1 synapses. A) Psilocybin (n=13) increased the AMPA:NMDA ratio in anhedonic animals compared to vehicle controls (n = 13; p = 0.0004). Individuals data points represent AMPA:NMDA for each animal. B) There were no differences in synaptic strength between cohorts in either treatment group. Group mean +/- SEM are also represented. *** p < 0.001, ns = not significant

4.2 Aim 2. Psilocybin will have anti-anhedonic and synaptic strengthening effects even when 5HT2Rs are blocked.

4.2.a. Prediction 1. Pretreatment with ketanserin, a 5-HT2A/C antagonist, will not block the anti-anhedonic effects of psilocybin in rodents following chronic multimodal stress.

Here, I tested if blocking activation of 5-HT2Rs would prevent psilocybin from restoring SP or FU preference. As in Aim 1, CMMS was used to induce an anhedonic phenotype in mice. To determine the role of the 5-HT2R in the anti-depressant like response of psilocybin, ketanserin (2 mg/kg), a 5-HT2A/C antagonist, was administered

60 minutes prior to either vehicle or psilocybin injection (Figure 2). Ketanserin injected animals were compared to vehicle injected animals described in Aim 1. Exposure to

CMMS decreased SPT across all groups (F2,68 = 60.26, p < 0.0001; Figure 8). There was a significant CMMS x Psilocybin interaction (F2,68 = 4.50, p = 0.015; Figure 8).

Psilocybin increased SP compared to post-stress levels regardless of pretreatment with vehicle (n = 13, p = 0.002) or ketanserin (n = 7, p = 0.04).

Figure 8. Ketanserin does not block psilocybin's antidepressant-like effect. Psilocybin increased sucrose preference (SP) in anhedonic mice pre-treated with ketanserin (n = 7, p = 0.04). Neither, ketanserin alone (n = 6, p = 0.87) nor vehicle alone (n = 12, p = 0.078) had an effect on SP following CMMS exposure. The height of the bars represent group means and the error bars illustrate standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

As with SPT, exposure to CMMS decreased preference for FU across all groups

(F2,30 = 43.41, p < 0.0001; Figure 9). There was a significant stress x psilocybin interaction (F2,30 = 4.26, p = 0.024). Multiple comparisons revealed significant increases in FU preference following psilocybin injection in mice pre-treated with vehicle (p =

0.0012) or ketanserin (p = 0.0024). Anhedonic mice injected with vehicle alone (p =

0.43) or ketanserin alone (p = 0.072) did not recover FU preference following CMMS exposure.

To further confirm the effects of psilocybin, a 2-way ANOVA with ketanserin and psilocybin as factors was run on the post-treatment behavioral data. Psilocybin increased hedonic behavior compared to vehicle treated rodents (Figure 10). Looking at the final SPT data, there was a significant effect of psilocybin (F1,34 = 5.83, p = 0.021) but no effect of ketanserin (F1,34 = 0.72, p = 0.40). There was also a significant effect of psilocybin on final FUST results (F1, 15 = 12.15, p = 0.0033) and no effect of ketanserin

(F1, 15 = 0.56, p 0.47).

Figure 9. Pre-treatment with ketanserin does not prevent psilocybin induced recovery of Female Urine Preference. Exposure to CMMS significantly reduced preference for FU across all groups: vehicle- vehicle (n = 6, p = 0.013), vehicle-ketanserin (n = 4, p = 0.0012), vehicle-psilocybin (n = 5, p = 0.0012), ketanserin-psilocybin (n = 4, p = 0.0012). Psilocybin increased FU preference following CMMS in mice pre-treated with vehicle (p = 0.0012) or ketanserin (p = 0.0024). Mice injected with vehicle after pretreatment with vehicle (p = 0.043) or ketanserin (p = 0.072) had FU preference no different from post-stress preference. The height of the bars represent group means and the error bars illustrate standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

Figure 10. Psilocybin treatment increases hedonic behavior compared to vehicle treated rodents. Examining just the post-treatment hedonic behavior shows psilocybin increased (A) sucrose preference (p = 0.021) and (B) female urine preference (p = 0.0033) regardless of pretreatment.

The mouse head twitch response (HTR) has been used to measure the hallucinatory effect of psychedelic compounds153,154,217,218. Head twitches were quantified in a subset of mice across all 4 experimental groups. There was a significant increase in

HTR in mice injected with vehicle then psilocybin (n = 7; F3,25 = 6.96, p = 0.0015; Figure

11a). Mice injected with ketanserin prior to receiving psilocybin (n = 8) had HTR comparable to those exhibited by vehicle controls (n = 7; p = 0.40) or ketanserin controls

(n = 7; p = 0.26; Figure 11a), which, as predicted, had none or few head twitches quantified.

To see if the number of head twitches had an effect on SPT, I divided the animal injected with psilocybin into high (HTR > 7) and low (HTR < 7) HTR groups (threshold was based on the maximum number of HTR observed in control animals). A 2-way repeated measures ANOVA revealed no effect of HTR (F1,12 = 0.53, p = 0.48) but a significant effect of time (F2,24 = 38.9, p < 0.0001). Following CMMS, increased sucrose preference was seen in both HTR > 7 (p = 0.0005) and HTR < 7 (p = 0.038) groups

(Figure 11b). There were no significant correlations between number of head twitches and final SPT (R2 = 0.094. p = 0.29) or FUST (R2 = 0.23, p = 0.19; Figure 11c). These data suggest 5-HT2A activation does not account for the variance seen in restoration of hedonic behavior following CMMS in psilocybin treated animals.

Figure 11. Head twitches in mice do not effect Sucrose or Female Urine Preference following chronic stress. A) Mice injected with vehicle 60 minutes prior to psilocybin (n = 7) exhibited significantly more head twitches than control mice injected with vehicle alone (n = 7, p = 0.0045) or ketanserin alone (n = 7, p = 0.0022). The number of head twitches in mice injected with ketanserin (n = 8) 60 minutes prior to psilocybin was not different from either vehicle controls (p = 0.40) or ketanserin-vehicle (p = 0.26). B) There was no effect of head twitches (p = 0.48) in mice injected with psilocybin, regardless of pretreatment condition. Sucrose preference was significantly increased following chronic multimodal stress in high head twitch mice (p = 0.0005) and low head twitch mice (p = 0.038). C) Head twitches were not correlated with sucrose preference (y = 0.54x + 64.70, R2 = 0.094, p = 0.29) or female urine preference (y = 0.82x + 71.03, R2 = 0.23, p = 0.19). Data shown in panels A and B are the individual data points along with the group means +/- SEM. Panel C includes individual data points per animal. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

To further demonstrate that ketanserin was still active at the time of psilocybin injection, behavior in an open field was analyzed for 90 minutes over 30 minute bins for distance travelled and time in center. With regard to distance, there was a significant interaction between time x treatment (F4,42 = 3.17, p = 0.023; Figure 12a) with ketanserin

(2 mg/kg) pretreated mice exhibiting hypolocomotion compared to vehicle-control mice

(p = 0.0019) and psilocybin (5 mg/kg) injected mice (p = 0.0059) at the 0-30 minute time point. Both vehicle-psilocybin (p = 0.0050) and ketanserin-psilocybin (p = 0.0008) were hypolocomotive compared to control mice at the 30-60 minute timepoint (Figure 12a).

There was a significant effects of treatment on duration of time spent in the center of the arena (F2,21 = 7.085, p = 0.0045; Figure 12b). Mice pretreated with ketanserin spent less time in the center of the arena compared to control mice at the 0-30 minute (p = 0.017) and 30-60 minute (p =0.0001) timepoints. Mice injected with psilocybin alone displayed decreased center time compared to control mice at the 30-60 minute (p = 0.0077) timepoint (Figure 12b). These locomotion findings are consistent with our predictions based on the literature and suggest ketanserin is present and physiologically active 60 minutes after injection at the time psilocybin is administered.

Despite pretreatment with ketanserin, psilocybin restored hedonic behavior following CMMS. To confirm the expected activity of ketanserin 60 minutes after administration, psilocybin induced head twitches and changes in locomotion were analyzed. As expected, the HTR was increased following psilocybin injection in mice pretreated with vehicle but not ketanserin. Ketanserin pretreated mice showed more hypolocomtion 0-30 minutes after psilocybin injection than vehicle pretreated mice, indicating ketanserin activity at the time of psilocybin injection.

Figure 12. Ketanserin pretreatment promotes hypolocomotion compared to Psilocybin and Vehicle control conditions. A) Mice (n = 9) injected with ketanserin (ket; 2 mg/kg) 60 minutes prior to receiving a psilocybin (psil; 5 mg/kg) injection were hypolocomotive during the 0-30 minute time period compared to vehicle-psil (veh-psil; n = 9; p = 0.0059) and veh-control (n = 6; p = 0.0019) injected animals. Both ket-psil (p = 0.0008) and veh-psil (p = 0.0050) mice travelled less than control mice during the 30-60 minute time period. There were no differences between treatment groups are the 60-90 minute time point. B) Mice pretreated with ketanserin before psilocybin, spent less time in the center of the arena compared to controls (p = 0.017) at 0-30 minutes. Both ket-psil (p = 0.0001) and veh-psil (p = 0.0077) injected mice spent less time in the center of the arena, compared to controls, during the 30-60 minutes of testing. There were no differences between groups at the 60-90 minute timepoint. Figures displays the group means +/- SEM at each time bin. **p < 0.01 ket- psil v. veh-psil. ##p < 0.01 and ###p < 0.001 control comparisons.

4.2.b. Prediction 2. Pretreatment with ketanserin will not block psilocybin induced restoration of synaptic strength following chronic multimodal stress.

Next, I tested if activation of the 5-HT2Rs was necessary for psilocybin to restore synaptic strength. Animals were sacrificed following final hedonic assessment of SP and brain slices prepared for electrophysiological measurement of hippocampal synaptic strength, as described in depth in the methods section. A comprehensive analysis of all groups revealed a significant effect of psilocybin (F1,34 = 34.79, p < 0.0001; Figure 13).

Multiple comparisons revealed significant differences between ketanserin-vehicle and ketanserin-psilocybin (p = 0.0002) and vehicle-vehicle and vehicle-psilocybin (p =

0.0003) groups (Figure 13).

The increases in synaptic strength were driven by increases in the AMPA component of the fEPSP. A 2-way ANOVA of the AMPA component normalized to fiber volley (FV) revealed a significant effect of psilocybin (F1,34 = 4.38, p = 0.044, Figure

14A). However, there was no effect of psilocybin across groups looking at either

NMDA/FV (F1,34 = 2.077, p = 0.16; Figure 14B) or FV alone (F1,34 = 0.29, p = 0.59;

Figure 14C).

Figure 13. Ketanserin pretreatment does not block psilocybin from increasing synaptic strength. Pretreatment with ketanserin, 60 minutes prior to injection of psilocybin, does not block psilocybin induced increases in synaptic strength (p < 0.0001). Vehicle-psilocybin (n = 13) injected animals had significantly higher AMPA:NMDA than vehicle-vehicle controls (n = 12, p = 0.0003). Ketanserin-psilocybin (n = 7), injected animals also had significantly higher AMPA:NMDA than ketanserin-vehicle (n = 6, p = 0.0002). Individual data points per animal are shown along with group means +/- SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

Figure 14. Psilocybin increases the AMPA/FV component of fEPSP. A) Psilocybin treated mice had higher AMPA/FV than vehicle treated mice (p = 0.044). B) Psilocybin treatment had no effect on the NMDA/FV component of the fEPSP (p = 0.016). C) There was no effect of psilocybin on fiber volley (p = 0.59). Individual data points per animal are shown along with group means +/- SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns = not significant

Looking at the behavior and electrophysiology together, there was a significant correlation between the change in SP between baseline and final SP and synaptic strength

(R2 = 0.18, p = 0.0072; Figure 15). The difference from baseline SP was used to emphasize the stress induced changes in hedonic behavior. In this metric, a small change in SPT from baseline indicates restoration of hedonic behavior while a larger change is indicative of anhedonia or ineffective antidepressant response.

Figure 15. Synaptic strength is correlated with changes in Sucrose Preference. Changes in sucrose preference between baseline and post-treatment measurements are significantly correlated with TA-CA1 synaptic strength (y = 1.725x – 44.06, R2 = 0.18, p = 0.0072).

4.3 Aim 3. Psilocin will acutely potentiate the TA-CA1 synapse.

4.3.a. Prediction 1. Bath application of psilocin in hippocampal brain slices will potentiate the field EPSP response.

Physiologically relevant concentrations of psilocin were washed on to hippocampal tissue for 45 minutes. There was no change in the fEPSP from baseline after addition of 10 nM psilocin to the acsf (n = 8; t = 0.31, df = 7, p = 0.77; Figure 16a).

There was also no effect on fEPSP after increasing psilocin concentration to 30 nM (n =

5; t = 1.26, df = 4, p = 0.28; Figure 16b). Lastly, there was no change in fEPSP following

45 minutes of 100 nM psilocin wash-in (n = 29; t = 0.82, df = 28, p = 0.42; Figure 16c).

Figure 16. Physiologically relevant doses of psilocin did not potentiate the TA-CA1 synapse.

A) Washing on 10 nM of psilocin for 45 minutes had no effect on the fEPSP (n = 8; p = 0.77). B) Addition of 30 nM psilocin also had no effect on the fEPSP (n = 5; p = 0.28). C) 100 nM of psilocin washed onto the slice for 45 minutes had no effect on the fEPSP (n = 29; p = 0.42). Data presented over time illustrates the mean TA-CA1 fEPSP per minute normalized to baseline +/- SEM. Group data represent mean TA-CA1 fEPSP normalized to baseline per slice taken over the last 10 minutes of baseline or psilocin wash on +/- SEM. The n = number of slices averaged. Data represented over time are the mean of the AMPA/FV components per minute of wash-in of the fEPSP normalized to the mean baseline AMPA/FV +/- SEM. Group data are the individual means for each slice over the last 10 minutes of baseline or drug wash-in period.

4.3.b. Prediction 2. Ketanserin will not block psilocin’s ability to potentiate the field

EPSP response at the TA-CA1 synapse.

Ketanserin (10 µM) was washed onto the slice after baseline for 10 minutes prior to the addition of psilocin (100 nM) to elucidate the role of 5-HT2Rs. Ketanserin had no impact on the effects of psilocin on fEPSP (n = 13; t = 1.43, df = 12, p = 0.18; Figure

17). However, there was a significant difference between baseline and post-psilocin in the slices treated with vehicle (n = 10; t = 5.032, df = 9, p = 0.0007). There was no difference between mean fEPSPs from the last 10 minutes of ketanserin + psilocin and vehicle + psilocin (n = 10) treated hippocampal slices (t = 1.46, df = 21, p = 0.16; Figure

17). Baseline fEPSPs did not differ between groups (t = 0.35, df = 21, p = 0.73).

Figure 17. Ketanserin does not alter the effect of psilocin on TA-CA1 fEPSP. There were no differences in fEPSPs after 45 min wash-in of ketanserin (10 µM) + psilocin (100 nM; n = 13) or vehicle + psilocin (100 nM; n = 10; p = 0.16). There were also no differences between groups in baseline fEPSPs (p = 0.73). There was a significant difference between the last 10 minutes of baseline compared to the last 10 minutes of vehicle+psilocybin wash (p = 0.0007) Data represented over time are the mean of the AMPA/FV components per minute of wash-in of the fEPSP normalized to the mean baseline AMPA/FV +/- SEM. Group data are the individual means for each slice over the last 10 minutes of baseline or drug wash-in period.

5. Discussion

5.1 Psilocybin has an anti-anhedonic like effect in the rodent chronic stress model of depression.

To the best of my knowledge, this is the first data suggesting the classical psychedelic psilocybin has rapid antidepressant-like effects in the chronic stress model of anhedonia in C57/B6J mice. The behavioral effects of psilocybin were first seen 24 hours after injection with FUST (Figure 5) and 48 hours after injection with the SPT (Figure 4).

Previous studies have used the FST to assess the antidepressant like effects of psilocybin.

One study using the Wistar-Kyoto rat strain reported decreased immobility in the FST, a marker of the antidepressant effect, 7 days after a single psilocybin (1 mg/kg) administration compared to saline which persisted for 35 days113. A separate study reported no effects of psilocybin (0.05 or 2.0 mg/kg) on immobility time in FST in either the flinders resistant or flinders sensitive rat strain216. Decreased immobility time in FST was also not seen with a psilocin rich alkaloid extract injected 30 minutes prior in NMRI mice219.

A major problem reconciling these data are the different timepoints and strains used to assess the effects of psilocybin. Each study reported FST findings at different time points ranging from 30 minutes post injection219, to 4-24 hours216 or 7 to 35 days113.

The one human study reporting the antidepressant effects of psilocybin in treatment resistant depression assessed these effects 7 days post psilocybin administration190. While the Hibicke et al.113 findings are consistent with these timepoints, we know from previous studies using SSRIs that the timing of effects in the FST may not directly correlate to the timeline of therapeutic efficacy in humans214. The timepoints reported here are consistent

73 with our previous findings using other compounds known to elicit an antidepressant response in rodents following chronic stress211,220. The choice of C57/B6 mouse strain was also chosen based on the published findings that chronic stress decreases in TA-CA1 synaptic strength are correlated with SP199. The flinders sensitive line, for example, used in Jefsen et al.216 is known to have decreased expression of 5-HT2AR221,222, which could explain the null result if 5-HT2AR is necessary for psilocybin to decrease immobility time in FST. Similar to the strain inconsistency, I found no effect of psilocybin in

B6129/F1 mice, a mouse strain reported to have higher sensitivity to stress than C57/B6 mice223. Unlike the unstressed animals usually used in FST studies, the mice I used to demonstrate the effects of psilocybin were exposed to CMMS and required to demonstrate an anhedonic phenotype. The 65% threshold dividing preference from anhedonia in the SPT and FUST was based on previously published findings211 and reward preference data generated from many cohorts of animals run through chronic stress exposure in the Thompson lab. Stress based animal models of depression provide greater validity when it comes to modeling complex human behaviors214. Generally, like humans, rodents reliably demonstrate preference for rewarding experiences under baseline conditions. Similar to stressful life-events preceding MDD or precipitating a major depressive episode, exposure to chronic but not acute stress decreases the rodent response to rewarding stimuli215,224,225. Furthermore, unlike the FST where the response to SSRIs may be observed minutes after acute administration226, SSRIs must be administered chronically following chronic stress induced anhedonia for restoration of hedonic preference to be observed57,212. Hence, the time-courses of the development of

74 phenotypic anhedonia and length of treatment with SSRIs to remission is more consistent with stress-based models of depression than those demonstrated by FST.

5.2. Psilocybin has an anti-anhedonic effect even when 5HT2Rs are blocked.

Pretreatment with ketanserin, a 5-HT2A/C antagonist, did not prevent psilocybin from restoring hedonic behavior assessed via SPT (Figure 8) or FUST (Figure 9). While this is consistent with my hypothesis, it is inconsistent with what might be predicted based on human studies linking the 5-HT2AR receptor with the psychedelic experience97,167,227. A recent PET study demonstrated the positive non-linear relationship between psilocin plasma levels, 5-HT2AR occupancy and intensity of hallucinations227.

Further analysis of the psilocybin treated TRD cohort revealed that the positive quality of the individuals’ experience could predict improvements in depression scores97. Lastly, ketanserin has been shown to block the mind-altering effects of psilocybin in healthy controls167. Although the 5-HT2AR may be necessary for the psychedelic experience, it is not clear if it is necessary for the antidepressant effect. My data, in a preclinical rodent model of depression, suggest it might not be.

Psilocybin is recognized as a pan-serotonergic agonist (Table 2). Rodent locomotor studies have implicated other 5HTRs, such as 5HT-2C and 5-HT1A, in psilocybin’s actions159. This finding agrees with what is known about the binding profile of psilocin. Outside of 5-HT2Rs, psilocin has a high affinity for 5-HT1Rs. Agonism of

5HT-1B has been shown to produce reductions in immobility in the FST similar to tricyclic antidepressants228,229, while antagonism blocked paroxetine and imipramine from increasing mobility time in the tail suspension test229. The 5HT-1BR is also necessary for fluoxetine to exert an antidepressant response in mice following chronic

75 stress57. The mind-altering effects of psilocybin may be mediated through the 5-HT2R, but its antidepressant actions may occur via 5-HT1R activation.

The time between ketanserin and psilocybin injections was carefully chosen based on previous studies in rodents114,217 and humans167. But were the 5-HT2Rs actually blocked at the time of psilocybin injection? To ensure ketanserin’s efficacy, I measured behaviors known to be sensitive to psilocybin and psilocybin + ketanserin. As previously described, the rodent HTR has been used to test the hallucinogenic properties of known and novel psychedelic compounds since the 1960s152. Psilocybin induces HTR in wild- type mice over a range of doses from 0.3 mg/kg to 4.8 mg/kg, but not in 5-HT2A knockout mice159. The number of head twitches observed in the ketanserin-psilocybin injected animals were not different from control animals, but vehicle-psilocybin injected animals had a significantly HTR than controls (Figure 11). Psilocybin (4.8 mg/kg) is also known to have effects on locomotion, decreasing the distance travelled, between 20-40 minutes after injection, and center duration, between 0-60 minutes, in a novel open field environment159. Ketanserin alone also has hypolocomotive effects compared to saline vehicle controls230. In a separate cohort of unstressed mice, mice treated with ketanserin

60 minutes prior to psilocybin (5 mg/kg) demonstrated hypolocomotion and decreased center time at 0-30 minutes compared to vehicle-psilocybin or saline control injected animals (Figure 12).

These findings are consistent with ketanserin’s known effects on psilocybin, suggesting that 5-HT2Rs are antagonized at the time of psilocybin injection and for at least 30 minutes after. Unfortunately, I cannot confirm that ketanserin antagonized 5-

HT2Rs for the duration of time of psilocybin’s activity. The mouse behaviors I was able

76 to observe and quantify are only a proxy for the human psychedelic experience. Future pharmacology studies measuring the concentrations overtime of ketanserin and psilocin in the mouse brain are needed. However, human studies have shown that 60 minute pretreatment with ketanserin effectively blocks the mind altering effects of psilocybin and attenuates psilocybin EEG changes166.

5.3 Psilocybin increases TA-CA1 synaptic strength following CMMS

Consistent with my prediction, I have shown that psilocybin increases TA-CA1 synaptic strength following CMMS (Figure 7). Furthermore, pretreatment with ketanserin did not prevent psilocybin from increasing synaptic strength (Figure 13). The TA-CA1 is a unique hippocampal region in that it is stress sensitive199,213 and responsive to treatment199,211. We have shown that exposure to chronic stress199 or corticosterone213, the endogenous stress hormone, decreases synaptic strength, measured by AMPA:NMDA, through decreases in the AMPA component of the fEPSP response. These decreases are correlated with sucrose preference199,213, my primary measure of hedonic behavior, and known to be attenuated by treatment with standard SSRIs199 and a novel rapid-acting antidepressant211. Similarly, I have found lower AMPA:FV in untreated stressed animals compared to psilocybin injected animals with recovered SP (Figure 14). I have also reported a significant correlation between the change in SP from baseline and synaptic strength (Figure 15).

Various rodent stress models have been shown to produce morphological and functional changes in depression relevant brain regions that are consistent with human literature231. Chronic but not acute stress, for example, is known to decrease the size of the dendritic arborization of hippocampal CA1 pyramidal neurons232,233. Similar findings

77 have been found following chronic restraint stress in the medial prefrontal cortex234,235. In agreement with these findings, human MRI studies have repeatedly found decreases in hippocampal and prefrontal cortical volumes in MDD236,237. These structural changes are consistent with the decreases in TA-CA1 AMPA:NMDA we have seen historically199,211,213 and I have reported here. Extending beyond the hippocampus, in addition to inducing an anhedonic phenotype in mice, chronic stress has been shown to decrease hippocampal input to the nucleus accumbens, a key node in the mesolimbic reward pathway, while successful SSRI treatment restores hippocampal input to the accumbens212. Analogous findings of decreased activity in the nucleus accumbens have been reported in human anhedonia238. Together the data implicate a hypofunction in excitatory neurotransmission in anhedonia following exposure to chronic stress.

Unifying data from human and animal studies of stress and depression, the excitatory synapse hypothesis of depression posits a global hypofunction of glutamatergic neurotransmission underlying MDD239. Increased BDNF levels and synaptic plasticity following SSRI administration in vivo and in vitro support this hypothesis240. As does the finding that ketamine, a drug modulating glutamatergic activity, has an antidepressant effect in rodents220,241 and humans60. Ketamine has also been used to reverse chronic stress induced decreases in spine density and neuronal activity in the PFC which occur with anhedonia241. When administered at subanesthetic doses, ketamine increases glutamate levels and gamma oscillatory power in the PFC242. High frequency gamma activity drives LTP, a form of synaptic strengthening which may compensate for hypoactivity or weakened synapses found following stress in animals displaying an anhedonic phenotype243,244. But not all antidepressants induce increases in gamma power.

Neither psilocybin nor SSRIs increase gamma oscillations, but both are known to decrease alpha activity166,245.

If global decreases in excitatory transmission are underlying depression, then drugs promoting excitation and synaptic strengthening should have therapeutic effects.

This occurs slowly when endogenous 5HT levels are modulated with traditional antidepressants like SSRIs. It can also occur more rapidly with ECT or DBS. Ketamine is the only approved drug known to improve depressive symptoms within hours. But the short lived effects of ketamine suggest its mechanisms of action may be different. The therapeutic time course for psilocybin is longer than ketamine (7 days) but much shorter than SSRIs. Its antidepressant effects, however, have been reported as far out as 3-6 months, which is far beyond the 14 days reported for ketamine. The changes in AMPA signaling I demonstrated with psilocybin are consistent with those following chronic

SSRI treatment. But how these changes are initiated and maintained remain unknown.

Future studies should explore psilocybin’s effects on specific signaling cascades implicated in depression and the sustained antidepressant response. Using 5-HT1B knock out mice, Cai et al57 has shown that the 5-HT1B receptor is necessary for fluoxetine to exert an antidepressant response in mice following chronic stress. My data suggest the 5-

HT2A/C receptor may not be necessary for psilocybin’s antidepressant effects following chronic stress. Psilocin has appreciable affinities for the 5-HT1Rs (Table 2). In fact,

Halberstadt et al have linked some of the behavioral effects of psilocin to 5-HT1Rs159. It is possible that 5-HT1Bs are necessary for psilocybin to exert antidepressant effects in mice following chronic stress. Investigating the synaptic and behavioral effects of psilocybin in the 5-HT1B knockout mouse would be incredibly informative. Similarly,

79 replicating my experiments using the 5-HT2A knockout mouse156 would further support my conclusions that psilocybin’s antidepressant effects are independent of 5-HT2Rs.

5.4. Psilocin failed to acutely potentiate the TA-CA1 synapse.

Acute application of psilocin did not potentiate the TA-CA1 local field potential as predicted. Although there was a slight significant depression with the addition of vehicle + 100 nM psilocin, this is likely due to slice instability than the effect of psilocin alone since there was no difference between the final fEPSP mean magnitude between the ketanserin+psilocin and vehicle+psilocin slices. It was previously shown that acutely washing imipramine (2 µM), a TCA, fluoxetine (20 µM) or citalopram (10 µM), both

SSRIs, potentiates the TA-CA1 response by increasing the amplitude of the AMPA component of the response in a 5-HT1B dependent manner57. It was also shown that a 5-

HT1B agonist alone potentiates the TA-CA1 response in both stressed and unstressed mice, with greater potentiation seen in stressed mice57. However, direct application of 5-

HT to hippocampal brain slices induces depression of the TA-CA1 synapse246. This difference may be explained by location, as 5-HT elevations under SSRI application are more synapse specific whereas bath application of 5-HT increases the general concentration. It could also be due to regional concentration differences between endogenous 5-HT availability versus the general exogenous increases that occur during drug wash in experiments.

Psilocin is known to be unstable in solution, rapidly oxidizing at the 4-OH position247. When psilocin is prepared for injection, it is mixed with tartaric acid and kept at a pH below 7 to increase stability159,247. We cannot mimic these conditions during acute electrophysiology experiments because it would jeopardize the health of the brain

80 tissue. Therefore, before concluding that psilocin has no acute effects on hippocampal tissue, I wanted to be sure sufficient levels of psilocin were reaching the tissue to induce a response. Partnering with Dr. John Hamlyn, psilocin concentrations were measured at various time points over the course of the electrophysiological experiment. Mass spectrometry data demonstrated that although adequate amounts of psilocin were added to the ACSF reservoir at the beginning of the experiment (Figure A1), almost none was present after 60 minutes (Figure A2). Furthermore, there was a difference in concentration levels between the reservoir and the eluate at the same timepoint, suggesting an appreciable amount of psilocin is taken up by the tissue, lost to adherence to plastic tubing in the apparatus or both (Figure A3). Future studies should utilize higher concentrations of psilocin to compensate for the rapid oxidation and differential concentrations between the reservoir and eluate. Direct infusion of psilocin to the recording chamber, bypassing the reservoir may also help prevent degradation, as well as utilization of alternate ACSF preparations containing no or lower levels of sodium bicarbonate. Ultimately, if these methods produce appreciable psilocin induced potentiation or depression, further mass spectrometry studies will be needed to confirm the presence and quantify the concentration of psilocin over the duration of the experiment.

5.5. Summary

Having received “break through” therapeutic status from the FDA, psilocybin is poised to become the first psychedelic compound approved for treatment of MDD248.

Following Carhart-Harris et al’s ground-breaking study reporting rapid acting antidepressant effects in a cohort of patients with treatment resistant depression190, more

81 carefully controlled double blinded studies attempting to replicate the findings and further understand the time-course of the antidepressant effect and mechanism are underway. The greatest limitation of psilocybin treatment is the psychedelic effects, the alterations of consciousness that last 3-6 hours108. Because of these side effects, Johnson et al197 recommend that psilocybin treatment sessions be conducted in a carefully controlled environment with psychological support before, during and after psilocybin administration. These factors greatly increase the potential cost of psilocybin therapy and patient burden. It also limits the potential generalizability and availability of treatment to patients suffering from MDD. If, however, the psychedelic effects were mostly eliminated, there might no longer be a need for intensive psychological support in a controlled environment for up to 6 hours during psilocybin treatment, making psilocybin therapy more affordable and accessible.

To date, most of the preclinical and human studies of psilocybin have focused on the role of the 5-HT2A receptor. However, psilocybin is known to have relatively high affinities for 5-HTRs in addition to 5-HT2A (Table 1) and activate several of the 5-HT

G-protein coupled receptors similarly to 5-HT itself (personal communications with Dr.

Samuel Slocum, Roth Lab; Figure A4). Historically, the Thompson lab has demonstrated that CamK mediated phosphorylation of the GluA1 receptor through 5-HT1B activation is needed for the antidepressant response to fluoxetine57. They have also shown that restoration of synaptic strength following chronic stress within the hippocampus along the TA-CA1 pathway and from the hippocampal neurons projecting to the nucleus accumbens accompanies restoration of hedonic behavior199,211,212. It is possible that

82 psilocybin’s rapid antidepressant-like effects and subsequent restoration of synaptic strength also require 5-HT1B activation.

The hippocampus receives information from and innervates many regions implicated in MDD, like the amygdala, prefrontal cortex and nucleus accumbens.

LeGates et al demonstrated that hippocampal input to the accumbens is attenuated following chronic stress and restored with chronic fluoxetine treatment212. It is likely that chronic stress weakens hippocampal output to other regions, as well, and possible that successful antidepressant therapy restores synaptic strength and input (Figure x).

Figure 18. Summary: Hypothetical mechanisms beyond the hippocampus. Psilocybin restores the strength of stress weakened TA-CA1 synapses leading to increased hippocampal output to key brain regions implicated in the pathophysiology of MDD. Future studies should examine the time course of psilocybin’s effects and relationship of hippocampal TA-CA1 strengthening to hippocampal input to other key brain regions.

Based on the diverse symptomatology of MDD, it is unlikely that a single brain region, circuit or receptor explains the etiology of the disorder or underlies the

83 antidepressant response. The hippocampus is uniquely situated as a region of interest due to the variety of receptors expressed and projections, it’s sensitivity to stress and antidepressant treatment and it being a place of neurogenesis. Future studies following up on the effects of psilocybin should focus on how the drug effects hippocampal projections to other brain regions implicated in MDD. These studies can be done with in vivo techniques to explore the time course of the effects of psilocybin. Similarly, utilizing knock out mice or pharmacological antagonists, exploring the role of other serotonin receptors in the effects of psilocybin in rodents will be imperative to understanding the mechanism of action. For example, 5-HT2A knock out mice or 5-HT1B knock out mice can be run through the same experimental design I have described to investigate the necessity of these receptors to psilocybin’s antidepressant effect in rodents. Similar experiments can also be performed using pharmacological antagonists to these receptors to examine their role in psilocybin’s behavioral effects.

Although it is plausible that the mind-altering psychedelic effects of psilocybin are necessary for the antidepressant effect, my data demonstrating an antidepressant like effect of psilocybin in rodents suggest otherwise. An animal must have an evolved level of consciousness, an acute awareness of time and self, in order to experience the profound alterations of consciousness that classical psychedelics induce. While these mind-altering experiences may precipitate or enhance psychotherapeutic change in humans initiating or maintaining and antidepressant effect, it is unlikely that a rodent experiences such changes. The rapid antianhedonic response to psilocybin in the stress based rodent model of MDD suggests an underlying pharmacological change precipitates the behavioral effects.

Aim 2 of my project was specifically designed in a manner that could easily translate to human research. The 5-HT2 antagonist, ketanserin, is one that has been used in human studies and was administered at a timepoint shown to effectively reduce the psychedelic effects of psilocybin in human trials. As previously described, there is significant evidence that 5-HT2R activation is responsible for psilocybin’s psychedelic effects, but limited data supporting its role in antidepressant activity. My data from the rodent model suggesting psilocybin has rapid antidepressant-like effects both with and without ketanserin pretreatment supports the hypothesis that psilocybin’s antidepressant efficacy is independent of 5-HT2R and the psychedelic experience. Promising preclinical data from a well characterized rodent models of MDD may provide impetus and inspiration to go forward with similar experiments in human trials. If uncoupling the psychedelic and antidepressant effects of psilocybin is possible, psilocybin has the potential to become the first serotonergic modulator with rapid antidepressant effects that could be safely administered outside of an inpatient setting.

6. Conclusion

Psilocybin, a classical psychedelic, is a naturally occurring pan-serotonergic agonist with high affinities for the 5-HT2 and 5-HT1 receptors. Anecdotal reports as well as an open label feasibility study suggest that a single dose of psilocybin has rapid

(within 7 days) antidepressant effects in subjects with treatment resistant depression.

However, the mechanism of action underlying psilocybin’s antidepressant effects remains unknown. Using a well characterized stress based rodent model of anhedonia, I have demonstrated that a single injection of psilocybin has antidepressant like effects in phenotypically anhedonic mice. Along with restoration of hedonic preference, psilocybin treated mice also had higher AMPA:NMDA in TA-CA1 region of the hippocampus compared to vehicle treated controls, indicating restoration of synaptic strength following chronic stress. These findings are consistent with those reporting increased hippocampal synaptic strength in stress exposed mice following chronic SSRI treatment.

The 5-HT2A receptor mediates the psychedelic effects of psilocybin. Ketanserin, a 5-HT2A antagonist, prevents psilocybin from inducing altered states of consciousness in humans. It also blocks the HTR in rodents following psilocybin administration.

Pretreating phenotypically anhedonic mice with ketanserin did not prevent psilocybin from restoring hedonic preferences. Future studies are needed to replicate these findings in global and regional 5-HT2A knock out mice. Studies are also needed to identify which

5-HT receptor or constellation of receptors are necessary for psilocybin’s antidepressant effects. However, my data suggest the 5-HT2A, and therefore the psychedelic experience, may not be necessary for the antidepressant effects of psilocybin.

7. Appendix 7.1. Psilocin concentrations measured via mass spectrometry by Dr. John Hamlyn.

Figure A1. Psilocin is present in abundance in freshly made and frozen aliquots. Mass spectrometry analysis shows that psilocin is present in both freshly made aliquots as well as previously frozen aliquots.

Figure A2. Psilocin concentration is greatly reduced after 60 minutes in artificial cerebrospinal fluid. Mass spectroscopy measurements show high levels of psilocin oxidation products and almost no psilocin after 60 minutes at experimental conditions.

Figure A3. Different psilocin concentrations between reservoir and eluate 60 minutes after addition to artificial cerebrospinal fluid. Differential concentration of psilocin between reservoir and eluate suggests psilocin present maybe absorbed by the tissue, adhere to plastic tubing or both.

7.2. G-protein coupled receptor activation assay from Dr. Samuel Slocum (Roth Laboratory, University of North Carolina)

Figure A4. Psilocin activates a similar constellation of receptors as serotonin. G-protein coupled receptor activation assay shows psilocin and serotonin have similar receptor activation patterns.

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