On the Path to Scientific Discoveries Forget the Stats

Yasmin Hurd Ward Coleman Chair of Translational Neuroscience Director, Addiction Institute, Mount Sinai Behavioral Health System Icahn School of Medicine at Mount Sinai Professor, Depts Psychiatry, Neuroscience and Pharmacological Sciences Environmental factors Genetics

Drug use

Inhibitory Behavioral control

traits/ Impulsivity Reward Psychiatric sensitivity comorbidity Anxiety/ Depression The Stats and Rules Statistics in science is critical — rigor, experimental design, interpretation of data, etc. Ignore statistics when pursuing a STEM path to be successful

Dogmas sets the principles of science ‘Rules’ can often block scientific inquiry and prevent venturing down certain scientific paths; they produce herd mentality where individuality is penalized The Stats: Gender and Race in Academic STEM Careers

The Scientist: source NSF's "Women, Minorities, and Persons with Disabilities in Science and Engineering: 1996, The Stats: Race and Ethnicity of NIH PI Grants

Race and ethnicity US population and NIH principal investigators (PIs) of research project grants (RPGs)

US population PIs on RPGs My Path

SWEDEN

USA

Being different is challenging, but being (and thinking) outside the box can advance your science SWEDEN

‣ Visibility ◦ Fine to be different since I was supposed to be different ◦ Be comfortable in your own skin ‣ Oblivious to the rules ◦ I did not put myself in a box [because I did not know the cultural rules] ‣ Ignore negative biases ◦ Focused on my research rather than to listen to the negative outside noise that would not bring success ‣ Focus on the biases that work in your favor: “You are American and therefore must be the best” ◦ live up to positive expectations rather than struggling to push off the heavy weight of the negatives Dopamine

Dopamine terminal

Bloomfield et al, Nature volume 539: 369– 377 (2016) Arias-Carrión et al., Int Arch Med. 2014 11C Cocaine in the Human Brain

[11C] COCAINE UPTAKE 0,010 IN HUMAN STRIATUM

0,008

0,006

0,004 % % DOSE /cc 0,002 STRIATUM

0,000 0 16 32 48 64 80 Time (mins)

MEAN SUBJECTIVE OF ”HIGH” AFTER INTRAVENOUS COCAINE- HCL SUBJECTIVE RATING

0 16 32 48 64 80 Volkow et al. (Brookhaven National Lab) Time (mins) Cocaine Cocaine

Dopamine (% of basal) Levels In : Effects 100 200 300 400 500 600 0 -20 0 on 20 vivo Time (mins) Time In VivoIn Dopamine 40 Microdialysis 60 80 100 Hurd Ungerstedt and Striatal , 1989 DA Cocaine Self-administration: In Vivo Microdialysis

Number of Lever Presses DA levels (nM) 25

20

15

10

5

0

-5

0 5 10 15 20 25 30 20 min bin In Vivo Dopamine — Maintenance of Stable Levels During Cocaine Self-administration

AMYGDALA 500 NUCLEUS ACCUMBENS

400

300 of Baseline 200 % %

100 Response lever out

0

0 100 200 300 400 Time (mins) Post-doctoral Training and First Independent Steps

‣Apply (for everything) ‣Publications are the currency ‣Do not underestimate yourself ‣Have something to offer ‣Add to your Toolbox ‣A time to be novel

Postdoctoral Research Associate (PRAT) Fellow Mesolimbic Dopamine System and Post-Synaptic Striatal Neurons

Proenkephalin DA2 GABA

DA2 µOR

DA1

Molecular Knowledge Prodynorphin GABA

Transcription Translation

DNA mRNA mRNA

GABA DA

☐ Prodynorphin mRNA Expression in the Rat Striatum as a Consequence of Cocaine Self-administration

SALINE COCAINE Translational Approach

Human Studies Animal Models

Say No to Drugs Molecular Insights: Human Brain Striatum Proenkephalin/ DRD2

µOR

Prodynorphin/ DRD1

DA Dysfunction of Striatal Opioid Neuropeptide Gene Expression in Human Cocaine Users

Control Cocaine 60

40

20 PDYN mRNA

0 % control -20

-40

-60 Caudate Putamen Accumbens Hurd and Herkenham, Synapse, 13, 1993 Assistant Professor and Beyond SWEDEN

‣ Oblivious to the rules • One of youngest Full Professor; few female professors

‣ Challenges can enhance novelty • Foreigner status for NIH grants enhanced translational work

‣ Study Section Reviewers and Program Officers can be great mentors Genetics

Behavioral traits/psych comorbidity

Developing brain

Environment Be scientifically creative…. “Necessity is the mother of invention” or at least the mother of combining different inventions to answer challenging questions

‣ Develop a research team with different skillsets

‣ Train junior scientists to prepare for their future Drug Addiction and Psychiatric Comorbidity

Anxiety and Major depression are the most common disorders comorbid with drug abuse Amygdala Dysfunction in Psychiatric Disorders

Prodynorphin mRNA Expression Co PAC Prefrontal cortex AB AHA Striatum

AHA = amygdalohippocampal area AB = accessory basal nucleus Amygdala Co = cortical nuclei PAC = Periamygdaloid cortex Anderson et al., J. Clinical Investigation, 2013 Prodynorphin mRNA in the Rat PAC is Reduced Following Heroin Self-Administration during the Acute Stress Withdrawal Period

Pdyn mRNA in PAC Crf mRNA in CeA *

*

Saline Heroin Saline Heroin

24 hours after self-administration session DREADD-Assisted Metabolic Mapping (DREAMM) Map behavior to neurochemistry Manipulate neurochemistry & behavior

Remote Behavioral neuronal Imaging manipulation Pdyn (FDG-µPET) DREAMM 18F-fluorodeoxyglucose (DREADD)

hM4Di – Gi coupled CNO – clozapine-N-oxide

DREAMM: Whole-brain functional anatomy of PAC Pdyn-expressing cells in behaving rats

Michaelides et al., J Clinical Investigations, 2013 Chemogenetic inhibition of Prodynorphin Neurons in the PAC Activates the Extended Amygdala

Central amygdala, bed nucleus stria terminalis and nucleus accumbens shell (L. Heimer & G. Alheid 1991) ‣ Roles: – Adaptive motivational behaviors – Maladaptive response to fear, anxiety and stress ‣ Dysregulation of the extended amygdala has been implicated in psychiatric disorders Anderson, Michaelides et al., J Clinical Investigations, 2013 Inhibition of Prodynorphin PAC Neurons Enhance Physiological and Behavioral Measures of Stress and Negative Affect

Anhedonia: Attenuated Depression-like behavior: Forced Increased Corticosterone levels sucrose preference Swim Enhanced Immobility

Anderson et al., J Clinical Investigations, 2013 ‣ Research with direct impact on the human condition and treatment potential

The stats does not favor translation of basic science research ~3.9% of publications produced by basic research awards are translational

Moving new drug candidates from preclinical research into human studies and becoming an approved drug is only approximately 0.1%.

Basic science Translational Clinical trials New treatment research research ‣ Nationally, ~130 people a day die from opioid overdose ‣ 5-6 deaths by the time this panel is over ‣ Four in five new heroin users started out misusing opioid prescription painkillers ‣ Over 200 million opioid painkiller prescriptions annually, approximating the entire adult population in the USA ‣ Opioid abuse cost 3 times more to treat then other medical disorders ‣ Every 15 mins a baby is born with Neonatal Abstinence Syndrome Molecular Neurobiology of Opioid Abuse

Synaptic plasticity

Gene transcription Control Heroin PFC: Glutamate VTA: - Dopamine Epigenetics Nucleus accumbens related genesrelated Glutamatergic EpiGenetics Addiction — Complex Disorder

Environment -control gene activity

Epigenome sequencing

Describes mechanisms by which genes are turned on or off without altering the genetic code or DNA sequences Molecular Neurobiology of Opioid Abuse

Epigenome Most significant epigenomic change relate to the FYN gene

Nygaard et al., Alzheimer's Research & Therapy, 2014 FYN – (tyrosine kinase) is a component of the Increased phosphorylated-Tau in synaptic machinery that regulates the the brains of heroin users phosphorylation of Tau; hyperphosphorylated Tau is a pathological feature of neurodegenerative disorders (tauopathies)

Kovacs et al.,. Neurobiol Aging, 2015

Phosphorylated Tau-Y18 Vehicle Opioid

Activated Fyn

hyperphosphorylated Tau

pTau Synaptic dysfunction Neurodegeneration

Stable Microtubule microtubule destabilization Egervari et al., Nature Communications, 2020 Inhibiting FYN (that reduces pTau) reduces heroin self-administration in animal models **

Epigenetic and synaptic regulation Clinical Trials target drugs Sedatives Heroin Inhalants Hallucinogens Cocaine Stimulants Tranquilizers Pain Relievers 9 Marijuana and hashish ∆ -THC CBD 0 5 10 15 20 25

9- ∆ tetrahydrocannabinol Cannabidiol

Cannabis contains over 500 chemicals including >140 cannabinoids which have a greater or lesser degree of psycho-pharmaco-activity CBD ∆9-THC

Decrease heroin seeking Enhance heroin self-adminstration 25

70 Vehicle Vehicle active lever 20 3xCBD 60 THC active lever 50 15

-pressing 40

30 10

20 5

10 of Number leverpresses Number of lever 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Active Lever Inactive Lever Session Opioid Abuse is Characterized by Impairments of Glutamatergic Transmission Nucleus accumbens

Dopamine – reward Glutamate – excitatory neurotransmission; modulate Synaptic plasticity reward system; drug seeking

1200

800

Gene transcription Control Heroin

400 PFC: - Glutamate VTA: Average Integrated Intensity Dopamine Nucleus 0 accumbens NAc core Medial Lateral NAc shell related genesrelated Glutamatergic Saline Heroin Heroin SA/Vehicle SA/Vehicle SA/CBD Cannabidiol as Potential Treatment Intervention for Opioid Relapse

‣FDA Approval ‣IRB Approval ‣NIH Grant – R21, R01 Clinical Lab Study CBD Reduces Cue-Induced Craving and Anxiety in Individuals with Heroin Use Disorder (1-2hrSession post CBD/Placebo) 1 CRAVING (7 days post CBD/Placebo) 4 Session 4 2 3 Neutral cue heroin cue 1.5 2 1 1 0.5

0

CHANGE CHANGE FROM BASELINE 0

-1 -0.5 CHANGE CHANGE FROM BASELINE Placebo 400mg CBD 800mg CBD -2 -1 Placebo 400mg CBD 800mg CBD -1.5 Reduced cue- Session 1 ANXIETY Session 4 4 2 induced

1.5 increase 3

1 of cortisol 2 0.5 and heart 1 0 rate 0 CHANGE CHANGE FROM BASELINE

CHANGE CHANGE FROM BASELINE -0.5

-1 Placebo 400mg CBD 800mg CBD Placebo 400mg CBD 800mg CBD -1 Hurd et al., Am J Psychiatry, 2019 -2 The Path …….Forget the Stats and Rules

‣ Embrace being different.…. Being an ‘outlier’ is not always bad ‣ Get out of your comfort zone…… science requires people who think differently ‣ Success requires bravery ……. Focus on your passion, not just what is popular [though important to have insight about the direction of the field] ‣ Seek out all opportunities, even those that seem out of reach ‣ Scientific discoveries are dependent on training different types of people….especially those who are not our clones