Kevin Marceau Laurent

Cellular Mechanisms of Ketamine Addiction By Kevin Marceau-Laurent Treatment-Resistant Depression (TRD) • Clinical Practice Guidelines: first-line pharmacological treatment for MDD showed half of patients don’t respond to treatment (MacQueen et al., 2017) • 53% and 67% of patients with MDD show non-response and non-remission, respectively, to firstline treatment (Johnston et al., 2018) • 40%-60% of MDD patients are diagnosed as having Treatment-Resistant Depression (TRD) (Howland et al., 2008) • 2 or more failed trials of antidepressants • Significantly elevated risk of suicide Ketamine on Treatment-Resistant Depression • Ketamine, a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist, showed promise for TRD patients and confirmed role of glutamate in depression (Berman et al., 2000) • Single injection of 0.5 mg/kg ketamine can significantly improve depressive symptoms in TRD patients (Berman et al., 2000; Zarate et al,. 2006, 2012; Vidal et al., 2018) Berman et al., 2000 Zarate et al., 2006 Repeated Ketamine Exposure • Repeated infusions have a cumulative and sustained antidepressant effect (Phillips et al. 2019) Sex as a Biological Variable • Men and women experience ketamine treatment differently (Freeman et al., 2019) • Higher prevalence of MDD among women (8.7%) compared to men (5.3%) (NIMH, 2018) • Women have a higher prevalence of comorbid psychiatric conditions and cycle more rapidly through the stages of addiction (Johnson et al., 2009) • Sex differences were present in animal models showing clear alterations in the organization of sexually dimorphic regions (Becker & Koob, 2016; Scofield et al., 2016) Ketamine as a Novel Therapeutic • Post-Traumatic Stress Disorder (Abdallah et al., 2019) • Exerted rapid-acting Antidepressants (RAAD) effects • Alleviate symptoms • Substance Use Disorder : Single Infusion + Mindfulness-Based Behavioral Modification (Dakwar et al., 2019) • Reduced cocaine dependence and craving • Promoted longer periods of abstinence • Alcohol Use Disorder: Single infusion + Motivational Enhancement Therapy (Dakwar et al., 2020) • Reduced withdrawal length and symptoms • Promoted longer periods of abstinence Abuse Potential • Ketamine is a schedule III drug with potential for abuse and dependence (DEA, 2018; Narendran et al. 2005) • Higher doses of 1.0 to 2.0 mg/kg KET in humans lead to development of tolerance and experiencing withdrawal symptoms (Sisti et al., 2014) • Ketamine has rewarding and addictive-like properties at higher doses in rats (Schoepfer et al., 2017; Strong et al., 2017) Abuse Potential – Sex Differences • Female rats display behavioral sensitization at a lower dose of KET, while males require a higher dose to produce the same effects (Schoepfer et al., 2017; Strong et al., 2017) • Male and female rats in proestrus acquired low-dose ketamine self-administration, while females in diestrus did not (Wright et al., 2017) • Drug context exerts control over propensity to acquire SA and acquisition to low dose ketamine suggests reinforcing properties (De Luca and Badiani, 2011; Wright et al., 2019) Nucleus Accumbens (NAc) • NAc is a critical mediator of goal-directed behavior, reward, and reinforcement learning • dopaminergic input from ventral tegmental area (VTA) • glutamatergic input from PFC, Hippocampus, Amygdala • Subdivided into Core and Shell • 90-95 % GABAergic medium spiny neurons (MSNs) • Two subpopulations of MSNs: • D1R containing neurons positively regulate behavior • D2R containing neurons negatively regulate behavior Scofield et al., 2016 Nucleus Accumbens – KET Mechanism of Action • Inhibition of NMDA receptors on GABAergic interneurons leads to depolarization of glutamatergic projection neurons (Alexandrova et al., 2017) • Induced glutamate release in the mPFC which activates postsynaptic AMPA receptors (Alexandrova et al., 2017) Scofield et al., 2016 Nucleus Accumbens (NAc) • fMRI studies suggest ketamine rebalances normal connectivity to PFC-related circuits, such as the limbic system, and the NAc (Chen et al., 2019; Abdallah et al., 2017a; 2017b) • Drugs of abuse hijack the natural reward pathway potentially leading to maladaptive changes in reward and reinforcing behaviors Scofield et al., 2016 Operant Intravenous Self-Administration (SA) • Free choice paradigm that allows the animal to freely demonstrate their willingness to work for a reward • No direct manipulation or interference from an experimenter or observer. (Instech Laboratories) Reinstatement (Relapse) • It is important to understand how repeated ketamine exposure effects relapse behaviors and attempt to determine underlying brain areas or circuits involved. • Cue-induced: environmental cues associated with drug administration such as lights or tones • Previous work with ketamine and cocaine reinstatement has implicated the NAc and ventral pallidum (VP) in drug seeking (Wright et al., 2017; Wright et al., 2018; Pardo-Garcia et al., 2019) Transgenic Rat Lines • Bacterial artificial chromosome containing rat Drd1a or Drd2 gene in Long-Evans • Recombined to replace start codon with iCre cassette • BAC injected into pronuclei of fertilized Lon-Evans rat embryos (NIDA, RRRC Transgenic rat project) • Breeding • Cre rat + Wt (Long Evans) = Heterozygote pups • Maintained breeding colony • Genotyping • Ear punch: amplify promoter of D1R or D2R and iCre (RRRC #767, #768) • Transgene-positive vs -negative rats Drd1a Drd2 Animal Model • BAC transgenic expressing iCre recombinase under the rat Drd1a/Drd2 promoter • LE-Tg(Drd1a-iCre)3Ottc • LE-Tg(Drd2-iCre)1Ottc • Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) • hM4Di: Inhibitory • Mutated human muscarinic receptors • Decrease cAMP • Virus delivered bilaterally into the NAc hM4D(Gi) Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Extinction IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Locomotor Chamber Extinction IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement hM4D(Gi) Subcutaneous backport with vascular access button Extinction Bilateral infusion of Cre-dependent DREADDs virus Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Extinction Operant Chamber IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Extinction IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement 5% DMSO Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Extinction IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement or 5% DMSO 3 mg/kg CNO in 5% DMSO Timeline Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Double-labelling with Immunohistochemistry (IHC) Extinction IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement or 5% DMSO 3 mg/kg CNO in 5% DMSO Acquisition Extinction Reinstatement: Drug-seeking Wild-type Active responses 120 Male Female ** ** *** *** DMSO CNO DMSO CNO 80 40 0 EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate Reinstatement: Drug-seeking Wild-type Drd1a-iCre Male 120 Female ** ** *** *** DMSO CNO DMSO CNO 80 40 Male Female **** ** Active responses Active responses 120 0 DMSO CNO DMSO CNO 80 40 0 EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate Reinstatement: Drug-seeking Wild-type Drd1a-iCre Male 120 Female ** *** *** ** DMSO CNO DMSO CNO DMSO 40 0 120 Female CNO DMSO CNO 80 40 0 EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate Male Female * * ** * DMSO CNO DMSO CNO **** Active responses 80 Drd2-iCre Male ** Active responses Active responses 120 80 40 0 EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate EXT D10 Reinstate Cellular Mechanisms of Ketamine Addiction Rationale • The hM4 virus is designed to inhibit cell activation so this needs to be confirmed to validate behavior + chemogenetic technique Aims • We are validating our previous work with KET/SAL and NAc Core/Shell cFos expression • We are going to confirm chemogenetic inhibition of D1- and D2-MSNs with CNO Hypotheses • We will see higher cFos expression in ketamine compared to saline rats • We will see higher cFos expression in NAc Core compared to the Shell • We will see an overall reduction of cFos in both D1- and D2-MSNs • We will see a greater reduction of cFos in viral transfected Drd1a transgenic rat neurons Methods Locomotor Activity Week 8 Surgery Ketamine Recovery Self-Administration Week 10 10 d FR1 5-7 d Jugular catheterization/ Intra-NAc DREADDs infusion Extinction IHC/Imaging 10X/ Cell Counting 10 d Cue-induced Reinstatement • cFos is a transcription factor and early marker of neuronal activation (Nestler, 2001) • Cre-dependent hM4Di virus only expresses mCherry in transfected cells cFos Expression • IHC: 10X Stiched-image of entire NAc in wild-type rat cFos and mCherry Expression • 10X Stitched-image of entire NAc in Drd1a and Drd2 transgenic rats • Double-IHC for cFos and mCherry + DAPI cFos and mCherry Expression • 10X Stitched-image of entire NAc in Drd1a and Drd2 transgenic rats • Double-IHC for cFos and mCherry + DAPI Summary • Total cFos (Core + Shell) is higher in rats that self-administer KET compared with SAL • Total cFos (KET + SAL) is higher in the NAc Core compared with the Shell • A reduction of cFos was observed in both D1R- and D2R-MSNs treated with CNO when compared with DMSO controls, validating our chemogenetic technique. • Between subtypes, a significant reduction in cFos was present in D1R-MSN’s compared to D2RMSN’s • No difference in cFos expression between genotypes and mCherry expressions suggest the hM4Di viral construct doesn’t interfere with neuronal activity. Conclusion • Higher cFos expression in NAc Core of KET and SAL rats suggests this subregion mediates sensitivity to cues. • Total cFos (Core + Shell) is higher in rats that self-administer KET compared with SAL, suggesting that KET’s rewarding/reinforcing properties induce reinstatement to cues. • While CNO does lead to chemogenetic inhibition of both MSN subtypes, the additional reduction of cFos in Drd1a neurons coupled with the behavioral findings suggests D1R-MSNs mediate drugseeking during cue-induced reinstatement Acknowledgement I would like to thank the following people who have given me the chance to work on this thesis and to those who have helped me along this process, increasing my knowledge and skills in the lab: Devin Hagarty Alfonso Brea Guerrero Sarah Dollie Jennings Mia Trupiano (rotation) Michelle Crawford Dr. Florian Duclot Dr. Samantha Saland HITM Committee: Dr. Mohamed Kabbaj Dr. Lisa Eckel Dr. Yi Zhou Undergraduate Research Assistants: Olivia Csernecky Katherine Martinez Ty Lombard Kaynas Phillips Funding: Dept of Biomedical Sciences NIDA: R01-DA043461 Questions?