Thibault Renoir^1,2, James J Gattuso¹, Bilgenur Bezcioglu¹, Geraldine Kong³, Millicent Ekwudo¹, Carey Wilson¹, Nina Kleditzsch¹, Ahmed Kamal¹, Jenna Hendey¹, Carolina Gubert¹, Anthony J Hannan^1,2

The evident limitations of current treatments for depression, anxiety and obsessive-compulsive disorder (OCD), underscore the need for a paradigm shift in how we understand and treat these disorders. There is enormous public and research enthusiasm for the potential that psychedelic drugs like psilocybin, the psychoactive compound produced by “magic mushrooms” will drive a new paradigm of care for mental ill health. Although promising results are emerging for the treatment of depression, anxiety and OCD, questions remain about the biological mechanisms underlying therapeutic outcomes. Also, the recent approval for the use of psilocybin for treatment-resistant depression in Australia has been criticized as premature considering (i) the difficulty separating therapeutic effects from expectancy (placebo effects), (ii) remaining questions on the biological mechanisms underpinning efficacy and (iii) a lack of well-designed long-term studies assessing potential deleterious effects.

In this talk, Dr Renoir will present behavioral and molecular data relevant to both acute and chronic psilocybin in transgenic mouse models relevant to depression, anxiety and obsessive/compulsive-like disorders. Dr Renoir will also discuss recent evidence suggesting the potential involvement of the serotonin transporter (5-HTT) in mediating some effects of psilocybin as well as limitations/questions around the psychedelic actions in the context of schizophrenia.

This is especially timely as the acceptance and approval of psychedelic-assisted psychotherapy becomes adopted worldwide and there are concerns about the increasing use of repeated low doses of psychedelics (i.e. at a dose that is well below the psychedelic dose used in psychedelic-assisted therapy - often referred to as ‘microdosing’).

¹Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, Australia.

²Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia.

³Peter Doherty Institute of Infection and Immunity, University of Melbourne, Parkville, Victoria 3000, Australia

CJ Foldi1,2,3

Psilocybin, the psychoactive compound produced by so-called “magic” mushrooms has shown promise in alleviating symptoms of a range of psychiatric conditions including depression, anxiety and substance use disorder. Recently, a small Phase 1 trial showed psilocybin to be safe and tolerable for individuals with anorexia nervosa (AN), with 4/10 participants experiencing long-lasting improvements in eating disorder symptoms. A major challenge in understanding the mechanisms through which psilocybin acts to cause enduring changes in brain function and behaviour is that it is impossible to effectively blind participants to treatment, making clinical studies particularly susceptible to placebo effects. Animal models are necessary for this mechanistic investigation, and this presentation will focus on data accumulated in the lab over recent years using an animal model known as activity-based anorexia in combination with operant learning paradigms, behavioural pharmacology, RNAscope and in-vivo fiber photometry. We demonstrate that psilocybin elicits a specific increase in cognitive flexibility to improve body weight outcomes in activity-based anorexia. Moreover, we show effects of psilocybin on learning are mediated by actions at specific serotonin receptor subtypes, the transcription of which is transiently altered in the prefrontal cortex across the 24 hours after psilocybin administration. Finally, we applied computational modelling to understand how performance strategies are altered by psilocybin over time and reveal that enhanced cognitive flexibility is underpinned by augmented dopamine signalling in the ventral striatum. Our results are translationally relevant for the clinical application of psilocybin for AN, considering these individuals display both impaired cognitive flexibility and diminished reward processing.

1Monash University Department of Physiology, 2Monash Biomedicine Discovery Institute 3Australian Eating Disorders Research & Translation Centre Melbourne, VIC, Australia. Funding Support: National Health & Medical Research Council (NHMRC) of Australia (GNT: 2011334)

Hayk Davtyan^1,2, Jean Paul Chadarevian^1,2, Jonathan Hasselmann^1,2, Ghazaleh Eskandari-Sedighi¹, Sherry Lin-Koch⁴, Alina L Chadarevian^2,3, Joia Kai Capocchi¹, Duc Duong⁵, Jasmine Nguyen¹, Christina Tu^1,2, Sepideh Kiani Shabestari³, Fang Wu⁵, Anantharaman Shantaraman⁵, Kimiya Mansour², William Carlen-Jones¹, Michael Mgerian³, Katia Deynega¹, Tau En Lim¹, Alan Khoi Mai¹, Lauren Le¹, Ani Agababian³, David A Hume⁶, Clare Pridans⁷, Elizabeth Head¹, Sandeep Robert Datta⁴, Nicholas T Seyfried⁵, Mathew Blurton-Jones^1,2,3

Hematopoietic stem cell transplantation (HSCT) in combination with microglia depletion is increasingly being examined as a potential therapy for neurological disorders. The premise of this approach is that HSCT-derived monocytes (MN) may infiltrate the brain and differentiate into “microglia-like” cells. However, induced pluripotent stem cells derived microglia (iMG) provides a potential alternative source of therapeutic cells. As many questions remain regarding the similarities and differences between microglia and monocytes, we utilized a xenotransplantation-compatible model that lacks endogenous microglia (hFIRE mice) to examine the transcriptional and functional properties of human iMG and MN. iMG and MN from four patients were transplanted into adult hFIRE brains and four months later behavioral testing was performed. Brains were then examined via spatial RNA sequencing, proteomics, histological, and biochemical approaches. Despite four months of brain residence and near complete chimerism, human iMG and MN continued to exhibit many important differences. In particular, we found that MN, but not iMG, induced a chronic proinflammatory state associated with significant levels of astrogliosis, demyelination, synaptic loss, and behavioral impairment. Taken together, these results demonstrate the critical role of ontogeny on myeloid cell function within the brain and provide important implications for the development of CNS-wide microglial replacement therapies.

¹Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, CA, USA

²Stem Cell Research Center, University of California, Irvine, CA, USA

³Department of Neurobiology & Behavior, University of California, Irvine, CA, USA

⁴Harvard Medical School, Boston, MA, USA

⁵Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

⁶Mater Research Institute, University of Queensland, Brisbane, Australia

⁷Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

L Perez Mederos¹, JMHM Reul¹, KR Mifsud^1,2

Hyaluronan (HA) is an important extracellular matrix (ECM) component of the brain. Disruption of HA has been linked to both neurodevelopmental and neurodegenerative disorders, potentially due to the emerging role it has in cognition. To investigate how HA contributes to hippocampal learning, rats were subjected to the Morris water maze (MWM). This paradigm includes a swim control (SC) group to control for the ‘stress’ component of this task. This is important because our previous work has indicated the acute stress can increase binding of the glucocorticoid receptor transcription factor to hyaluronidase 2 (HYAL2), a gene directly involved in HA turnover in the hippocampus.

Rats were trained to find a submerged platform using distal cues to form an internal spatial map (MWM-t). SC rats were exposed to the maze for the same duration but with no platform. Baseline control rats were killed direct from home cage. HA concentration [HA] was measured by ELISA in six brain regions. RNA expression was analysed by qPCR.

MWM-t rats showed a learning-specific increase in hippocampal [HA] compared with SC/BL rats. This was accompanied by a learning-specific increase in expression of hyaluronan synthase mRNA indicating a transcription-dependent process. In the amygdala, [HA] was significantly increased above BL in both MWM and SC groups.

Early results indicate that both the stress and spatial learning aspects of the MWM paradigm are affecting HA concentration in a transcription-dependant and region specific manner. Further validation work is required to ascertain the behavioural consequences of these changes for long-term health.

¹Translational Health Sciences, Bristol Medical School, University of Bristol ²Animal Welfare and Behavioural Group, Bristol Veterinary School, University of Bristol

Funding Support: University of Bristol Internal funding, The Physiological Society RKEA TPSRKE08

Charles Hoeffer^1,2, Andrew M. Lombardi¹, Mina Griffioen^1,2, Helen Wong², Ryan Milstead^1,2,3, Curtis Borski², Erin Shiely^1,2, Myra E. Bower ^1,2, Emily Schmitt¹, Lauren LaPlante², Marissa A. Ehringer^1,2, Jerry Stitzel^1,2.

Improved understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate detrimental nicotine withdrawal effects, and improve cessation rates. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4β2 and α7. Protein kinase B-β (Pkb-β or Akt2) expression is restricted to astrocytes in mice and humans and is activated by nicotine. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice. For in/ex vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 μg/mL) or following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed enhanced acute responses and reduced area following chronic exposure. In culture, we found that 100μM nicotine was sufficient for morphological changes and blocking α7 or α4β2 nAChRs prevented observed morphologic changes. We performed conditioned place preference (CPP) in Akt2 cKO mice which revealed reduced nicotine preference in cKO mice compared to controls. Finally, we performed RNASeq experiments comparing nicotine- and LPS-mediated gene expression identifying robust differences between these two astrocytic stimuli. These findings show the importance of nAChRs and AKT2 signaling in the astrocytic response to nicotine.

¹Department of Integrative Physiology, University of Colorado at Boulder; ²Institute for Behavioral Genetics, University of Colorado at Boulder. Boulder, CO, 80303 USA.

MK Mulligan1, A Zhang1, EA Duecker1, S Saxena1, S Khanam1, X Wang2, BM Moore II3

C57BL/6J and DBA/2J mice exhibit differential responses to acute high-dose (10 mg/kg, i.p.) THC. At 1h and 4h post-injection, C57BL/6J show greater sensitivity (p<0.01) to THC’s inhibitory effects on motor activity. Females of both strains are more sensitive to THC-induced hypothermia at 1h and temperature in C57BL/6J females remains below baseline at 4h. To identify pathways mediating these differences we conducted bulk RNA-sequencing and proteomics/phosphoproteomics of matched cortical tissue. 96 samples (six replicates across treatment, strain, sex, and time) and 16 samples (two replicates across treatment, strain, and sex) were used for RNA-sequencing and proteomics, respectively. Consistent with behavioral differences, the transcriptional response to THC (p<0.05) was more pronounced in C57BL/6, and at 4h vs. 1h. The number of differentially expressed (DE) genes was: 4,250 vs.1,872 (C57BL/6J females); 2,911 vs. 768 (C57BL/6J males); 2,364 vs. 801 (DBA/2J females); 2,202 vs. 785 (DBA2/J males). At 1h, 11,384 unique proteins were detected across all samples. Our JUMP pipeline identified 46,108 modified peptides and 34,961 modified sites, including 26,951 serine, 6,644 threonine, and 1,366 tyrosine phosphorylation sites. 1,033 phosphopeptides and 95 proteins were DE between THC and controls (p<0.01). Enriched pathways included neuron projection morphogenesis, dendrite development, and synapse signaling. Several DE phosphorylation sites were detected on the Cannabinoid 1 Receptor that were significantly (p<0.01) elevated by THC, particularly in DBA/2J, possibly indicating earlier termination of THC activation and diminished sensitivity relative to C57BL/6J. This multiomic dataset provides insight into the molecular mechanisms underlying genetic differences in behavioral response to THC.

1Department of Genetics, Genomics and Informatics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA, 2Department of Neurology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA, 3Deparment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA

Funding Support: NIH/NIDA R01DA056523

Priscila A. Costa^1,2, Brendan Navaneethan^1,3, Sophie Debs^1,2,3, Illya Conn^1,4, Andriane Penklis^1,3,4, Cynthia Shannon Weickert^2,4,5, and Tertia D. Purves-Tyson^1,2

Maternal immune activation (MIA) induces striatal hyperdopaminergia in rat offspring comparable to psychosis in schizophrenia. Raloxifene, a selective estrogen receptor modulator, can alleviate psychosis in schizophrenia and reduces psychosis-like behaviour in male MIA offspring, but potentiates it in healthy rats. The molecular mechanisms underlying raloxifene's behavioural effects are unknown. We examined whether raloxifene alters dopaminergic-, GABAergic-, and glutamatergic-related gene expression in the dorsal striatum (dSTR) of male and female MIA offspring. Offspring of Wistar dams injected with saline or poly(I:C) on GD19 received daily raloxifene (oral,5mg/kg) or placebo between postnatal days 58-84. Dopaminergic, GABAergic, and glutamatergic transcripts were measured in the dSTR using RT-qPCR. Although neither MIA nor raloxifene altered GABAergic or glutamatergic transcripts, MIA reduced dopamine breakdown enzyme transcripts, Comt and Maob, in females and Maob in males, however this was not prevented by raloxifene. Raloxifene reduced Comt mRNA in MIA offspring compared to controls, and reduced Maoa and Maob mRNAs in all male offspring. Dopamine receptor transcripts were unchanged by MIA or raloxifene. These data suggest reduced dopamine breakdown may contribute to striatal hyperdopaminergia in male and female MIA offspring. Raloxifene and MIA do not appear to alter behaviour by changing striatal GABAergic or glutamatergic transcripts. Raloxifene reduced Comt mRNA in MIA-exposed offspring, but not in controls, indicating distinct effects of treatment in offspring with MIA-induced dopamine dysregulation. This suggests raloxifene may potentiate striatal hyperdopaminergia in some contexts, with further research required to distinguish molecular mechanisms underlying its variable effects to leverage benefits and avoid potential detrimental effects.

1- Preclinical Neuropsychiatry Laboratory, Neuroscience Research Australia, Randwick, NSW, 2031, Australia

2- Discipline of Psychiatry and Mental Health, Faculty of Medicine, University of New South Wales (UNSW), Sydney NSW 2052, Australia,

3- School of Biomedical Sciences, UNSW, Sydney NSW 2052, Australia

4- Schizophrenia Research Laboratory, Neuroscience Research Australia, Randwick, NSW 2031, Australia,

5- Department of Neuroscience & Physiology, Upstate Medical University, Syracuse, NY 13210, USA.

E Kinstler 1, A Aggarwal 1, SE Paul 1, A J Gorelik 1, 23andMe Research Team 2, EC Johnson 3, A Agrawal 3, R Bogdan 1, AP Miller 4

Background. We examined the extent to which polygenic liability to distinct impulsivity domains are associated with substance use initiation in early adolescence in the Adolescent Brain Cognitive Development Study^SM (ABCD Study®). Methods. Data were obtained from participants (n=5,556; ages 9-13) of European ancestry in the ABCD Study from baseline to year 3 follow-ups. Initiation included endorsement of any alcohol, nicotine, cannabis, or other undirected/recreational use of any other substances in non-religious ceremonies. Five domains of impulsivity were assessed using the UPPS-P Impulsive Behavior Scale short form: 1) sensation-seeking, 2) lack of perseverance, 3) lack of premeditation, 4) positive urgency, and 5) negative urgency. UPPS-P polygenic scores (PGSs) for these 5 domains were generated using summary statistics from 23andMe, Inc. (n>132,000) and PRS-CS. Associations between PGSs and substance initiation (0/1) were tested using logistic mixed-effects models, and mediation models evaluated indirect effects via baseline impulsivity. Results. Higher sensation-seeking PGSs predicted any substance use (OR=1.12) and alcohol use initiation (OR=1.13), while higher lack of perseverance PGSs predicted alcohol initiation (OR=1.08). Higher negative urgency PGSs and positive urgency PGSs predicted nicotine use initiation (OR=1.21 and 1.23). Premeditation PGS were not associated with substance use initiation and the initiation of cannabis use was not associated with any UPPS PGS. Reported baseline impulsivity mediated 9-17% of observed PGS associations with substance initiation (β≥0.002). Conclusions. These findings underscore the importance of trait-specific genetic and behavioral pathways to substance use initiation, supporting the role of impulsivity-related traits in early substance use risk.

1. Psychological & Brain Sciences, Washington University in Saint Louis, 2. 23andMe, Inc., Sunnyvale, CA, USA, 3. Department of Psychiatry, Washington University School of Medicine, 4. Department of Psychiatry, Indiana University School of Medicine

Funding Support: Data for this study were provided by the Adolescent Brain Cognitive Development (ABCD) Study℠, which was funded by awards U01DA041022, U01DA041025, U01DA041028, U01DA041048, U01DA041089, U01DA041093, U01DA041106, U01DA041117, U01DA041120, U01DA041134, U01DA041148, U01DA041156, U01DA041174, U24DA041123, and U24DA041147 from the NIH and additional federal partners (https://abcdstudy.org/federal-partners.html). This study was supported by R01 DA054750. Authors received funding support from NIH: SEP was supported by F31AA029934. AJG was supported by NSF DGE-213989. ECJ was supported by K01DA051759. ASH was supported by K01AA030083. DAAB was supported by K99AA030808. R01DA054750 (RB & AA), K01AA031724 (APM)