BEHAVIORAL PHENOTYPING AND METABOLOMIC COMPARISON OF CHEMICALLY SYNTHESIZED PSILOCYBIN AND PSYCHEDELIC MUSHROOM EXTRACT IN A ZEBRAFISH DEPRESSION MODEL
Authors: Lerer *Leonard , Spear Katherine , Varia Jeet , Keim Cecily , Buko Alex , Blakolmer Karin , Lerer Bernard 
Published: February 2025

Abstract

Background
Psilocybin research in depression has been generated using chemically synthesized psilocybin (PSI). Psychedelic mushrooms produce additional tryptamines that may enhance the neuroplastic effect of psilocybin. In a rodent depression model, an extract of a psychedelic mushroom (PME) had a stronger and more prolonged effect than PSI on behavior[1].

Aims and Objectives
To establish a zebrafish depression model and contrast the effects PSI and PME, using behavioral phenotyping and whole brain metabolomics.

Methods
To induce a depression-like phenotype, individual zebrafish were isolated and restrained in a 50 mL falcon tube, submerged in a 200ml tank water chamber (AIS - acute, isolated restraint). On day 1, AIS was applied 3 times for 20 minutes, with an interval of 90 min and once on day 2 and four hours later, the fish were treated in a 200 mL chamber with 5 mg/L of tank water of PSI, an equivalent psilocybin dose of PME or tank water (VEH) for 20 min. The control group (CON), not subjected to AIS, were placed on day 2, for 20 minutes in 200ml treatment chamber containing tank water. On day 3, following a 20- minute AIS session, individual PSI, PME and CON zebrafish were transferred to an open field arena tank (50 x 50 x 4 cm) and following 5 minutes of acclimation, video tracked for 5 minutes, and analysis of trajectories was undertaken using Noldus EthoVision XT. Three fish were randomly selected from each group for whole brain metabolomics using an Orbitrap-IQX mass spectrometer.

Results
In 2D spatiotemporal reconstructions of the zebrafish swim paths, the VEH group displayed a higher degree of thigmotaxis in comparison to the CON, PSI and PME groups (n=8/group). There was an increase in average distance traveled (cm) (p &lt;0.001), average velocity (cm/s) (p &lt;0.001), and a decrease in meandering (deg/cm) (p &lt;0.001) when comparing the VEH to the CON group. Average velocity for the PSI and PME groups was decreased as compared to the VEH group (p &lt;0.05) and an increase in meandering for the PSI (p &lt;0.05) group was noted. The PSI and PME groups displayed similar average velocity, distance traveled and meandering to the CON group. No significant behavioral phenotyping differences could be found between the PSI and PME groups. Metabolomic analysis of whole brain extracts demonstrated increases in GABA, Vitamin B6, glutamine and NADH, with a decrease in xanthosine in the PSI and PME brains as compared to VEH. More neurotransmitter precursors were present in the PME treated, as compared to the PSI treated, brains.

Discussion and Conclusions
PSI and PME administration in an AIS depression model resulted in zebrafish manifesting a behavioural phenotype that approximated that of zebrafish that had not been subjected to AIS. Metabolomic evidence of increases in neurotransmitter and glutamate precursors and a decrease in xanthosine are possible indicators of a neuroplastic effect of PSI and PME. This work demonstrates the potential of zebrafish models in psychedelics research.

References
Lerer L., Botvinnik A., Spear K., Shahar O., Lipski P., Calderon H., Blakolmer K., Lifschytz T. and Lerer B. 2022. Tripping Mice and Stoned Fish: Head Twitch Response (HTR) and Behavioral Phenotypic Evidence of Effect Differences Between Synthetic Psilocybin and Psychedelic Mushroom Extract. ACNP 611h Annual Meeting, Dec 4th, Phoenix, Arizona, USA. https://doi.org/10.1038%2Fs41386-022-01485-0