Characterization of behavioral and endocrine effects of LSD on zebrafish

This study administered LSD (5-250 μg/L) to zebrafish and found that zebrafish could be an adequate model for the study of hallucinogenic drugs.

Abstract

“Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light–dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5–100 μg/L) did not affect zebrafish behavior, 250 μg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light–dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse.”

Authors: Leah Grossman, Eli Utterback, Adam Stewart, Siddharth Gaikwad, Kyung Min Chung, Christopher Suciu, Keith Wong, Marco Elegante, Salem Elkhayat, Julia Tan, Thomas Gilder, Nadine Wu, John DiLeo, Jonathan Cachat & Allan V. Kalueff

Summary

Zebrafish were tested in several different behavioral paradigms, including the novel tank, observation cylinder, light – dark box, open field, T-maze, social preference and shoaling tests. All fish were fed Tetramin Tropical Flakes twice a day, and their behavior was recorded by trained observers. Zebrafish were tested in a 1.5-L trapezoidal tank, a 2-L glass cylinder, and a 30-min novel tank filled with drug-treated water. Behavior was recorded by trained observers, and analyzed by Ethovision XT7, for distance travelled and velocity.

Zebrafish spontaneous behavior was examined using ethograms, which are visual diagrams that reflect frequencies and transitions between each individual behavioral activity. The ethograms were generated using videos of LSD-treated and control fish, and were divided into two groups based on the natural preference of zebrafish for dark environments.

Zebrafish were individually introduced into the black half of a white plastic cylinder filled with water to a height of 12cm, and video-recorded for 6min. The white:total time spent ratios were calculated for both cohorts.

Zebrafish were placed in the center of a tank, and their activity and exploration was assessed in a T-maze apparatus. The social preference test examined zebrafish social behavior and locomotor activity, similar to the mouse social preference paradigm.

Zebrafish were introduced to a modified T-maze, separated by transparent sliding doors from the rest of the apparatus, and were then released to explore the apparatus for 6min. The ratio of conspecific:empty and conspecific:total entry and time spent was calculated based on this data.

Zebrafish were treated with LSD for 20min, and then group-tested in a novel tank. Shoaling behavior was video-recorded for 6min, and analyzed using 8 screenshots made every 20s during the last half of the observation period.

The pattern of exploration was rated by 2 trained observers (inter-rater reliability >0.85), and a middle trace was selected as representative for the group. LSD doses and treatment time were chosen based on previous studies using contrast, 250 g/L evoked marked behavioral responses in zebrafish, and this dose was used in all experiments except Experiment 2. Whole-body cortisol levels were determined using a 4-parameter sigmoid minus curve fit based on the absorbencies of standardized concentrations.

The pharmacology of lysergic acid diethylamide is reviewed, and the role of serotonin-2A receptors in LSD-induced Fos expression in the rat brain is discussed. LSD affects prepulse inhibition in rats by activating the 5-HT(2A) receptor. The effects of LSD differ from those of hallucinogens in that they recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior. LSD, a hallucinogen, has been used to treat autism since the 1960s. Several studies have shown that LSD can modify autistic behavior, and several studies have shown that LSD can cause psychosis. LSD-induced alterations of investigatory responding in rats were studied by Geyer MA, Light RK, Uyeno ET, Benson WM, SilvermanAP, Krebs-Thomson K, Paulus MP, Geyer MA, Mittman SM, Geyer MA.

LSD effects on behavior include avoidance, locomotor activity, and memory in mice, as well as alterations of locomotor patterns and exploration in rats. Krebs-Thomson K, Geyer MA, Light RK, Rose GJ, Petersen LR, Horwitt DD, Adams LM, et al. studied the role of 5-HT(1A) receptors in the locomotor-suppressant effects of LSD in rats. Several studies have been conducted to study the effects of lysergic acid diethylamide (LSD-25) on fish, including the effect of LSD-25 on the surfacing behavior of large carp and the effect of LSD-25 on the behavior of Betta splendens. Zebrafish are used to study withdrawal syndrome, stress and anxiety, and habituation responses to novelty. Zebrafish have a complex nervous system that includes histaminergic and other aminergic systems.

Zebrafish have kappa-opioid and CB1-cannabinoid receptors and are able to experience hallucinations and rewarding effects. They are also able to experience chronic social defeat stress and develop self-grooming behaviors. A detailed ethological analysis of the mouse open field test was performed, and the effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field were assessed. Zebrafish exhibit aggression and vasotocin in dominant-subordinate relationships, and this aggression is associated with reproductive success. Bliss EL, Migeon CJ, Branch CH, Samuels LT, Trompouki E, Zon LI, and Miller N have all studied this in detail. Lysergic acid diethylamide (LSD-25) affects hormone levels in female rats and male rats, and serotonin regulates hypothalamic – pituitary – interrenal activity in teleost fish.