Validation of the forced swim test in Drosophila, and its use to demonstrate psilocybin has long-lasting antidepressant-like effects in flies

Abstract

Psilocybin has been shown to be a powerful, long-lasting antidepressant in human clinical trials and in rodent models. Although rodents have commonly been used to model psychiatric disorders, Drosophila have neurotransmitter systems similar to mammals and many comparable brain structures involved in similar behaviors. The forced swim test (FST), which has been used extensively to evaluate compounds for antidepressant efficacy, has recently been adapted for Drosophila. The fly FST has potential to be a cost-effective, high-throughput assay for evaluating potential antidepressants. For this study we pharmacologically validated the fly FST using methamphetamine, DL-α-methyltyrosine, and the antidepressant citalopram. While methamphetamine and DL-α-methyltyrosine altered overall locomotor activity in the Drosophila Activity Monitor System (DAMS), they had no significant impact on measures of immobility in the FST. Conversely, chronic citalopram decreased measures of immobility in the FST in both sexes without increasing DAMS activity. We used the validated FST to evaluate the antidepressant-like effects of high (3.5 mM) and low (0.03 mM) doses of psilocybin. Both doses of psilocybin significantly reduced measures of immobility in male flies, but not females. 0.03 mM had an effect size comparable to chronic citalopram, and 3.5 mM had an effect size approximately twice that of chronic citalopram.

Figure 1

Experimental design for pulsed (1×) and chronic (5×) dosing. One-day old male and female flies were placed individually into transparent tubes plugged with a loose wad of cotton on one end, and with transparent food medium and a plastic cap on the other end. Food medium was either vehicle, METH (5.0 mM), αMT (3.0 mM), CIT (0.3, 1.0, or 2.5 mM), PSI (0.03 mM, 3.5 mM), KET (1.0 mM), or WAY (1.0 mM). 1× indicates that flies were fed for 24 h on treatment food under group conditions, then transferred into a tube containing vehicle medium (described above). 5× indicates that flies were fed for 5 consecutive days on treatment food in tube as described above. Flies were placed into the DAMS in groups of n = 16 for each sex in each treatment group, and kept at constant light conditions for at least 5 days while being monitored. All other flies were kept under constant light conditions for 5 days, then tested in the FST so that n = 7 for each sex in each treatment group, but the individual data points in the FST represent the mean behaviors of 16 flies. A total of 896 flies were used to generate n = 7 for males (448) and females (448) for each treatment group (112/group).

Figure 2

Differences in CS sub-strains in the DAMS and FST. The locomotor activity and forced swim responses of 6-day-old flies from two distinct CS lineages (CS^CLWU and CS^X) were compared under control and PSI (0.03 mM) 5× conditions, 5 days after administration. Data were evaluated using 2-way ANOVA; *p < 0.05, ***p < 0.001, ****p < 0.0001. (A) When fed vehicle medium, CS^X flies of both sexes were more active than CS^CLWU, and males of both strains were less active than females. PSI increased locomotor activity in female CS^X flies. No other relevant differences were observed. (B) CS^CLWU males were significantly more immobile than CS^CLWU females and CS^X males. CS^CLWU flies had significantly more bouts of immobility than CS^X. No other relevant differences were observed.

Figure 3

Locomotor activity as measured by DAMS for METH, αMT and CIT. The changes in overall locomotor activity caused by dopaminergic modulators METH (5.0 mM) 5× and αMT (3.0 mM) 5× were compared with those caused by SSRI CIT (0.3, 1.0, and 2.5 mM) 5×. Data were evaluated with 2-way ANOVA; ****p < 0.0001. (A) n = 16 for each group. Control males were significantly less active than control females. METH significantly increased locomotor activity in both males and females. (B) n = 16 for each group. Control males were significantly less active than control females. αMT significantly decreased locomotor activity in both males and females. (C) n = 16 for each group but females CIT (0.3 mM), in which n = 15. Control males were significantly less active than control females. CIT (2.5 mM) significantly decreased locomotor activity in females, but no other differences were observed among groups.

Figure 4

Use of METH, αMT, and CIT to establish predictive validity of FST. The changes in metrics of immobility in the forced swim caused by dopaminergic modulators METH (5.0 mM) 5× and αMT (3.0 mM) 5× were compared with those caused by SSRI CIT (3.0 mM) 5×. Sexes were evaluated independently with one-way ANOVA and Holm-Sidak post-hoc; n = 7 for each group, and each of 7 data points is the mean of 16 flies. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CIT significantly decreased male immobility vs vehicle, but no other relevant differences were observed. CIT increased latency to first immobility in females and males vs vehicle, but no other relevant differences were observed. CIT significantly decreased bouts of immobility in males vs vehicle, but no other relevant differences were observed.

Figure 5

Locomotor activity as measured by DAMS for PSI, WAY, and KET. The changes in overall locomotor activity caused by low (0.03 mM) 1× and high (3.5 mM) 1× dose PSI were assessed, as were those caused by PSI (0.03 mM) 5×, WAY (1.0 mM) 5×, and KET (1.0 mM) 5×. Data were evaluated with 2-way ANOVA; *p < 0.05, **p < 0.01, ****p < 0.0001. (A) Female PSI (3.5 mM) n = 15; all other groups n = 16. Control males were significantly less active than control females. 3.5 mM PSI significantly decreased locomotor activity in females. 3.5 mM and 0.03 mM PSI significantly increased locomotor activity in males. No other relevant differences were observed. (B) Female vehicle n = 15; all other groups n = 16. Control males were significantly less active than control females. KET significantly decreased locomotor activity vs vehicle in females vs vehicle and PSI (0.03 mM). PSI, WAY, and KET significantly decreased locomotor activity in males. Both sexes given KET were significantly less active than males given PSI. No other relevant differences were observed.

Figure 6

Comparison of PSI with CIT in the FST. The changes in metrics of immobility in the forced swim caused by SSRI CIT (3.0 mM) 5× and 1× were compared with those caused by PSI (3.5 mM) 1× and (0.03 mM) 1×. Sexes were evaluated independently with one-way ANOVA and Holm-Sidak post-hoc; n = 7 for each group, and each of 7 data points is the mean of 16 flies. *p < 0.05, **p < 0.01, ****p < 0.0001. Males given CIT 5×, and both high and low PSI were significantly less immobile than vehicle, but no differences were observed in males given CIT 1× or in any females. Males, and females given CIT 5×, and males given both high PSI and low PSI displayed greater latency to first immobility vs vehicle. Males and females given CIT 5×, and males given both high PSI and low PSI displayed significantly fewer bouts of immobility than vehicle.