Tryptophan Hydroxylase 2 Deficiency Modifies the Effects of Fluoxetine and Pargyline on the Behavior, 5-HT- and BDNF-Systems in the Brain of Zebrafish (Danio rerio)

Abstract

The mechanisms of resistance to antidepressant drugs is a key and still unresolved problem of psychopharmacology. Serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) play a key role in the therapeutic effect of many antidepressants. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT synthesis in the brain. We used zebrafish (Danio rerio) as a promising model organism in order to elucidate the effect of TPH2 deficiency caused by p-chlorophenylalanine (pCPA) on the alterations in behavior and expression of 5-HT-related (Tph2, Slc6a4b, Mao, Htr1aa, Htr2aa) and BDNF-related (Creb, Bdnf, Ntrk2a, Ngfra) genes in the brain after prolonged treatment with two antidepressants, inhibitors of 5-HT reuptake (fluoxetine) and oxidation (pargyline). In one experiment, zebrafish were treated for 72 h with 0.2 mg/L fluoxetine, 2 mg/L pCPA, or the drugs combination. In another experiment, zebrafish were treated for 72 h with 0.5 mg/L pargyline, 2 mg/L pCPA, or the drugs combination. Behavior was studied in the novel tank diving test, mRNA levels were assayed by qPCR, 5-HT and its metabolite concentrations were measured by HPLC. The effects of interaction between pCPA and the drugs on zebrafish behavior were observed: pCPA attenuated “surface dwelling” induced by the drugs. Fluoxetine decreased mRNA levels of Tph2 and Htr2aa genes, while pargyline decreased mRNA levels of Slc6a4b and Htr1aa genes. Pargyline reduced Creb, Bdnf and Ntrk2a genes mRNA concentration only in the zebrafish treated with pCPA. The results show that the disruption of the TPH2 function can cause a refractory to antidepressant treatment.

Effects of fluoxetine on fish: What do we know and where should we focus our efforts in the future?

“…The novel tank test allows the evaluation of the vertical exploratory behavior of a fish introduced into a new environment (e.g., a new aquarium), considering several different parameters such as swimming distance, speed, latency for free swimming (time between introduction and movement/ exploration of the aquarium), freezing behavior, time and number of entries at different areas of the tank (e.g., bottom, middle, top). In general, several studies that used D. rerio (embryos and adults) and O. latipes (medaka) adults, testing concentrations from 0.004 to 10,000 μg/L and different exposure periods (from 3 min to 30 days), showed decreased stress levels after fluoxetine exposure (Abreu et al, 2016a;Ansai et al, 2016;Evsiukova et al, 2021;Farias et al, 2020;Giacomini et al, 2016;Pittman and Hylton, 2015;Pittman and Ichikawa, 2013;Singer et al, 2016;Vera-Chang et al, 2018Wong et al, 2013). Overall, an increase in the exploratory behavior, with fish swimming longer at the top of the tank, decreases in freezing, lower latency to enter the top area and higher mean speed has been reported.…”

“…This decreased mRNA expression may impair neurotransmission, leading to behavioral changes (Dzieweczynski et al, 2016;Pelli and Connaughton, 2015;Winder et al, 2012). Evsiukova et al (2021) observed that a 72-h exposure to 200 μg/L fluoxetine (and the combined effect of 200 μg/L fluoxetine with 2000 μg/L an irreversible TPH2 inhibitor p-chlorophenylalanine (pCPA)), resulted in a significant decrease in the mRNA levels of tryptophan hydroxylase 2 (tph2) and serotonin receptor 2A (htr2aa) genes in the brain of adult D. rerio exposed. Theodoridi et al (2017) reported up-regulated levels of serotonin receptor 2b (htr2b) in the brain of adult D. rerio (dominant and submissive) exposed, for 2 h, to 5000 μg/L fluoxetine.…”

Age-Related Alterations in the Level and Metabolism of Serotonin in the Brain of Males and Females of Annual Turquoise Killifish (Nothobranchius furzeri)

“…The deficit of the TPH activity results in a dramatic decrease in the 5-HT and 5-HIAA levels in the brain of mice [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. An irreversible inhibitor of TPH, p-chlorophenylalanine, dramatically decreases the 5-HT and 5-HIAA levels in the brain of zebrafish [ 46 , 47 ]. Secondly, an MAO inhibitor, pargyline, dramatically increases the 5-HT level and decreases the 5-HIAA concentration in the brain of zebrafish [ 46 , 47 ].…”

“…An irreversible inhibitor of TPH, p-chlorophenylalanine, dramatically decreases the 5-HT and 5-HIAA levels in the brain of zebrafish [ 46 , 47 ]. Secondly, an MAO inhibitor, pargyline, dramatically increases the 5-HT level and decreases the 5-HIAA concentration in the brain of zebrafish [ 46 , 47 ]. Therefore, the age-related decrease in TPH activity and increase in MAO activity together accelerate the observed age-dependent decrease in the 5-HT levels in killifish brain.…”

Tryptophan Metabolism in Depression: A Narrative Review with a Focus on Serotonin and Kynurenine Pathways

“…Another recent study highlights the relationship between TPH2 expression and BDNF levels. The administration of a pargyline (MAO inhibitor) in zebrafish treated with an irreversible inhibitor of TPH2 (p-chlorophenylalanine) led to reduced BDNF levels, revealing an interdependence between 5-HT and BDNF systems in the antidepressant response [ 112 ]. The interaction between 5-HT neurotransmission and the BDNF-related pathways was also supported by another study.…”