Research articleSex differences in adult zebrafish anxiolytic-like responses to diazepam and melatonin
Introduction
Sex differences are an important variable in biomedical research, long recognized to modulate complex behaviors in both humans and animals [1,2]. In mammals, females often express more empathy and socialization [3], whereas males display higher aggression [[4], [5], [6]], modulated by steroids [7,8]. Sex differences are also common in human psychiatric disorders, as women are more prone to anxiety and depression [9,10], while men are more likely to develop autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) [11,12]. Paralleling these data, female rodents are also more vulnerable to depression-like states induced by chronic stress [13,14], whereas males display more social deficits in some experimental models of ASD [15]. Multiple other sex behavioral differences exist in these models [[16], [17], [18]], calling for further translational cross-species research on sex differences in the central nervous system (CNS) traits.
Pharmacological responses, including drug responsivity, metabolism and/or adverse side-effects, also differ between sexes [[19], [20], [21]]. For example, women are more sensitive to selective serotonin reuptake inhibitors (SSRIs), and men - to tricyclic antidepressants [22]. In rodents, females respond better to chronic clomipramine [23] or acute ketamine [24] and nicotine [25]. In contrast, only males demonstrate anxiolytic-like responses to diazepam [26], and show higher cocaine and nicotine addiction [27]. Diazepam is a widely used benzodiazepine anxiolytic drug, often prescribed to treat anxiety spectrum disorders, sleep deficits and related comorbidities [28,29]. Melatonin is an important hormone, also widely used as an anxiolytic medication [30,31]. Notably, these two clinically relevant anxiolytic drugs also show sex differences in their action in humans [32,33], necessitating further analyses of their effects in terms of individual differences and their potential role in ‘personalized medicine’. However, the exact nature and associated risks of sex differences in pharmacological responses to CNS drugs remain poorly understood [34], thus, meriting further clinical and experimental studies.
Animal models are an important tool able to generate translational insights into human disorders [[35], [36], [37]], including studies of anxiety and depression [[38], [39], [40]], as well as testing anxiolytic and antidepresant drugs in vivo [[41], [42], [43]]. In rodents, a benzodiazepine receptor agonist diazepam evokes overt anxiolytic-like [[44], [45], [46]] and amnestic effects [47,48], whereas melatonin is anxiolytic [[49], [50], [51]], but promnestic [[52], [53], [54]]. Furthermore, in line with clinical findings, both diazepam and melatonin also display sex differences in their behavioral effects in rodents [26,55].
Complementing rodent models, the zebrafish (Danio rerio) is rapidly becoming an important novel organism in translational neuroscience and pharmacology research [56,57], including its excellent potential for CNS drug screening in high- and medium-throughput batteries [31,58]. Prior studies have tested diazepam and melatonin in both adult and larvae zebrafish [[59], [60], [61], [62], [63]], supporting evolutionarily conserved mechanisms of their action. For example, diazepam is anxiolytic in both adult [61,64] and larval zebrafish [65], and melatonin shows similar effects in adult fish [66,67]. To examine potential sex differences in CNS responses to these clinically important anxiolytic drugs in a promising model organism with high-throughput drug screening potential, here we assess locomotion and anxiety-like behaviors in drug-free and drug-treated male and female adult zebrafish.
Section snippets
Animals
A total of 150 adult 1-year old zebrafish (75 male and 75 female) of the wild-type short-fin strain were housed 1 fish/L in 50-L tanks equipped with biological filters, under constant aeration and a natural (14 h light: 10 h dark) photoperiod (lights on at 8:00 am). Water temperature was maintained at 27±0.4 ◦C; pH 7.0 ± 0.2, with dissolved oxygen kept at 6.0 ± 0.1 mg/L, total ammonia at < 0.01 mg/L, total hardness at 6 mg/L, and alkalinity at 22 mg/L CaCO3. Animal experimentation reported here
Results
In the mixed-sex cohorts tested in Experiment 1, female zebrafish showed increased locomotion, as assessed by higher absolute turn angle (reflecting turning irrespective of direction), as well as lower frequency and duration of freezing, compared to males (Fig. 2A, Table 1). These female fish also spent less time in top of the novel tank test, demonstrating higher anxiety-like behavior (Fig. 2A). Females from the same-sex cohorts also spent less time in top of the tank, presenting a similar
Discussion
The present study aimed to examine potential sex differences in zebrafish activity and anxiety (Experiment 1), as well as their responsivity to anxiolytic drugs diazepam and melatonin (Experiment 2). The rationale for this study, as already mentioned, was based on overt sex differences reported for human and rodent stress responses [[75], [76], [77]], as well as on their sex differences in responses to both diazepam and melatonin [26,32,33,55], the two common anxiolytic agents selected here for
Contributors disclosure
R.G., A.C.V.V.G., L.M., M.S.A., K.A.D. and A.V.K. conceptualized the experiments, wrote the manuscript and prepared the figures. R.G., L.M., and M.S.A conducted the experimental procedures. All authors have discussed the results, and have read and approved the manuscript for publication.
Declaration of competing interest
None.
Acknowledgments
A.C.V.V.G. is supported by the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) research fellowships 17/2551-0001-269-0. A.V.K. is supported by the Russian Science Foundation (RSF) grant 19-15-00053. He is the Chair of the International Zebrafish Neuroscience Research Consortium (ZNRC) that coordinated this multi-laboratory collaborative project. K.A.D. is supported by the President of Russia Graduate Fellowship and the Special Productivity Fellowship for PhD students from
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