Model of voluntary ethanol intake in zebrafish: Effect on behavior and hypothalamic orexigenic peptides

Abstract

Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period of time to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically-relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake.

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides

“…Adult zebrafish ( Danio rerio ) of the AB strain were bred in our facility (Rockefeller University, NY) following standard procedures [44], from breeding pairs purchased from ZIRC (Eugene, Oregon) as described in our recent report [37]. The facility was fully accredited by AAALAC.…”

“…To perform WISH, the larvae were rehydrated in a series of PBST-methanol solutions (1:3, 1:1, 3:1), with 10 min in each, followed by a wash in PBST (4 × 5 min), PB (2 × 5 min), and DEPC water (1 × 5 min). They were then processed for WISH using the same procedures as previously described [37], with the temperature of the hybridization and post-hybridization washes increased to 70° C. After color developing, the larvae were fixed for 30 min in 4% PF, and their yolk was removed. They were then immersed in 50% glycerol in PBS for 1 h, then transferred into 100% glycerol overnight at 4° C in dark, afterwards, mounted with 100% glycerol.…”

“…For this procedure, the embryos were exposed at 24 hpf to 0.0 or 0.5% ethanol for 2 h ( n = 7/group/set), with BrdU incorporated at 48 hpf as described above, and the larvae were allowed to grow and were sacrificed at 50 dpf when the density of their peptide neurons was sufficiently high to reveal double labeling. The brains were dissected, fixed in 4% PF immediately at 4° C overnight, then cryoprotected in 25% sucrose at 4° C for 24–48 h, and then frozen at −80° C. For processing, 40 µm serial sagittal sections were cut using a cryostat, and alternate free-floating sections were prepared for digoxigenin-labeled ISH of GAL or OX, which was performed using the same procedures described in our recent publication [37], except for a lower probe concentration (2.5 µl/ml) that revealed enough but not too strong a signal to allow double labeling with BrdU immunofluorescence. After the ISH signal was visualized in NBT/BCIP, the sections were then further processed for BrdU immunofluorescence using the same procedures as described [45, 48], BrdU immunoreactivity was revealed by rat anti-BrdU (1:100) and Cy3-conjugated donkey anti-rat secondary antibody (1:100).…”

“…While there are no studies in this species investigating the effect of early ethanol exposure on these orexigenic peptides, there is evidence that embryonic ethanol impairs a number of neurotransmitter systems in zebrafish, causing a reduction in dopamine and serotonin levels in adult fish [16] and altering the expression of nicotinic acetylcholine receptors and µ-opioid receptor encoding gene [15]. In our recent study describing a model of voluntary ethanol consumption in adult zebrafish [37], we demonstrated that intake of ethanol increases in the expression of the orexigenic peptides, GAL and OX, in the hypothalamus, suggesting the existence of a positive relationship between ethanol and these neuropeptides in zebrafish, similar to that described in rodents [38]. Whereas there are no reports in zebrafish examining the direct effects of these peptides on ethanol consumption, there is evidence in teleosts that central injection of GAL or OX can significantly increase the intake of food [39–41].…”

Orexin/Hypocretin System: Role in Food and Drug Overconsumption

“…While OX activity is inhibited by acute alcohol at a very high dose that promotes sleep (Sharma, Sahota, & Thakkar, 2014), similar to its inhibition after a physiological rise in glucose levels (Burdakov et al, 2005), gene expression and peptide levels of OX are significantly increased by alcohol administered at lower doses (Morganstern et al, 2010). Similarly, in response to voluntary intake, OX gene expression is stimulated during the first 30 minutes following the start of intake (Barson et al, 2015; Lawrence et al, 2006; Sterling, Karatayev, Chang, Algava, & Leibowitz, 2015) but is subsequently unaffected or reduced along with OX peptide levels over the next several hours (Morganstern et al, 2010; Olney et al, 2015, 2017). This time-dependent shift in the effects of alcohol on OX, a stimulation followed by a suppression, may help to explain why individuals engage in alcohol binge sessions of a discrete duration (Carnicella et al, 2014), with drinking continuing until blood alcohol levels are sufficiently high to alter behavior and then ceasing until blood levels have significantly declined.…”