Repeated microdoses of LSD do not alter anxiety or boldness in zebrafish.

The therapeutic use of lysergic acid diethylamide (LSD) has resurfaced in the last decade, prompting further scientific investigation into its effectiveness in many animal models. Zebrafish (Danio rerio) are a popular model organism in medical sciences and are used to examine the repeated administration of pharmacological compounds. Previous zebrafish research found acute LSD altered behaviour and cortisol levels at high (250 µg/L) but not low (5-100 µg/L) levels. In this study, we used a motion tracking system to record and analyze the movement patterns of zebrafish after acute and repeated 10-day LSD exposure (1.5 µg/L, 15 µg/L, 150 µg/L) and after seven days of withdrawal. The open-field and novel object approach tests were used to examine anxiety-like behaviour, boldness, and locomotion. In the acute experiments we observed a significant decrease in high mobility with 1.5 µg/L, 15 µg/L, and 150 µg/L of LSD compared to the control and a decrease in velocity with 1.5 and 15 µg/L. In repeated experiments, there were no significant differences in the levels of anxiety, boldness, or locomotion between all LSD groups and controls immediately after 10-day treatment or after withdrawal.

ß-Carboline (FG-7142) modulates fear but not anxiety-like behaviour in zebrafish.

The ß-Carboline FG-7142 is a partial inverse agonist at the benzodiazepine allosteric site on the GABA-A receptor that induces anxiogenic, proconvulsant, and appetite-reducing effects in many species, including humans. Seizure-kindling effects have been well studied, however anxiogenic properties are relatively unexplored. This study aimed to investigate concentration-dependent effects of FG-7142 on anxiety-like behaviour and fear responses in zebrafish (Danio rerio) using the open-field test (OF) and novel object approach test (NOA). A U-shaped distribution was found with maximal responses in increased immobility and reduced distance moved at 10 µM in the NOA but not the OF. Follow up experiments demonstrated a lack of effect in repeated OF testing and no changes in opercular movements. Furthermore, the effect of FG-7142 was reversed with ethanol treatment. These results suggest that FG-7142 elicits a 'freezing' response in zebrafish via the introduction of novelty, suggesting fear-induction. These findings indicate that FG-7142 may act as an agent to promote acute fear responses in zebrafish.

Cannabinoid type-2 receptors modulate terpene induced anxiety-reduction in zebrafish.

Terpenes are the most extensive and varied group of naturally occurring compounds mostly found in plants, including cannabis, and have an array of potential therapeutic benefits for pathological conditions. The endocannabinoid system can potently modulate anxiety in humans, rodents, and zebrafish. The 'entourage effect' suggests terpenes may target cannabinoid CB1 and CB2 receptors, among others, but this requires further investigation. In this study we first tested for anxiety-altering effects of the predominant 'Super-Class' terpenes, bisabolol (0.001%, 0.0015%, and 0.002%) and terpinolene (TPL; 0.01%, 0.05%, and 0.1%), in zebrafish with the open field test. Bisabolol did not have an effect on zebrafish behaviour or locomotion. However, TPL caused a significant increase in time spent in the inner zone and decrease in time spent in the outer zone of the arena indicating an anxiolytic (anxiety decreasing) effect. Next, we assessed whether CB1 and CB2 receptor antagonists, rimonabant and AM630 (6-Iodopravadoline) respectively, could eliminate or reduce the anxiolytic effects of TPL (0.1%) and ß-caryophyllene (BCP; 4%), another super-class terpene previously shown to be anxiolytic in zebrafish. Rimonabant and AM630 were administered prior to terpene exposure and compared to controls and fish exposed to only the terpenes. AM630, but not rimonabant, eliminated the anxiolytic effects of both BCP and TPL. AM630 modulated locomotion on its own, which was potentiated by terpenes. These findings suggest the behavioural effects of TPL and BCP on zebrafish anxiety-like behaviour are mediated by a selective preference for CB2 receptor sites. Furthermore, the CB2 pathways mediating the anxiolytic response are likely different from those altering locomotion.

Innate colour preference in zebrafish (Danio rerio).

Innate (natural) colour preference in animals is used for a variety of behavioural neuroscience purposes in many animal models. In zebrafish, colour preference is often used in combination with place preference testing and some memory tests. However, baseline colour preference seems to differ in the few studies examining this innate behaviour. This necessitates a protocol for reliable colour preference testing to establish preferences prior to using more complex behavioural paradigms. This procedure involves an aquatic plus maze with a central neutral zone and 4 coloured zones: red, green, yellow, blue. Adult zebrafish spent significantly more time in the blue zone compared to the red and yellow zones. There were no sex differences in colour preference. This procedure is a rapid, affordable, straightforward, and effective method to establish baseline colour preference.

Examining behavioural test sensitivity and locomotor proxies of anxiety-like behaviour in zebrafish.

This study assessed the sensitivity of four anxiety-like behaviour paradigms in zebrafish: the novel tank dive test, shoaling test, light/dark test, and the less common shoal with novel object test. A second goal was to measure the extent to which the main effect measures are related to locomotor behaviours to determine whether swimming velocity and freezing (immobility) are indicative of anxiety-like behaviour. Using the well-established anxiolytic, chlordiazepoxide, we found the novel tank dive to be most sensitive followed by the shoaling test. The light/dark test and shoaling plus novel object test were the least sensitive. A principal component analysis and a correlational analysis also showed the locomotor variables, velocity and immobility, did not predict the anxiety-like behaviours across all behaviour tests.

Effects of ocean acidification on dopamine-mediated behavioral responses of a coral reef damselfish.

We investigated whether CO2-induced ocean acidification (OA) affects dopamine receptor-dependent behavior in bicolor damselfish (Stegastes partitus). Damselfish were kept in aquaria receiving flow through control (pH ~ 8.03; pCO2 ~ 384 µatm) or OA (pH ~ 7.64; CO2 ~ 1100 µatm) seawater at a rate of 1 L min-1. Despite this relatively fast flow rate, fish respiration further acidified the seawater in both control (pH ~7.88; pCO2 ~ 595 µatm) and OA (pH ~7.55; pCO2 ~ 1450 µatm) fish-holding aquaria. After five days of exposure, damselfish locomotion, boldness, anxiety, and aggression were assessed using a battery of behavioral tests using automated video analysis. Two days later, these tests were repeated following application of the dopamine D1 receptor agonist SKF 38393. OA-exposure induced ceiling anxiety levels that were significantly higher than in control damselfish, and SKF 38393 increased anxiety in control damselfish to a level not significantly different than that of OA-exposed damselfish. Additionally, SKF 38393 decreased locomotion and increased boldness in control damselfish but had no effect in OA-exposed damselfish, suggesting an alteration in activity of dopaminergic pathways that regulate behavior under OA conditions. These results indicate that changes in dopamine D1 receptor function affects fish behavior during exposure to OA. However, subsequent measurements of seawater sampled using syringes during the daytime (~3-4 pm local time) from crevasses in coral reef colonies, which are used as shelter by damselfish, revealed an average pH of 7.73 ± 0.03 and pCO2 of 925.8 ± 62.2 µatm; levels which are comparable to Representative Concentration Pathway (RCP) 8.5 predicted end-of-century mean OA levels in the open ocean. Further studies considering the immediate environmental conditions experienced by fish as well as individual variability and effect size are required to understand potential implications of the observed OA-induced behavioral effects on damselfish fitness in the wild.

Zebrafish (Danio rerio) shoaling in light and dark conditions involves a complex interplay between vision and lateral line.

We know little about how - or even if in some species - fish shoal in darkness. We hypothesized that 'dark shoaling' occurs in zebrafish and therefore must depend upon lateral line sensory input. Shoaling in groups of five adult zebrafish was analyzed with motion tracking software. We measured average inter-individual distance, time near the arena wall (thigmotaxis zone) and total distance traveled under normal room light, and in near-complete darkness (infrared light at 850 nm). These observations were repeated in fish treated with cobalt chloride (CoCl2), which ablates lateral line function. In untreated controls, dark shoaling was reduced compared to in light, but nonetheless still present. Elimination of lateral line sensory input by CoCl2 treatment similarly reduced, but did not eliminate, shoaling under both light and dark. Our findings indicate that normal zebrafish shoaling in light or dark requires both visual and lateral line inputs, with neither alone sufficient for normal shoaling.

Effects of super-class cannabis terpenes beta-caryophyllene and alpha-pinene on zebrafish behavioural biomarkers.

Terpenes possess a wide range of medicinal properties and are potential therapeutics for a variety of pathological conditions. This study investigated the acute effects of two cannabis terpenes, ß-caryophyllene and α-pinene, on zebrafish locomotion, anxiety-like, and boldness behaviour using the open field exploration and novel object approach tests. ß-caryophyllene was administered in 0.02%, 0.2%, 2.0%, and 4% doses. α-pinene was administered in 0.01%, 0.02%, and 0.1% doses. As α-pinene is a racemic compound, we also tested its (+) and (-) enantiomers to observe any differential effects. ß-caryophyllene had only a sedative effect at the highest dose tested. α-pinene had differing dose-dependent effects on anxiety-like and motor variables. Specifically, (+)-α-pinene and (-)-α-pinene had significant effects on anxiety measures, time spent in the thigmotaxis (outer) or center zone, in the open field test, as well as locomotor variables, swimming velocity and immobility. (+ /-)-α-pinene showed only a small effect on the open field test on immobility at the 0.1% dose. This study demonstrates that α-pinene can have a sedative or anxiolytic effect in zebrafish and may have different medicinal properties when isolated into its (+) or (-) enantiomers.

Continuous light (relative to a 12:12 photoperiod) has no effect on anxiety-like behaviour, boldness, and locomotion in coho salmon (Oncorhynchus kisutch) post-smolts in recirculating aquaculture systems at a salinity of either 2.5 or 10 ppt.

There is increased interest in rearing salmon in Recirculating Aquaculture Systems (RAS), where environmental conditions can be tightly controlled to optimize growth. Photoperiod and salinity are two important parameters that can be manipulated in RAS. A longer photoperiod permits more time for feeding, while intermediate salinities may reduce the energetic costs of ionoregulation, both of which may enhance growth. However, little is known about how rearing at different photoperiods and salinity affect behaviour, an understudied but important research topic for intensive fish rearing. To address this, we examined the behavioural effects of two salinities and two photoperiod regimes in coho salmon (Oncorhynchus kisutch) post-smolts reared continuously for 120 days in a RAS. Fish were reared on a photoperiod of either 12 h light:12 h dark (12:12), or 24 h light (24:0) at salinities of 2.5 and 10 ppt. To investigate behavioural differences associated with these treatments, we quantified: i) movement in an open-field test, ii) exploratory behaviour/boldness using a novel object approach test, and iii) anxiety-like behaviour with a light/dark test. The 24:0 groups displayed no differences in boldness/anxiety-like behaviour and locomotion relative to the 12:12 groups at their respective salinities. Taken together, fish reared under continuous light (24:0) show negligible behavioural alterations compared to fish reared under normal light dark conditions (12:12).

Zebrafish aversion to infrasound in an open field test.

Aquatic species are capable of detecting infrasound (sub-20 Hz frequencies) which may be a source of anthropogenic pollution and have a detrimental impact on the environmental fitness of fish. Infrasound is generated by infrastructure, producing acoustic frequency peaks that are not discernible by humans. The presence of these frequencies may therefore impact the environmental wellbeing of aquatic laboratory animals, which are often housed in spaces adjacent to facilities producing infrasound. To investigate the potential impact of infrasound, we used wild-type zebrafish (Danio rerio) and exposed them to short periods of infrasound at either 5, 10, 15, or 20 Hz, or 0 Hz as a control group. A motion-tracking software system was used to monitor fish movement in an open field test and arena location, distance moved, and immobility were quantified. There was a significant effect of 15 Hz which caused the fish to spend more time away from the infrasound source. The 20 Hz group also spent significantly less time in the zone closest to the speaker. There were no differences in distance moved or immobility between infrasound and control groups. These findings demonstrate that 15 Hz infrasound has aversive effects on zebrafish, causing them to move away from the sound source. To enhance environmental enrichment and wellbeing of aquatic laboratory animals, sources of infrasound pollution should be investigated and mitigated.

Robust behavioural effects in response to acute, but not repeated, terpene administration in Zebrafish (Danio rerio).

Terpenes are fragrant aromatic compounds produced by a variety of plants, most notably cannabis and hops. With increasing legalization of cannabis there is a need to better understand the behavioural effects of terpenes and ultimately their therapeutic value. Our study investigated the dose-dependent impact of three terpenes (limonene 0.25, 0.5, 0.75%; ß-myrcene 0.001, 0.01, 0.1%; and 0.0001, 0.001, 0.00125% linalool) on zebrafish (Danio rerio) behaviour when exposed both acutely and repeatedly over a 7-day period. Anxiety-like behaviour, boldness, and locomotion were assessed using the open field test and the novel object approach test. In the acute dosing experiment, limonene and ß-myrcene exposed groups demonstrated a significant decrease in locomotion, a decrease in anxiety-like behaviour, and an increase in boldness, while linalool treatment groups demonstrated only minor alterations in locomotion. Moreover, repeated exposure to limonene (0.39%) or ß-myrcene (0.0083%) for a seven day period did not result in any significant behavioural effects. In conclusion, our study provides support for an anxiolytic and sedative effect in zebrafish in response to acute limonene and ß-myrcene exposure that is no longer present after one week of repeated exposure.

Analysis of the potential behavioral impact of methanol when used as a solvent: Dataset from zebrafish (Danio rerio) behavioral research.

Toxicants are commonly administered to experimental organisms using solvents as vehicles. One common vehicle for dissolving toxicants is methanol (CH3OH), a solvent which on its own is capable of altering physiology and behavior high concentrations. This dataset describes behavioral results in zebrafish (Danio rerio) individually exposed to methanol (0.25%, 2.5% vol/vol), or control water, for 30 min prior to behavioral testing. Zebrafish were placed into an open field arena to examine locomotion and zone preference, which was recorded and quantified with motion-tracking software (EthoVision XT). Time spent in the outer ("thigmotaxis") zone of the arena is a proxy for increased anxiety-like behavior in zebrafish. Additionally, a novel object was placed into the center of the arena to quantify relative increases in boldness/exploration between the methanol and control groups. There were no differences in time spent in any zone of the arena or distance travelled between either group, in either test.

The dose makes the poison: Non-linear behavioural response to CO2-induced aquatic acidification in zebrafish (Danio rerio).

CO2-induced aquatic acidification is predicted to affect fish neuronal GABAA receptors leading to widespread behavioural alterations. However, the large variability in the magnitude and direction of behavioural responses suggests substantial species-specific CO2 threshold differences, life history and parental acclimation effects, experimental artifacts, or a combination of these factors. As an established model organism, zebrafish (Danio rerio) can be reared under stable conditions for multiple generations, which may help control for some of the variability observed in wild-caught fishes. Here, we used two standardized tests to investigate the effect of 1-week acclimatization to four pCO2 levels on zebrafish anxiety-like behaviour, exploratory behaviour, and locomotion. Fish acclimatized to 900 µatm CO2 demonstrated increased anxiety-like behaviour compared to control fish (~480 µatm), however, the behaviour of fish exposed to 2200 µatm CO2 was indistinguishable from that of controls. In addition, fish acclimatized to 4200 µatm CO2 had decreased anxiety-like behaviour; i.e. the opposite response than the 900 µatm CO2 treatment. On the other hand, exploratory behaviour did not differ among any of the pCO2 exposures that were tested. Thus, zebrafish behavioural responses to elevated pCO2 are not linear; with potential important implications for physiological, environmental, and aquatic acidification studies.

Vision of conspecifics decreases the effectiveness of ethanol on zebrafish behaviour.

Aquatic organisms in pharmacology and toxicology research are often exposed to compounds in isolation prior to physiological or behavioural testing. Recent evidence suggests that the presence of conspecifics during a stressful event can modulate behavioural outcomes (called 'social buffering') when testing occurs within the same context. It is unknown, however, whether the social environment during exposure interacts with the efficacy of anxiety-altering substances when subsequently tested in the absence of conspecifics. In this study, zebrafish were individually exposed to habitat water or ethanol (1.0% vol/vol) while untreated conspecifics were visually present or absent during dosing. Using the novel object approach test, a validated test of boldness and anxiety-like behaviour, we observed significantly greater effects of ethanol in isolated fish, compared to fish with a view of conspecifics during dosing. These results were not explained by altered locomotion during exposure, which might otherwise increase drug uptake. This highlights the need to consider the social environment during exposure when conducting and interpreting behavioural research involving drug or toxicant exposure.

Shoaling, boldness, anxiety-like behavior and locomotion in zebrafish (Danio rerio) are altered by acute benzo[a]pyrene exposure.

Environmental exposure to crude oil and/or its derivatives in fishes can negatively impact survival, morphology and physiology, but relatively little focus has been on behavior. Exposures can influence prey-predator interactions, courtship and other vital behaviors, leading to individual or population disruption at toxicant levels well below those producing morphological or physiological changes. The few behavioral studies of polycyclic aromatic hydrocarbons (PAHs) on fish behavior have yielded highly inconsistent results, likely relating to chronic vs. acute treatment. A few studies report lethargy and decreased exploratory behavior, while others indicate increased anxiety and greater exploratory behavior with PAH exposure. In our study on zebrafish (Danio rerio), we hypothesized that even relatively brief (30 min) exposure to the PAH benzo[a]pyrene (B[a]P) would impact group shoaling and individual behaviors in open field and novel object exploration tests. Exposures comprised measured concentrations of 1.0 µM, 10 µM, or 100 µM, B[a]P. Compared to controls, inter-individual distance (IID) was significantly increased by 100 µM B[a]P, but not by 1.0 µM or 10 µM B[a]P. Total distance moved by shoals was decreased significantly at B[a]P concentrations of 1.0 µM, 10 µM and 100 µM. In the open field test of individual locomotion and anxiety-like behavior, time spent in the thigmotaxis zone along the walls of the circular test arena (a proxy for anxiety-like behavior), was decreased at 100 µM. In the novel object approach test to investigate boldness, time spent near the object was significantly increased by both 10 µM and 100 µM B[a]P. Collectively, these data indicate a complex suite of changes in zebrafish including altered shoal dynamics, decreased anxiety, increased boldness, and decreased locomotion associated with exposure to B[a]P.

Opposing effects of acute and repeated nicotine exposure on boldness in zebrafish.

Nicotine is an addictive compound that activates neuronal nicotinic acetylcholine receptors (nAChRs) and causes behavioural effects that vary with dose, schedule of administration, and animal model. In zebrafish (Danio rerio), acute doses of nicotine have been consistently found to have anxiolytic properties, whereas, chronic exposure elicits anxiogenic effects. To date, however, studies on repeated nicotine administration and the effects of nicotine withdrawal have not been well explored using this model. In this study, we administered nicotine with three different dosing regimens: 1. Single exposures of a "high" dose (25, 50, 100, or 400 mg/L) for 3 minutes. 2. Single exposures to a "low" dose (2.5, 5, or 20 mg/L) for one hour. 3. Repeated one-hour exposure to a "low" dose (2.5, 5, or 20 mg/L) for 21 days. The novel object approach test was used to examine boldness based on the tendency of the fish to explore a novel object. Acutely, nicotine significantly increased the time spent approaching the object with both three-minute and onehour durations of exposure, indicating increased boldness. Conversely, after repeated nicotine exposure for 21 days, fish spent less time approaching the object suggesting a decrease in boldness. Distance moved was unaffected one hour after repeated nicotine exposure, yet decreased after a two-day withdrawal period. Our work suggests that nicotine can have opposing effects on boldness that vary based on dosage and schedule of exposure.

Behavioural responses of the hagfish Eptatretus stoutii to nutrient and noxious stimuli.

The suitability of a traditional testing paradigm (e.g. choice chamber) for assessing chemosensory behaviour in the Pacific hagfish, Eptatretus stoutii, was examined. Actively-swimming hagfish, tested at night, showed no preference for any region of a T-maze in the absence of a stimulus, but in the presence of an olfactory food cue, spent significantly more time in the zone where the cue was placed. Conversely, hagfish avoided spending time in the zone the fish anaesthetic 3-amino benzoic acid ethylester (MS-222) was placed, and demonstrated significantly more reversal responses in which the fish moved its body backwards. These data suggest that hagfish are an amenable model species for laboratory testing of behaviour.

The submerged plus maze as an assay for studying anxiety-like behaviour in fish.

The elevated plus maze is a commonly used and well-validated test of anxiety-related behaviour in rodents. The use of fish in behavioural neuroscience paradigms is increasing, necessitating an equivalent test for studying anxiety-like behaviour in fish. Because behaviour in the elevated plus maze is driven by aversion to open space, the submerged plus maze described here uses transparent walls to elicit similar behaviour in fish. The tendency of fish to explore or avoid the sections of the maze containing transparent walls is used as proxy for anxiety level. This submerged plus maze was designed and validated for convict cichlid (Amatitlania nigrofasciata) fish.

Neurodevelopmental and Metabolomic Responses from Prenatal Coexposure to Perfluorooctanesulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats.

Methylmercury (MeHg) and perfluorooctanesulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory, and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain as well as delayed reflexes and innate behavioral responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behavior. However, other developmental test results suggested antagonism, as PFOS-only and MeHg-only juveniles had increased hyperactivity and thigmotaxic behavior, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioral observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g., lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.

Repeated ethanol exposure increases anxiety-like behaviour in zebrafish during withdrawal.

Zebrafish (Danio rerio) are quickly becoming an important model organism in behavioural neuroscience and drug addiction research. Conditioned place preference studies show that drugs of abuse produce responses in zebrafish that are similar to mammalian animal models. Repeated administration of ethanol in zebrafish results in withdrawal-induced behavioural responses that vary with dose and exposure duration, requiring additional investigation. Here, we examine the effects of ethanol withdrawal on anxiety-like behaviours in adult zebrafish after a 21-day ethanol dosing schedule at either 0.4% or 0.8%. Anxiety-like behaviour was measured with the novel object approach test; this test involves placing a fish in a circular arena with a novel object in the centre and observing the amount of exploration of the object. We found increased anxiety-like behaviour during ethanol withdrawal. This study adds to the growing body of literature that validates the zebrafish as a model organism in the field of behavioural neuroscience and addiction.