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).

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.

Acid-base physiology, neurobiology and behaviour in relation to CO2-induced ocean acidification.

Experimental exposure to ocean and freshwater acidification affects the behaviour of multiple aquatic organisms in laboratory tests. One proposed cause involves an imbalance in plasma chloride and bicarbonate ion concentrations as a result of acid-base regulation, causing the reversal of ionic fluxes through GABAA receptors, which leads to altered neuronal function. This model is exclusively based on differential effects of the GABAA receptor antagonist gabazine on control animals and those exposed to elevated CO2 However, direct measurements of actual chloride and bicarbonate concentrations in neurons and their extracellular fluids and of GABAA receptor properties in aquatic organisms are largely lacking. Similarly, very little is known about potential compensatory mechanisms, and about alternative mechanisms that might lead to ocean acidification-induced behavioural changes. This article reviews the current knowledge on acid-base physiology, neurobiology, pharmacology and behaviour in relation to marine CO2-induced acidification, and identifies important topics for future research that will help us to understand the potential effects of predicted levels of aquatic acidification on organisms.

Dopamine D1 receptor activation leads to object recognition memory in a coral reef fish.

Object recognition memory is the ability to identify previously seen objects and is an adaptive mechanism that increases survival for many species throughout the animal kingdom. Previously believed to be possessed by only the highest order mammals, it is now becoming clear that fish are also capable of this type of memory formation. Similar to the mammalian hippocampus, the dorsolateral pallium regulates distinct memory processes and is modulated by neurotransmitters such as dopamine. Caribbean bicolour damselfish (Stegastes partitus) live in complex environments dominated by coral reef structures and thus likely possess many types of complex memory abilities including object recognition. This study used a novel object recognition test in which fish were first presented two identical objects, then after a retention interval of 10 min with no objects, the fish were presented with a novel object and one of the objects they had previously encountered in the first trial. We demonstrate that the dopamine D1-receptor agonist (SKF 38393) induces the formation of object recognition memories in these fish. Thus, our results suggest that dopamine-receptor mediated enhancement of spatial memory formation in fish represents an evolutionarily conserved mechanism in vertebrates.

Episodic-like memory in zebrafish.

Episodic-like memory tests often aid in determining an animal's ability to recall the what, where, and which (context) of an event. To date, this type of memory has been demonstrated in humans, wild chacma baboons, corvids (Scrub jays), humming birds, mice, rats, Yucatan minipigs, and cuttlefish. The potential for this type of memory in zebrafish remains unexplored even though they are quickly becoming an essential model organism for the study of a variety of human cognitive and mental disorders. Here we explore the episodic-like capabilities of zebrafish (Danio rerio) in a previously established mammalian memory paradigm. We demonstrate that when zebrafish were presented with a familiar object in a familiar context but a novel location within that context, they spend more time in the novel quadrant. Thus, zebrafish display episodic-like memory as they remember what object they saw, where they saw it (quadrant location), and on which occasion (yellow or blue walls) it was presented.

Modulation of distal calcium electrogenesis by neuropeptide Y1 receptors inhibits neocortical long-term depression.

In layer 5 neocortical pyramidal neurons, backpropagating action potentials (bAPs) firing at rates above a critical frequency (CF) induce supralinear Ca²âº influx and regenerative potentials in apical dendrites. Paired temporally with an EPSP, this Ca²âº influx can result in synaptic plasticity. We studied the actions of neuropeptide Y (NPY), an abundant neocortical neuropeptide, on Ca²âº influx in layer 5 pyramidal neurons of somatosensory neocortex in Sprague Dawley and Wistar rats, using a combination of somatic and dendritic intracellular recordings and simultaneous Ca²âº imaging. Ca²âº influx induced by trains of bAPs above a neuron's CF was inhibited by NPY, acting only at the distal dendrite, via Y1 receptors. NPY does not affect evoked synaptic glutamate release, paired synaptic facilitation, or synaptic rundown in longer trains. Extracellular Cs⁺ did not prevent NPY's postsynaptic effects, suggesting it does not act via either G-protein-activated inwardly rectifying K⁺ conductance (G(IRK)) or hyperpolarization-activated, cyclic nucleotide-gated channels. NPY application suppresses the induction of the long-term depression (LTD) normally caused by pairing 100 EPSPs with bursts of 2 bAPs evoked at a supracritical frequency. These findings suggest that distal dendritic Ca²âº influx is necessary for LTD induction, and selective inhibition of this distal dendritic Ca²âº influx by NPY can thus regulate synaptic plasticity in layer 5 pyramidal neurons.

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.

Sex, drugs, and zebrafish: Acute exposure to anxiety-modulating compounds in a modified novel tank dive test.

This study investigated the effects of anxiogenic and anxiolytic drugs on zebrafish (Danio rerio) behaviour using a modified novel tank dive test with higher walls and a narrower depth. Zebrafish were administered chondroitin sulfate, beta-carboline, delta-9-tetrahydrocannabinol (THC), ethanol, and beta-caryophyllene, and their behaviours were evaluated for geotaxis, swimming velocity, and immobility. Both anxiogenic and anxiolytic compounds generally increased bottom-dwelling behaviour, suggesting that the tank's modified dimensions significantly influence zebrafish responses. EC50 values for ethanol showed a lower threshold for velocity reduction compared to zone preference. Chondroitin sulfate uniquely caused a sex-specific increase in male swimming velocity, whereas no other sex-differences were observed with any compound. Interestingly, the presence of drug-treated fish did not alter the behaviour of observer fish, suggesting limited social buffering effects. The findings underscore the complexity of zebrafish behavioural phenotypes and highlight the need for considering tank dimensions and multiple behavioural parameters to accurately assess the effects of anxiety-modulating drugs. This study demonstrates the utility of the modified novel tank dive test in providing nuanced insights into the behavioural effects of different pharmacological agents in zebrafish.

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.

Dopamine modulates synaptic plasticity in dendrites of rat and human dentate granule cells.

The mechanisms underlying memory formation in the hippocampal network remain a major unanswered aspect of neuroscience. Although high-frequency activity appears essential for plasticity, salience for memory formation is also provided by activity in ventral tegmental area (VTA) dopamine projections. Here, we report that activation of dopamine D1 receptors in dentate granule cells (DGCs) can preferentially increase dendritic excitability to both high-frequency afferent activity and high-frequency trains of backpropagating action potentials. Using whole-cell patch clamp recordings, calcium imaging, and neuropeptide Y to inhibit postsynaptic calcium influx, we found that activation of dendritic voltage-dependent calcium channels (VDCCs) is essential for dopamine-induced long-term potentiation (LTP), both in rat and human dentate gyrus (DG). Moreover, we demonstrate previously unreported spike-timing-dependent plasticity in the human hippocampus. These results suggest that when dopamine is released in the dentate gyrus with concurrent high-frequency activity there is an increased probability that synapses will be strengthened and reward-associated spatial memories will be formed.

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.

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.

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.

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.

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.

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.

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.