Acute effect of Δ-9-tetrahydrocannabinol on neuromuscular transmission and locomotive behaviors in larval zebrafish.

Given the increasing trend of cannabis use for recreational and therapeutic purposes, a comprehensive examination of cannabis effects is warranted. The principal psychoactive constituent of cannabis, Δ-9-tetrahydrocannabinol (THC), is a potent disrupter of neurodevelopment. Nevertheless, the impact of acute exposure to THC on developing motor systems is not well-investigated. In this study, using a neurophysiological whole cell patch clamp approach we demonstrated that a 30-min exposure to THC can alter spontaneous synaptic activities at neuromuscular junctions of 5-day post-fertilized zebrafish. An increased frequency of synaptic activity and altered decay kinetic properties were documented in the THC-treated larvae. Locomotive behaviors, including swimming activity rate and C-start escape response to sound were also affected by THC. Although the THC-treated larvae displayed hyperactivity of their basal swimming levels, their escape response rate to sound stimuli was reduced. These findings suggest that acute exposure to THC can disrupt neuromuscular transmission and locomotor-driven responses in developing zebrafish.NEW & NOTEWORTHY Acute exposure to THC alters motor neuron-muscle communication and motor behaviors in developing zebrafish. Our neurophysiology data indicated that the properties of spontaneous synaptic activity at neuromuscular junctions, such as decay component of acetylcholine receptors and frequency of synaptic events, were affected by a 30-min exposure to THC. Hyperactivity and reduced responsiveness to the sound stimulus were also observed in the THC-treated larvae. Exposure to THC during early developing stages may induce motor dysfunction.

Impact of Copper Nanoparticles and Copper Ions on Transcripts Involved in Neural Repair Mechanisms in Rainbow Trout Olfactory Mucosa.

Olfactory mucosa is well known for its lifelong ability for regeneration. Regeneration of neurons and regrowth of severed axons are the most common neural repair mechanisms in olfactory mucosa. Nonetheless, exposure to neurotoxic contaminants, such as copper nanoparticles (CuNPs) and copper ions (Cu2+), may alter the reparative capacity of olfactory mucosa. Here, using RNA-sequencing, we investigated the molecular basis of neural repair mechanisms that were affected by CuNPs and Cu2+ in rainbow trout olfactory mucosa. The transcript profile of olfactory mucosa suggested that regeneration of neurons was inhibited by CuNPs. Exposure to CuNPs reduced the transcript abundances of pro-inflammatory proteins which are required to initiate neuroregeneration. Moreover, the transcript of genes encoding regeneration promoters, including canonical Wnt/ß-catenin signaling proteins and developmental transcription factors, were downregulated in the CuNP-treated fish. The mRNA levels of genes regulating axonal regrowth, including the growth-promoting signals secreted from olfactory ensheathing cells, were mainly increased in the CuNP treatment. However, the reduced transcript abundances of a few cell adhesion molecules and neural polarity genes may restrict axonogenesis in the CuNP-exposed olfactory mucosa. In the Cu2+-treated olfactory mucosa, both neural repair strategies were initiated at the transcript level. The stimulation of repair mechanisms can lead to the recovery of Cu2+-induced olfactory dysfunction. These results indicated CuNPs and Cu2+ differentially affected the neural repair mechanism in olfactory mucosa. Exposure to CuNP had greater effects on the expression of genes involved in olfactory repair mechanisms relative to Cu2+ and dysregulated the transcripts associated with stem cell proliferation and neural reconstitution.

The Combined Effect of Copper Nanoparticles and Microplastics on Transcripts Involved in Oxidative Stress Pathway in Rainbow Trout (Oncorhynchus Mykiss) Hepatocytes.

Copper nanoparticles (CuNPs) and microplastics (MPs) are two emerging contaminants of freshwater systems. Despite their co-occurrence in many water bodies, the combined effects of CuNPs and MPs on aquatic organisms are not well-investigated. In this study, primary cultures of rainbow trout hepatocytes were exposed to dissolved Cu, CuNPs, MPs, or a combination of MPs and CuNPs for 48 h, and the transcript abundances of oxidative stress-related genes were investigated. Exposure to CuNPs or dissolved Cu resulted in a significant increase in the transcript abundances of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). Exposure to CuNPs also led to an upregulation in the expression of Na+/K+ ATPase alpha 1 subunit (ATP1A1). Microplastics alone or in combination with CuNPs did not have a significant effect on abundances of the target gene transcripts. Overall, our findings suggested acute exposure to CuNPs or dissolved ions may induce oxidative stress in hepatocytes, and the Cu-induced effect on target gene transcripts was not associated with MPs.

Expression and Development of TARP γ-4 in Embryonic Zebrafish.

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPAR-related proteins, known as TARPs. Little is known about the role of TARPs during development or about their function in nonmammalian organisms. Here, we report on the presence of TARP γ-4 in developing zebrafish. We find that zebrafish express 2 forms of TARP γ-4: γ-4a and γ-4b as early as 12 h post-fertilization. Sequence analysis shows that both γ-4a and γ-4b shows great level of variation particularly in the intracellular C-terminal domain compared to rat, mouse, and human γ-4. RT-qPCR showed a gradual increase in the expression of γ-4a throughout the first 5 days of development, whereas γ-4b levels were constant until day 5 when levels increased significantly. Knockdown of TARP γ-4a and γ-4b via either splice-blocking morpholinos or translation-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles. Morphant larvae displayed reduced bouts of swimming. Whole-cell patch-clamp recordings of AMPAR-mediated currents from Mauthner cells showed a reduction in the frequency of mEPCs but no change in amplitude or kinetics. Together, these results suggest that γ-4a and γ-4b are required for proper neuronal development.

Effect of copper nanoparticles and copper ions on the architecture of rainbow trout olfactory mucosa.

Olfactory epithelial cells are in direct contact with myriad environmental contaminants which may consequently disrupt their structure and function. Copper ions (Cu2+) and copper nanoparticles (CuNPs) are two types of olfactory neurotoxicants. However, their effects on the structure of olfactory epithelium are largely uninvestigated. The density of olfactory goblet cells in CuNP- and Cu2+ - exposed rainbow trout was assessed using light microscopy throughout time. In both copper (Cu) treatments, the number of goblet cells increased initially over the 24 h exposure and then recovered to normal throughout the 96 h exposure. These data suggested the 96 h exposure to Cu contaminants interfered with protective barrier provided by goblet cells. Nonetheless, lamellar and epithelial thickness of olfactory rosette did not change in the Cu-exposed fish. The gene transcript profile of olfactory mucosa studied by RNA-seq indicated Cu2+ and CuNPs differentially targeted the molecular composition of cell junctions. In the Cu2+ treatment, reduced mRNA abundances of tight junctions, adherens junction, desmosomes and hemidesmosomes, suggest that Cu2+-exposed olfactory mucosal cells had weak junctional complexes. In the CuNP treatment, on the other hand, the transcript abundances of cell junction compositions, except adherens junction, were upregulated. Transcripts associated with gap junctional channels were increased in both Cu treatments. The elevated transcript levels of gap junctions in both Cu treatments suggested that the demand for intercellular communication was increased in the Cu-exposed olfactory mucosa. Overall, our findings suggested that Cu2+ induced greater adverse effects on the molecular composition of olfactory cell junctions relative to CuNPs. Impairment of junctional complexes may disrupt the structural integrity of olfactory mucosa.

Oil sands process-affected water impairs the olfactory system of rainbow trout (Oncorhynchus mykiss).

Oil sands process-affected water (OSPW), a byproduct of the extraction of bitumen in the surface mining of oil sands, is currently stored in massive on-site tailings ponds. Determining the potential effects of OSPW on aquatic ecosystems is of main concern to oil sands companies and legislators concerned about the reclamation of mining sites. In the present study, the interaction of OSPW with the chemosensory system of rainbow trout was studied. Using an electro-olfactography (EOG) technique, a 24 h inhibition curve was established and concentrations that inhibit the olfactory system by 20% and 80% (IC20 and IC80) were estimated at 3% and 22% OSPW, respectively. To study the interaction of exposure time and concentration along with the mechanism of the toxic effects, rainbow trout were exposed to 3% and 22% OSPW for 2, 24, and 96 h. An EOG investigation of olfactory sensitivity demonstrated a positive interaction between exposure time and concentration of OSPW concentration, because an increase in either or both elevated the inhibitory effect. To investigate whether or not structural damage of the olfactory epithelium could account for the observed inhibitory effects of OSPW on fish olfaction, the ultrastructure of the olfactory epithelium of exposed fish was investigated using scanning electron microscopy (SEM) and light microscopy (LM). The SEM micrographs showed no changes in the structure of the olfactory epithelium. The light micrographs revealed an increase in the number of mucous cells in 22% OSPW. The results of the present study demonstrated that exposure to OSPW impairs the olfactory system of rainbow trout and its effects increase gradually with increasing exposure time. The present study demonstrated that structural epithelial damage did not contribute to the inhibitory effects of OSPW on the olfactory system.

Recovery of rainbow trout olfactory function following exposure to copper nanoparticles and copper ions.

In response to environmental information received by olfactory sensory neurons (OSNs), fish display different behaviors that are crucial for reproduction and survival. Damage to OSNs from direct exposure to environmental contaminants can disrupt fish olfaction. Copper nanoparticles (CuNPs) are neurotoxic contaminants which can impair fish olfactory function. However, it is uncertain if CuNP-induced olfactory dysfunction is reversible. Here, we compared the recovery of rainbow trout olfactory mucosa after being exposed to CuNPs or dissolved copper (Cu2+). Following a 96 h exposure to CuNPs or Cu2+, recovery was tested 14 min and 7 days after exposure using electro-olfactography (EOG). Results indicated the 14 min recovery period was not sufficient to improve the olfactory sensitivity in either Cu treatment. After 7 days of transition to clean water, olfactory mucosa was able to recover from Cu2+-induced dysfunction, while no recovery was observed in the CuNP-exposed OSNs. This olfactory dysfunction in the CuNP treatment was observed when no Cu was significantly accumulated in the olfactory mucosa after the recovery period. The transcript abundances of a subset of genes involved in olfactory signal transduction (OST) were downregulated in the CuNP-exposed fish after the 7-day recovery period. These results revealed that odorant reception through OST cascade remained impaired over the recovery period in the CuNP-treated OSNs. The ion regulation gene transcripts were not dysregulated in either Cu treatment, which suggests that neural ion balance was not affected following the recovery period. Collectively, our findings revealed the CuNP-induced olfactory dysfunction was irreversible after the 7-day recovery period. Given the importance of olfaction in crucial aspects of fish life, it is likely that the CuNP-induced impairment of odorant reception pose risks to the survival of fish.

Mechanism of copper nanoparticle toxicity in rainbow trout olfactory mucosa.

Chemosensory perception is crucial for fish reproduction and survival. Direct contact of olfactory neuroepithelium to the surrounding environment makes it vulnerable to contaminants in aquatic ecosystems. Copper nanoparticles (CuNPs), which are increasingly used in commercial and domestic applications due their exceptional properties, can impair fish olfactory function. However, the molecular events underlying olfactory toxicity of CuNPs are largely unexplored. Our results suggested that CuNPs were bioavailable to olfactory mucosal cells. Using RNA-seq, we compared the effect of CuNPs and copper ions (Cu2+) on gene transcript profiles of rainbow trout (Oncorhynchus mykiss) olfactory mucosa. The narrow overlap in differential gene expression between the CuNP- and Cu2+-exposed fish revealed that these two contaminants exert their effects through distinct mechanisms. We propose a transcript-based conceptual model that shows that olfactory signal transduction, calcium homeostasis, and synaptic vesicular signaling were affected by CuNPs in the olfactory sensory neurons (OSNs). Neuroregenerative pathways were also impaired by CuNPs. In contrast, Cu2+ did not induce toxicity pathways and rather upregulated regeneration pathways. Both Cu treatments reduced immune system pathway transcripts. However, suppression of transcripts that were associated with inflammatory signaling was only observed with CuNPs. Neither oxidative stress nor apoptosis were triggered by Cu2+ or CuNPs in mucosal cells. Dysregulation of transcripts that regulate function, maintenance, and reestablishment of damaged olfactory mucosa represents critical mechanisms of toxicity of CuNPs. The loss of olfaction by CuNPs may impact survival of rainbow trout and impose an ecological risk to fish populations in contaminated environments.

Rainbow trout (Oncorhynchus mykiss) chemosensory detection of and reactions to copper nanoparticles and copper ions.

Copper is known to interfere with fish olfaction. Although the chemosensory detection and olfactory toxicity of copper ions (Cu2+) has been heavily studied in fish, the olfactory-driven detection of copper nanoparticles (CuNPs)-a rapidly emerging contaminant to aquatic systems-remains largely unknown. This study aimed to investigate the olfactory response of rainbow trout to equitoxic concentrations of CuNPs or Cu2+ using electro-olfactography (EOG, a neurophysiological technique) and olfactory-mediated behavioural assay. In the first experiment, the concentration of contaminants known to impair olfaction by 20% over 24 h (EOG-based 24-h IC20s of 220 and 3.5 µg/L for CuNPs and Cu2+, respectively) were tested as olfactory stimuli using both neurophysiological and behavioural assays. In the second experiment, to determine whether the presence of CuNPs or Cu2+ can affect the ability of fish to perceive a social cue (taurocholic acid (TCA)), fish were acutely exposed to one form of Cu-contaminants (approximately 15 min). Following exposure, olfactory sensitivity was measured by EOG and olfactory-mediated behaviour within a choice maze was recorded in the presence of TCA. Results of neurophysiological and behavioural experiments demonstrate that rainbow trout can detect and avoid the IC20 of CuNPs. The IC20 of Cu2+ was below the olfactory detection threshold of rainbow trout, as such, fish did not avoid Cu2+. The high sensitivity of behavioural endpoints revealed a lack of aversion response to TCA in CuNP-exposed fish, despite this change not being present utilizing EOG. The reduced response to TCA during the brief exposure to CuNPs may be a result of either olfactory fatigue or blockage of olfactory sensory neurons (OSNs) by CuNPs. The observed behavioural interference caused by CuNP exposure may indicate that CuNPs have the ability to interfere with other behaviours potentially affecting fitness and survival. Our findings also revealed the differential response of OSNs to CuNPs and Cu2+.

Comparative tissue distribution and depuration characteristics of copper nanoparticles and soluble copper in rainbow trout (Oncorhynchus mykiss).

The continuously growing number of products containing nanoparticles (NPs) makes their presence in the environment inevitable, and given the well-known toxicity of dissolved metals, concerns regarding the toxicity of metal-based NPs have been raised. Whether metal-based NPs present similar or different toxicological effects compared with metal salts is an emerging field. In the present study, rainbow trout were intraperitoneally injected with CuSO4 or copper NPs (CuNPs) to investigate tissue distribution and depuration characteristics. Fish injected with Cu showed an initial accumulation of Cu in the liver, kidney, gills, intestine, and carcass. The Cu concentration in the liver of CuNP-injected fish increased over time. It appears as though CuNPs accumulated in the liver at a greater rate than they were excreted. In livers of fish injected with CuSO4 , the Cu concentration appeared to increase and reach an equilibrium, suggesting that copper was accumulated and excreted at the same rate. The possibility that CuNPs can accumulate at a higher rate than it is excreted in the liver warrants further investigation. The present study demonstrates the possibility of dietary uptake of CuNPs because elevated Cu concentrations were observed in carcass, gills, kidney, and intestine of fish gavaged with CuNPs. In conclusion, the results suggest that dietary CuNPs are taken up by the gut and preferentially accumulate in the liver. Environ Toxicol Chem 2019;38:80-89. © 2018 SETAC.

An epithelium is not just an epithelium: Effects of Na, Cl, and pH on olfaction and/or copper-induced olfactory deficits.

While the toxic effects of several substances on fish olfaction are well known, studies on how water chemistry affects contaminant-induced olfactory toxicity are rare. In the present study, the effect of water pH or Na concentration on fish olfactory response and Cu-induced olfactory toxicity was investigated. Also, the effects of two sodium salts, NaCl and NaNO3, on olfaction were studied. Juvenile rainbow trout were exposed to 6 and 32 µg/L Cu, each under five different conditions (pH 9, pH 6.5, 20 or 40 mg/L sodium added, or culture water), for 10 days before characterizing fish olfactory response using electro-olfactography (EOG). The results demonstrated that reducing the pH to 6.5 or adding 20 or 40 mg/L Na impairs the fish response to a standard olfactory cue. None of the water treatments were protective against, or synergic with, the toxic effect of Cu on the olfactory system. Of the two Na salts, NaCl caused significantly higher impairment than NaNO3. The results of the present study demonstrate that water quality modifies contaminant-induced olfactory toxicity, but differently than what is known for other tissues (i.e. gill).