Behavioral effects of developmental exposure to JWH-018 in wild type and disrupted in schizophrenia 1 (disc1) mutant zebrafish

Synthetic cannabinoids can cause acute adverse psychological effects, but the potential impact when exposure happens before birth is unknown. Use of synthetic cannabinoids during pregnancy may affect fetal brain development, and such effects could be moderated by the genetic makeup of an individual. Disrupted in schizophrenia 1 (DISC1) is a gene with important roles in neurodevelopment which has been associated with psychiatric disorders in pedigree analyses. Using zebrafish as a model, we investigated (1) the behavioral impact of developmental exposure to JWH-018 (a common psychoactive synthetic cannabinoid) and (2) whether disc1 moderates the effects of JWH-018. As altered anxiety responses are seen in a several psychiatric disorders, we focused on zebrafish anxiety-like behavior. Zebrafish embryos were exposed to JWH-018 from one to six days post-fertilization. Anxiety-like behavior was assessed using forced light/dark and acoustic startle assays in larvae, and novel tank diving in adults. Compared to controls, developmentally exposed zebrafish larvae had impaired locomotion during the forced light/dark test, but anxiety levels and response to startle stimuli was unaltered. Adult zebrafish developmentally exposed to JWH-018 spent less time on the bottom of the tank, suggesting decreased anxiety. Loss-of-function in disc1 increased anxiety but did not alter sensitivity to JWH-018. Results suggest developmental exposure to JWH-018 has behavioral impact in zebrafish, which is not moderated by disc1.


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In contrast to tobacco smoking, where prevalence during pregnancy has dropped from 14.6 to 10.6%

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To breed zebrafish, we placed them in breeding tanks which had either perforated floors or a 153 container with marbles to isolate eggs from progenitors. We moved the animals to breeding tanks in 154 the evening and collected eggs the following morning. Eggs were incubated in Petri dishes at 28°C 155 until five dpf. If reared, larvae were moved to the recirculating system at six dpf and fed with 156 commercial fry food.

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In response to abrupt sound/vibration stimuli zebrafish larvae execute a fast, non-associative learning 199 escape response. This response has been extensively characterized and involves one of two distinct 200 motor behaviors: a short-latency C-bend of the tail, initiating within 5-15 milliseconds of the 201 stimulus, or a slower, long-latency C-bend response initiating within 20-80 milliseconds. These two 202 motor behaviors use different, possibly overlapping neuronal circuitry [35] but in this study they 203 were measured jointly, since a high-speed camera was not available.

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When the abrupt sound/vibration stimuli are given repeatedly, zebrafish exhibit iterative reduction 205 in the magnitude of the response, commonly known as habituation. Habituation is the mechanism 206 by which the nervous system filters irrelevant stimuli. It is evolutionarily conserved and present in a 207 wide range of species from invertebrates, such as Aplysia and Drosophila, to vertebrates such as As proof of concept, we replicated the experiment by Best and colleagues [38], where 50 stimuli were 218 given using 1, 5 and 20 seconds inter-stimulus intervals (ISI). Following the habituation paradigm

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[36], shorter ISI led to faster habituation [Effect of ISI: χ2(2)=19.04, p<0.0001] ( Figure S1).   Figure S2) and filmed for five minutes. Their behavior 231 was tracked using EthoVision system (Noldus, Netherlands) and data were outputted in one-minute 232 time-bins. Care was taken to ensure that experimental groups were randomized during testing.

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Behavioral testing was conducted between 9 am and 2 pm.

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We next assessed the response to repeated startle stimuli at six dpf. There were no significant 266 differences between 3 µM JWH-018 treated and control larvae in distance travelled before and during 267 the stimuli (p>0.05) (Figure 3).

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In contrast to JWH-018 and THC, exposure to nicotine produced an increase in distances travelled.

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There was a qualitative difference between control and treated zebrafish in the slopes during light 297 periods, as nicotine-treated zebrafish seemed to recover faster, suggesting an anxiolytic effect of 298 nicotine. However the difference between nicotine treated and control zebrafish was not significant

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Alterations in the typical response to light and dark periods can be used to study the anxiety-like

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In order to disentangle these scenarios, liquid chromatography-mass spectrometry analyses could be

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Fish that were not developmentally treated with nicotine showed a small increase in preference when 508 conditioned with 5 µM nicotine. By contrast, fish exposed to 0.15 µM nicotine from two to seven days 509 showed an increased change in preference [Interaction between CPP condition and developmental

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To analyze genotype or treatment differences in the total distance that zebrafish travelled in the tank,

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we fitted the data to a linear mixed model with the total distance travelled during one minute as 550 response variable, time, genotype and/or treatment as fixed effects, and fish ID as random effects.

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To analyze genotype or treatment differences in the number of transitions that zebrafish made between 552 the top and the bottom of the tank, we fitted the data to a generalized linear mixed model with time, genotype or/and treatment as fixed effects, and fish ID as random effects.