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Venlafaxine in embryos stimulates neurogenesis and disrupts larval behavior in zebrafish William Andrew Thompson, Victoria Arnold, and Mathilakath M. Vijayan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04099 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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Venlafaxine in embryos stimulates neurogenesis and disrupts larval behavior in zebrafish

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William A. Thompson1, Victoria I. Arnold2 and Mathilakath M. Vijayan1,*

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Department of Biological Sciences, University of Calgary. 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 2 Water Resources, The City of Calgary, P.O. Box 2100, Stn. M, Calgary, AB, Canada T2P 2M5 Manuscript Format: Abstract word count: Manuscript word count: Small figures (300 words): Large figures (600 words): Small tables (300 words): Large tables (600 words): Word-equivalent: References: Supporting information: Pages: Figures: Tables:

Research Article – 7,000 word-equivalent limit. 216 3,669 900 1200 300 6,286 46 270 words 29 5 1

*Corresponding Author [email protected] Running title: Venlafaxine disrupts zebrafish development

Keywords: antidepressant; neurodevelopment; developmental programming; locomotion; cortisol

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Abstract

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Venlafaxine, a widely prescribed antidepressant, is a selective serotonin and

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norepinephrine re-uptake inhibitor in humans, and this drug is prevalent in municipal

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wastewater effluents. While studies have shown that this drug affects juvenile fish

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behavior, little is known about the developmental impact on non-target aquatic animals.

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We tested the hypothesis that venlafaxine deposition in the egg, mimicking maternal

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transfer of this antidepressant, disrupts developmental programming using zebrafish

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(Danio rerio) as a model. Embryos (1-4 cell stage) were microinjected with either 1 or 10

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ng venlafaxine, which led to a rapid reduction (90%) of this drug in the embryo at hatch.

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There was a concomitant increase in the concentration of the major metabolite o-

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desmethylvenlafaxine during the same period. Embryonic exposure to venlafaxine

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accelerated early development, increased hatching rate and produced larger larvae at 5

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days post fertilization. Also, there was an increase in neuronal birth in the hypothalamus,

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dorsal thalamus, posterior tuberculum, and the preoptic region, and this corresponded

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with a higher spatial expression of nrd4, a key marker of neurogenesis. The venlafaxine-

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exposed larvae were less active and covered shorter distance in a light and dark

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behavioral test compared to the controls. Overall, zygotic exposure to venlafaxine

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disrupts early development, including brain function, and compromises larval behavior,

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suggesting impact of this drug on developmental programming in zebrafish.

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Introduction

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Venlafaxine (trade name Effexor) is a selective serotonin and norepinheprine

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reuptake inhibitor (SSNRI) highly prescribed for the treatment for depression in humans.1

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The majority of the drug is excreted from humans and detectable in waterways receiving

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municipal wastewater effluent (MWWE).2 Recent studies have shown that exposure to

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venlafaxine at environmentally relevant levels impact non-target aquatic animals,

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including fish.3-5 For instance, exposure of juvenile fish to venlafaxine affects the stress

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response and feeding behavior in rainbow trout (Oncorhynchus mykiss).3,5 This may

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include changes in brain function as this antidepressant modulates concentrations of

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monoamines, including serotonin (5-HT), norepinephrine (NE), and dopamine (DA) in

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the midbrain of trout.5 Also, venlafaxine exposure in juvenile fathead minnow

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(Pimephales promelas) altered the expression of genes associated with neuronal action

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potentials, neuron sheath formation and growth,6 suggesting changes in brain function as

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a possible cause for altered stress, feeding and agonistic behaviors.5,7,8

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Most studies have focused on exposure of juveniles or adults to venlafaxine, often

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overlooking the impact associated with exposure during the early critical developmental

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windows, which are highly sensitive to contaminant impact leading to changes in

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developmental programming.9,10 Indeed, a recent study showed that contaminants are

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transferred from the mother to eggs in oviparous fishes,11 which may expose embryos to

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toxicants during the early critical window of development.9 Venlafaxine and its

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metabolite O-desmethylvenlafaxine (ODV), which is equipotent to the parent compound

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in inhibiting 5-HT and NE reuptake,12 are consistently detected in MWWE.2,13

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Consequently, they are considered pseudo-persistent contaminants in fish,14 and

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venlafaxine has also been shown to accumulate in fish tissues, including the brain, liver,

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and muscle.4 However, no study has examined either the deposition of venlafaxine in the

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embryos or its impact on early development in fish. The zygotic deposition of the drug

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may be either as a consequence of maternal transfer or uptake from the external

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environment in fishes. Nonetheless, venlafaxine deposition in the embryos may be an

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important route in affecting early developmental phenotypes, but this has not been tested

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before.

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Against this backdrop, we tested the hypothesis that venlafaxine deposition in the

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embryo disrupts brain development and behavior in zebrafish. We mimicked zygotic

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venlafaxine deposition by microinjecting 1 and 10 ng of venlafaxine into 1-4 cell stage

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zebrafish embryos. We chose microinjection, as opposed to water exposure, to precisely

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control the zygotic dosage administered. These concentrations were chosen as

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preliminary studies demonstrated that they did not affect the mortality of the embryo. A

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rough calculation using tissue levels and environmental concentrations from previous

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studies2,4,13 suggest that our dosage may be approximately 50 x higher than a theoretical

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estimate based on environmental levels. However, this rough estimate may vary in the

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wild given that maternal transfer could also contribute to zygotic deposition, in addition

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to direct uptake from the environment. A key objective was to test whether venlafaxine

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perturbed brain development and if this was reflected in altered behavioral phenotypes in

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larval zebrafish. Larval movement was recorded in response to cycles of light and dark,

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which can induce periods of hypo- and hyper-activity in zebrafish.15,16 We assessed

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anxiolytic effects of venlafaxine using thigmotaxis, a measurement of the anxiety

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response in larval zebrafish.16,17 Early developmental changes monitored include

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somitogenesis, head-trunk angle, and hatch rate, while brain development was assessed

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by the birth of new neurons during peak neurogenesis in different regions of the

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brain.16,18 Also, neurogenesis was further confirmed by measuring the spatial abundance

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of the key neuronal gene markers, neuroD4 (nrd4) and orthopedia B (otpB) using whole

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mount in-situ hybridization. Nrd4 is specifically associated with neuronal differentiation

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in the midbrain and hindbrain of zebrafish,19 while otpB is specific to dopaminergic

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neuronal differentiation.20

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Materials and Methods

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Animals

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Zebrafish (TL strain) were maintained in 10L tanks on aquatic habitats

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recirculation systems (Pentair Aquatic Habitats, Apopka, FL, USA). Fish were held at a

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light cycle of 14:10 light: dark, fed twice daily on weekdays, and once daily on

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weekends. Water was maintained at 28.5°C, with a pH of 7.5, and a conductivity of

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~770µS. Three adult males and three adult females were placed in a breeding trap

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overnight, and eggs were collected the following morning, with each replicate consisting

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of a new breeding cycle.

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Venlafaxine injection

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1 nL of 1 ng and 10 ng venlafaxine solutions (Venlafaxine HCl [Sigma-Aldrich]

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mixed with 0.025% phenol red for visual confirmation of injection) were injected into the

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yolk of zebrafish embryos at the 1-4 cell stage using a Narishige IM 300 Microinjector

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(Narshige Group, Japan) under a Leica M165 Dissecting Scope (Leica Microsystems,

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Wetzlar, Germany). Glass capillary needles (Sutter Instruments, Novato, CA) were pulled

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before use. An injection control solution was also used, containing solely the 0.025% of

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phenol red in water. Preliminary studies confirmed that there was no difference in the

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development and behavior of zebrafish between the injection and no injection controls.

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To complete this study, approximately 1000 embryos were injected for each treatment

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group and raised in embryo media (5mM NaCl; 0.17mM KCl; 0.33mM CaCl2; 0.33mM

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MgSO4) in 60 x 15 mm petri dishes held at 28.5°C

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Venlafaxine and O-desmethylvenlafaxine measurement

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To confirm injection concentrations, zebrafish embryos were collected

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immediately following injection, and at 24 and 48 hpf. Each sample consisted of 20

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embryos/larvae, and were collected in 10 x 75 mm culture tubes, flash frozen on dry ice,

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and stored at -20°C. This was repeated 3 times, for a total of 60 embryos per treatment.

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After adding venlafaxine-d6 and O-desmethylvenlafaxine-d6 as internal standards

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(Cerilliant Corporation, Round Rock, TX, USA), the samples were homogenized as

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described in Grabicova et al.4 Subsequent to centrifugation, the supernatant was reduced

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to approximately 250µL under a gentle stream of nitrogen at 40°C then diluted with

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17.6mL of Milli-Q water adjusted to pH 2 with concentrated sulfuric acid. Solid-phase

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extraction was performed similar to previous reports21 using Oasis MCX 150 mg

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cartridges (Waters Corporation, Milford, MA, USA). After sample loading, the cartridges

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were washed with 3 mL of pH 2 Milli-Q water, and then eluted with 2 x 6 mL 5%

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NH4OH in methanol. The extracts were reduced in volume under a gentle stream of

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nitrogen at 40°C and reconstituted in 500µL of 30:70 methanol water, syringe filtered

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using 13 mm x 0.2µm WWPTFE syringe filters (Pall Canada Ltd., Missassauga, ON,

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Canada) and stored at -20°C. Analysis by liquid chromatography tandem mass

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spectrometry was similar to that described by Lajeunesse et al.22 using a 1260 HPLC

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connected to a 6460 tandem mass spectrometer equipped with a Jet Stream electrospray

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ionization source (Agilent Technologies, Santa Clara, CA, USA). Spike and recovery

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tests were performed to verify the performance of the extraction method, and the average

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recoveries were 75% and 66%, with a coefficient of variation (n=3) of 9% and 4%, for

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venlafaxine and ODV, respectively.

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Developmental markers and body length

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For developmental assessment, zebrafish were viewed under a Nikon H550L

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microscope (Nikon, Tokyo, Japan) and images taken with a Nikon DS Ri1 camera.

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Embryos were placed in a petridish containing 0.66% low melting agarose (Sigma-

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Aldrich) for imaging. Somites, head trunk angle (HTA), and body lengths were obtained

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using methods described previously.23 Somites (n= 3-12 embryos) were counted at 10,

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12, and 15 h time points, respectively. HTA was obtained using dechorionated embryos

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euthanized with an overdose of MS-222 (Sigma-Aldrich; 0.5 g/L; buffered with sodium

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bicarbonate), with measurements taken at 24, 30, and 36 hpf (n= 4-11 zebrafish

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embryos). Larval lengths were taken at 48 and 120 hpf on hatched fish using NIS-

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Elements Basic Research software (Version 3.10; NIS). This analysis was replicated

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three times, totaling 16 larvae for each group. Embryos were recorded for hatching

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success daily from 48 hpf onwards, and hatch rate (%) was calculated as: total hatch/total

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number of embryos x 100 (n= 4 batches of injections).

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Behavior

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Zebrafish (120 hpf) behavior and activity level was assessed using either a

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light/dark cycle or thigmotaxis as described previously.16 Briefly, at 104 hpf, zebrafish

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were loaded into a 96-well plate in 300 µL of embryo media, and allowed to acclimate

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over night at 28.5°C. At 120 hpf, zebrafish were placed into a zebrabox tracking system

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(Viewpoint, Lyon, France) and allowed to acclimate for an additional hour, following

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which they were exposed to alternating 7.5-min cycles of light and dark for an hour.16

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Total distance moved in each of these light cycles (light and dark) were calculated for

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each individual larva from 3 replicate plates (Total n= 103-107 larvae). To measure

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thigmotaxis, or the boldness of an individual, 104 hpf larvae were placed in a 24-well

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plate in 500 µL of embryo media and allowed to acclimate overnight at 28.5°C. The well

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width allowed 2 distinct arenas, with the inner arena being 1-body length from the edge

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of the well. Zebrafish larvae were allowed to acclimate for 1 h in total darkness, before

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exposure to 1 min of light and subsequent recording of their movement in the well over

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30 min. Thigmotaxis (%) was calculated as the distance traveled in the outside

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arena/(outside + inside arena) x 100. The study was replicated 4 times (n= 20 larvae)

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Neurogenesis

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We assessed neurogenesis in zebrafish embryo as described previously,16,18 using

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5-ethynyl-2’-deoxyuridine (EdU; a thymidine analogue; Molecular Probes™), HuC (A

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mouse anti-human neuronal protein; A21271; Molecular Probes™), Alexa Fluor goat

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anti-mouse 488 IgG (A11001; Molecular Probes™) and the nuclear stain DAPI (D1306;

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Molecular Probes™). Regions sectioned were the hypothalamus, posterior tuberculum,

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dorsal thalamus, and the pallium and preoptic region (Figure SI2, Supporting

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Information). Images were obtained using a Leica DM6000 fluorescence microscope

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(Leica Microsystems, Wetzlar, Germany). Images were overlaid and total (HuC tagged)

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and newborn neurons at 24 hpf (EdU incorporation) were counted using Photoshop

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(Adobe Version 6), and cells confirmed by observing DAPI staining of the nucleus.

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Percent neurogenesis was calculated as new neurons/total neurons x 100 per brain

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region.16

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Gene expression

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In-situ hybridization of whole mount embryos at 48 hpf was assessed as described

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previously.16,24 Probes for nrd4 and otpB were synthesized using an in vitro transcription

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reaction (16; Table S1, Supporting information). Images were taken using a Nikon 550L

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(Nikon, Tokyo, Japan) dissecting scope on the lateral and dorsal views of the organisms.

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Images were aligned using Photoshop, and visually scored for changes in transcript

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abundance in specific brain regions.19

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Statistical analysis

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All data is shown as mean± S.E.M and statistical analysis was carried out using

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either one-way or two-way ANOVA with a Tukey’s post hoc test to compare

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significance. All percentages were transformed (arc sin transformation) prior to analysis,

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while untransformed data is shown in figures and tables. A significance level of 0.05 was

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taken in all data sets. Prism 6.0 (Graphpad Software, CA, USA) was used for all

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statistical analysis.

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Results

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Venlafaxine and O-desmethylvenlafaxine content in embryos

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Immediately following microinjection, zygotic venlafaxine content was measured

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to be 0.4 and 4.2 ng in the 1 and 10 ng venlafaxine injected embryos, respectively (Figure

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1A). By 24 hpf, these concentrations fell to 0.17 and 1.6 ng per embryo, further

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decreasing to 0.065 and 0.62 ng per embryo at 48 hpf in the 1 and 10 ng venlafaxine

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groups, respectively (Figure 1B). Immediately following injection, only the 10 ng

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injected group had detectable levels of the major metabolite ODV present (approximately

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4 pg per embryo). By 24 hpf, ODV was recorded at 7 and 27 pg per embryo in the 1 and

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10 ng venlafaxine injected fish respectively, with these values rising to 16 pg and 120 pg

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at 48 hpf.

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Early developmental effects

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Venlafaxine treatment at 1 ng and 10 ng increased the hatch rate (at 48 hpf) by

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15% and 32%, respectively, compared to the control embryos (p=0.0013 and p