Subscriber access provided by LAURENTIAN UNIV
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
s:
Page 1 of 31
Environmental Science & Technology
1 2
Venlafaxine in embryos stimulates neurogenesis and disrupts larval behavior in zebrafish
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
William A. Thompson1, Victoria I. Arnold2 and Mathilakath M. Vijayan1,*
1
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
41 42
Abstract
ACS Paragon Plus Environment
Environmental Science & Technology
Thompson et al. 43
Venlafaxine, a widely prescribed antidepressant, is a selective serotonin and
44
norepinephrine re-uptake inhibitor in humans, and this drug is prevalent in municipal
45
wastewater effluents. While studies have shown that this drug affects juvenile fish
46
behavior, little is known about the developmental impact on non-target aquatic animals.
47
We tested the hypothesis that venlafaxine deposition in the egg, mimicking maternal
48
transfer of this antidepressant, disrupts developmental programming using zebrafish
49
(Danio rerio) as a model. Embryos (1-4 cell stage) were microinjected with either 1 or 10
50
ng venlafaxine, which led to a rapid reduction (90%) of this drug in the embryo at hatch.
51
There was a concomitant increase in the concentration of the major metabolite o-
52
desmethylvenlafaxine during the same period. Embryonic exposure to venlafaxine
53
accelerated early development, increased hatching rate and produced larger larvae at 5
54
days post fertilization. Also, there was an increase in neuronal birth in the hypothalamus,
55
dorsal thalamus, posterior tuberculum, and the preoptic region, and this corresponded
56
with a higher spatial expression of nrd4, a key marker of neurogenesis. The venlafaxine-
57
exposed larvae were less active and covered shorter distance in a light and dark
58
behavioral test compared to the controls. Overall, zygotic exposure to venlafaxine
59
disrupts early development, including brain function, and compromises larval behavior,
60
suggesting impact of this drug on developmental programming in zebrafish.
61 62 63 64 65
Introduction
2
ACS Paragon Plus Environment
Page 2 of 31
Page 3 of 31
Environmental Science & Technology
Thompson et al. 66
Venlafaxine (trade name Effexor) is a selective serotonin and norepinheprine
67
reuptake inhibitor (SSNRI) highly prescribed for the treatment for depression in humans.1
68
The majority of the drug is excreted from humans and detectable in waterways receiving
69
municipal wastewater effluent (MWWE).2 Recent studies have shown that exposure to
70
venlafaxine at environmentally relevant levels impact non-target aquatic animals,
71
including fish.3-5 For instance, exposure of juvenile fish to venlafaxine affects the stress
72
response and feeding behavior in rainbow trout (Oncorhynchus mykiss).3,5 This may
73
include changes in brain function as this antidepressant modulates concentrations of
74
monoamines, including serotonin (5-HT), norepinephrine (NE), and dopamine (DA) in
75
the midbrain of trout.5 Also, venlafaxine exposure in juvenile fathead minnow
76
(Pimephales promelas) altered the expression of genes associated with neuronal action
77
potentials, neuron sheath formation and growth,6 suggesting changes in brain function as
78
a possible cause for altered stress, feeding and agonistic behaviors.5,7,8
79
Most studies have focused on exposure of juveniles or adults to venlafaxine, often
80
overlooking the impact associated with exposure during the early critical developmental
81
windows, which are highly sensitive to contaminant impact leading to changes in
82
developmental programming.9,10 Indeed, a recent study showed that contaminants are
83
transferred from the mother to eggs in oviparous fishes,11 which may expose embryos to
84
toxicants during the early critical window of development.9 Venlafaxine and its
85
metabolite O-desmethylvenlafaxine (ODV), which is equipotent to the parent compound
86
in inhibiting 5-HT and NE reuptake,12 are consistently detected in MWWE.2,13
87
Consequently, they are considered pseudo-persistent contaminants in fish,14 and
88
venlafaxine has also been shown to accumulate in fish tissues, including the brain, liver,
3
ACS Paragon Plus Environment
Environmental Science & Technology
Thompson et al. 89
and muscle.4 However, no study has examined either the deposition of venlafaxine in the
90
embryos or its impact on early development in fish. The zygotic deposition of the drug
91
may be either as a consequence of maternal transfer or uptake from the external
92
environment in fishes. Nonetheless, venlafaxine deposition in the embryos may be an
93
important route in affecting early developmental phenotypes, but this has not been tested
94
before.
95
Against this backdrop, we tested the hypothesis that venlafaxine deposition in the
96
embryo disrupts brain development and behavior in zebrafish. We mimicked zygotic
97
venlafaxine deposition by microinjecting 1 and 10 ng of venlafaxine into 1-4 cell stage
98
zebrafish embryos. We chose microinjection, as opposed to water exposure, to precisely
99
control the zygotic dosage administered. These concentrations were chosen as
100
preliminary studies demonstrated that they did not affect the mortality of the embryo. A
101
rough calculation using tissue levels and environmental concentrations from previous
102
studies2,4,13 suggest that our dosage may be approximately 50 x higher than a theoretical
103
estimate based on environmental levels. However, this rough estimate may vary in the
104
wild given that maternal transfer could also contribute to zygotic deposition, in addition
105
to direct uptake from the environment. A key objective was to test whether venlafaxine
106
perturbed brain development and if this was reflected in altered behavioral phenotypes in
107
larval zebrafish. Larval movement was recorded in response to cycles of light and dark,
108
which can induce periods of hypo- and hyper-activity in zebrafish.15,16 We assessed
109
anxiolytic effects of venlafaxine using thigmotaxis, a measurement of the anxiety
110
response in larval zebrafish.16,17 Early developmental changes monitored include
111
somitogenesis, head-trunk angle, and hatch rate, while brain development was assessed
4
ACS Paragon Plus Environment
Page 4 of 31
Page 5 of 31
Environmental Science & Technology
Thompson et al. 112
by the birth of new neurons during peak neurogenesis in different regions of the
113
brain.16,18 Also, neurogenesis was further confirmed by measuring the spatial abundance
114
of the key neuronal gene markers, neuroD4 (nrd4) and orthopedia B (otpB) using whole
115
mount in-situ hybridization. Nrd4 is specifically associated with neuronal differentiation
116
in the midbrain and hindbrain of zebrafish,19 while otpB is specific to dopaminergic
117
neuronal differentiation.20
118
Materials and Methods
119
Animals
120
Zebrafish (TL strain) were maintained in 10L tanks on aquatic habitats
121
recirculation systems (Pentair Aquatic Habitats, Apopka, FL, USA). Fish were held at a
122
light cycle of 14:10 light: dark, fed twice daily on weekdays, and once daily on
123
weekends. Water was maintained at 28.5°C, with a pH of 7.5, and a conductivity of
124
~770µS. Three adult males and three adult females were placed in a breeding trap
125
overnight, and eggs were collected the following morning, with each replicate consisting
126
of a new breeding cycle.
127
Venlafaxine injection
128
1 nL of 1 ng and 10 ng venlafaxine solutions (Venlafaxine HCl [Sigma-Aldrich]
129
mixed with 0.025% phenol red for visual confirmation of injection) were injected into the
130
yolk of zebrafish embryos at the 1-4 cell stage using a Narishige IM 300 Microinjector
131
(Narshige Group, Japan) under a Leica M165 Dissecting Scope (Leica Microsystems,
132
Wetzlar, Germany). Glass capillary needles (Sutter Instruments, Novato, CA) were pulled
133
before use. An injection control solution was also used, containing solely the 0.025% of
134
phenol red in water. Preliminary studies confirmed that there was no difference in the
5
ACS Paragon Plus Environment
Environmental Science & Technology
Thompson et al. 135
development and behavior of zebrafish between the injection and no injection controls.
136
To complete this study, approximately 1000 embryos were injected for each treatment
137
group and raised in embryo media (5mM NaCl; 0.17mM KCl; 0.33mM CaCl2; 0.33mM
138
MgSO4) in 60 x 15 mm petri dishes held at 28.5°C
139
Venlafaxine and O-desmethylvenlafaxine measurement
140
To confirm injection concentrations, zebrafish embryos were collected
141
immediately following injection, and at 24 and 48 hpf. Each sample consisted of 20
142
embryos/larvae, and were collected in 10 x 75 mm culture tubes, flash frozen on dry ice,
143
and stored at -20°C. This was repeated 3 times, for a total of 60 embryos per treatment.
144
After adding venlafaxine-d6 and O-desmethylvenlafaxine-d6 as internal standards
145
(Cerilliant Corporation, Round Rock, TX, USA), the samples were homogenized as
146
described in Grabicova et al.4 Subsequent to centrifugation, the supernatant was reduced
147
to approximately 250µL under a gentle stream of nitrogen at 40°C then diluted with
148
17.6mL of Milli-Q water adjusted to pH 2 with concentrated sulfuric acid. Solid-phase
149
extraction was performed similar to previous reports21 using Oasis MCX 150 mg
150
cartridges (Waters Corporation, Milford, MA, USA). After sample loading, the cartridges
151
were washed with 3 mL of pH 2 Milli-Q water, and then eluted with 2 x 6 mL 5%
152
NH4OH in methanol. The extracts were reduced in volume under a gentle stream of
153
nitrogen at 40°C and reconstituted in 500µL of 30:70 methanol water, syringe filtered
154
using 13 mm x 0.2µm WWPTFE syringe filters (Pall Canada Ltd., Missassauga, ON,
155
Canada) and stored at -20°C. Analysis by liquid chromatography tandem mass
156
spectrometry was similar to that described by Lajeunesse et al.22 using a 1260 HPLC
157
connected to a 6460 tandem mass spectrometer equipped with a Jet Stream electrospray
6
ACS Paragon Plus Environment
Page 6 of 31
Page 7 of 31
Environmental Science & Technology
Thompson et al. 158
ionization source (Agilent Technologies, Santa Clara, CA, USA). Spike and recovery
159
tests were performed to verify the performance of the extraction method, and the average
160
recoveries were 75% and 66%, with a coefficient of variation (n=3) of 9% and 4%, for
161
venlafaxine and ODV, respectively.
162
Developmental markers and body length
163
For developmental assessment, zebrafish were viewed under a Nikon H550L
164
microscope (Nikon, Tokyo, Japan) and images taken with a Nikon DS Ri1 camera.
165
Embryos were placed in a petridish containing 0.66% low melting agarose (Sigma-
166
Aldrich) for imaging. Somites, head trunk angle (HTA), and body lengths were obtained
167
using methods described previously.23 Somites (n= 3-12 embryos) were counted at 10,
168
12, and 15 h time points, respectively. HTA was obtained using dechorionated embryos
169
euthanized with an overdose of MS-222 (Sigma-Aldrich; 0.5 g/L; buffered with sodium
170
bicarbonate), with measurements taken at 24, 30, and 36 hpf (n= 4-11 zebrafish
171
embryos). Larval lengths were taken at 48 and 120 hpf on hatched fish using NIS-
172
Elements Basic Research software (Version 3.10; NIS). This analysis was replicated
173
three times, totaling 16 larvae for each group. Embryos were recorded for hatching
174
success daily from 48 hpf onwards, and hatch rate (%) was calculated as: total hatch/total
175
number of embryos x 100 (n= 4 batches of injections).
176
Behavior
177
Zebrafish (120 hpf) behavior and activity level was assessed using either a
178
light/dark cycle or thigmotaxis as described previously.16 Briefly, at 104 hpf, zebrafish
179
were loaded into a 96-well plate in 300 µL of embryo media, and allowed to acclimate
180
over night at 28.5°C. At 120 hpf, zebrafish were placed into a zebrabox tracking system
7
ACS Paragon Plus Environment
Environmental Science & Technology
Thompson et al. 181
(Viewpoint, Lyon, France) and allowed to acclimate for an additional hour, following
182
which they were exposed to alternating 7.5-min cycles of light and dark for an hour.16
183
Total distance moved in each of these light cycles (light and dark) were calculated for
184
each individual larva from 3 replicate plates (Total n= 103-107 larvae). To measure
185
thigmotaxis, or the boldness of an individual, 104 hpf larvae were placed in a 24-well
186
plate in 500 µL of embryo media and allowed to acclimate overnight at 28.5°C. The well
187
width allowed 2 distinct arenas, with the inner arena being 1-body length from the edge
188
of the well. Zebrafish larvae were allowed to acclimate for 1 h in total darkness, before
189
exposure to 1 min of light and subsequent recording of their movement in the well over
190
30 min. Thigmotaxis (%) was calculated as the distance traveled in the outside
191
arena/(outside + inside arena) x 100. The study was replicated 4 times (n= 20 larvae)
192
Neurogenesis
193
We assessed neurogenesis in zebrafish embryo as described previously,16,18 using
194
5-ethynyl-2’-deoxyuridine (EdU; a thymidine analogue; Molecular Probes™), HuC (A
195
mouse anti-human neuronal protein; A21271; Molecular Probes™), Alexa Fluor goat
196
anti-mouse 488 IgG (A11001; Molecular Probes™) and the nuclear stain DAPI (D1306;
197
Molecular Probes™). Regions sectioned were the hypothalamus, posterior tuberculum,
198
dorsal thalamus, and the pallium and preoptic region (Figure SI2, Supporting
199
Information). Images were obtained using a Leica DM6000 fluorescence microscope
200
(Leica Microsystems, Wetzlar, Germany). Images were overlaid and total (HuC tagged)
201
and newborn neurons at 24 hpf (EdU incorporation) were counted using Photoshop
202
(Adobe Version 6), and cells confirmed by observing DAPI staining of the nucleus.
8
ACS Paragon Plus Environment
Page 8 of 31
Page 9 of 31
Environmental Science & Technology
Thompson et al. 203
Percent neurogenesis was calculated as new neurons/total neurons x 100 per brain
204
region.16
205
Gene expression
206
In-situ hybridization of whole mount embryos at 48 hpf was assessed as described
207
previously.16,24 Probes for nrd4 and otpB were synthesized using an in vitro transcription
208
reaction (16; Table S1, Supporting information). Images were taken using a Nikon 550L
209
(Nikon, Tokyo, Japan) dissecting scope on the lateral and dorsal views of the organisms.
210
Images were aligned using Photoshop, and visually scored for changes in transcript
211
abundance in specific brain regions.19
212
Statistical analysis
213
All data is shown as mean± S.E.M and statistical analysis was carried out using
214
either one-way or two-way ANOVA with a Tukey’s post hoc test to compare
215
significance. All percentages were transformed (arc sin transformation) prior to analysis,
216
while untransformed data is shown in figures and tables. A significance level of 0.05 was
217
taken in all data sets. Prism 6.0 (Graphpad Software, CA, USA) was used for all
218
statistical analysis.
219 220
Results
221
Venlafaxine and O-desmethylvenlafaxine content in embryos
222
Immediately following microinjection, zygotic venlafaxine content was measured
223
to be 0.4 and 4.2 ng in the 1 and 10 ng venlafaxine injected embryos, respectively (Figure
224
1A). By 24 hpf, these concentrations fell to 0.17 and 1.6 ng per embryo, further
225
decreasing to 0.065 and 0.62 ng per embryo at 48 hpf in the 1 and 10 ng venlafaxine
9
ACS Paragon Plus Environment
Environmental Science & Technology
Thompson et al. 226
groups, respectively (Figure 1B). Immediately following injection, only the 10 ng
227
injected group had detectable levels of the major metabolite ODV present (approximately
228
4 pg per embryo). By 24 hpf, ODV was recorded at 7 and 27 pg per embryo in the 1 and
229
10 ng venlafaxine injected fish respectively, with these values rising to 16 pg and 120 pg
230
at 48 hpf.
231
Early developmental effects
232
Venlafaxine treatment at 1 ng and 10 ng increased the hatch rate (at 48 hpf) by
233
15% and 32%, respectively, compared to the control embryos (p=0.0013 and p
