DOI: 10.5061/dryad.xpnvx0kkr
title: Behavior and brain size of larval zebrafish exposed to environmentally relevant concentrations of betamethylamino- L-alanine (BMAA)

description: Harmful algal blooms (HABs) release toxic compounds in water and are
 increasing in frequency worldwide. The neurotoxin
 β-methylamino-L-alanine (BMAA) is released by HABs and has
 garnered much attention over the past twenty years due to
 its association with human neurodegenerative disorders, but its
 effects on wildlife are still largely unknown. This study
 characterized the effects of chronic exposure to environmentally
 relevant concentrations of BMAA on the behavior and brain size
 of developing zebrafish (Danio rerio). Zebrafish were
 continuously exposed to 0, 1, 10, or 100 μg/L waterborne BMAA
 between 0- and 5-days post-fertilization (dpf) before the onset
 of exogenous feeding. At 5 dpf, locomotion and responses to vibrational
 and visual stimuli were assessed. Following behavioral testing,
 larvae body and brain size were measured. Survival between 0 and
 5 dpf did not differ between treatments. Moreover, BMAA exposure
 did not affect thigmotaxis, startle response
 magnitude, habituation to repeated presentation of vibrational
 startling stimuli, or relative brain size. A moderate increase in
 overall activity was observed in larvae exposed to 10 ug/L BMAA
 under light, but this effect was not seen in dark conditions, indicating
 that visual processing may have been affected by chronic BMAA
 exposure. Thus, our results show that passive continuous exposure
 to environmentally relevant concentrations of BMAA prior to first
 feeding in zebrafish does not affect overall brain development,
 locomotion, anxiety, and motor neuron-mediated reflexes, but
 suggest targeted neurotoxicity within the visual system.
description: Zebrafish were exposed to four BMAA treatments: 0 μg/L (control),
 1 μg/L, 10 μg/L, and 100 μg/L BMAA within one hour after spawning.
 Mortalities were recorded for each group at 6-8 hours post fertilization
 (hpf), 24 hpf, and every morning thereafter until 5 days post
 fertilization (dpf). At 4 dpf, 6 larvae per treatment group were
 transferred into 24 multi-well plates for acclimation before testing for
 behaviour at 5 dpf using a DanioVision observation chamber (Noldus
 Information Technology, Wageningen, The Netherlands). Larval behavior was
 recorded with a Basler Ace camera with Gigabit Ethernet interface and
 infrared filter (1280 × 1024 resolution). The observation chamber was
 under constant infrared illumination, with the camera output fed into a
 standard PC system with Ethovision software (Noldus Information
 Technology). Ethovision was used to detect objects that were darker
 than the background, with a sensitivity setting of 113, and minimum object
 size of 5 pixels. The tracking rate was 25 samples/s. To minimize noise,
 input filters were applied. A minimum displacement of 0.2 mm was required
 before movement was registered between samples, and data was considered
 “missing” if &gt;15 mm of movement was recorded between samples.
 Various larval behaviours were analyzed. Following
 behavioral measurements, a subset of euthanized larvae was fixed with 4%
 paraformaldehyde at 4°C for 24 hrs before cryoprotection and sectioning.
 Prior to sectioning, larvae embedded in 4.4% agarose blocks were imaged to
 determine their standard length (mm) using the segmented line tool in
 ImageJ (Schindelin et al., 2012). The head of each larva was cut into 20
 μm thick coronal sections and mounted onto slides. Micrographs of each
 section were taken using a Nikon 90i microscope and the
 area (mm<sup>2</sup>) of the brain in each section was
 determined by contour tracing using ImageJ. To estimate brain volume, the
 olfactory bulbs served as the rostral limit of the brain, while the first
 wrapping of developing cartilage of the vertebral column around the spinal
 cord demarcated the caudal endpoint of the brain. The volume
 (mm<sup>3</sup>) of each brain segment was determined by
 multiplying brain area by twice the section thickness (40 μm) to account
 for the use of alternate sections. The volumes of the individual segments
 were then summed to get the total brain volume
 (mm<sup>3</sup>), which was an estimate of absolute brain
 size. We used the residuals calculated from the linear relationship
 between standard length (mm) and brain volume (mm<sup>3</sup>)
 to represent relative brain size.