Qussay Al-Jubouri

Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish

ABSTRACT Research has recently demonstrated that larval zebrafish show similar molecular responses to nociception to those of adults. Our study explored whether unprotected larval zebrafish exhibited altered behaviour after exposure to noxious chemicals and screened a range of analgesic drugs to determine their efficacy to reduce these responses. This approach aimed to validate larval zebrafish as a reliable replacement for adults as well as providing a high-throughput means of analysing behavioural responses. Zebrafish at 5 days post-fertilization were exposed to known noxious stimuli: acetic acid (0.01%, 0.1% and 0.25%) and citric acid (0.1%, 1% and 5%). The behavioural response of each was recorded and analysed using novel tracking software that measures time spent active in 25 larvae at one time. Subsequently, the efficacy of aspirin, lidocaine, morphine and flunixin as analgesics after exposure to 0.1% acetic acid was tested. Larvae exposed to 0.1% and 0.25% acetic acid spent less time active, whereas those exposed to 0.01% acetic acid and 0.1–5% citric acid showed an increase in swimming activity. Administration of 2.5 mg l−1 aspirin, 5 mg l−1 lidocaine and 48 mg l−1 morphine prevented the behavioural changes induced by acetic acid. These results suggest that larvae respond to a noxious challenge in a similar way to adult zebrafish and other vertebrates and that the effect of nociception on activity can be ameliorated by using analgesics. Therefore, adopting larval zebrafish could represent a direct replacement of a protected adult fish with a non-protected form in pain- and nociception-related research. Highlighted Article: Larval zebrafish can be used as a model for the study of pain and nociception and thus represent a valid replacement of a protected adult vertebrate.

Automated electrical stimulation and physical activity monitoring of zebrafish larvae

Zebrafish (Danio rerio) is accepted as an efficient model to aid in research over a broad range of human diseases and drug discovery. However, obtaining evidences of pain perception similarity between adults and larvae is still a considerable scientific challenge. This paper presents an automated test-bed for electrical stimulation and physical activity monitoring of zebrafish larvae. It comprises of infrared digital camera and 25 test arenas along with the required software and hardware circuitry. Unlike existing systems, the developed system is fully automatic and does not require any manual adjustments. In addition, it is capable of estimating and monitoring physical activity of multiple larvae at various levels of painful stimulation. Performance of the developed testbed is assessed experimentally using various stimulation levels for numerous test batches of zebrafish larvae. The obtained results demonstrated accurate tracking and monitoring of multiple larvae responses for various levels of painful stimulation.

An automatic pattern detection method for behavioral analysis of zebrafish larvae

Zebrafish has becomes a popular biological model for studies in pain, stress and welfare. However, automated assessment of nociceptive thresholds in larval zebrafish remains a challenge for biomedical researchers. This paper presents a new automatic pattern detection method for behavioral analysis of zebrafish larvae. The proposed method divides each arena in the test-bed mesh into an inner and outer zone with the aim of detecting patterns of fish behavior in the outer zones (also called thigmotaxis or wall hugging) that is considered one of the most common behavioral patterns studied in anxiety models. Three distinct groups of fish larvae are used as test subjects in this study. These groups are exposed to electric stimulation using different voltage levels. Poststimulation behaviors of the subjects under test are recorded using an infrared sensitive camera and analyzed. The obtained results demonstrated a noticeable change in the larval behavior in terms of the number of detected patterns in the outer zones of the arena cells. These findings confirm the validity of the proposed pattern detection method as a new metric to assess nociceptive thresholds for behavioral analysis of larvae.

Impact of stress, fear and anxiety on the nociceptive responses of larval zebrafish

Both adult and larval zebrafish have been demonstrated to show behavioural responses to noxious stimulation but also to potentially stress- and fear or anxiety- eliciting situations. The pain or nociceptive response can be altered and modulated by these situations in adult fish through a mechanism called stress-induced analgesia. However, this phenomenon has not been described in larval fish yet. Therefore, this study explores the behavioural changes in larval zebrafish after noxious stimulation and exposure to challenges that can trigger a stress, fear or anxiety reaction. Five-day post fertilization zebrafish were exposed to either a stressor (air emersion), a predatory fear cue (alarm substance) or an anxiogenic (caffeine) alone or prior to immersion in acetic acid 0.1%. Pre- and post-stimulation behaviour (swimming velocity and time spent active) was recorded using a novel tracking software in 25 fish at once. Results show that larvae reduced both velocity and activity after exposure to the air emersion and alarm substance challenges and that these changes were attenuated using etomidate and diazepam, respectively. Exposure to acetic acid decreased velocity and activity as well, whereas air emersion and alarm substance inhibited these responses, showing no differences between pre- and post-stimulation. Therefore, we hypothesize that an antinociceptive mechanism, activated by stress and/or fear, occur in 5dpf zebrafish, which could have prevented the larvae to display the characteristic responses to pain.

Occurrence density index for behavior classification of zebrafish larvae

Larval zebrafish are proving to be promising subjects for research that is not subject to legal requirements. However, the behavior of this animal has not been fully explored by researchers yet. This paper proposes a new occurrence-density index (ODI) for behavioral analysis and classification of zebrafish larvae. The ODI is identified through a multistage process that includes (i) mapping of the testbed arena into a virtual arena, (ii) projection of the original objects trajectory into the virtual arena, (iii) assessment of the occurrence-density, and (iv) calculation the proposed index. The results obtained from this pilot study clearly demonstrated a promising ability to classify behaviors of zebrafish larvae. The ODI will therefore not only provide a new classification feature to the known set of features for fish behavior analysis but also explain and distinguish different behavioral traits.

Towards automated monitoring of adult zebrafish

Over the last two decades, zebrafish (Danio rerio) have emerged as an efficient model to aid in the research of a broad range of human diseases as well as such diverse applications as environmental modelling and drug discovery. Economically, the large number, low price and low maintenance requirements of this fish species encouraged its use for research. In addition to this, the study of zebrafish is being used to improve the understanding of fish physiology, with implications for fish welfare. In order to thoroughly model the behaviour, development and growth of these fish, it is important to be able to scrutinise the characteristics of individual fish as they respond to a range of stimuli, and to this end off-line fish recognition and on-line tracking using video data is employed. Tracking and identifying such small and fast-moving objects is a challenge, and this paper seeks to address this using a behavioural analysis approach. Utilising single high resolution camera and two low-cost synchronised video cameras, the proposed systems captures front (face) and side (profile) pictures of each isolated fish as they swim past a given marker. The acquired images are then subject to three separate processing routes in order to satisfy three complementary but distinct objectives. Initially, fish face and profile features are extracted to aid the identification of individual fish. Then, for each fish identified, behavioural features such as the frequency and intensity of the operculum beat rate or breathing cycle are quantified in order to assess aspects of the fish welfare. Additionally, the volume of each fish is estimated based on its profile dimensions, enabling the weight of the fish to be monitored throughout its lifetime. This paper presents preliminary experimental considerations and findings of this on-going research project. Results to date have been both encouraging and promising, validating the approach and the experimental configuration adopted.

Identification of tail curvature malformation in zebrafish embryos

Biologists and pharmacologists commonly use zebrafish embryos during the testing of drugs. The testing of these substances is a tedious and painstaking process, carried out manually by trained experts who determine whether the embryos have been deformed or killed as a result of administering the chemical. This paper proposes a novel automatic system for the detection and classification of abnormal curvature of the zebrafish larvae tail (up or down) using a number of geometric features. The most important part of this system is the feature extraction technique, which is done depending on the shape of the tail by applying several arithmetic equations. The proposed system also addresses the orientation problem of the embryos, is part of a larger system for the classification of multiple classes of malformations, and demonstrated a high performance compared to published automated techniques.