Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson’s Disease Model

The present work successfully demonstrated the locomotor assessment of the established 6-OHDA-induced, adult zebrafish-based PD model. The entire experiment involved three major steps: pre-ICV microinjection preparations, ICV microinjection of zebrafish, and locomotor assessment. To ensure the healthy recovery of adult zebrafish following the ICV microinjection procedure and good experimental outcome, some good practices for each step have been recommended in the present study.

Pre-ICV microinjection preparation: Animal selection was best performed the day before the experiment. The gender was identified and the length of the fish was measured. Male adult zebrafish with a standardized length of 3.2-3.7 cm were placed in a separate experimental tank. Additionally, the fish should undergo 24 h fasting period to avoid regurgitation during anaesthesia³³. A fish tank (with its four walls covered with white paper) with a standing water tank set up should be prepared before the experiment to lessen the exterior stress and help the recovery process of the zebrafish. All chemicals were prepared fresh before the start of each experiment as they could deteriorate rapidly over time and become unstable at room temperature^34,35.

Microinjection of 6-OHDA:Clean and gentle handling of zebrafish should be performed throughout the process to prevent the introduction of unnecessary injury and infection to the fish. The fish should be placed on top of a wet sponge and kept in a moist condition to avoid drying out³⁶. A small incision was made using a sterile needle with firm and appropriate force to avoid extra pressure that may crack the zebrafish skull. This incision should allow entry of the microcapillary into the brain cavity. The microcapillary was then lowered until a depth of 1,200 µm from the entry point, which is in between two hemispheres-the telencephalon and tectum (Figure 1B,C). The entry point was chosen between these hemispheres to prevent any additional laceration of neurons³⁷. This technique involved the use of a microinjector, the pressure and timing of the delivery should be calibrated to ensure delivery of 0.5 µL of neurotoxin. This calibration can be performed by measuring the size of the droplet formed on the filter paper³⁸. Our practice usually involved the following set up of programmable parameters whereby the intensity of injection was lowered with each subsequent injection (injection pressure: 4000 hPa, duration of injection: 0.3 s, and compensation pressure: 10 hPa). In order to avoid the neurotoxin from leaking out of the brain cavity, a 20 s interval was applied between injection and withdrawal of the microcapillary³⁵. Due to the small size of the capillary, the microcapillary may be blocked after each injection. As such, the microcapillary should be essentially flushed prior to the next injection so as to clear the blockage and ensure that the intensity of injection is sufficient to yield the desired volume of 0.5 µL of 6-OHDA. The fish would then be transferred into a recovery tank maintained at 28 ± 1.0 °C. If the fish fails to recover within 30 s, flush out its gill and mouth with distilled water until full recovery of muscle movements occurs.

Locomotor Assessment: To ensure good locomotor assessment on 6-OHDA-induced adult zebrafish, the behavioral study should be conducted within the same time frame for each time point. Each behavioral study should allow for a minimum of 2 min acclimatization period and should be performed within 4 h³⁹. The present experiment was carried out in the early morning between 8 am to 12 pm as zebrafish were more active during this period⁴⁰. A longer acclimatization period is necessary if zebrafish show any abnormal behavior with clear signs of stress and anxiety (freezing and erratic behavior)⁴¹. However, to avoid inconsistency in the swimming behavior of the earlier and the last batch of recordings, the acclimatization should not exceed 10 min³⁰. For the open field test, an experimental tank of any size, color, shape, and texture could be used for recordings that range from a minimum of 5 to 30 min^42,43. Zebrafish behavior is greatly influenced by the temperature of its surroundings. Small fluctuations of more than 4 °C could greatly impact the swimming speed⁴⁴. Hence, the temperature of water in the experimental tank should be strictly maintained under a controlled temperature of 28 ± 1.0 °C using a commercial heater, and the water level was kept around 12 cm deep throughout the experiment. The walls of the tank were covered with white paper to create contrast between the test subject and experimental arena as well as to reduce external stimuli that may cause an unprompted reaction from test subjects⁴⁵. The fish from each group were tested individually in accordance with the current standard practice for zebrafish neurobehavioural research^23,37,46. Given the zebrafish propensity to social interactions, there is concern that isolation during the testing period might impact their behavior⁴⁷. However, the current experimental setup was limited to a maximum of 10 min per trial and this short period of isolation was found to not have any effect on the locomotor activity of adult zebrafish⁴⁸. In order to provide accurate data collection for the behavioral study, the assessment was performed by randomly selecting the zebrafish from different experimental groups (i.e., alternating two zebrafish from either the sham and 6-OHDA lesioned group until n = 6) during the open tank test⁴⁹. The recorded videos were analyzed using a video tracking system that is commonly used for rodents' behavioral tracking. As zebrafish is an emerging animal model, the behavioral tests conducted using zebrafish are usually adapted from the established scientific literature on rodents⁵⁰. Here, we demonstrated the ability of the video tracking software to automatically track zebrafish in the experimental arena and effectively compute the desired parameters. The video tracking software stood out from the other available software due to the variety of video files supported by the software, regular update package releases, and support provided for different operating systems⁵¹.

One of the limitations of existing animal-based PD models is the lack of mechanistic similarities that mimic the motor impairment as observed after dopaminergic neuronal loss in the substantia nigra pars compacta of the human brain⁵². The emergence of the adult zebrafish-based PD model may, however, address this particular limitation. As observed in the present study, the reduced swimming speed corresponded to our previous cellular findings whereby more than 85% dopaminergic neuronal loss in the ventral diencephalon of 6-OHDA-induced adult zebrafish model three days postlesion²². It appears that specific ablation of dopaminergic neurons in this area of interest is required to disrupt the descending motor signaling from the brain, causing slowness in movement⁵³. L. J. Caldwell, et al.²³ who performed ICV injection of 6-OHDA at the optic tectum (point of entry), for instance, observed only changes in zebrafish shoaling and mating behavior. Ablation of DpN in ventral Dn is crucial as the population of DpN in the Dn of adult zebrafish acts as the only dopamine source for zebrafish motor neurons. This is analogous to the human substantia nigra⁵⁴. The present study also observed faster-swimming speed and longer distance traveled by lesioned zebrafish in later postlesion time points, indicating continued and ultimately full restoration of dopamine signaling that governs zebrafish swimming behavior. These findings thus validated the capability of newly regenerated dopaminergic neurons in regaining their functional activities in lesioned adult zebrafish.

The current application route of 6-OHDA involved a slightly invasive injection paradigm that required insertion of microcapillary deep into the brain, toward the lesion area of ventral Dn. This method is slightly laborious in comparison to peripheral injection and needs to be performed within 3 min per fish to reduce the risk of mortality following injection. As such, prior practice of ICV injection is required to ensure that the method can be performed within the critical duration at the targeted area (Dn). In order to achieve a valid locomotor assessment of adult zebrafish, the open tank test is limited to only 4 h of assessment period per day. Hence, prior planning is required in an experimental framework that involves a large number of animals whereby extra time should be allocated to ensure that the setup met the minimum requirements for each recording (e.g., temperature and water depth). This planning is especially crucial in time-based experiments such as that of the current study, as each recording needs to be performed on the intended timepoint. The present experimental setup was limited to the study of two swimming parameters that specifically assessed the zebrafish motor function. Other behavioral parameters such as shoaling and anxiety-like behavior, however, required other experimental setups and different analytical methods. In summary, this is a reproducible and useful method to study the DpN neuroregeneration process in 6-OHDA-induced adult zebrafish which may yield important insights into cell replacement treatment strategies against PD.