The protocol is significant in demonstrating zebrafish motor impairment and a subsequent recovery, following intracerebroventricular injection of neurotoxin 6-hydroxydopamine at the ventral diencephalon. This technique demonstrates changes in the functional effects, that correspond to the specific ablation of dopaminergic neurons in the ventral diencephalon of 6-OHDA lesioned adult zebrafish, using a very simple setup. Future studies on the mechanisms underlying neuroregeneration, as well as the intrinsic and extrinsic factors that modulate the process, may provide important insights into cell replacement treatment strategies against Parkinson's disease.
Maintain the fish in an aerated distilled mineralized water tank with one gram per liter commercial sea salt, under a control temperature of 28, plus or minus one degree Celsius. House a maximum of 25 fish per 45-liter tank and expose them to a 14-hour light and 10-hour dark photoperiod. Feed the fish at least twice a day with food pellets, supplemented with freeze dried worms.
Prepare a stock solution of Tricaine methanesulfonate, by dissolving two and a half grams of Tricaine methanesulfonate and five grams of sodium bicarbonate in 250 milliliters of distilled water. Dilute two milliliters of stock solution to make 200 milliliters of working anesthesia solution. Freshly prepare 99.96 millimolar 6-hydroxydopamine, by first dissolving 0.2 milligrams of ascorbic acid in one milliliter of 0.9%weight by volume sterile filtered sodium chloride.
Filter the solution with a 0.2 micron filter. Then add 25 milligrams of 6-hydroxydopamine, in powder form, into the solution. Position the anesthetized fish on a water-soaked sponge, placed under a stereo microscope and wet the fish regularly.
Identify the position for injection, based on the intersection between the metopic suture, coronal suture and sagittal suture, connecting the parietal and frontal skull of the zebrafish brain. Then make a small hole of one millimeter squared, using a sharp 27 gauge needle. Lower the microcapillary injector at a 60 degree angle, until it reaches a depth of 1200 micrometers from the cranial roof of the zebrafish skull.
Press the Z-limit to fix the position. Set the initial injection pressure to 4, 000 hectopascal. Duration of injection at 2.3 seconds.
Compensation pressure to 10 hectopascal. Lower the intensity of injection, with each subsequent injection. Inject 0.5 microliters of 99.96 millimolar neurotoxin 6-hydroxydopamine or 0.9%weight by volume saline, in the sham control group, and let the microcapillary rest for 20 seconds.
Continue to wet the fish with distilled water throughout the process, to prevent drying. Slowly remove the microcapillary and resuscitate the fish under running distilled water. Place the fish in an isolated recovery tank and remove any distractions that can potentially disturb the recovery process.
Flush the microcapillary before the next injection, to clear the blockage and ensure that the intensity of injection is sufficient to yield the desired volume of 0.5 microliters of 6-hydroxydopamine. Perform locomotor assessment of zebrafish individually, via the open tank test, at day three and day 30, post 6-hydroxydopamine. Place the experimental tank, with its walls covered with white paper, on a raised platform.
Illuminate the tank from the bottom, using a light source. Fill the tank with 80 to 90%distilled water and maintain the temperature at 28, plus or minus one degree Celsius. Measure the temperature using a thermometer and regulate it using a commercial aquarium heater.
After a minimum of two minutes of acclimatization, record the fish swimming behavior from a planned view on the two-dimensional plane of the experimental area, using a video camera for five minutes. To avoid inconsistencies, in different batches of recordings, do not exceed 10 minutes of acclimatization. Analyze the videos using video tracking software with the open tank protocol, for the acquisition of distance traveled and mean speed of each subject.
The present experiment assessed the changes in adult zebrafish swimming behavior, following intracerebroventricular microinjection with 6-hydroxydopamine. The main brain region of interest was the ventral diencephalon, made up of the preoptic area, posterior tuberculum and hypothalamus. More than 85%of the tyrosine hydroxylase immunoreactive dopaminergic neurons in the ventral diencephalon were ablated on day three postlesion.
The number of tyrosine hydroxylase immunoreactive dopaminergenic neurons then increase by more than 50%at day 14 postlesion, before achieving full regeneration 30 days postlesion. Analysis of zebrafish swimming behavior, using video tracking software, indicated that both the mean speed and distance traveled of the lesioned group, on day three postlesion, were significantly reduced to less than 45%when compared to sham. The lesioned group exhibited recovery of motor function, 30 days postlesion, with no significant difference of either the mean speed or the distance traveled, when compared to sham.
It is important to have the locomotor assessment performed within a fixed timeframe. For example, 8:00 AM to 12:00 PM.And the water temperature maintained at 28 degrees Celsius, throughout the experiment. Other behavioral tests, like shoaling and anxiety-like behaviors, can be performed, to explore additional changes or functional effects of dopaminergic neurons ablation, following 6-OHDA injection of the adult zebrafish brain.
