I am deeply indebted to Cammi Thornton, for your confidence in my ability to carry out this research project, guidance along the way and unwavering patience. Also, thank you, Zach Miller, for allowing me to be a part of your graduate training and for your knowledge, leadership, and humor that made the rest of my research project worthwhile. Biochemistry within me; thank you also for your time and wisdom in helping me create the final product of this thesis.
The experience of an SMBHC education has truly allowed me to grow academically and personally. Thanks to my friends and family whose words of encouragement gave me the strength to finish this project. Natural compounds including cannabidiol, CBD and Tapinanthus globiferous, TG, extracts were screened for their anti-seizure activity.
Seizure activity after morpholino injection for normal conditions (a), higher temperature (b) and increased morpholino amounts (c) …………22 Figure 8. Percentage of fluorescent embryos after morpholino injections for normal conditions (10 ng MO at 28°C) ), higher temperature (10 ng MO at 34°C), and increased MO amounts at.
INTRODUCTION
- Epilepsy
- Dravet Syndrome
- Scn1Lab gene
- Zebrafish as a model
- Morpholino
- Natural products with hypothesized anti-seizure activity
- Study Goals
People with DS require constant care; the condition has a major impact on the lives of patients and the quality of life of the family. One of the leading monogenic causes of DS involves mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Voltage-gated sodium channels (VGSC) are essential for neuronal excitability by initiating and propagating the rising phase of the action potential ( Frank et al., 2003 ).
However, by using morpholino and chemical mutagenesis we can develop model organisms with the unique characteristics of DS; this allows the study of the molecular and behavioral basis of this specific disease. Furthermore, the embryos develop outside the parent organism, facilitating microscopic visualization during early development. The movement of zebrafish can be monitored via the ViewPoint Zebrabox, which allows researchers to analyze behavioral aspects of zebrafish seizures.
For example, in scn1Lab -/- mutants, the fish show darker pigmentation, lack of a swim bladder, slight curvature of the body and seizures (Mayo Clinic, 2018). The use of morpholino oligos allows for the transient knockdown of the SCN1A gene by blocking translation at the ribosome. Instead, they work by inhibiting the biological activity of the target RNA until that RNA is naturally degraded.
One of the main reasons for this effort is related to the treatment of refractory epilepsy such as DS.
METHODS AND MATERIALS
- Zebrafish culture and egg collection
- Chemically-induced seizures
- Morpholino injection
- a Survival and florescent checks
- b Higher temperature exposure
- Viewpoint data collection and analysis
- Transgenic scn1Lab mutants
- a Exposure assay
Junk debris and unfertilized/dead eggs were removed after transferring the eggs to a petri dish; this was done using a transfer pipette. The water in the Petri dish was then replaced with zebrafish water (60 ppm Instant Ocean, pH 7.5) and placed in an incubator at 28°C for five days. Once dosing was complete, the plates were covered with aluminum foil and returned to the incubator.
After a 24-hour exposure to the dosing solutions (6 dpf), morphological changes were noted to determine whether TG extracts had any toxic side effects, including a reaction to touch, pericardial edema, yolk sac edema, a curved body axis, and/or an uninflated swim bladder (Dietrich, 2017). Phenol red (0.5 L) was added to the morpholine solution to ensure visibility of the injected solution. MOs were labeled with green fluorescent protein (GFP) to ensure that MOs were incorporated into the injected fish.
After transferring larvae to a 96 well plate at 4 dpf, a subset of larvae was placed in an incubator at 34.0C for 10 minutes and then returned to the incubator at 28.0C. The ZebraBox software creates an Excel sheet that includes the duration each larva spends in the inactive (<5 mm/sec), small (5-9 mm/sec) and large (>9 mm/sec) movements over the 45 minutes spent period at intervals of 15 minutes. Movement that meets the large activity requirement is indicative of seizure activity and was the data used to test the morpholino discount's effectiveness.
After final recording at 6 dpf, larvae were euthanized with buffered MS-222 and placed in RNA later and stored at -80.0C. Data collected by ViewPoint ZebraBox were analyzed using GraphPad Prism 5.0 (La Jolla, CA). Photographs were taken to characterize physical differences between heterozygous and homozygous scn1Lab mutants.
After plating, the plate was covered with aluminum foil and placed in an incubator at 28 °C. Behavior was monitored using the ViewPoint ZebraBox before and after treatment; large duration data from 6 dpf and 7 dpf were used for data analysis. A Viewpoint ZebraBox monitored larval movements for 15 min with 100% lights on as described in Section 2.4.
RESULTS
- Chemically-induced seizures
- Dravet Syndrome induced by morpholino
- a Survival and fluorescent checks
- b ViewPoint data and analysis
- scn1Lab mutant zebrafish
Figure A shows the injection of control MO into two embryos, and panel B shows the injection of scn1Lab-MO. Non-injected fish were not included in the table because they did not receive an injection of morpholine and therefore did not fluoresce. Experiments were performed under normal conditions (n=60-90/treatment group; 3 experiments included in the data set), elevated temperatures, and increased amounts of MO (n=60/treatment group; 1 experiment included in the data set).
Calculations were determined by taking the number of live fish at 4 dpf and dividing by the total number of fish in that group at 0 dpf. Data under normal conditions (28C), elevated temperature conditions (34C for 10 min), and increased amounts of morpholino are depicted in Figure 6. and t survival follows in g morp. hol in o in jecti on for normal conditions. ncr eas ed morp hol in o amounts. rial included in data set) was performed. The results for three trials of morpholino injections under normal conditions are shown in Figure 7A.
Exposure of the fish to a heat of 34C failed to increase gross locomotion in the scn1Lab-MO injected group. Increased amounts of morpholino did not significantly increase gross locomotion compared to the non-injected group. Zebrafish larval behavior was analyzed using the ViewPoint Zebrabox (45 min recording with 100% light) to record the duration of major activity (>9 mm/sec) at 4 dpf, 5 dpf and 6 dpf.
Eggs were collected at 0 dpf and determination between WT/heterozygous and homozygous was performed at 4 dpf. The percentage of scn1Lab -/- fish was calculated by taking the number of scn1Lab -/- fish and dividing that number by the total number of eggs. Scn1Lab -/- shows darker pigmentation towards the head region and along the edges of the body, as well as the absence of a swim bladder.
The percentage of large activity duration from scn1Lab +/- to scn1Lab -/- (heterozygous/homozygous) was calculated bold 4, 5, 6 and 7 dpf. However, none of the concentrations of CBD (0.075 and 0.3 mg/L) showed a significant decrease in the duration of high activity compared to control. Scn1Lab mutants that were homozygous (-/-) and heterozygous (+/-) were in a ViewPoint ZebraBox (15 min recording with 100% light).
The duration of grand activity after a 24 and 48 exposure to CBD and clemizole was analyzed to determine if CBD shows promise in reducing seizure-like activity (n=6).
DISCUSSION
However, previous research conducted by Devinsky et al. 2017) showed that CBD may show promise in reducing seizure-like activity in human DS patients. These phenotypes are characteristic features of scn1Lab mutant zebrafish as observed in previous research (Sourbron et al., 2016; Zhang et al., 2015). The digital image of the morpholino injection showed us that the MO was successfully injected into the embryo.
However, the knockdown was considered a failure because behavioral experiments using ViewPoint data showed that the MO-injected zebrafish showed no increase in the duration of high activity compared to the uninjected fish. From research by Zhang et al. 2015), an automated video-based behavioral tracking system was used to simultaneously monitor and quantify the locomotor activity of free-swimming scn1Lab morphants. However, in our experiments, the scn1Lab group showed no increase in movement.
The control MO and non-injected groups should have shown similar duration at high activity, but they did not. Also, in our results we did not see larval sensitivity to hyperthermia as observed in Zhang et al. 2015) also performed higher resolution video recording to capture more subtle larval attack behavior that was not detected by the automated tracker. Based on previous research, we hypothesized that CBD would be an effective anticonvulsant agent against DS (Cunha et al., 1980; Devinsky et al., 2017).
Experimental results obtained from ViewPoint data showed that both 0.075 mg/L CBD (0.25 M) and 0.3 mg/L CBD (1 M) did not significantly decrease the duration of high activity. In the homozygous group, clemizole decreased the duration of major activity by 6 dpf, but lost its efficacy by 7 dpf. Although clemizole did not significantly decrease gross activity movement in our experiment, there was a decrease in gross movement compared to the control group.
Clemizole seemed to lose its efficacy at 7 dpf in the homozygous group; this may be due to natural metabolism of the drug. Although CBD failed to significantly reduce large activity movement in our experiment, Cunha et al. 1980) found that CBD administration inhibited the effect of PTZ in rats, reduced the astrocytic hyperplasia, reduced neuronal damage in the hippocampus caused by the seizures and selectively reduced the expression of the NR1 subunit of NMDA ( Cunha et al., 2015). No significant central nervous system side effects, or effects on vital signs or mood were seen at doses up to 1,500 mg/day (p.o.) or 30 mg (i.v.) in both acute and chronic administration (Devinsky et al., 2014).
Retrieved February 2018, from https://www.ninds.nih.gov/Disorders/All-Disorders/Dravet-Syndrome-Information-Page. 34; Efficacy and tolerability of the new antiepileptic drugs I: Treatment of new epilepsy Report of the Therapeutics and Technology Assessment Subcommittee and Quality Standards Subcommittee of the American Academy of Neurology and the American Epilepsy Society. "Neurology. 34; De novo loss-Function mutations in CHD2 cause a febrile myoclonic epileptic encephalopathy that shares features with Dravet syndrome." The American Journal of Human Genetics.
