Your willingness to accept me into your laboratory has provided me with an unparalleled experience of personal and professional growth. I am indebted to Cammi Thornton for your confidence in my ability to carry out this research project, for your guidance along the way, and for your unyielding patience. Thank you also, Zach Miller, for allowing me to be part of your graduate education and for your knowledge, leadership, and humor that made the rest of my research project worthwhile.
Biochemistry in me; I also thank you for your time and wisdom in helping to create the final product of this dissertation. The experience of an SMBHC education has truly allowed me to grow academically and personally. Thank you to my friends and family, whose words of encouragement gave me the strength to complete this project.
Natural compounds including extracts of cannabidiol, CBD, and Tapinanthus globiferous, TG, were screened for their anticonvulsant activity. 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 amounts of MO at.
INTRODUCTION
- Epilepsy
- Dravet Syndrome
- Scn1Lab gene
- Zebrafish as a model
- Morpholino
- Natural products with hypothesized anti-seizure activity
- Study Goals
People with DS need constant care; the condition has a major impact on the lives of the patients as well as the family's quality of life. One of the primary monogenic causes of DS includes 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 ).
Furthermore, embryos develop outside the mother organism facilitating microscopic visualization during early development. Zebrafish movement can be tracked through the ViewPoint Zebrabox, which enables researchers to analyze aspects of seizure behavior in zebrafish. Therefore, researchers usually perform genetic mapping and positional cloning experiments, which allow researchers to pinpoint the location of the associated gene and identify the mutation responsible.
The use of morpholino oligos enables transient knockdown of the SCN1A gene by blocking translation on the ribosome. Instead, they work by inhibiting the biological activity of a target RNA until that RNA is naturally degraded. The 25 base morpholino oligo binds strongly and specifically to its complementary 25 base pair target site in the RNA strand because each morpholino ring positions one of the standard DNA bases (GeneTools, 2018).
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
Unwanted debris and unfertilized/dead eggs were removed after the eggs were transferred 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 placed back into the incubator.
After a 24-h exposure to the dosing solutions (6 dpf), morphological changes were observed to determine whether the TG extracts had any toxic side effects, including a touch response, pericardial edema, yolk sac edema, a curved body axis and/or a non-inflated swim bladder (Dietrich, 2017). Phenol red (0.5 µL) was added to the morpholino 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 were placed in an incubator at 34.0°C for 10 min and then returned to the incubator at 28.0°C. ZebraBox software creates an excel sheet that includes the duration each larva spends in inactive (<5 mm/sec), small (5-9 mm/sec), and large (>9 mm/sec) movements over 45 minutes. periods with intervals of 15 minutes. Movement that meets the requirement of large activity is indicative of seizure activity and was the data that was used to test the effectiveness of the morpholino knockdown.
After the final recording at 6 dpf, larvae were euthanized with buffered MS-222 and placed in RNAlater and stored at −80.0°C. Data collected from the ViewPoint ZebraBox were analyzed with GraphPad Prism 5.0 (La Jolla, CA). Images were taken to characterize physical differences between heterozygous and homozygous scn1Lab mutants.
After plating, the plate was covered with aluminum foil and placed inside the incubator at 28°C. Behavior was tracked using the ViewPoint ZebraBox before and after treatment; long duration data from 6 dpf and 7 dpf were used for data analysis. The Viewpoint ZebraBox tracked larval movements for 15 min with the lights on at 100% 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).
Counts 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 (28°C), elevated temperature conditions (34°C for 10 min) and increased amounts of morpholino are depicted in Figure 6. Experiments under normal conditions (n=60-90/treatment group; 3 experiments included in dataset) , higher temperature and increased MO amounts. n=60/treatment group; 1 trial included in the data set) was performed.
The results for three experiments with morpholino injections under normal conditions are shown in Figure 7A. Exposing the fish to a heat of 34°C failed to increase gross locomotion in the scn1Lab-MO-injected group. Increased amounts of morpholino did not significantly increase greater locomotion compared to the non-injected group.
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 dividing the number of scn1Lab -/- fish by the total number of eggs. The percentage duration of high activity scn1Lab +/- to scn1Lab -/- (heterozygous/homozygous) was calculated fat 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 high activity following a 24 and 48 exposure to CBD and clemizole was analyzed to determine whether CBD shows promise in reducing seizure-like activity (n=6).
DISCUSSION
However, previous research conducted by Devinsky et al. 2017) suggested that CBD may be promising in reducing seizures in DS patients. These phenotypes are characteristic features of scn1Lab mutant zebrafish, as observed in previous studies (Sourbron et al., 2016; Zhang et al., 2015). Digital imaging of the morpholino injection told us that the MO was successfully injected into the embryo.
However, the knockdown appeared to fail because behavioral experiments using ViewPoint data showed that MO-injected zebrafish did not show an increase in the duration of high activity compared to non-injected fish. In a study 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 did not show an increase in locomotion.
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 μM) and 0.3 mg/L CBD (1 μM) 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.
