Anesthetic and sedative drugs are small compounds known to bind to hundreds of proteins. One intriguing binding partner of propofol is the motor domain of a neuronal mitochondrial transport kinesin, kif5A. Here, we used zebrafish wild type (WT) and kif5Aa knockout (KO) larval behavioral assays to assess anesthetic sensitivity and combined that with zebrafish primary neuronal cell culture to probe for alterations in mitochondrial motility. We found that the loss of kif5Aa increases behavioral sensitivity to propofol and etomidate, with etomidate hypersensitivity greater than propofol. In contrast, kif5Aa KO animals were resistant to the behavioral effects of dexmedetomidine. Finally, WT and kif5Aa KO larvae responded similarly to the behavioral effects of ketamine. Propofol inhibited the anterograde motility of mitochondria in WT zebrafish neurons, while etomidate inhibited mitochondrial motility in both anterograde and retrograde directions; neither drug altered mitochondrial motility in the kif5Aa knockout (KO) neurons. In contrast, dexmedetomidine enhanced retrograde mitochondrial motility in both WT and kif5Aa KO animals. Finally, ketamine had little significant effect on mitochondrial motility in either mutant or WT animals. These data demonstrate that each anesthetic/sedative drug affects the motor protein machinery uniquely and is associated with unique changes in behavior. Understanding how different anesthetic compounds alter neuronal motor proteins will be important in defining how anesthetics alter neuronal signaling and energetic dynamics.

Zebrafish husbandry

All zebrafish experiments complied with the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). The adult zebrafish were maintained in an aquatic facility overseen by the University Laboratory Animal Resources (ULAR) at the University of Pennsylvania. 

All zebrafish were maintained at 28 degree C with a 13-11 light-dark cycle as previously described. Adult fish were maintained using standard husbandry conditions. Knockout (KO) and wild-type (WT) siblings were maintained in the same conditions until experimentation. 

Genotyping the kif5Aa KO line

Genomic DNA was obtained from adult animals using the fin-clipping method. The PCR identifying the kif5Aa^sa7168 KO animals was run using previously described primers and digested using Dra III. 

kKif5Aasa7168heterozygous zebrafish were mated, and before behavioural testing, the larvae were separated based on known visual phenotypes: loss of the swim bladder and increased pigmentation. “WT” was a combination of WT and heterozygous animals, as they were indistinguishable.

The kif5Aa heterozygous line was outcrossed into a stable Tg(actb1:Has.GLRX:roGFP) line (available on Zfin.com under the name p312Tg, ID name ZDB-ALT-140508-36). In this line t, the roGFP is targeted to the mitochondria and labels all mitochondria within the zebrafish.  

Drug exposures

We tested four sedative/anesthetics: propofol, etomidate, dexmedetomidine, and ketamine. Each drug was diluted into E3 embryo water for the behavior experiments described previously. Briefly, propofol was diluted to 100 mM from a 100 mM DMSO stock in E3 by sonicating for 5 minutes (min); absorbance at 270nm was used to confirm the concentration. Etomidate and dexmedetomidine were solubilized directly by vortexing. Finally, ketamine was diluted in an E3 with 55 mM HEPES at a pH of 8, which is required for improved uptake by the larvae.

For cell culture, all drugs were initially diluted to a 2x concentration using 1x PBS. This was then diluted to the final drug concentration in complete cell culture media to ensure adequate nutrition for the cells.

Zebrafish 5 dpf behavioural assay

Behavior experiments were performed as previously described. Briefly, we used the DanioVision behavioral system with EthoVision software (Noldus) to measure the distance the WT and kif5Aa KO larvae moved spontaneously (SM) over 4 min or in 1 second after an elicited acoustic stimulus (EM). The experiments were 2 hours within the dark enclosure of the behaviour system, with the EM at the highest setting every 30 minutes. All experiments used 96-well glass plates (Chemglass Life Sciences, CG-1910) at 28 degree C.

Primary zebrafish neuronal cell culture

This protocol is based on a previously described zebrafish neuronal cell culture assay. Briefly, we used a 96-well tissue culture plate with a glass-like polymer coverslip bottom (Cellvis, P96-1.5P) coated with Entactin-Collagen-Laminin (EMD Millipore, 08-110) at 10-20 mg/mL and stored at 4 degree C overnight.

Twenty 5 dpf zebrafish WT siblings and kif5Aa KO larvae containing the Grx ro-GFP2 were euthanized on ice until the heart stopped beating. Brains were dissected and placed in 500 mL Hank's base salt solution with 1% penicillin/streptomycin on ice. 200 mL of Accutase (Sigma, A6964) was added for dissociation and incubated for 10 min in a 37 degree C water bath. Brains were triturated using a glass pipette for 2-4 min or until there were few visible clumps of cells. This was key to decreasing aggregation and promoting attachment and axon outgrowth during cell culturing. The dissociation reaction was stopped using 700 mL of complete media. Complete media consisted of 0.22 mm sterile-filtered Leibovitz-15, 2.5mM Glutamax I, 15 ng/mL epidermal growth factor, 10% fetal bovine serum, 1% penicillin/streptomycin, and 5% zebrafish embryo extract. The embryo extract was created as previously described.

The sample was centrifuged at 1500 RPM for 6 min at 4 degree C. The supernatant was discarded,d and 100-125 mL of complete media was added to resuspend the pellet. The cell mixture was plated using 50-75 mL per well. The cells were incubated at 28 degree C for 2 days.

Imaging

 Neurons were imaged using a Leica DMI4000 with a Yokagawa CSU-X1 spinning disk confocal attachment at 63x.

The anaesthetic concentration chosen was the EC50s previously determined: propofol 3 mM, etomidate 30 mM, dexmedetomidine 10 nM, and ketamine 130 mM. The cells were incubated in the drug for at least 30 min before imaging. Neurons that could be traced back to a specific cell body were imaged every 3 seconds for 5 minutes for a total of 101 images (initial image plus imaging for the next five minutes). Five to ten cells were imaged per well.

An open-source ImageJ plugin, KymoToolBox, defines mitochondrial motility. Supplemental Table 1 is a summary of the mitochondria traced. Briefly, each movie contained a range of 1-6 axons. The axons were highlighted, and a kymograph was created, on which individual mitochondria were traced. The number of traceable mitochondria varied between movies; therefore, the range of mitochondria traced is given in Supplemental Table 1. Any movie with fewer than 5 traceable mitochondria was discarded. ImageJ was then able to calculate the distance moved for the mitochondria. Total mitochondrial movement is herein defined as any movement, retrograde plus anterograde, including zero movement. No minimum motility down the axon was used.

Statistics

For behavior studies, the distance moved for 8-12 larvae per drug concentration per genotype was averaged and counted as a single experimental value (n). The experiment was performed at least 4 times on separate days to ensure technical and biological replication. Each animal was exposed to a single drug and not reused. For the baseline movement assays, 40-41 experiments at 30 minutes were analyzed, and significance was tested using the Mann-Whitney test for nonparametric data. Hill curves were created using 6-8 drug concentrations, including a no-drug (ND) control. The distance moved was normalized using the ND as 100% movement. Hill curves were created using GraphPad Prism. As with prior studies, the Hill curves were constrained to a top value of 100% and a bottom ovalue f 0%. The EC50s with 95% confidence intervals (CI) were calculated and plotted. EC50 comparison p-values were determined using a sum of squares F test.

All experiments were performed at least in triplicate for the mitochondrial motility assays. The motility data were,e in most cases, normally distributed. Therefore, a two-way ANOVA using Bonferroni’s correction for multiple comparisons determined significance. For all groups, significance is labeled: not significant (n.s., p > 0.05), p ≤ 0.05 (), p ≤ 0.01 (), p ≤ 0.001 (), p ≤ 0.0001.