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White, MD, PhD Institution: University of Pennsylvania, Perelman School of Medicine Application: Reserve – One Informative Addition ($3,000) 1. Question and Rational Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in Tetrahymena pyriformis.1–3 We propose an independent test of this interaction in larval zebrafish (Danio rerio), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements. Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints.4,5 Prior work in Xenopus tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics.6 We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish. Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models. 2. Experimental Design and Analysis EpoB dose and exposure pilot. Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or 1, 3, 10, or 30 nM EpoB, centered on the 10 nM concentration effective in Tetrahymena.3 Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and spontaneous movement (SM) and elicited movement (EM) will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published Tetrahymena study. Full isoflurane curves will not be performed at each EpoB concentration. Definitive isoflurane concentration-response experiment. At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method.4 Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence. Two complementary quantitative endpoints will be measured: SM, defined as total distance traveled during a four-minute observation period, and EM following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. SM will serve as the primary behavioral endpoint, with both EC50 and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals. EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC50 curve is from parallel displacement of the concentration-response relationship. Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes. Definitive experiments will comprise four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment). Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold. 3. Why Zebrafish are an Informative Addition This experiment directly addresses a central uncertainty identified in the RFP: reduced anesthetic sensitivity versus altered induction kinetics. Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,1,2 while the Tetrahymena EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves.3 Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies. Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior. Our laboratory has observed pronounced changes in both anesthetic EC50 and concentration-response slope during pharmacological antagonism,5 making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC50. Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity. Finally, larval zebrafish bridge the biological gap between Tetrahymena and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions. 4. Interpretation, Feasibility and Timeline A reproducible rightward EC50 shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction. The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within approximately 10 weeks of study initiation, followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report. 5. Budget Item Requested Laboratory/Institutional Contribution Epothilone B and preparation reagents $500 Routine laboratory reagents/equipment Zebrafish breeding and husbandry $400 Established colony and aquatic facility Anesthetic/behavioral assay consumables $300 Isoflurane, equipment, tracking system, major equipment Personnel effort: behavioral experiments, data analysis $1,800 PI supervision and additional laboratory personnel effort Total $3,000 Major equipment, infrastructure, and PI effort contributed 6. References (1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. eNeuro 2024, 11, ENEURO.0291-24.2024. DOI: 10.1523/ENEURO.0291-24.2024. (2) Huang, Y.; Qiu, Z.; Yu, X.; et al. Brain-Penetrant Microtubule-Stabilizer Epothilone B Delays Isoflurane-Induced Unconsciousness in Mice. Neuropharmacology 2026, 287, 110834. DOI: 10.1016/j.neuropharm.2026.110834. (3) Yu, X.; Qiu, Z.; Nashrah, F.; et al. Microtubule-Stabilizer Epothilone B Counteracts Anesthetic-Induced Slowed Swimming in Tetrahymena pyriformis. Exp. Cell Res. 2026, 457, 114938. DOI: 10.1016/j.yexcr.2026.114938. (4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896. (5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943. (6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. DOI: 10.1021/ja311171u.83:T2924,Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish Applicant: Elizabeth R. White, MD, PhD Institution: University of Pennsylvania, Perelman School of Medicine Application: Reserve – One Informative Addition ($3,000) 1. Question and Rational Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in Tetrahymena pyriformis.1–3 We propose an independent test of this interaction in larval zebrafish (Danio rerio), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements. Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints.4,5 Prior work in Xenopus tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics.6 We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish. Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models. 2. Experimental Design and Analysis EpoB dose and exposure pilot. Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or 1, 3, 10, or 30 nM EpoB, centered on the 10 nM concentration effective in Tetrahymena.3 Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and spontaneous movement (SM) and elicited movement (EM) will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published Tetrahymena study. Full isoflurane curves will not be performed at each EpoB concentration. Definitive isoflurane concentration-response experiment. At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method.4 Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence. Two complementary quantitative endpoints will be measured: SM, defined as total distance traveled during a four-minute observation period, and EM following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. SM will serve as the primary behavioral endpoint, with both EC50 and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals. EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC50 curve is from parallel displacement of the concentration-response relationship. Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes. Definitive experiments will comprise four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment). Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold. 3. Why Zebrafish are an Informative Addition This experiment directly addresses a central uncertainty identified in the RFP: reduced anesthetic sensitivity versus altered induction kinetics. Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,1,2 while the Tetrahymena EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves.3 Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies. Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior. Our laboratory has observed pronounced changes in both anesthetic EC50 and concentration-response slope during pharmacological antagonism,5 making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC50. Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity. Finally, larval zebrafish bridge the biological gap between Tetrahymena and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions. 4. Interpretation, Feasibility and Timeline A reproducible rightward EC50 shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction. The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within approximately 10 weeks of study initiation, followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report. 5. Budget Item Requested Laboratory/Institutional Contribution Epothilone B and preparation reagents $500 Routine laboratory reagents/equipment Zebrafish breeding and husbandry $400 Established colony and aquatic facility Anesthetic/behavioral assay consumables $300 Isoflurane, equipment, tracking system, major equipment Personnel effort: behavioral experiments, data analysis $1,800 PI supervision and additional laboratory personnel effort Total $3,000 Major equipment, infrastructure, and PI effort contributed 6. References (1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. eNeuro 2024, 11, ENEURO.0291-24.2024. DOI: 10.1523/ENEURO.0291-24.2024. (2) Huang, Y.; Qiu, Z.; Yu, X.; et al. Brain-Penetrant Microtubule-Stabilizer Epothilone B Delays Isoflurane-Induced Unconsciousness in Mice. Neuropharmacology 2026, 287, 110834. DOI: 10.1016/j.neuropharm.2026.110834. (3) Yu, X.; Qiu, Z.; Nashrah, F.; et al. Microtubule-Stabilizer Epothilone B Counteracts Anesthetic-Induced Slowed Swimming in Tetrahymena pyriformis. Exp. Cell Res. 2026, 457, 114938. DOI: 10.1016/j.yexcr.2026.114938. (4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896. (5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943. (6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. DOI: 10.1021/ja311171u. 84:T9c62,{"type":"doc","content":[{"type":"heading","attrs":{"id":"b326f3d6-9379-498f-9271-ee900563e082","data-toc-id":"b326f3d6-9379-498f-9271-ee900563e082","textAlign":"left","level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish"}]},{"type":"paragraph","attrs":{"id":"47c4a2ac-a6df-4ad3-ae74-a3d25b65387e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Applicant:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Elizabeth R. White, MD, PhD"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Institution:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" University of Pennsylvania, Perelman School of Medicine"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Application:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Reserve – One Informative Addition ($3,000)"}]},{"type":"heading","attrs":{"id":"1cb3be69-7e23-4cf6-9ce7-0761cedd6ba5","data-toc-id":"1cb3be69-7e23-4cf6-9ce7-0761cedd6ba5","textAlign":"left","level":2},"content":[{"type":"text","text":"1. Question and Rational"}]},{"type":"paragraph","attrs":{"id":"1ba0ea10-dee0-4980-b976-d46c6a1f1ac1","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena pyriformis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"1–3"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" We propose an independent test of this interaction in larval zebrafish ("},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Danio rerio"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements."}]},{"type":"paragraph","attrs":{"id":"9146e807-a282-4a9a-9641-331f0242f5db","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"4,5"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Prior work in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Xenopus"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"6"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":" Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models."}]},{"type":"heading","attrs":{"id":"45dfd00e-4649-4d19-aaca-cd56dc89a46c","data-toc-id":"45dfd00e-4649-4d19-aaca-cd56dc89a46c","textAlign":"left","level":2},"content":[{"type":"text","text":"2. Experimental Design and Analysis"}]},{"type":"paragraph","attrs":{"id":"f2f5853f-2410-41d7-b647-9c847406d1b6","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"EpoB dose and exposure pilot."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"1, 3, 10, or 30 nM EpoB"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", centered on the 10 nM concentration effective in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"3"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"underline"}],"text":"spontaneous movement (SM)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"underline"}],"text":"elicited movement (EM)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" study. Full isoflurane curves will not be performed at each EpoB concentration."}]},{"type":"paragraph","attrs":{"id":"5def17dc-5de1-43fd-b6d9-1c223bc23dbd","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Definitive isoflurane concentration-response experiment."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"4"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence."}]},{"type":"paragraph","attrs":{"id":"d6c9ce4f-1711-4f9f-a7db-c594e7b209e7","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Two complementary quantitative endpoints will be measured: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"SM"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", defined as total distance traveled during a four-minute observation period, and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"EM"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"SM will serve as the primary behavioral endpoint, with both EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":" and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" curve is from parallel displacement of the concentration-response relationship. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes."}]},{"type":"paragraph","attrs":{"id":"34d95ff9-6088-45d8-a505-3637ee501393","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Definitive experiments will comprise "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":". Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold."}]},{"type":"heading","attrs":{"id":"23c5dd94-9102-4311-b5d2-044a21cec114","data-toc-id":"23c5dd94-9102-4311-b5d2-044a21cec114","textAlign":"left","level":2},"content":[{"type":"text","text":"3. Why Zebrafish are an Informative Addition"}]},{"type":"paragraph","attrs":{"id":"bb1d4145-238d-4a17-9d43-21836e130c49","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"This experiment directly addresses a central uncertainty identified in the RFP: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"reduced anesthetic sensitivity versus altered induction kinetics"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":". Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"1,2"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" while the "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"3"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies."}]},{"type":"paragraph","attrs":{"id":"fef1e7f5-1d55-4ef9-95be-e76fddc858ba","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Our laboratory has observed pronounced changes in both anesthetic EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" and concentration-response slope during pharmacological antagonism,"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"5"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"."}]},{"type":"paragraph","attrs":{"id":"f96be8c5-2f03-4004-b6c9-11dfefea7d44","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity."}]},{"type":"paragraph","attrs":{"id":"a8604fb6-6e76-48a5-8c59-7e19e2e420c2","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Finally, larval zebrafish bridge the biological gap between "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions."}]},{"type":"heading","attrs":{"id":"ab931b7c-9272-4183-994d-e3bb12572339","data-toc-id":"ab931b7c-9272-4183-994d-e3bb12572339","textAlign":"left","level":2},"content":[{"type":"text","text":"4. Interpretation, Feasibility and Timeline"}]},{"type":"paragraph","attrs":{"id":"490355ed-aeaa-4a47-8f15-eeb1d7aca706","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"A reproducible rightward EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction."}]},{"type":"paragraph","attrs":{"id":"5c17c496-7927-4f9b-977f-a93b20e1ae31","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"approximately 10 weeks of study initiation"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report."}]},{"type":"heading","attrs":{"id":"d1d9f65f-8d71-4a1d-b6c9-4cf8620ece32","data-toc-id":"d1d9f65f-8d71-4a1d-b6c9-4cf8620ece32","textAlign":"left","level":2},"content":[{"type":"text","text":"5. 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References"}]},{"type":"paragraph","attrs":{"id":"2a299515-bc4c-4b22-8ef1-62de25bed169","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"eNeuro"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2024"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"11"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", ENEURO.0291-24.2024. 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Exp."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2025"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"220"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", e67896."}]},{"type":"paragraph","attrs":{"id":"5ffdf27d-760b-4605-8c6e-7faec7d42d20","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Sci. Rep."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2024"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"14"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", 15943."}]},{"type":"paragraph","attrs":{"id":"2156885f-67b3-4bb9-904e-8f6fff8e675b","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"J. Am. Chem. Soc."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2013"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"135"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", 5389–5398. DOI: 10.1021/ja311171u."}]},{"type":"paragraph","attrs":{"id":"8c7e2bb0-8b56-42f1-91ac-2579632d875a","class":null,"textAlign":null}}]}85:T2924,Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish Applicant: Elizabeth R. White, MD, PhD Institution: University of Pennsylvania, Perelman School of Medicine Application: Reserve – One Informative Addition ($3,000) 1. Question and Rational Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in Tetrahymena pyriformis.1–3 We propose an independent test of this interaction in larval zebrafish (Danio rerio), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements. Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints.4,5 Prior work in Xenopus tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics.6 We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish. Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models. 2. Experimental Design and Analysis EpoB dose and exposure pilot. Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or 1, 3, 10, or 30 nM EpoB, centered on the 10 nM concentration effective in Tetrahymena.3 Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and spontaneous movement (SM) and elicited movement (EM) will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published Tetrahymena study. Full isoflurane curves will not be performed at each EpoB concentration. Definitive isoflurane concentration-response experiment. At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method.4 Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence. Two complementary quantitative endpoints will be measured: SM, defined as total distance traveled during a four-minute observation period, and EM following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. SM will serve as the primary behavioral endpoint, with both EC50 and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals. EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC50 curve is from parallel displacement of the concentration-response relationship. Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes. Definitive experiments will comprise four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment). Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold. 3. Why Zebrafish are an Informative Addition This experiment directly addresses a central uncertainty identified in the RFP: reduced anesthetic sensitivity versus altered induction kinetics. Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,1,2 while the Tetrahymena EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves.3 Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies. Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior. Our laboratory has observed pronounced changes in both anesthetic EC50 and concentration-response slope during pharmacological antagonism,5 making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC50. Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity. Finally, larval zebrafish bridge the biological gap between Tetrahymena and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions. 4. Interpretation, Feasibility and Timeline A reproducible rightward EC50 shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction. The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within approximately 10 weeks of study initiation, followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report. 5. Budget Item Requested Laboratory/Institutional Contribution Epothilone B and preparation reagents $500 Routine laboratory reagents/equipment Zebrafish breeding and husbandry $400 Established colony and aquatic facility Anesthetic/behavioral assay consumables $300 Isoflurane, equipment, tracking system, major equipment Personnel effort: behavioral experiments, data analysis $1,800 PI supervision and additional laboratory personnel effort Total $3,000 Major equipment, infrastructure, and PI effort contributed 6. References (1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. eNeuro 2024, 11, ENEURO.0291-24.2024. DOI: 10.1523/ENEURO.0291-24.2024. (2) Huang, Y.; Qiu, Z.; Yu, X.; et al. Brain-Penetrant Microtubule-Stabilizer Epothilone B Delays Isoflurane-Induced Unconsciousness in Mice. Neuropharmacology 2026, 287, 110834. DOI: 10.1016/j.neuropharm.2026.110834. (3) Yu, X.; Qiu, Z.; Nashrah, F.; et al. Microtubule-Stabilizer Epothilone B Counteracts Anesthetic-Induced Slowed Swimming in Tetrahymena pyriformis. Exp. Cell Res. 2026, 457, 114938. DOI: 10.1016/j.yexcr.2026.114938. (4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896. (5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943. (6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. DOI: 10.1021/ja311171u. 86:T9c62,{"type":"doc","content":[{"type":"heading","attrs":{"id":"b326f3d6-9379-498f-9271-ee900563e082","data-toc-id":"b326f3d6-9379-498f-9271-ee900563e082","textAlign":"left","level":1},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"14pt","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish"}]},{"type":"paragraph","attrs":{"id":"47c4a2ac-a6df-4ad3-ae74-a3d25b65387e","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Applicant:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Elizabeth R. White, MD, PhD"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Institution:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" University of Pennsylvania, Perelman School of Medicine"},{"type":"hardBreak","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}]},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Application:"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Reserve – One Informative Addition ($3,000)"}]},{"type":"heading","attrs":{"id":"1cb3be69-7e23-4cf6-9ce7-0761cedd6ba5","data-toc-id":"1cb3be69-7e23-4cf6-9ce7-0761cedd6ba5","textAlign":"left","level":2},"content":[{"type":"text","text":"1. Question and Rational"}]},{"type":"paragraph","attrs":{"id":"1ba0ea10-dee0-4980-b976-d46c6a1f1ac1","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena pyriformis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"1–3"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" We propose an independent test of this interaction in larval zebrafish ("},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Danio rerio"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements."}]},{"type":"paragraph","attrs":{"id":"9146e807-a282-4a9a-9641-331f0242f5db","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"4,5"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Prior work in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Xenopus"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"6"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":" Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models."}]},{"type":"heading","attrs":{"id":"45dfd00e-4649-4d19-aaca-cd56dc89a46c","data-toc-id":"45dfd00e-4649-4d19-aaca-cd56dc89a46c","textAlign":"left","level":2},"content":[{"type":"text","text":"2. Experimental Design and Analysis"}]},{"type":"paragraph","attrs":{"id":"f2f5853f-2410-41d7-b647-9c847406d1b6","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"EpoB dose and exposure pilot."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"1, 3, 10, or 30 nM EpoB"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", centered on the 10 nM concentration effective in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"3"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"underline"}],"text":"spontaneous movement (SM)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"underline"}],"text":"elicited movement (EM)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" study. Full isoflurane curves will not be performed at each EpoB concentration."}]},{"type":"paragraph","attrs":{"id":"5def17dc-5de1-43fd-b6d9-1c223bc23dbd","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"Definitive isoflurane concentration-response experiment."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"4"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence."}]},{"type":"paragraph","attrs":{"id":"d6c9ce4f-1711-4f9f-a7db-c594e7b209e7","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Two complementary quantitative endpoints will be measured: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"SM"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", defined as total distance traveled during a four-minute observation period, and "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"EM"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"SM will serve as the primary behavioral endpoint, with both EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":" and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" curve is from parallel displacement of the concentration-response relationship. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes."}]},{"type":"paragraph","attrs":{"id":"34d95ff9-6088-45d8-a505-3637ee501393","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Definitive experiments will comprise "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":". Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold."}]},{"type":"heading","attrs":{"id":"23c5dd94-9102-4311-b5d2-044a21cec114","data-toc-id":"23c5dd94-9102-4311-b5d2-044a21cec114","textAlign":"left","level":2},"content":[{"type":"text","text":"3. Why Zebrafish are an Informative Addition"}]},{"type":"paragraph","attrs":{"id":"bb1d4145-238d-4a17-9d43-21836e130c49","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"This experiment directly addresses a central uncertainty identified in the RFP: "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"reduced anesthetic sensitivity versus altered induction kinetics"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":". Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"1,2"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" while the "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"3"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies."}]},{"type":"paragraph","attrs":{"id":"fef1e7f5-1d55-4ef9-95be-e76fddc858ba","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" Our laboratory has observed pronounced changes in both anesthetic EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" and concentration-response slope during pharmacological antagonism,"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"superscript"}],"text":"5"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"."}]},{"type":"paragraph","attrs":{"id":"f96be8c5-2f03-4004-b6c9-11dfefea7d44","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity."}]},{"type":"paragraph","attrs":{"id":"a8604fb6-6e76-48a5-8c59-7e19e2e420c2","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"Finally, larval zebrafish bridge the biological gap between "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions."}]},{"type":"heading","attrs":{"id":"ab931b7c-9272-4183-994d-e3bb12572339","data-toc-id":"ab931b7c-9272-4183-994d-e3bb12572339","textAlign":"left","level":2},"content":[{"type":"text","text":"4. Interpretation, Feasibility and Timeline"}]},{"type":"paragraph","attrs":{"id":"490355ed-aeaa-4a47-8f15-eeb1d7aca706","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"A reproducible rightward EC"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"subscript"}],"text":"50"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction."}]},{"type":"paragraph","attrs":{"id":"5c17c496-7927-4f9b-977f-a93b20e1ae31","class":"MsoNormalCxSpMiddle","textAlign":"justify"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"approximately 10 weeks of study initiation"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report."}]},{"type":"heading","attrs":{"id":"d1d9f65f-8d71-4a1d-b6c9-4cf8620ece32","data-toc-id":"d1d9f65f-8d71-4a1d-b6c9-4cf8620ece32","textAlign":"left","level":2},"content":[{"type":"text","text":"5. 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References"}]},{"type":"paragraph","attrs":{"id":"2a299515-bc4c-4b22-8ef1-62de25bed169","class":null,"textAlign":"left"},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"eNeuro"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2024"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"11"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", ENEURO.0291-24.2024. DOI: 10.1523/ENEURO.0291-24.2024."}]},{"type":"paragraph","attrs":{"id":"1ceb7173-e848-4077-8ee9-a99e0ba2118f","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(2) Huang, Y.; Qiu, Z.; Yu, X.; et al. Brain-Penetrant Microtubule-Stabilizer Epothilone B Delays Isoflurane-Induced Unconsciousness in Mice. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Neuropharmacology"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2026"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"287"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", 110834. DOI: 10.1016/j.neuropharm.2026.110834."}]},{"type":"paragraph","attrs":{"id":"ae2b7192-239d-4296-be28-603e234eb901","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(3) Yu, X.; Qiu, Z.; Nashrah, F.; et al. Microtubule-Stabilizer Epothilone B Counteracts Anesthetic-Induced Slowed Swimming in "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Tetrahymena pyriformis"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":". "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Exp. Cell Res."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2026"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"457"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", 114938. DOI: 10.1016/j.yexcr.2026.114938."}]},{"type":"paragraph","attrs":{"id":"0b3c0156-564e-459c-add8-0a556d053bfc","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"J. Vis. 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M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"Sci. Rep."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2024"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"14"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", 15943."}]},{"type":"paragraph","attrs":{"id":"2156885f-67b3-4bb9-904e-8f6fff8e675b","class":"MsoNormal","textAlign":null},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":"(6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"J. Am. Chem. Soc."},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":" "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"bold"}],"text":"2013"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", "},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}},{"type":"italic"}],"text":"135"},{"type":"text","marks":[{"type":"textStyle","attrs":{"fontSize":"","fontFamily":"\"Times New Roman\",serif","color":""}}],"text":", 5389–5398. 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Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish

Applicant: Elizabeth R. White, MD, PhD
Institution: University of Pennsylvania, Perelman School of Medicine
Application: Reserve – One Informative Addition ($3,000)

1. Question and Rational

Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in Tetrahymena pyriformis.^1–3 We propose an independent test of this interaction in larval zebrafish (Danio rerio), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements.

Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints.^4,5 Prior work in Xenopus tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics.⁶ We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish. Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models.

2. Experimental Design and Analysis

EpoB dose and exposure pilot. Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or 1, 3, 10, or 30 nM EpoB, centered on the 10 nM concentration effective in Tetrahymena.³ Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and spontaneous movement (SM) and elicited movement (EM) will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published Tetrahymena study. Full isoflurane curves will not be performed at each EpoB concentration.

Definitive isoflurane concentration-response experiment. At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method.⁴ Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence.

Two complementary quantitative endpoints will be measured: SM, defined as total distance traveled during a four-minute observation period, and EM following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. SM will serve as the primary behavioral endpoint, with both EC₅₀ and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals. EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC₅₀ curve is from parallel displacement of the concentration-response relationship. Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes.

Definitive experiments will comprise four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment). Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold.

3. Why Zebrafish are an Informative Addition

This experiment directly addresses a central uncertainty identified in the RFP: reduced anesthetic sensitivity versus altered induction kinetics. Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,^1,2 while the Tetrahymena EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves.³ Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies.

Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior. Our laboratory has observed pronounced changes in both anesthetic EC₅₀ and concentration-response slope during pharmacological antagonism,⁵ making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC₅₀.

Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity.

Finally, larval zebrafish bridge the biological gap between Tetrahymena and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions.

4. Interpretation, Feasibility and Timeline

A reproducible rightward EC₅₀ shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction.

The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within approximately 10 weeks of study initiation, followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report.

5. Budget

Item	Requested	Laboratory/Institutional Contribution
Epothilone B and preparation reagents	$500	Routine laboratory reagents/equipment
Zebrafish breeding and husbandry	$400	Established colony and aquatic facility
Anesthetic/behavioral assay consumables	$300	Isoflurane, equipment, tracking system, major equipment
Personnel effort: behavioral experiments, data analysis	$1,800	PI supervision and additional laboratory personnel effort
Total	$3,000	Major equipment, infrastructure, and PI effort contributed

6. References

(1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. eNeuro 2024, 11, ENEURO.0291-24.2024. DOI: 10.1523/ENEURO.0291-24.2024.

(2) Huang, Y.; Qiu, Z.; Yu, X.; et al. Brain-Penetrant Microtubule-Stabilizer Epothilone B Delays Isoflurane-Induced Unconsciousness in Mice. Neuropharmacology 2026, 287, 110834. DOI: 10.1016/j.neuropharm.2026.110834.

(3) Yu, X.; Qiu, Z.; Nashrah, F.; et al. Microtubule-Stabilizer Epothilone B Counteracts Anesthetic-Induced Slowed Swimming in Tetrahymena pyriformis. Exp. Cell Res. 2026, 457, 114938. DOI: 10.1016/j.yexcr.2026.114938.

(4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896.

(5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943.

(6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. DOI: 10.1021/ja311171u.

88:T4d3e,

Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish

Applicant: Elizabeth R. White, MD, PhD
Institution: University of Pennsylvania, Perelman School of Medicine
Application: Reserve – One Informative Addition ($3,000)

1. Question and Rational

2. Experimental Design and Analysis

3. Why Zebrafish are an Informative Addition

4. Interpretation, Feasibility and Timeline

5. Budget

Item	Requested	Laboratory/Institutional Contribution
Epothilone B and preparation reagents	$500	Routine laboratory reagents/equipment
Zebrafish breeding and husbandry	$400	Established colony and aquatic facility
Anesthetic/behavioral assay consumables	$300	Isoflurane, equipment, tracking system, major equipment
Personnel effort: behavioral experiments, data analysis	$1,800	PI supervision and additional laboratory personnel effort
Total	$3,000	Major equipment, infrastructure, and PI effort contributed

6. References

(4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896.

(5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943.

(6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. DOI: 10.1021/ja311171u.

89:T425,The proposal targets the Athanatos reserve slot ($3,000) for one informative addition to the epothilone B / isoflurane replication call. It proposes to test whether EpoB pretreatment shifts the isoflurane concentration-response relationship in 5 dpf larval zebrafish, using a validated sealed 96-well volatile anesthetic assay with spontaneous movement (SM) as primary and tap-evoked movement (EM) as secondary endpoints, reporting EC50 and Hill slope with 95% CIs. A small EpoB dose-tolerance pilot (1-30 nM) precedes a definitive experiment (n=48/concentration/treatment across 4 biological replicates). Strengths include tight alignment with the RFP's stated interest in equilibrated concentration-response assessment separating pharmacodynamics from induction kinetics, established lab infrastructure and prior publications in the exact assay, and reasonable budget with substantial institutional cost-sharing. Weaknesses include limited detail on preregistration, blinding/randomization procedures, animal welfare statements, and formal power calculations.8a:T2924,Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish Applicant: Elizabeth R. White, MD, PhD Institution: University of Pennsylvania, Perelman School of Medicine Application: Reserve – One Informative Addition ($3,000) 1. Question and Rational Does epothilone B (EpoB) alter the concentration-response relationship for isoflurane anesthesia in an intact vertebrate? EpoB has been reported to decrease isoflurane sensitivity in rats and mice, measured as increased latency to loss of righting reflex (LORR), and to attenuate isoflurane-induced slowing in Tetrahymena pyriformis.1–3 We propose an independent test of this interaction in larval zebrafish (Danio rerio), an intact vertebrate model that permits quantitative, equilibrated anesthetic concentration-response measurements. Our laboratory is well versed in administering volatile and intravenous general anesthetics to larval zebrafish and has characterized isoflurane and sevoflurane anesthesia, as well as pharmacological anesthetic antagonism using complementary spontaneous and stimulus-evoked behavioral endpoints.4,5 Prior work in Xenopus tadpoles also provides precedent that epothilone-mediated microtubule stabilization can modify anesthetic concentration-response behavior in an aquatic vertebrate, although that study used epothilone D and different anesthetics.6 We hypothesize that EpoB pretreatment will alter the isoflurane concentration-response relationship in larval zebrafish. Zebrafish are a vertebrate model that provides specific pharmacokinetic advantages over current measurements obtained from rodent models. 2. Experimental Design and Analysis EpoB dose and exposure pilot. Because EpoB exposure has not been characterized behaviorally in larval zebrafish, a small preliminary study will establish a tolerated, behaviorally neutral exposure before the definitive experiment. At 4 days post fertilization (dpf), larvae will be exposed to vehicle or 1, 3, 10, or 30 nM EpoB, centered on the 10 nM concentration effective in Tetrahymena.3 Larvae will be monitored during exposure for evidence of toxicity or abnormal behavior, and spontaneous movement (SM) and elicited movement (EM) will be assessed in the absence of anesthetic. This pilot will establish an appropriate exposure duration and EpoB concentration; 10 nM will be preferred if well tolerated and behaviorally neutral because it permits direct comparison with the published Tetrahymena study. Full isoflurane curves will not be performed at each EpoB concentration. Definitive isoflurane concentration-response experiment. At 5 dpf, larvae pretreated with the selected EpoB concentration or matched vehicle will undergo isoflurane concentration-response testing using our validated sealed 96-well volatile-anesthetic exposure method.4 Treatment groups will be studied concurrently with variable plate position and testing order. Behavioral measurements will be automated and treatment-blinded. Isoflurane concentrations will span the complete behavioral concentration-response range. Our method maintains stable aqueous volatile-anesthetic concentrations during behavioral testing, permitting assessment after equilibration at defined external concentrations. This is a distinct advantage to more adequately assess pharmacodynamic effects of isoflurane resistance as these measurements rather than the pharmacokinetics of induction or emergence. Two complementary quantitative endpoints will be measured: SM, defined as total distance traveled during a four-minute observation period, and EM following a standardized mechanical tap stimulus. EpoB-alone and vehicle-alone conditions will establish baseline behavior. Full concentration-response curves will be fit by nonlinear regression. SM will serve as the primary behavioral endpoint, with both EC50 and Hill slope as principal pharmacodynamic parameters of interest and reported with 95% confidence intervals. EM concentration-response will provide a complementary secondary endpoint. This analysis will prevent a potentially false assumption that a rightward shift of the EC50 curve is from parallel displacement of the concentration-response relationship. Altered anesthetic potency accompanied by changes in slope, or baseline behavior may indicate qualitatively different resistance phenotypes. Definitive experiments will comprise four independent biological replicates with 12 individual larvae per treatment in each replicate (n = 48/concentration/treatment). Each larva will be retained as an individual observation after normalization to replicate-matched vehicle controls, with biological replicate incorporated into the statistical analysis. Historical datasets from our established volatile-anesthetic assays and pilot data inform expected variability. Effect sizes and confidence intervals will be emphasized rather than relying solely on a binary significance threshold. 3. Why Zebrafish are an Informative Addition This experiment directly addresses a central uncertainty identified in the RFP: reduced anesthetic sensitivity versus altered induction kinetics. Published rodent EpoB studies measured time to LORR during induction at a fixed isoflurane concentration,1,2 while the Tetrahymena EpoB study measured swimming during exposure to 4–5% isoflurane rather than generating EpoB-dependent isoflurane concentration-response curves.3 Induction latency necessarily reflects anesthetic uptake and distribution in addition to pharmacodynamic sensitivity. In contrast, larval zebrafish can equilibrate at maintained anesthetic concentrations, permitting the “equilibrated concentration-response assessment” specifically identified in the RFP as a useful extension of the existing studies. Importantly, full concentration-response analysis provides substantially more information than determining whether EpoB changes an induction time or response at a single threshold. The proposed approach will quantitatively determine not only whether EpoB shifts isoflurane potency, but whether the phenotype is characterized by a parallel displacement of the concentration-response relationship or by altered Hill slope, maximal effect, or baseline behavior. Our laboratory has observed pronounced changes in both anesthetic EC50 and concentration-response slope during pharmacological antagonism,5 making curve shape an informative feature of anesthetic resistance rather than simply a means of estimating an EC50. Zebrafish additionally provide multiple quantitative behavioral endpoints rather than a single binary LORR or withdrawal endpoint. SM and EM independently assess suppression of spontaneous behavior and responsiveness to an external stimulus across the anesthetic concentration range. Quantitative baseline measurements are particularly important for a microtubule-modifying compound because EpoB-induced changes in locomotion could otherwise be misinterpreted as altered anesthetic sensitivity. Finally, larval zebrafish bridge the biological gap between Tetrahymena and rodents while allowing large cohorts and quantitative pharmacology in an intact vertebrate nervous system. Volatile and non-volatile anesthetics can also be administered continuously through the surrounding medium. Thus, if an EpoB-dependent phenotype is identified, future studies can efficiently determine whether it is selective for isoflurane or volatile anesthetics or extends to structurally and pharmacologically distinct anesthetic classes under closely matched exposure conditions. 4. Interpretation, Feasibility and Timeline A reproducible rightward EC50 shift without altered baseline behavior would support generalization of EpoB-associated isoflurane resistance to another vertebrate and to an equilibrated concentration-response endpoint. A parallel shift would be pharmacologically distinct from antagonistic phenotypes typically dominated by altered Hill slope, maximal response, or endpoint-specific effects; the latter outcomes would suggest a more complex interaction requiring additional investigation. Conversely, a precise null effect would help define the limits of the reported EpoB-isoflurane interaction. The White Laboratory has established zebrafish husbandry, automated behavioral tracking, volatile-anesthetic exposure infrastructure, and extensive experience studying anesthetic concentration-response and antagonism in zebrafish. Existing equipment, colony infrastructure, PI supervision, and routine laboratory resources will be contributed. We anticipate completion of the experimental work and analysis within approximately 10 weeks of study initiation, followed by release of the detailed experimental protocol, animal-level data, analysis, and a concise report. 5. Budget Item Requested Laboratory/Institutional Contribution Epothilone B and preparation reagents $500 Routine laboratory reagents/equipment Zebrafish breeding and husbandry $400 Established colony and aquatic facility Anesthetic/behavioral assay consumables $300 Isoflurane, equipment, tracking system, major equipment Personnel effort: behavioral experiments, data analysis $1,800 PI supervision and additional laboratory personnel effort Total $3,000 Major equipment, infrastructure, and PI effort contributed 6. References (1) Khan, S. K.; Huang, Y.; Timuçin, D.; et al. Microtubule-Stabilizer Epothilone B Delays Anesthetic-Induced Unconsciousness in Rats. eNeuro 2024, 11, ENEURO.0291-24.2024. DOI: 10.1523/ENEURO.0291-24.2024. (2) Huang, Y.; Qiu, Z.; Yu, X.; et al. Brain-Penetrant Microtubule-Stabilizer Epothilone B Delays Isoflurane-Induced Unconsciousness in Mice. Neuropharmacology 2026, 287, 110834. DOI: 10.1016/j.neuropharm.2026.110834. (3) Yu, X.; Qiu, Z.; Nashrah, F.; et al. Microtubule-Stabilizer Epothilone B Counteracts Anesthetic-Induced Slowed Swimming in Tetrahymena pyriformis. Exp. Cell Res. 2026, 457, 114938. DOI: 10.1016/j.yexcr.2026.114938. (4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896. (5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943. (6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. 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8f:T3efa,

Applicant: Elizabeth R. White, MD, PhD
Institution: University of Pennsylvania, Perelman School of Medicine
Application: Reserve – One Informative Addition ($3,000)

1. Question and Rational

2. Experimental Design and Analysis

3. Why Zebrafish are an Informative Addition

4. Interpretation, Feasibility and Timeline

5. Budget

Item	Requested	Laboratory/Institutional Contribution
Epothilone B and preparation reagents	$500	Routine laboratory reagents/equipment
Zebrafish breeding and husbandry	$400	Established colony and aquatic facility
Anesthetic/behavioral assay consumables	$300	Isoflurane, equipment, tracking system, major equipment
Personnel effort: behavioral experiments, data analysis	$1,800	PI supervision and additional laboratory personnel effort
Total	$3,000	Major equipment, infrastructure, and PI effort contributed

6. References

(4) Hough, M. K.; Plasencia, D. M.; White, E. R. Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation. J. Vis. Exp. 2025, 220, e67896.

(5) Plasencia, D. M.; Rodgers, L. H.; Knighton, A. R.; Eckenhoff, R. G.; White, E. R. Antagonism of Propofol Anesthesia by Alkyl-Fluorobenzene Derivatives. Sci. Rep. 2024, 14, 15943.

(6) Emerson, D. J.; et al. Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia. J. Am. Chem. Soc. 2013, 135, 5389–5398. DOI: 10.1021/ja311171u.

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Drafts

Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish

Drafts

Drafts

Quantitative Testing of Epothilone B Modulation of Isoflurane Sensitivity in Zebrafish

Key Insights

Funding Progress

Peer Reviews

Related Funding

Epothilone B and anesthetic sensitivity

Nonprofit Recipient