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Xenopus laevis
Chapter 25
Xenopus laevis Laura A. Jansen University of Virginia, Charlottesville, VA, United States
GENERAL DESCRIPTION
Oocyte Isolation
Xenopus laevis (Fig. 25.1), also known as the African clawed frog, has played a significant role in understanding the causes of epilepsy, and in identifying and characterizing effective treatments. This has largely been accomplished through the use of the oocytes of Xenopus, which possess many qualities that make them well-suited for the study of ion channels, neurotransmitter receptors, and membrane transporters involved in the generation of epileptic seizures. The Xenopus tadpole has also been used in important toxicology studies of antiepileptic drugs and as a model of chemically induced seizures. In this chapter, the use of Xenopus as a model in the study of epilepsy and seizures will be detailed, as well as the advantages and limitations of its use.
For studies using oocytes, mature female Xenopus of at least 100 g are preferred. Oocytes are generally obtained by surgical laparotomy performed under anesthesia. The anesthetic of choice is a 0.2% solution of MS-222 (Tricaine methanesulfonate), in which the frogs are immersed for a period of about 15 min. The anesthetic is directly absorbed through the skin of the frog. The appropriate depth of anesthesia results in loss of the righting reflex, and absence of response to toe pinch. A small incision is made through the skin and underlying fascia on one side of the abdomen near the inguinal region, after which the ovary, a large, dark, gelatinous structure, should be easily visualized. A small section of ovary is resected using sharp scissors, and immediately placed into OR-2 solution (96 mM NaCl, 2 mL KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4). The incision can then be closed with a silk suture. The same frog may be used again for oocyte harvest in 1–2 months, using alternating sides for a recommended total of up to six surgeries. An alternative to survival surgery that is preferred by some animal care committees is bilateral laparotomy under deep MS-222 anesthesia, followed by a terminal procedure, such as decapitation. After resection, the ovary is washed, and follicular cells surrounding the oocytes are removed, either manually under a microscope or after incubation in collagenase. The timing of exposure of the ovaries to collagenase is important, as too short of an exposure results in difficulty removing the follicular cells, while too long an exposure causes the oocyte membrane to soften. After collagenase treatment, the oocytes are washed and mature, stage V–VI oocytes are selected for experimentation. Oocytes in these mature stages are identified by their large size (1–1.2 mm in diameter) and distinct animal and vegetal poles. The animal pole, containing the nucleus, should be brown in color, while the vegetal pole, containing the “yolk,” should be cream in color, with a lighter band separating the two poles in the more mature oocytes. The selected oocytes should be stored in an incubator at 18°C in ND-96 solution (96 mM NaCl, 2 mL KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.4) until use.
METHODS OF GENERATION Animal Breeding and Care One of the factors contributing to the popularity of Xenopus in research is the relative ease with which they are maintained in a laboratory setting. Xenopus laevis, which are airbreathing aquatic amphibians, are native to South Africa. Wild-caught Xenopus and laboratory-bred animals can be obtained from commercial suppliers, or breeding colonies may be maintained onsite. Xenopus are housed in aquatic tanks at a density of 1 adult frog per 3–4 L of water. Either static or circulating water systems can be used, but the water must be treated to remove chlorine and chloramine. The ideal water temperature for Xenopus is between 18 and 22°C, with a light-dark cycle of 12 h each. Laboratory-bred Xenopus may be fed a synthetic diet, while wild-caught animals prefer meat. The most common disease affecting Xenopus is red leg, manifested by subcutaneous hemorrhages and swelling. Red leg is caused by opportunistic Aeromonas hydrophila infection in stressed frogs, and is treated with antibiotics, and isolation of affected animals (Goldin, 1992; Green, 2002; Schultz and Dawson, 2003; Smart and Krishek, 1995). Models of Seizures and Epilepsy. http://dx.doi.org/10.1016/B978-0-12-804066-9.00025-0 Copyright © 2017 Elsevier Inc. All rights reserved.
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FIGURE 25.1 Adult female Xenopus laevis.
Expression of Exogenous Proteins in Xenopus Oocytes One of the main advantages in the use of Xenopus as an experimental model is the relative ease by which their oocytes express and incorporate exogenous proteins. The use of Xenopus oocytes in this manner was first described by Gurdon et al. (1971), when they described the translation of hemoglobin by oocytes after injection of RNA isolated from rabbit reticulocytes. The specific use of oocytes to express and characterize neuronal ion channels was first reported by Miledi and coworkers in 1982. In their work, RNA isolated from the Torpedo electric organ injected into oocytes resulted in the synthesis of functional nicotinic acetylcholine receptors, while injection of chick brain RNA produced expression of functional GABAA receptors (Barnard et al., 1982; Miledi et al., 1982). Several methods exist for inducing exogenous protein expression in Xenopus oocytes, including injection of RNA, DNA, or suspensions of membrane vesicles into the oocyte. General procedures for oocyte injection will be described first, followed by specifics regarding use of RNA, DNA, or membrane vesicles for injection.
General Procedures for Oocyte Injection Oocytes used for injection should have smooth, intact membranes, and regular pigmentation. Injections should be done in calcium-free media. The injection set up generally consists of a stereomicroscope, a nonheat generating light source, a nanoinjector coupled to a micromanipulator, and a container that holds and stabilizes the oocytes during injection. An injection pipette with an opening of 20–40 µm is fabricated from capillary glass, and is filled with the substance to be injected. Each oocyte is then sequentially impaled with the injection pipette and the appropriate volume of solution released by the nanoinjector. Slight swelling of the oocyte should be seen during injections. Oocytes that
exhibit loss of membrane integrity or significant leakage of intracellular contents should be discarded. After about 5 s the injection pipette is slowly removed, and the oocytes allowed to briefly recover in calcium-free media before return to standard ND-96 solution (Goldin, 1992).
Expression of Exogenous Proteins Via RNA Injection Cytoplasmic injection of RNA followed by oocyte-mediated translation, posttranslational processing, transport, and insertion of the protein into the cell membrane represents the most common method used in the study of epilepsyassociated ion channels and receptors in Xenopus oocytes. RNA may either be directly isolated from tissue (e.g., brain, cultured neurons, etc.) or, more commonly, specific RNAs corresponding to the native or mutant protein of interest may be synthesized. In order for RNAs to be translated by oocytes, the vector must contain a promoter for DNAdependent RNA polymerase, such as T7, and a poly-A tail. Including noncoding sequences from the X enopus β-globin gene increases expression in oocytes (Shin et al., 1998). The RNA must be prepared and manipulated under RNAse free-conditions to prevent degradation. Volumes of up to 100 nL are injected into the cytoplasm of the oocyte, and following a 24–48h period of incubation, expression of the protein of interest can be detected for at least a week (Goldin, 1992). Relative advantages of the expression of exogenous proteins via RNA injection include the relative ease of the techniques involved, high levels of protein expression, and the ability to influence the composition of multisubunit proteins through use of set ratios of subunit RNA amount. Relative disadvantages include the extra steps needed to synthesize oocyte-compatible RNA, the need to maintain an RNAse-free environment, and dependence on the translational, posttranslational, trafficking, and assembly mechanisms of the oocytes that may not accurately reflect those of the host tissue.
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Expression of Exogenous Proteins Via DNA Injection As opposed to cytoplasmic injection of RNA into oocytes, DNA may be injected directly into the nucleus to produce expression of foreign proteins. The DNA may take the form of plasmid DNA or may be DNA isolated directly from another cell or organism. The procedure of injecting DNA into the oocyte nucleus requires more skill than for cytoplasmic injections. Smaller injection pipettes (<20 µm) and smaller volumes (10–20 nL) are used for nuclear injections. Relative advantages to the use of DNA include ease of sample preparation, and greater stability of DNA, as compared with RNA. Disadvantages include a lesser ability to control expression levels and subunit ratios than with RNA injection, and dependence on the oocyte machinery for transcription, in addition to the processing steps necessary after RNA injection (Goldin, 1992; Smart and Krishek, 1995).
Expression of Exogenous Proteins Via Injection of Membrane Vesicles (Microtransplantation) Although this technique is less well-known that that of protein expression after injection of oocytes with RNA or DNA, it has an extensive history, and a number of unique advantages. It was initially developed to aid in the study of nicotinic acetylcholine receptors from the Torpedo electroplaque. In this technique, membrane vesicles were prepared from the electric organ, and injected into the cytoplasm of Xenopus oocytes. Within 2 h after injection, nicotinic acetylcholine receptor currents could begin to be detected in the oocytes. The appearance of these currents did not depend on RNA synthesis, but rather arose from the incorporation into the oocyte membrane of Torpedo membrane vesicles harboring native receptors. The nicotinic currents obtained in this manner exhibited physiologic and pharmacologic properties characteristic of these receptors, as determined in other experimental paradigms (Marsal et al., 1995). This technique has since been expanded and modified to allow analysis of ion channels and receptors from multiple sources (Fig. 25.2). Membrane vesicles, obtained by tissue homogenization and differential centrifugation, may be prepared from fresh or frozen brain specimens obtained from any species (including human), from cultured cells, or even from nonnervous system tissues, such as muscle and placenta (Díaz et al., 2008; Eusebi et al., 2009; Jansen et al., 2008; Miledi et al., 2002; Palma et al., 2011, 2003; Sanna et al., 1996). The procedure of injection of oocytes with membrane vesicle preparations is very similar to that of cytoplasmic injection of RNA, although without the need for maintenance of RNAse-free conditions. Membrane protein incorporation is able to be detected within a few hours, and remains stable for several days (Eusebi et al., 2009). As membrane-imbedded receptors from the source tissue are
FIGURE 25.2 Schematic depicting the technique of receptor microtransplantation into Xenopus oocytes.
directly incorporated into the oocyte plasma membrane, there is no dependence on the transcriptional, translational, posttranslational, protein trafficking, or assembly machinery of the oocyte. Therefore, it his likely the measured functional properties reflect better those of the receptors in situ than those obtained after RNA or DNA injection.
Analysis Methods The function of wild type and mutant proteins can be analyzed in a number of ways after their expression in Xenopus oocytes. In the study of epilepsy, this system has been largely used in the characterization of ion channels, neurotransmitter receptors, and membrane transporters that will be the subjects of focus here, although characterization of the function of any type of cellular protein is possible.
Two Electrode Voltage Clamp Electrophysiology The most common method of assessment of the pharmacologic and physiologic properties of native and mutant ion channels in Xenopus oocytes is two electrode voltage clamp electrophysiology (TEVC) electrophysiology. Both voltage-gated and ligand-gated channels may be studied in this way. This technique is the least technically challenging of the electrophysiological methods of analysis, and usually generates robust and reproducible data. In TEVC the o ocyte is bathed in an oocyte Ringer’s solution (e.g., 82.5 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM HEPES). Under low-level magnification, the oocyte is impaled by two glass electrodes, a voltage-sensing electrode, and a current-passing electrode. These electrodes should be of low resistance (0.5–2 MΩ), and filled with a 3 M KCl solution. The resting membrane potential of a healthy
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o ocyte is generally in the range of –40 to –60 mV. A TEVC amplifier is used to clamp the voltage at a given level, and channel currents may then be activated using either voltage steps or through application of pharmacologic agonists. Currents generated by oocytes expressing exogenous channels following RNA injection are often greater than 1 µA, while currents from exogenous channels following DNA or membrane vesicle injection are usually smaller (as small as a few nA). Oocytes are usually very stable during the recording process, and may be maintained for hours (Guan et al., 2013; Smart and Krishek, 1995). Several factors must be considered in the analysis of currents measured from oocytes by TEVC. Many of these are due to the inherent physical properties of the oocyte. While the very large size of Xenopus oocytes promotes ease in injection and placement of recording and stimulating electrodes, it also results in a large membrane capacitance (180–250 nF) that must be overcome during applied changes in membrane voltage (Smart and Krishek, 1995). This is further exacerbated by a highly convoluted membrane surface with deep invaginations that are not visible to the naked eye. For this reason, analysis of channels with fast kinetics or rapid desensitization is difficult with TEVC. The large membrane capacitance also contributes to high levels of noise, negligible when analyzing currents on the order of microampere, but much more of an issue when trying to analyze nanoampere size currents. Another critical factor related to the size and complexity of the oocyte membrane is the effect this has on the effective concentration of agents used to activate and inhibit ligand-gated channels. Apparent EC50 and IC50 values are generally much higher when measured in oocytes, as compared with cultured mammalian cells or brain slices, due to delayed accessibility to the channels. However, relative concentration responses to agonists and antagonists are maintained, allowing comparison between wild-type and mutant channels, or between different pharmacologic agents. Finally, the large size of some currents measured in oocytes after RNA injection can actually be a detriment, leading to amplifier saturation.
Cut-Open Oocyte Electrophysiology Application of this technique may overcome many of the obstacles presented by TEVC, and can be applied to the study of ion channels expressed after RNA, DNA, or membrane vesicle injection (Kaneko et al., 1998; Stefani and Bezanilla, 1998). A specialized three-chamber apparatus is used, separating the oocyte into three zones: the upper zone from which current recordings are obtained, a middle zone that serves as a voltage “guard,” and a lower zone in which a portion of the oocyte is permeabilized by exposure to saponin. This procedure effectively decreases the membrane surface of the oocyte, allowing for more rapid voltage changes, and responses to extracellular solutions. This allows more accurate analysis of channel kinetics. In
addition, recording noise is much lower with this technique. Further, in contrast to the TEVC technique, in which the internal environment of the oocyte cannot be easily manipulated, with the cut-open oocyte technique a cannula can be inserted to perfuse the inside of the oocyte, with the intracellular solution varied to suit the experiment. This technique also has disadvantages, including the need for specialized equipment, increased need for technical expertise, and spatial voltage variation across the upper zone of the oocyte.
Macropatch Electrophysiology This technique is similar to the patch-clamp technique used in mammalian cells, with several adaptations to suit the oocyte (Goldin, 2006; Tammaro et al., 2008). A critical factor that differentiates this technique from the TEVC and cutopen oocyte techniques is that it requires the removal of the oocyte vitelline membrane that separates the plasma membrane from the follicular cells. Removal is performed manually, using sharp forceps after shrinking the oocyte in hypertonic solution. The technique may be difficult to master and tedious, with the resulting oocyte being much more fragile. A gigaohm seal is formed using a low resistance pipette (0.1– 1 MΩ), and recordings may be made either in cell-attached mode, or after patch excision. In cell-attached recordings, intracellular electrodes can be simultaneously utilized to voltage clamp the oocyte. Advantages of the oocyte macropatch technique include minimized capacitive transients ideal for examination of fast currents, and the ability to control both the intracellular and extracellular environments when using excised patches. Disadvantages include lower recording stability, as compared with the TEVC or cut-open oocyte techniques, and the requirement for high levels of channel expression, in order to increase the chance of obtaining patches with sufficiently large currents for analysis.
Single-Channel Electrophysiology Single channel electrophysiology may be performed on devitellinized oocytes, and is analogous in practice to that performed in mammalian cells (Goldin, 2006; Tammaro et al., 2008). The procedure is similar to that used in macropatch electrophysiology, with the exception of the need to use small diameter (and therefore higher resistance) electrodes. This results in the ability to resolve very fast events. It is best suited to the study of channels expressed at high density after RNA injection. The main disadvantage of this technique is its technical difficulty.
Measurement of Transporter Activity The activity and pharmacology of membrane transporters may also be analyzed in Xenopus oocytes. Methods to assess transporter function include measurement of the rate and extent of uptake of radiolabeled or fluorescently labeled substances, or use of electrophysiologic methods to measure currents generated by electrogenic
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transport (Cha et al., 1998; Mager et al., 1998; Seatter and Gould, 1999; Taylor et al., 1996).
UTILIZATION IN THE STUDY OF EPILEPSY Use in Characterizing Ion Channels Responsible for Control of Neuronal Excitability Many of the early reports using Xenopus oocytes as a model system involved their application as a tool to identify types of ligand-gated ion channels present in different neuronal systems. For example, injection of oocytes with mRNA isolated from human and rat cerebral cortex resulted in the appearance of currents mediated by sodium and potassium voltage-gated channels, and by serotonin, kainate, and GABA ligand-gated channels (Gundersen et al., 1984). Prior to the development of current DNA sequencing techniques, Xenopus oocytes were used in expression cloning of neuronal channels and receptors. The serotonin 5-HT1C receptor was cloned by construction of a cDNA library from mouse choroid plexus, followed by expression of the individual clones in oocytes, and testing for current responses to applied serotonin (Julius et al., 1988; Lübbert et al., 1987). As the identity of the genes responsible for the synthesis of both ligand- and voltage-gated ion channels became known, oocytes were frequently used for the expression of these channels, and characterization of their physiologic and pharmacologic properties. Oocyte studies were also instrumental in the identification of the subunit combinations necessary to replicate observed in vivo pharmacologic properties of multisubunit channels. In particular, subunit combinations responsible for the pharmacology of the GABAA receptor have been particularly well studied in oocytes. Understanding of GABAA receptor pharmacology is critical in the study of seizures and epilepsy, given its prominent role in mediating inhibition of neuronal activity. Work in oocytes has resulted in the identification of GABAA receptor subunit combinations necessary for the actions of benzodiazepines, barbiturates, and neurosteroids (Belelli et al., 2002; Blair et al., 1988; Campo-Soria et al., 2006; Parker et al., 1986; Sigel et al., 1990; Thompson et al., 1996). Finally, mutagenesis studies have allowed characterization of motifs crucial for the assembly, activity, and pharmacologic properties of many voltage- and ligandgated ion channels (Casula et al., 2001; Davies et al., 2001; Dunn et al., 1999; Greenfield et al., 2002)
Use in Characterizing Phenotype of Mutant Channels and Transporters Identified in Human Epilepsies Xenopus oocytes have been extensively used in the characterization of ion channel and membrane transporter
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mutations identified in human epilepsy. GABAA receptor gamma 2 subunit (GABRG2) mutations identified in genetic epilepsy with febrile seizures plus (GEFS+) were demonstrated to cause decreased channel function after expression in oocytes (Baulac et al., 2001; Harkin et al., 2002). In contrast, potassium channel KCNT1 mutations causing malignant migrating partial seizures of infancy (MMPSI), and other genetic epilepsies, were identified as gain-of-function rather than loss-of-function after TEVC and single channel analysis in oocytes (Barcia et al., 2012; Kim et al., 2014). Mutations in the SLC2A1 gene that encodes the GLUT1 transporter responsible for transport of glucose across the blood–brain barrier are a cause of generalized epilepsy. Xenopus oocytes have been used to demonstrate that mutations identified in individuals with GLUT1-related epilepsy result in impairment in glucose transport (Arsov et al., 2012). Other epilepsy-associated mutations that have undergone functional characterization in oocytes include SCN1A (Barela et al., 2006; Spampanato et al., 2001), KCNJ10 (Bockenhauer et al., 2009), KCNQ2 (Hunter et al., 2006; Orhan et al., 2014), GRIN2B (Lemke et al., 2014), KCNH1 (Simons et al., 2015), KCNMA1 (Yang et al., 2010), and CHRNA4 and CHRNB2 (Weltzin et al., 2016).
Use in Characterizing Human Epilepsies (Microtransplantation) The technique of microtransplantation of ion channels from human brain to the oocyte membrane by membrane vesicle injection has been used to characterize a number of human epilepsies that, by nature, are not easily amenable to standard electrophysiologic characterization. Palma, Eusebi, Miledi, and coworkers have extensively characterized properties of temporal lobe epilepsy tissue using this technique (Palma et al., 2002, 2007). Their findings in oocytes have been corroborated by studies in human epileptic brain slices (Ragozzino et al., 2005) and rodent epilepsy models (Mazzuferi et al., 2010). This technique has also been applied to the study of GABAA receptor pharmacology in surgically resected hypothalamic hamartomas (Li et al., 2011; Wu et al., 2007), focal cortical dysplasia (Jansen et al., 2008), and gliomas (Conti et al., 2011). Differences in pharmacology between GABAA receptors isolated from infants with epilepsy associated with infantile spasms, as compared with infants with epilepsy, but no spasms, have been identified using oocyte microtransplantation, an analysis that would have been extremely difficult to perform using other techniques (Fig. 25.3). In addition, because of the ability to utilize frozen postmortem tissues in this technique, analysis of ion channel properties associated with human genetic, neurodevelopmental, and neurodegenerative disorders has also been possible. Specifically, functional properties of GABAA, AMPA, kainate, and serotonin receptors have been characterized in postmortem brain
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FIGURE 25.3 Use of the receptor microtransplantation technique to demonstrate that cortical GABAergic pharmacology is altered in children with infantile spasms (IS). The degree of GABAA current enhancement by the alpha 1 subunit-selective benzodiazepine agonist zolpidem (A) and the neurosteroid 5α3α (B) is different in oocytes incorporating receptors from children with IS, as compared with those from children with epilepsy (EPI), but not IS. The number of patients examined is indicated in the bars. *P < 0.05. Representative current traces are shown in panels (C) and (D).
tissue from individuals with Angelman syndrome (Roden et al., 2010), autism (Limon et al., 2008), and Alzheimer’s disease (Miledi et al., 2004).
USE IN THERAPY AND BIOMARKER DEVELOPMENT Due to the rapidity and ease with which native and mutant proteins can be expressed and analyzed in Xenopus oocytes, they represent a valuable resource for the screening and identification of pharmacologic agents with potential to serve as therapeutics in epilepsy. This may be done in a low-throughput fashion, through the use of standard injection and recording techniques, or in a medium- to highthroughput fashion, through the use of automated systems (Kvist et al., 2011; Papke and Smith-Maxwell, 2009; Papke and Stokes, 2010; Pehl et al., 2004). For example, the use of standard oocyte techniques resulted in the identification of quinidine as a targeted therapy for epilepsy caused by activating mutations in KCNT1 (Milligan et al., 2014). Furthermore, the medium chain fatty acid decanoic acid, a component of MCT oil used in ketogenic diet therapy, was found to directly inhibit glutamate AMPA receptor activity as assayed in oocytes, opening an avenue to future antiepileptic drug discovery (Chang et al., 2015).
mammalian cell, in particular that of excitable cells, such as neurons. The membrane and intracellular compositions of amphibian oocytes are significantly different from those of mammalian neurons. Expression of exogenous proteins after RNA or DNA injection depends on the processing, assembly, and trafficking machinery of the oocyte that may result in structural and functional variances from proteins expressed in neurons. Oocytes must be maintained and analyzed at room temperature or below, resulting in alterations in channel kinetics and receptor turnover, as compared with mammalian body temperature. Many investigators experience seasonal variation in oocyte quality, particularly with wild-caught Xenopus (Goldin, 1992; Smart and Krishek, 1995; Weber, 1999). An additional important factor is that Xenopus oocytes express a number of endogenous receptors, channels, and transporters that may interfere with the analysis of exogenous ones (Mercado et al., 2001; Terhag et al., 2010; Weber, 1999). In particular, native oocytes express a calcium-activated chloride current (Barish, 1983) that can significantly interfere with the analysis of exogenous calcium channels, unless calcium-free extracellular solutions are used. Finally, similar to other in vitro models, sex-based differences are not able to be accurately assessed using oocytes.
LIMITATIONS
XENOPUS TADPOLES AS A MODEL IN THE STUDY OF EPILEPSY
In addition to the many advantages of the use of Xenopus oocytes in the study of channels and transporters underlying the pathophysiology of seizures and epilepsy, the model possesses a number of limitations that should be considered with its use. The oocyte milieu does not replicate that of any
Although most of the literature on the use of Xenopus as a model for the study of epilepsy focuses on the use of their oocytes, the study of Xenopus tadpoles has also provided important insights (Pratt and Khakhalin, 2013). The tadpoles are easy to generate and maintain, and the fact that
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drugs are absorbed through their skin makes administration simple. An extensive knowledge of the normal development of the Xenopus nervous system has allowed characterization of abnormalities that arise upon exposure of tadpoles to antiseizure drugs, including valproic acid and phenytoin, as well as other toxins (Dawson, 1991; Fort and Bantle, 1990; Gurvich et al., 2005; James et al., 2015). In addition, a model of seizures in Xenopus tadpoles has been developed. Addition of the convulsants pentylenetetrazole (PTZ), kainic acid, bicuculline, picrotoxin, 4-aminopyridine, or pilocarpine to the bath solution results in abnormal motor activity in tadpoles (e.g., tail bending, rapid circling, chaotic movements), followed by a period of decreased activity. This behavioral seizure activity is correlated with field potential oscillations and neuronal calcium spiking recorded in vivo (Bell et al., 2011; Hewapathirane et al., 2008). The effects of antiseizure medications can be easily tested in this model through their addition to the bath. Unfortunately, given the limited use of the tadpole model of seizures to date, a full assessment of its advantages, disadvantages, and applicability to human epilepsy is not possible.
INSIGHT INTO HUMAN DISORDERS Although Xenopus oocytes do not model human epilepsy per se, the ability to utilize this system to characterize ion channels and transporters associated with the pathophysiology of human epilepsy has provided crucial insight into this disorder. Through the use of Xenopus, substantial understanding has been achieved of the pharmacology and physiology of proteins responsible for the control of neuronal excitability, as well as how these properties are deranged by genetic mutations identified in human epilepsy. Importantly, the ability to screen rapidly potential therapeutic agents in oocytes for their effects on mutated channels identified in individual patients provides a pathway toward a “personalized medicine” approach to treating epilepsy.
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Eusebi, F., Palma, E., Amici, M., Miledi, R., 2009. Microtransplantation of ligand-gated receptor-channels from fresh or frozen nervous tissue into Xenopus oocytes: a potent tool for expanding functional information. Prog. Neurobiol. 88, 32–40. Fort, D.J., Bantle, J.A., 1990. Use of frog embryo teratogenesis assayXenopus and an exogenous metabolic activation system to evaluate the developmental toxicity of diphenylhydantoin. Fundam. Appl. Toxicol. 14, 720–733. Goldin, A.L., 1992. Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol. 207, 266–279. Goldin, A.L., 2006. Expression of ion channels in Xenopus oocytes. In: Clare, J.J., Trezise, D.J. (Eds.), Expression and Analysis of Recombinant Ion Channels: From Structural Studies to Pharmacological Screening. Wiley-VCH, Weinheim, Germany, pp. 1–25. Green, S.L., 2002. Factors affecting oogenesis in the South African clawed frog (Xenopus laevis). Comp. Med. 52, 307–312. Greenfield, L.J., Zaman, S.H., Sutherland, M.L., Lummis, S.C., Niemeyer, M.I., Barnard, E.A., Macdonald, R.L., 2002. Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement. Neuropharmacology 42, 502–521. Guan, B., Chen, X., Zhang, H., 2013. Two-electrode voltage clamp. Methods Mol. Biol. 998, 79–89. Gundersen, C.B., Miledi, R., Parker, I., 1984. Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopus oocytes. Nature 308, 421–424. Gurdon, J.B., Lane, C.D., Woodland, H.R., Marbaix, G., 1971. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233, 177–182. Gurvich, N., Berman, M.G., Wittner, B.S., Gentleman, R.C., Klein, P.S., Green, J.B.A., 2005. Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo. FASEB J. 19, 1166–1168. Harkin, L.A., Bowser, D.N., Dibbens, L.M., Singh, R., Phillips, F., Wallace, R.H., Richards, M.C., Williams, D.A., Mulley, J.C., Berkovic, S.F., Scheffer, I.E., Petrou, S., 2002. Truncation of the GABA(A)-receptor gamma2 subunit in a family with generalized epilepsy with febrile seizures plus. Am. J. Hum. Genet. 70, 530–536. Hewapathirane, D.S., Dunfield, D., Yen, W., Chen, S., Haas, K., 2008. In vivo imaging of seizure activity in a novel developmental seizure model. Exp. Neurol. 211, 480–488. Hunter, J., Maljevic, S., Shankar, A., Siegel, A., Weissman, B., Holt, P., Olson, L., Lerche, H., Escayg, A., 2006. Subthreshold changes of voltage-dependent activation of the K(V)7.2 channel in neonatal epilepsy. Neurobiol. Dis. 24, 194–201. James, E.J., Gu, J., Ramirez-Vizcarrondo, C.M., Hasan, M., Truszkowski, T.L.S., Tan, Y., Oupravanh, P.M., Khakhalin, A.S., Aizenman, C.D., 2015. Valproate-induced neurodevelopmental deficits in Xenopus laevis tadpoles. J. Neurosci. 35, 3218–3229. Jansen, L.A., Peugh, L.D., Ojemann, J.G., 2008. GABAA receptor properties in catastrophic infantile epilepsy. Epilepsy Res. 81, 188–197. Julius, D., MacDermott, A.B., Axel, R., Jessell, T.M., 1988. Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241, 558–564. Kaneko, S., Akaike, A., Satoh, M., 1998. Cut-open recording techniques. Methods Enzymol. 293, 319–331. Kim, G.E., Kronengold, J., Barcia, G., Quraishi, I.H., Martin, H.C., Blair, E., Taylor, J.C., Dulac, O., Colleaux, L., Nabbout, R., Kaczmarek, L.K., 2014. Human slack potassium channel mutations increase positive cooperativity between individual channels. Cell Rep. 9, 1661– 1672.
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Xenopus laevis Laura A. Jansen University of Virginia, Charlottesville, VA, United States
GENERAL DESCRIPTION
Oocyte Isolation
Xenopus laevis (Fig. 25.1), also known as the African clawed frog, has played a significant role in understanding the causes of epilepsy, and in identifying and characterizing effective treatments. This has largely been accomplished through the use of the oocytes of Xenopus, which possess many qualities that make them well-suited for the study of ion channels, neurotransmitter receptors, and membrane transporters involved in the generation of epileptic seizures. The Xenopus tadpole has also been used in important toxicology studies of antiepileptic drugs and as a model of chemically induced seizures. In this chapter, the use of Xenopus as a model in the study of epilepsy and seizures will be detailed, as well as the advantages and limitations of its use.
For studies using oocytes, mature female Xenopus of at least 100 g are preferred. Oocytes are generally obtained by surgical laparotomy performed under anesthesia. The anesthetic of choice is a 0.2% solution of MS-222 (Tricaine methanesulfonate), in which the frogs are immersed for a period of about 15 min. The anesthetic is directly absorbed through the skin of the frog. The appropriate depth of anesthesia results in loss of the righting reflex, and absence of response to toe pinch. A small incision is made through the skin and underlying fascia on one side of the abdomen near the inguinal region, after which the ovary, a large, dark, gelatinous structure, should be easily visualized. A small section of ovary is resected using sharp scissors, and immediately placed into OR-2 solution (96 mM NaCl, 2 mL KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4). The incision can then be closed with a silk suture. The same frog may be used again for oocyte harvest in 1–2 months, using alternating sides for a recommended total of up to six surgeries. An alternative to survival surgery that is preferred by some animal care committees is bilateral laparotomy under deep MS-222 anesthesia, followed by a terminal procedure, such as decapitation. After resection, the ovary is washed, and follicular cells surrounding the oocytes are removed, either manually under a microscope or after incubation in collagenase. The timing of exposure of the ovaries to collagenase is important, as too short of an exposure results in difficulty removing the follicular cells, while too long an exposure causes the oocyte membrane to soften. After collagenase treatment, the oocytes are washed and mature, stage V–VI oocytes are selected for experimentation. Oocytes in these mature stages are identified by their large size (1–1.2 mm in diameter) and distinct animal and vegetal poles. The animal pole, containing the nucleus, should be brown in color, while the vegetal pole, containing the “yolk,” should be cream in color, with a lighter band separating the two poles in the more mature oocytes. The selected oocytes should be stored in an incubator at 18°C in ND-96 solution (96 mM NaCl, 2 mL KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.4) until use.
METHODS OF GENERATION Animal Breeding and Care One of the factors contributing to the popularity of Xenopus in research is the relative ease with which they are maintained in a laboratory setting. Xenopus laevis, which are airbreathing aquatic amphibians, are native to South Africa. Wild-caught Xenopus and laboratory-bred animals can be obtained from commercial suppliers, or breeding colonies may be maintained onsite. Xenopus are housed in aquatic tanks at a density of 1 adult frog per 3–4 L of water. Either static or circulating water systems can be used, but the water must be treated to remove chlorine and chloramine. The ideal water temperature for Xenopus is between 18 and 22°C, with a light-dark cycle of 12 h each. Laboratory-bred Xenopus may be fed a synthetic diet, while wild-caught animals prefer meat. The most common disease affecting Xenopus is red leg, manifested by subcutaneous hemorrhages and swelling. Red leg is caused by opportunistic Aeromonas hydrophila infection in stressed frogs, and is treated with antibiotics, and isolation of affected animals (Goldin, 1992; Green, 2002; Schultz and Dawson, 2003; Smart and Krishek, 1995). Models of Seizures and Epilepsy. http://dx.doi.org/10.1016/B978-0-12-804066-9.00025-0 Copyright © 2017 Elsevier Inc. All rights reserved.
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FIGURE 25.1 Adult female Xenopus laevis.
Expression of Exogenous Proteins in Xenopus Oocytes One of the main advantages in the use of Xenopus as an experimental model is the relative ease by which their oocytes express and incorporate exogenous proteins. The use of Xenopus oocytes in this manner was first described by Gurdon et al. (1971), when they described the translation of hemoglobin by oocytes after injection of RNA isolated from rabbit reticulocytes. The specific use of oocytes to express and characterize neuronal ion channels was first reported by Miledi and coworkers in 1982. In their work, RNA isolated from the Torpedo electric organ injected into oocytes resulted in the synthesis of functional nicotinic acetylcholine receptors, while injection of chick brain RNA produced expression of functional GABAA receptors (Barnard et al., 1982; Miledi et al., 1982). Several methods exist for inducing exogenous protein expression in Xenopus oocytes, including injection of RNA, DNA, or suspensions of membrane vesicles into the oocyte. General procedures for oocyte injection will be described first, followed by specifics regarding use of RNA, DNA, or membrane vesicles for injection.
General Procedures for Oocyte Injection Oocytes used for injection should have smooth, intact membranes, and regular pigmentation. Injections should be done in calcium-free media. The injection set up generally consists of a stereomicroscope, a nonheat generating light source, a nanoinjector coupled to a micromanipulator, and a container that holds and stabilizes the oocytes during injection. An injection pipette with an opening of 20–40 µm is fabricated from capillary glass, and is filled with the substance to be injected. Each oocyte is then sequentially impaled with the injection pipette and the appropriate volume of solution released by the nanoinjector. Slight swelling of the oocyte should be seen during injections. Oocytes that
exhibit loss of membrane integrity or significant leakage of intracellular contents should be discarded. After about 5 s the injection pipette is slowly removed, and the oocytes allowed to briefly recover in calcium-free media before return to standard ND-96 solution (Goldin, 1992).
Expression of Exogenous Proteins Via RNA Injection Cytoplasmic injection of RNA followed by oocyte-mediated translation, posttranslational processing, transport, and insertion of the protein into the cell membrane represents the most common method used in the study of epilepsyassociated ion channels and receptors in Xenopus oocytes. RNA may either be directly isolated from tissue (e.g., brain, cultured neurons, etc.) or, more commonly, specific RNAs corresponding to the native or mutant protein of interest may be synthesized. In order for RNAs to be translated by oocytes, the vector must contain a promoter for DNAdependent RNA polymerase, such as T7, and a poly-A tail. Including noncoding sequences from the X enopus β-globin gene increases expression in oocytes (Shin et al., 1998). The RNA must be prepared and manipulated under RNAse free-conditions to prevent degradation. Volumes of up to 100 nL are injected into the cytoplasm of the oocyte, and following a 24–48h period of incubation, expression of the protein of interest can be detected for at least a week (Goldin, 1992). Relative advantages of the expression of exogenous proteins via RNA injection include the relative ease of the techniques involved, high levels of protein expression, and the ability to influence the composition of multisubunit proteins through use of set ratios of subunit RNA amount. Relative disadvantages include the extra steps needed to synthesize oocyte-compatible RNA, the need to maintain an RNAse-free environment, and dependence on the translational, posttranslational, trafficking, and assembly mechanisms of the oocytes that may not accurately reflect those of the host tissue.
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Expression of Exogenous Proteins Via DNA Injection As opposed to cytoplasmic injection of RNA into oocytes, DNA may be injected directly into the nucleus to produce expression of foreign proteins. The DNA may take the form of plasmid DNA or may be DNA isolated directly from another cell or organism. The procedure of injecting DNA into the oocyte nucleus requires more skill than for cytoplasmic injections. Smaller injection pipettes (<20 µm) and smaller volumes (10–20 nL) are used for nuclear injections. Relative advantages to the use of DNA include ease of sample preparation, and greater stability of DNA, as compared with RNA. Disadvantages include a lesser ability to control expression levels and subunit ratios than with RNA injection, and dependence on the oocyte machinery for transcription, in addition to the processing steps necessary after RNA injection (Goldin, 1992; Smart and Krishek, 1995).
Expression of Exogenous Proteins Via Injection of Membrane Vesicles (Microtransplantation) Although this technique is less well-known that that of protein expression after injection of oocytes with RNA or DNA, it has an extensive history, and a number of unique advantages. It was initially developed to aid in the study of nicotinic acetylcholine receptors from the Torpedo electroplaque. In this technique, membrane vesicles were prepared from the electric organ, and injected into the cytoplasm of Xenopus oocytes. Within 2 h after injection, nicotinic acetylcholine receptor currents could begin to be detected in the oocytes. The appearance of these currents did not depend on RNA synthesis, but rather arose from the incorporation into the oocyte membrane of Torpedo membrane vesicles harboring native receptors. The nicotinic currents obtained in this manner exhibited physiologic and pharmacologic properties characteristic of these receptors, as determined in other experimental paradigms (Marsal et al., 1995). This technique has since been expanded and modified to allow analysis of ion channels and receptors from multiple sources (Fig. 25.2). Membrane vesicles, obtained by tissue homogenization and differential centrifugation, may be prepared from fresh or frozen brain specimens obtained from any species (including human), from cultured cells, or even from nonnervous system tissues, such as muscle and placenta (Díaz et al., 2008; Eusebi et al., 2009; Jansen et al., 2008; Miledi et al., 2002; Palma et al., 2011, 2003; Sanna et al., 1996). The procedure of injection of oocytes with membrane vesicle preparations is very similar to that of cytoplasmic injection of RNA, although without the need for maintenance of RNAse-free conditions. Membrane protein incorporation is able to be detected within a few hours, and remains stable for several days (Eusebi et al., 2009). As membrane-imbedded receptors from the source tissue are
FIGURE 25.2 Schematic depicting the technique of receptor microtransplantation into Xenopus oocytes.
directly incorporated into the oocyte plasma membrane, there is no dependence on the transcriptional, translational, posttranslational, protein trafficking, or assembly machinery of the oocyte. Therefore, it his likely the measured functional properties reflect better those of the receptors in situ than those obtained after RNA or DNA injection.
Analysis Methods The function of wild type and mutant proteins can be analyzed in a number of ways after their expression in Xenopus oocytes. In the study of epilepsy, this system has been largely used in the characterization of ion channels, neurotransmitter receptors, and membrane transporters that will be the subjects of focus here, although characterization of the function of any type of cellular protein is possible.
Two Electrode Voltage Clamp Electrophysiology The most common method of assessment of the pharmacologic and physiologic properties of native and mutant ion channels in Xenopus oocytes is two electrode voltage clamp electrophysiology (TEVC) electrophysiology. Both voltage-gated and ligand-gated channels may be studied in this way. This technique is the least technically challenging of the electrophysiological methods of analysis, and usually generates robust and reproducible data. In TEVC the o ocyte is bathed in an oocyte Ringer’s solution (e.g., 82.5 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM HEPES). Under low-level magnification, the oocyte is impaled by two glass electrodes, a voltage-sensing electrode, and a current-passing electrode. These electrodes should be of low resistance (0.5–2 MΩ), and filled with a 3 M KCl solution. The resting membrane potential of a healthy
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o ocyte is generally in the range of –40 to –60 mV. A TEVC amplifier is used to clamp the voltage at a given level, and channel currents may then be activated using either voltage steps or through application of pharmacologic agonists. Currents generated by oocytes expressing exogenous channels following RNA injection are often greater than 1 µA, while currents from exogenous channels following DNA or membrane vesicle injection are usually smaller (as small as a few nA). Oocytes are usually very stable during the recording process, and may be maintained for hours (Guan et al., 2013; Smart and Krishek, 1995). Several factors must be considered in the analysis of currents measured from oocytes by TEVC. Many of these are due to the inherent physical properties of the oocyte. While the very large size of Xenopus oocytes promotes ease in injection and placement of recording and stimulating electrodes, it also results in a large membrane capacitance (180–250 nF) that must be overcome during applied changes in membrane voltage (Smart and Krishek, 1995). This is further exacerbated by a highly convoluted membrane surface with deep invaginations that are not visible to the naked eye. For this reason, analysis of channels with fast kinetics or rapid desensitization is difficult with TEVC. The large membrane capacitance also contributes to high levels of noise, negligible when analyzing currents on the order of microampere, but much more of an issue when trying to analyze nanoampere size currents. Another critical factor related to the size and complexity of the oocyte membrane is the effect this has on the effective concentration of agents used to activate and inhibit ligand-gated channels. Apparent EC50 and IC50 values are generally much higher when measured in oocytes, as compared with cultured mammalian cells or brain slices, due to delayed accessibility to the channels. However, relative concentration responses to agonists and antagonists are maintained, allowing comparison between wild-type and mutant channels, or between different pharmacologic agents. Finally, the large size of some currents measured in oocytes after RNA injection can actually be a detriment, leading to amplifier saturation.
Cut-Open Oocyte Electrophysiology Application of this technique may overcome many of the obstacles presented by TEVC, and can be applied to the study of ion channels expressed after RNA, DNA, or membrane vesicle injection (Kaneko et al., 1998; Stefani and Bezanilla, 1998). A specialized three-chamber apparatus is used, separating the oocyte into three zones: the upper zone from which current recordings are obtained, a middle zone that serves as a voltage “guard,” and a lower zone in which a portion of the oocyte is permeabilized by exposure to saponin. This procedure effectively decreases the membrane surface of the oocyte, allowing for more rapid voltage changes, and responses to extracellular solutions. This allows more accurate analysis of channel kinetics. In
addition, recording noise is much lower with this technique. Further, in contrast to the TEVC technique, in which the internal environment of the oocyte cannot be easily manipulated, with the cut-open oocyte technique a cannula can be inserted to perfuse the inside of the oocyte, with the intracellular solution varied to suit the experiment. This technique also has disadvantages, including the need for specialized equipment, increased need for technical expertise, and spatial voltage variation across the upper zone of the oocyte.
Macropatch Electrophysiology This technique is similar to the patch-clamp technique used in mammalian cells, with several adaptations to suit the oocyte (Goldin, 2006; Tammaro et al., 2008). A critical factor that differentiates this technique from the TEVC and cutopen oocyte techniques is that it requires the removal of the oocyte vitelline membrane that separates the plasma membrane from the follicular cells. Removal is performed manually, using sharp forceps after shrinking the oocyte in hypertonic solution. The technique may be difficult to master and tedious, with the resulting oocyte being much more fragile. A gigaohm seal is formed using a low resistance pipette (0.1– 1 MΩ), and recordings may be made either in cell-attached mode, or after patch excision. In cell-attached recordings, intracellular electrodes can be simultaneously utilized to voltage clamp the oocyte. Advantages of the oocyte macropatch technique include minimized capacitive transients ideal for examination of fast currents, and the ability to control both the intracellular and extracellular environments when using excised patches. Disadvantages include lower recording stability, as compared with the TEVC or cut-open oocyte techniques, and the requirement for high levels of channel expression, in order to increase the chance of obtaining patches with sufficiently large currents for analysis.
Single-Channel Electrophysiology Single channel electrophysiology may be performed on devitellinized oocytes, and is analogous in practice to that performed in mammalian cells (Goldin, 2006; Tammaro et al., 2008). The procedure is similar to that used in macropatch electrophysiology, with the exception of the need to use small diameter (and therefore higher resistance) electrodes. This results in the ability to resolve very fast events. It is best suited to the study of channels expressed at high density after RNA injection. The main disadvantage of this technique is its technical difficulty.
Measurement of Transporter Activity The activity and pharmacology of membrane transporters may also be analyzed in Xenopus oocytes. Methods to assess transporter function include measurement of the rate and extent of uptake of radiolabeled or fluorescently labeled substances, or use of electrophysiologic methods to measure currents generated by electrogenic
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transport (Cha et al., 1998; Mager et al., 1998; Seatter and Gould, 1999; Taylor et al., 1996).
UTILIZATION IN THE STUDY OF EPILEPSY Use in Characterizing Ion Channels Responsible for Control of Neuronal Excitability Many of the early reports using Xenopus oocytes as a model system involved their application as a tool to identify types of ligand-gated ion channels present in different neuronal systems. For example, injection of oocytes with mRNA isolated from human and rat cerebral cortex resulted in the appearance of currents mediated by sodium and potassium voltage-gated channels, and by serotonin, kainate, and GABA ligand-gated channels (Gundersen et al., 1984). Prior to the development of current DNA sequencing techniques, Xenopus oocytes were used in expression cloning of neuronal channels and receptors. The serotonin 5-HT1C receptor was cloned by construction of a cDNA library from mouse choroid plexus, followed by expression of the individual clones in oocytes, and testing for current responses to applied serotonin (Julius et al., 1988; Lübbert et al., 1987). As the identity of the genes responsible for the synthesis of both ligand- and voltage-gated ion channels became known, oocytes were frequently used for the expression of these channels, and characterization of their physiologic and pharmacologic properties. Oocyte studies were also instrumental in the identification of the subunit combinations necessary to replicate observed in vivo pharmacologic properties of multisubunit channels. In particular, subunit combinations responsible for the pharmacology of the GABAA receptor have been particularly well studied in oocytes. Understanding of GABAA receptor pharmacology is critical in the study of seizures and epilepsy, given its prominent role in mediating inhibition of neuronal activity. Work in oocytes has resulted in the identification of GABAA receptor subunit combinations necessary for the actions of benzodiazepines, barbiturates, and neurosteroids (Belelli et al., 2002; Blair et al., 1988; Campo-Soria et al., 2006; Parker et al., 1986; Sigel et al., 1990; Thompson et al., 1996). Finally, mutagenesis studies have allowed characterization of motifs crucial for the assembly, activity, and pharmacologic properties of many voltage- and ligandgated ion channels (Casula et al., 2001; Davies et al., 2001; Dunn et al., 1999; Greenfield et al., 2002)
Use in Characterizing Phenotype of Mutant Channels and Transporters Identified in Human Epilepsies Xenopus oocytes have been extensively used in the characterization of ion channel and membrane transporter
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mutations identified in human epilepsy. GABAA receptor gamma 2 subunit (GABRG2) mutations identified in genetic epilepsy with febrile seizures plus (GEFS+) were demonstrated to cause decreased channel function after expression in oocytes (Baulac et al., 2001; Harkin et al., 2002). In contrast, potassium channel KCNT1 mutations causing malignant migrating partial seizures of infancy (MMPSI), and other genetic epilepsies, were identified as gain-of-function rather than loss-of-function after TEVC and single channel analysis in oocytes (Barcia et al., 2012; Kim et al., 2014). Mutations in the SLC2A1 gene that encodes the GLUT1 transporter responsible for transport of glucose across the blood–brain barrier are a cause of generalized epilepsy. Xenopus oocytes have been used to demonstrate that mutations identified in individuals with GLUT1-related epilepsy result in impairment in glucose transport (Arsov et al., 2012). Other epilepsy-associated mutations that have undergone functional characterization in oocytes include SCN1A (Barela et al., 2006; Spampanato et al., 2001), KCNJ10 (Bockenhauer et al., 2009), KCNQ2 (Hunter et al., 2006; Orhan et al., 2014), GRIN2B (Lemke et al., 2014), KCNH1 (Simons et al., 2015), KCNMA1 (Yang et al., 2010), and CHRNA4 and CHRNB2 (Weltzin et al., 2016).
Use in Characterizing Human Epilepsies (Microtransplantation) The technique of microtransplantation of ion channels from human brain to the oocyte membrane by membrane vesicle injection has been used to characterize a number of human epilepsies that, by nature, are not easily amenable to standard electrophysiologic characterization. Palma, Eusebi, Miledi, and coworkers have extensively characterized properties of temporal lobe epilepsy tissue using this technique (Palma et al., 2002, 2007). Their findings in oocytes have been corroborated by studies in human epileptic brain slices (Ragozzino et al., 2005) and rodent epilepsy models (Mazzuferi et al., 2010). This technique has also been applied to the study of GABAA receptor pharmacology in surgically resected hypothalamic hamartomas (Li et al., 2011; Wu et al., 2007), focal cortical dysplasia (Jansen et al., 2008), and gliomas (Conti et al., 2011). Differences in pharmacology between GABAA receptors isolated from infants with epilepsy associated with infantile spasms, as compared with infants with epilepsy, but no spasms, have been identified using oocyte microtransplantation, an analysis that would have been extremely difficult to perform using other techniques (Fig. 25.3). In addition, because of the ability to utilize frozen postmortem tissues in this technique, analysis of ion channel properties associated with human genetic, neurodevelopmental, and neurodegenerative disorders has also been possible. Specifically, functional properties of GABAA, AMPA, kainate, and serotonin receptors have been characterized in postmortem brain
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FIGURE 25.3 Use of the receptor microtransplantation technique to demonstrate that cortical GABAergic pharmacology is altered in children with infantile spasms (IS). The degree of GABAA current enhancement by the alpha 1 subunit-selective benzodiazepine agonist zolpidem (A) and the neurosteroid 5α3α (B) is different in oocytes incorporating receptors from children with IS, as compared with those from children with epilepsy (EPI), but not IS. The number of patients examined is indicated in the bars. *P < 0.05. Representative current traces are shown in panels (C) and (D).
tissue from individuals with Angelman syndrome (Roden et al., 2010), autism (Limon et al., 2008), and Alzheimer’s disease (Miledi et al., 2004).
USE IN THERAPY AND BIOMARKER DEVELOPMENT Due to the rapidity and ease with which native and mutant proteins can be expressed and analyzed in Xenopus oocytes, they represent a valuable resource for the screening and identification of pharmacologic agents with potential to serve as therapeutics in epilepsy. This may be done in a low-throughput fashion, through the use of standard injection and recording techniques, or in a medium- to highthroughput fashion, through the use of automated systems (Kvist et al., 2011; Papke and Smith-Maxwell, 2009; Papke and Stokes, 2010; Pehl et al., 2004). For example, the use of standard oocyte techniques resulted in the identification of quinidine as a targeted therapy for epilepsy caused by activating mutations in KCNT1 (Milligan et al., 2014). Furthermore, the medium chain fatty acid decanoic acid, a component of MCT oil used in ketogenic diet therapy, was found to directly inhibit glutamate AMPA receptor activity as assayed in oocytes, opening an avenue to future antiepileptic drug discovery (Chang et al., 2015).
mammalian cell, in particular that of excitable cells, such as neurons. The membrane and intracellular compositions of amphibian oocytes are significantly different from those of mammalian neurons. Expression of exogenous proteins after RNA or DNA injection depends on the processing, assembly, and trafficking machinery of the oocyte that may result in structural and functional variances from proteins expressed in neurons. Oocytes must be maintained and analyzed at room temperature or below, resulting in alterations in channel kinetics and receptor turnover, as compared with mammalian body temperature. Many investigators experience seasonal variation in oocyte quality, particularly with wild-caught Xenopus (Goldin, 1992; Smart and Krishek, 1995; Weber, 1999). An additional important factor is that Xenopus oocytes express a number of endogenous receptors, channels, and transporters that may interfere with the analysis of exogenous ones (Mercado et al., 2001; Terhag et al., 2010; Weber, 1999). In particular, native oocytes express a calcium-activated chloride current (Barish, 1983) that can significantly interfere with the analysis of exogenous calcium channels, unless calcium-free extracellular solutions are used. Finally, similar to other in vitro models, sex-based differences are not able to be accurately assessed using oocytes.
LIMITATIONS
XENOPUS TADPOLES AS A MODEL IN THE STUDY OF EPILEPSY
In addition to the many advantages of the use of Xenopus oocytes in the study of channels and transporters underlying the pathophysiology of seizures and epilepsy, the model possesses a number of limitations that should be considered with its use. The oocyte milieu does not replicate that of any
Although most of the literature on the use of Xenopus as a model for the study of epilepsy focuses on the use of their oocytes, the study of Xenopus tadpoles has also provided important insights (Pratt and Khakhalin, 2013). The tadpoles are easy to generate and maintain, and the fact that
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drugs are absorbed through their skin makes administration simple. An extensive knowledge of the normal development of the Xenopus nervous system has allowed characterization of abnormalities that arise upon exposure of tadpoles to antiseizure drugs, including valproic acid and phenytoin, as well as other toxins (Dawson, 1991; Fort and Bantle, 1990; Gurvich et al., 2005; James et al., 2015). In addition, a model of seizures in Xenopus tadpoles has been developed. Addition of the convulsants pentylenetetrazole (PTZ), kainic acid, bicuculline, picrotoxin, 4-aminopyridine, or pilocarpine to the bath solution results in abnormal motor activity in tadpoles (e.g., tail bending, rapid circling, chaotic movements), followed by a period of decreased activity. This behavioral seizure activity is correlated with field potential oscillations and neuronal calcium spiking recorded in vivo (Bell et al., 2011; Hewapathirane et al., 2008). The effects of antiseizure medications can be easily tested in this model through their addition to the bath. Unfortunately, given the limited use of the tadpole model of seizures to date, a full assessment of its advantages, disadvantages, and applicability to human epilepsy is not possible.
INSIGHT INTO HUMAN DISORDERS Although Xenopus oocytes do not model human epilepsy per se, the ability to utilize this system to characterize ion channels and transporters associated with the pathophysiology of human epilepsy has provided crucial insight into this disorder. Through the use of Xenopus, substantial understanding has been achieved of the pharmacology and physiology of proteins responsible for the control of neuronal excitability, as well as how these properties are deranged by genetic mutations identified in human epilepsy. Importantly, the ability to screen rapidly potential therapeutic agents in oocytes for their effects on mutated channels identified in individual patients provides a pathway toward a “personalized medicine” approach to treating epilepsy.
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