Improving a Microtransplantation Assay Based on Neurolemma-Injected

Chapter 4

Downloaded by UNIV OF FLORIDA on November 30, 2017 | http://pubs.acs.org Publication Date (Web): October 24, 2017 | doi: 10.1021/bk-2017-1264.ch004

Realizing the Potential: Improving a Microtransplantation Assay Based on Neurolemma-Injected Xenopus Oocytes An Ex Vivo Approach To Study Ion Channels in Their Native State Steven B. Symington,1 Edwin Murenzi,2 Abigail C. Toltin,1 David Lansky,3 and J. Marshall Clark*,2,4 1Department

of Biology and Biomedical Sciences, Salve Regina University, Newport, Rhode Island 02850, United States 2Molecular and Cellular Biology Program, University of Massachusetts, Amherst, Massachusetts 01003, United States 3Precision Bioassay Inc., Burlington, Vermont 05401, United States 4Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, United States *E-mail: [email protected].

Microtransplantation of rat brain neurolemma into the plasma membrane of Xenopus laevis oocytes is an ex vivo method used to study channels and receptors in their native state using standard electrophysiological approaches. Here we show that oocytes injected with adult rat brain neurolemma elicited ion currents upon membrane depolarization, which were increased by DDT and pyrethroid insecticides. Neurolemma incorporation and oocyte health varied, however, limiting the usefulness of the assay. A collection of changes to the assay procedure, data acceptance criteria, and analysis method yield substantially improved precision and hence, assay performance.

© 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Microtransplantation of Rat Brain Tissue into Oocytes To Examine Native Ion Channels Traditional electrophysiological approaches are powerful tools to examine the adverse effects of both natural toxins and synthetic toxicants on ion channels. Approaches used to study various ion channels include external cell recordings, whole cell patch clamp and heterologous expression of cloned channels, such as cRNAs injected into Xenopus oocytes (1–3). The speed of electronic data collection matches that of the gating processes of endogenous ion channels and allows a direct assessment of the effects of drugs and neurotoxicants on physiologically germane channel kinetic events. Nevertheless, these techniques are usually not amenable to high throughput screening and the physiological relevance of cRNA-injected oocytes has been questioned. For example, cRNA based approaches do not usually include multiple isoforms, variation in the level of expression, variations in channel activity due to axillary regulatory proteins, exon splicing or post-translational modifications, all of which occur in vivo. Alternatively, functional biochemical data collected from nervous system tissues from in situ preparations, such as tissue slices and isolated presynaptic nerve terminals (synaptosomes) are easily obtained from the mammalian CNS and retain a variety of functional attributes for study, such as ion flux, membrane depolarization and neurotransmitter release. However, these systems utilize non-physiological means to evoke depolarizing conditions and data collection occurs over time intervals in excess of those occurring in the intact nervous system (4). The model system evaluated here combines the strengths of both electrophysiological and biochemical techniques and reduces or eliminates many of the limitations mentioned above. This technique, pioneered by Ricardo Miledi, supports incorporation of neuronal tissue or cell lines expressing ion channels from a variety of brain regions and species, including humans (Figure 1) into Xenopus oocytes (5–12). The incorporation of intact lipid rafts from rat brain (neurolemma fragments), which are integrated into the plasma membrane of Xenopus oocytes, yields functional native voltage-sensitive sodium channels (VSSC) and auxiliary regulatory proteins in what is arguably a more physiologically-relevant ex vivo system to evaluate the effects of neurotoxic compounds, such as DDT and pyrethroids (13). This approach allows intact lipid membrane fragments along with associated target proteins to be microtransplanted into and function in the plasma membrane of the oocyte (Figure 1). The advantages of such an approach are that: a) receptors are in their native state within a lipid environment that mimics that found in the intact brain; b) a wide variety of voltage- and ligand-gated ion channels, transporters, and pumps are simultaneously available for study; c) rapid assessment of currents post injection (1-2 hours post-injection) is practical; d) small amounts of tissue are required; e) the procedure works with fresh or frozen tissue; f) the method can be used to the study differences among species, ages or tissue/organ location; g) selective currents can be separated pharmacologically and/or electrophysiologically and h) the method is amenable 54 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


to rapid data acquisition using two electrode voltage clamp (TEVC) techniques in a high-throughput format (e.g., Roboocyte2® system) (14).

Figure 1. Overview of experimental procedure used to analyze the effects of insecticides on rat brain neurolemma tissue microtransplanted into Xenopus laevis oocytes. (A) Roboinject (B) Robocyte with perfusion system (14).

Disadvantages include that: a) the approach is relatively uncharacterized; b) complex currents can result and; c) there is variability in the level of and orientation of the neurolemma incorporated into the oocyte plasma membrane. Results published on the incorporation of acetylcholine receptors from Torpedo marmorata electric organs showed variability in the amplitude of responses on an oocyte-to-oocyte basis as well the magnitude of the effect in the oocytes expressing the necessary proteins (8–15). Such differences could be due to variation among neurolemma preparations, variation in oocyte age or health associated with parents, variation in injection technique, neurolemma orientation, or any combination of these. 55 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


In the research presented herein, we evaluate the utility of using Xenopus oocytes, which are injected with rat brain neurolemma, to characterize the action of three well-understood neurotoxic insecticides, DDT and two pyrethroids: deltamethrin and permethrin; all three share a common mode of action on mammalian VSSC. Demonstrating that these insecticides function at VSSCs in this assay system would support use of this assay system as a useful ex vivo approach. This paper describes the improvements made to the assay methods, such as assay protocols for improving oocyte health, the development of criteria for the selection of neurolemma-injected oocytes, and data normalization procedures that, in combination, substantially reduce the variability in this model system. With the modifications described, we demonstrate that this assay system is a useful ex vivo approach to characterize the mode/site of action of a variety of neuroactive chemicals with the sensitivity, reproducibility and precision necessary for regulatory toxicological evaluations.

Initial Neurolemma-Injected Oocyte Assay Isolating and Estimating a TTX-Sensitive Inward Current In previously published research (16), we investigated the utility of using Xenopus laevis oocytes injected with 90 day old (PND90) adult rat brain neurolemma as a toxicologically-relevant, high-throughput, electrophysiological approach for studying the action of neurotoxicants on native ion channels in an ex vivo assay (Figure 1). The resulting current following neurolemma microtransplantation and oocyte depolarization was a complex outward current (Figure 2A), a component of which was sensitive to tetrodotoxin (TTX), a specific blocker of many VSSCs (Figure 2A). This TTX-sensitive component of the outward current was abolished by replacing sodium chloride with choline chloride in the recording buffer (16). Additionally, with calcium-activated chloride channels blocked by niflumic acid (NFA), a TTX-sensitive ‘inward’ post-depolarizatoin current of approximately 200 nA was measurable and spontaneously inactivated over approximately 50 ms. Thus, the TTX-sensitive inward current evoked upon depolarization appeared to undergo channel activation and inactivation in a manner similar if not identical to that seen during heterologous expression of VSSC/beta subunit cRNAs following co-injection into oocytes (17–22). To generate the concentration-dependent response curves (CDRCs), rows of each assay plate were assigned to concentrations of insecticide with one neurolemma-injected oocytes in each well on a 96-well micotiter assay plate. The concentrations used ranged from 1x10-9 M to 1x10-6 M. Concentrations were assigned to rows as follows: Row B = 1x10-9 M, Row C = 5x10-9 M, Rox D = 1x10-8 M, Row E = 5x10-8 M, Row F = 1x10-7 M, Row G =5x10-7 M, Row H = 1x10-6 M. Each oocyte was exposed to insecticide for 5 minutes by perfusing them at a flowrate of 0.45 ml/min with using a roboflow perfusion system (Figure 2B). 56 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. Insecticide induced TTX-sensitive inward currents in rat brain neurolemma tissue microtransplanted into Xenopus laevis oocytes in the presence of NFA. (A) TTX-sensitive inward currents (right panel) are generated from the difference between the treatment trace and TTX-insensitive trace (left panel). (B) Sample plate setup for microtransplantation assay. Insecticides concentrations (1x10-9-1x10-6 M) were perfused using a roboflow perfusion system for 5 minutes at a flowrate of 0.45ml/min. Pyrethroids concentrations were administrered in rows B-H of the 96 wellplate each pyrethroid perfusion varied between asssay plates.

In order to determine the effects of the test insecticides (DDT, permethrin or deltamethrin) on TTX-sensitive inward current, neurolemma-injected oocytes were analyzed in the presence of NFA (16). TTX-sensitive inward currents were determined by subtracting the experimental traces in the presence of TTX from the individual (oocyte-specfic) total current, both in the presence of NFA (Figure 2A). TTX-sensitive area under the curve values (AUC, nA x 50 ms) were determined for individual inward current traces from each oocyte. Pseudo-replicate oocytes at 57 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


each concentration of pesticide in each row of the assay plate (Figure 2B) yielded a TTX-sensistive AUC value, which were then (for the initial calculation method) averaged and then normalized using the mean of the same-plate NFA-treated controls via:

The data used to produce a CDRC were calculated from the mean AUC +/- S.E. values from 3 or more different biological replicates, each consisting of neurolemma from a single cohort of adults rats paired with a preparation of oocytes. CDRC data were fitted to a sigmoidal concentration-dependent response curve (3 parameter logistic equation) using GraphPad Prism ver 6.0. The asymptote of maximum response, the curve shape (or slope), and EC50 values were estimated.

Confirmation of Expected Perfomance Effect of DDT on TTX-Sensitive Inward Current It is well established that DDT acts on VSSCs, slowing the processes of channel inactivation and deactivation (17, 18). This action gives rise to increased sodium ion influx, leading to neuronal membrane depolarizing and “negative after potentials”, which ultimately are associated with repetitive discharges in the nerve axon and a likely cause of the “DDT jitter” syndrome typical of DDT poisoning. This action is similar, if not identical, to the tremor or T-syndrome produced by Type I pyrethroids, such as permethrin (19–23). With the above in mind, we investigated the action of DDT on neurolemmainjected oocytes (16). The rationale for this approach was that if the action of DDT on the TTX-sensitive inward current measured from neurolemma-injected oocytes mimicked the action of DDT on heterologous expressed VSSCs, it would provide support of the toxicological relevancy of this ex vivo approach. Using the method described above, many of the issues/limitations of previously used approaches (e.g., heterologous expressed channels, non-neural tissues, biochemical assays) are reduced or eliminated, supporting more precise comparisons of the neurotoxicity of various chemicals under various conditions (i.e.; animal age). Using the microtransplantation approach to obtain CDRCs, DDT increased TTX-sensitive inward currents in a concentration-dependent manner by increasing Na+ influx during depolarization (Figure 3A and B) primarily by slowing the inactivation kinetics of VSSC (Figure 3C). The non-toxic DDT metabolite, DDE, however, resulted in no observable concentration-dependent response (16). These results indicate a clear structure-activity relationship for DDT versus DDE on the VSSC current that has been pharmacologically isolated with TTX and in the presence of NFA using rat brain neurolemma microtransplanted oocytes.

58 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. DDT increases TTX-sensitive current in rat brain neurolemma injected oocytes. (A) Neurolemma-injected oocytes in the presence of NFA and increasing concentrations of DDT. TTX-sensitive area-under the curve (AUC) values (nA x 50 ms) were determined from individual inward current traces during depolarization. (B) Concentration-dependent response curve illustrating the effect of DDT. Percent over control values were determined as follows: ((Treatment TTX-sensitive AUC – No treatment TTX-sensitive AUC)/No treatment TTX-sensitive AUC) x 100). (C) Inactivation tau values are increased in the presence of 10-6 M DDT. Adapted with permission from Murenzi et al., 2017 (16). The inactivation tau 0.5 (t0.5) values can be estimated from the current traces given in Figure 1C as 2.6 ms for untreated cells and 5.9 ms for DDT-treated cells, a 1.3-fold increase. In the present work, we determined the inactivation tau0.5 value in the presence of 1 µM DDT as 6.7 ms, a 1.5-fold increase compared to the 4.4 ms control value. These results are similar to those reported by Song et al. (24, 25), who report that 10 µM DDT slowed the inactivation kinetic of TTX-sensitive VSSCs in rat dorsal root ganglion cells following a 20 ms step depolarization. We conclude that this ex vivo assay is a toxicologically-relevant approach to examine native receptors in their endogenous states.

Effect of Pyrethroids on TTX-Sensitive Inward Current It is well also well established that pyrethroids modify VSSCs by slowing the inactivation and deactivation channel kinetic processes (26–28). This action also gives rise to increase sodium ion influx, leading to neuronal membrane depolarization, nervous system excitation, convulsions and death. The addition of increasing concentrations of permethrin (Figure 4A) or deltamethrin (Figure 4D) from 10-9 to 10-6 M resulted in an increase of TTX-sensitive inward current on VSSCs microtransplanted into Xenopus oocytes. Calculation of AUC values at each concentration and their normalization to percent over control values for each biological replicate using (1) allowed composite CDRCs to be similarly generated. Increasing concentrations of either permethrin (Figure 4B) or deltamethrin (Figure 4E) progressively increased the percent over control values. The increases in the percent over control values 59 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


were primarily due to the prolongation of inactivation process as seen at the 10-6 M concentration (Figure 4C for permethrin; Figure 4F for deltamethrin). These results illustrate that the pyrethroid effect on late current is similar to results previously obtained from other heterologous expression systems, supporting the notion that this ex vivo preparation is a toxicologically-relevant approach to examine native receptors in their endogenous states (13).

Figure 4. Effects of increasing concentrations of pyrethroids on depolarization-evoked, TTX-sensitive inward currents associated with rat brain neurolemma microtransplanted into Xenopus oocytes in the presence of NFA, a Ca2+-activated chloride channel blocker. A) Electrophysiological TTX-sensitive current traces illustrating the effects of increasing concentrations of permethrin. (B) Concentration-dependent response curve (CDRC) illustrating the effect of permethrin. (C) Effect of 10-6 M permethrin on the inactivation tau value. D) Electrophysiological TTX-sensitive current traces illustrating the effects of increasing concentrations of deltamethrin. (E) CDRC illustrating the effect of deltamethrin. (F) Effect of 10-6 M deltamethrin on the inactivation tau value. TTX-sensitive inward currents were determined by subtracting the experimental traces in the presence of TTX from the total current. Percent over control values were determined using equation 1. Inactivation tau values were obtained by fitting the late current traces to an exponential decay equation during inactivation using Origin (Ver 8.6, Origin Lab, Northampton, MA). An asterisk (*) indicates that the sample mean is significantly different from the mean of control treated (NFA) oocytes (one-sample Student’s t-test, 10-6 M: p

Improving a Microtransplantation Assay Based on Neurolemma-Injected

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