Neurotoxicology. 2016 Dec;57:270-281. doi: 10.1016/j.neuro.2016.10.007.

In vitro evaluation of pyrethroid-mediated changes on neuronal burst parameters using microelectrode arrays.

Mohana Krishnan Baskara, *, Prakhya Balakrishna Murthya, 1

aDepartment of Toxicology, International Institute of Biotechnology and Toxicology (IIBAT), Padappai – 601301, Tamil Nadu, India.

*Corresponding author. Department of Toxicology, International Institute of Biotechnology and Toxicology (IIBAT), Padappai–601301, Tamil Nadu, India. E-mail address: baskarmohanakrishnan@gmail.com

1Retired since October 2013; E-mail address: prakhya@yahoo.com

 

Abstract

Effects of pyrethroids (beta-cyfluthrin, bifenthrin, cypermethrin, deltamethrin, lambda-cyhalothrin, and permethrin) on the burst parameters (mean burst rate [MBR], percent spikes in burst [PSB], mean burst duration [MBD], mean spikes in burst [MSB], mean interspike interval in burst [MISIB], and mean interburst interval [MIBI]) have been investigated using the microelectrode array technique. Rat cortical neuronal networks (between 24 and 35 DIV) were exposed to the five accumulative concentrations of pyrethroids (0.01 µM, 0.1 µM, 1 µM, 10 µM, and 100 µM) after initially recording the baseline activity. When compared to the baseline, the burst parameter that had undergone the most change (either increase/decrease) at the initial concentrations was MBR, followed by MIBI and PSB. The other burst parameters (MSB, MBD, and MISIB) did not undergo much change (either increase/decrease) by the pyrethroids at the initial concentrations when compared to the baseline. The MBR of all pyrethroids rose at initial concentrations followed by decrease at higher concentrations. A drop in the MIBI accompanied the rise in the MBR. The rank orders of relative potency of pyrethroids on the IC50s of different burst parameters clearly distinguish type-1 pyrethroids (bifenthrin, permethrin) from the type-2 pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, lambda-cyhalothrin), with type-2 being more potent. The rank order of relative potency of pyrethroids based on the IC50 of MBR was beta-cyfluthrin > lambda-cyhalothrin > deltamethrin > cypermethrin > bifenthrin > permethrin.

 

Supplement:

Characterizing the neurotoxic effects using animal models remains as the trend in regulatory toxicity assessment. While there are a significant number of chemicals for which the neurotoxicity data is not available, the time consuming and expensive in vivo tests allow only less number of compounds to be tested with less mechanistic insights (Coecke et al., 2006). Hence, there is a need for the in vitro alternative methods that are more rapid, and provide a better mechanistic data for predicting the neurotoxicity potential of a compound. The in vitro alternative methods may also help in the providing information for the subsequent in vivo studies determining the neurotoxicity potential of a compound (Bal-Price et al., 2008).  On the other hand, the organization and functioning of the nervous system is very complex to be reproduced in an in vitro model. No single in vitro model captures the entire diverse mechanisms behind the effects caused by the chemicals affecting the central or peripheral nervous system (Bal-Price et al., 2010; Llorens et al., 2012). Hence, the in vitro neurotoxicity testing should involve a combination of in vitro tests utilizing various models to improve the predictability of the neurotoxicity potential of a chemical (Bal-Price et al., 2008).  While various in vitro models are available for neurotoxicity testing, the regulatory authorities have validated none of them as of today. Among the in vitro models for neurotoxicity assessment, one of the promising models is the microelectrode array (MEA) technique, which measures the functional activity of the neuronal network (Johnstone et al., 2010; Defranchi et al., 2011; Novellino et al., 2011).

MEA captures the spike trains of the neurons, which is the basic physiological function of the neurons. Neuronal spike train has two distinct features: individual spikes (action potentials) and bursts of spikes (high frequency of action potentials). Both spikes and bursts code the behavior of an organism. However, bursts are more significant in representing the behavior of an organism. This is because, when compared to the individual spikes, the information transmitted by bursts is more reliable, and bursts enhance the signal-to-noise ratio in the information transmission. However, spike rate can predict the behavior of an organism better than the bursts sometimes.  Taken together, both spikes and bursts are important in coding the behavior of an organism.

Generally, MEA studies screening the acute neurotoxicity of a chemical determine the changes in the spike rate; but determination of changes in the burst parameters is not standard. Defranchi et al. (2011) had noted that the analysis of burst parameters did not give any particular advantage for acute neurotoxicity assessment. In a recent study, Vassallo et al. (2017) did not find the burst parameters to be useful in screening the neurotoxicants.  However, certain MEA studies had indicated that the burst parameters bring out the differences between the groups of chemicals and may help to better define the chemical toxicity (Mack et al., 2014; Alloisio et al., 2015). Thus, the importance of the burst parameters in acute in vitro neurotoxicity screening and its relevance to the in vivo data is not clear and not yet determined. Hence, in our study we have made a preliminary approach in investigating the changes caused by the known neurotoxic compound pyrethroid, on the neuronal burst parameters. Primary cortical neuronal cultures (between 24 and 35 days in vitro) grown on the MEA chips were exposed to the five accumulative concentrations of pyrethroids (0.01, 0.1, 1, 10, and 100 µM) after recording the baseline activity. The burst parameters viz., mean burst rate (MBR, bursts per min), percent spikes in burst (PSB), mean burst duration (MBD, in sec), mean spikes in burst (MSB), mean interspike interval in burst (MISIB, in sec), and mean interburst interval (MIBI, in sec) were investigated. The definition of a burst is as previously described (Robinette et al., 2011). Burst is a set of at least four spikes occurring for a minimum duration of 0.02 sec. The bursts are separated from each other with a minimum duration of 0.1 sec. The maximum interval to start and end a burst is 0.01 and 0.075 sec, respectively. Bifenthrin and permethrin used in this study are type-1 pyrethroids, whereas beta-cyfluthrin, cypermethrin, deltamethrin and lambda-cyhalothrin are type-2 pyrethroids.

 

IC50s of the pyrethroids on the burst parameters, their relative potencies, and rank order of relative potencies

The IC50s (response corresponding to 50% of the baseline value) of the pyrethroids on the burst parameters, and the relative potencies of IC50s (calculated by having deltamethrin as the index chemical) are represented in Table 1. Rank order of relative potencies (RP) of pyrethroids on the IC50s of different burst parameters are as follows: for MBR, the rank order is, beta-cyfluthrin > lambda-cyhalothrin > deltamethrin > cypermethrin > bifenthrin > permethrin; for PSB, the rank order is, beta-cyfluthrin > cypermethrin > lambda-cyhalothrin > deltamethrin > permethrin > bifenthrin; for MBD, MISIB, and MIBI, the rank order is, lambda-cyhalothrin > deltamethrin > beta-cyfluthrin > cypermethrin > bifenthrin > permethrin; and for MSB, the rank order is, lambda-cyhalothrin > deltamethrin > beta-cyfluthrin > cypermethrin > permethrin > bifenthrin.

The rank order of relative potencies for the burst parameters based on the IC50 values clearly distinguishes type-1 pyrethroid from the type-2 pyrethroid, with type-2 being more potent.

 

Table 1. IC50s of pyrethroids on burst parameters and their relative potencies (RP)

RP of a pyrethroid on the IC50 of the burst parameter = IC50 of the index chemical (deltamethrin)/IC50 of the respective pyrethroid.

 

 

Pyrethroid-mediated changes in the burst parameters

All the burst parameters underwent concentration-dependent changes by the pyrethroids.

Among the burst parameters analyzed, the parameters that had undergone much change (either increase/decrease) at the initial concentrations were MBR, MIBI, and the PSB. MBR of all the pyrethroids increased initially at lower concentrations, and thereafter decreased at higher concentrations. The increase in the MBR was higher for type-1 pyrethroids than for the type-2 pyrethroids (though the MBR was higher for lambda-cyhalothrin than the bifenthrin at 0.1 µM, when averaged among the concentrations 0.01 and 0.1 µM, the MBR was higher for bifenthrin than the Lambda-cyhalothrin). Moreover, an increase in the MBR was observed up to 1 µM for permethrin and bifenthrin (both are type-1 pyrethroids), whereas no type-2 pyrethroids showed an increase in the MBR beyond 0.1 µM. This may be due to the difference in the sodium channel holding time between the type-1 and type-2 pyrethroids. Type-2 pyrethroids keep the sodium channels opened for longer time, causing depolarization and conduction block, whereas type-1 pyrethroids hold the sodium channels opened for a relatively short time, causing repetitive firing (Ray and Fry, 2006). This also points out the difference in the potencies of the tested pyrethroids. The type-1 pyrethroid permethrin, being less potent among the pyrethroids tested, keeps up the MBR for longer time, followed by the next type-1 pyrethroid, bifenthrin. All type-2 pyrethroids, which are more potent than the type-1 pyrethroids, inhibit the MBR earlier (Fig. 1).

The drop in the MIBI accompanies the pyrethroid-mediated increase in the MBR at lower concentrations. All pyrethroids (except cypermethrin) decreased MIBI, which might have contributed to the initial increase in the MBR. Apart from MBR, MIBI, and PSB, the other burst parameters, MSB, MBD, and MISIB did not undergo much change (either increase/decrease) in the initial lower concentrations (Fig. 1).

 

 

Fig. 1.  Pyrethroid-mediated changes in the burst parameters. Percent changes in the burst parameters with respect to the baseline (regarded as 0%) were represented. Values are expressed as mean±SEM.  Complete drop in the burst parameter is at ˗100 in the Y-axis. n = 4 for beta-cyfluthrin, and n = 3 for rest of the pyrethroids.

 

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