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ADME PK

eCiphr®Neuro

Assessment of Neuronal Activity Using Microelectrode Array

  • The eCiphr®Neuro assay uses primary cultures of rat cortical neurons.
  • Cyprotex’s neuronal assay uses high throughput microelectrode array (MEA) technology to monitor electrophysiological activity.
  • Neurons grown on microelectrode arrays recapitulate many features of neurons in vivo, including spontaneous activity (spiking and bursting), plasticity, organization and responsiveness to a wide range of neurotransmitters and pharmacological agonists/antagonists1.
  • This technology provides a unique in vitro system for preclinical drug discovery, neurotoxicity assessment and disease modeling.
The unique capabilities of MEAs to provide functional measurements of network activity, including spontaneous activity, evoked activity, and responses to pharmacological challenges, therefore offers an advantage over other potential screening approaches that rely on biochemical or structural endpoints.

1Robinette BL et al., (2011) Front Neuroeng 4; 1-9

Protocol

eCiphr®Neuro assay protocol

Cell Type Primary rat cortical neurons
Analysis Platform Maestro 48-well MEA system (Axion BioSystems)
Test Article Concentrations Four concentrations in triplicate (dependent on customer requirements)
Quality Controls Negative control: 0.2% DMSO (vehicle)
Positive controls: picrotoxin and domoic acid (at single concentration)
Data Delivery Firing rate (spikes/second)
Burst rate (bursts/second)
Number of spikes in burst
Percent of isolated spikes
Coefficient of variation (CV) of the inter-spike intervals (ISI)
Burst duration
Normalized IQR (inter-quartile range) burst duration
Interburst interval
Mean ISI-distance (measure of synchrony)
Normalized Median Absolute Deviation (MAD) burst spike number
Median ISI/Mean ISI

Data

Data from Cyprotex's eCiphr®Neuro assay

 
Figure 1
Rat cortical neurons stained with Hoechst 33342 and βIII-tubulin/DyLight® 488 and imaged with an ArrayScan VTi.
Figure 2
Change in spontaneous spike activity in rat cortical neurons after drug treatment.

The spontaneous spike activity is recorded in rat cortical neurons using Axion Biosystems microelectrode array Maestro platform. The spike train data is extracted from baseline and post dose measurements and converted to numerical values using a custom Matlab script to characterize firing and burst organization. The negative control 0.2% DMSO (vehicle) caused no change in activity, burst characteristics or synchrony. A distinct pattern of change affecting spike activity, burst characteristics and synchrony is observed with GABAA antagonists picrotoxin and gabazine. A different but significant pattern of activity can be seen with other proconvulsant toxins such as strychnine, a glycine receptor antagonist. Meanwhile complete abolishment of spike activity is observed with the neurotoxin, domoic acid.

Figure 3
Raster plots of spike activity in five individual electrodes before and after 1 hr treatment with 10µM picrotoxin.

Five representative electrodes out of the 16 electrodes in a well are shown over a 150 sec time span. The recorded spike activity of rat cortical neurons is represented by the raster plots which illustrate the structure of typical baseline spike activity for a well compared to its structure following a 10µM dose with the GABAA antagonist picrotoxin. The qualitative visual differences in the dynamics of the spike train are quantified through computation of the spike train features as seen in Figure 2.

CompoundChemical classNeurological effect in vivoeCiphr®Neuro
0.2% DMSO Vehicle None No effect
Gabazine GABAA antagonist Seizurogenic2 Seizurogenic
Bicuculline GABAA antagonist Seizurogenic2 Seizurogenic
Picrotoxin GABAA antagonist Seizurogenic3 Seizurogenic
Pentylenetetrazole (PTZ) GABAA antagonist Seizurogenic4 Seizurogenic
Tutin GABAA antagonist Seizurogenic5 Seizurogenic
GABA GABAA agonist Decreases neural activity6 Decreased activity
Tetrodotoxin Sodium channel blocker Neurotoxic7 Neurotoxic
Aminopyridine Potassium channel blocker Seizurogenic8 Seizurogenic
Domoic Acid Glutamate signalling Neurotoxic9 Neurotoxic
L-Glutamate Glutamate agonist Increases neural activity10 Increased activity
Strychnine Glycine receptor antagonist Seizurogenic11 Seizurogenic
Acetaminophen NSAID None No effect
Ibuprofen NSAID None No effect
Table 1
Comparison of eCiphr®Neuro data with neurological effects observed in vivo.

A number of compounds with a range of neurological effects were tested in the eCiphr®Neuro assay using rat cortical neurons. A good correlation was seen with drugs tested in this in vitro assay with their known in vivo effects. Different patterns of change affecting spike activity, burst characteristics and synchrony are observed in GABAA antagonists and other proconvulsants as illustrated in Figure 2.

Posters on eCiphr®Neuro Research

References

1 Robinette BL et al, (2011) Front Neuroeng 4; Article 1
2 Margineau DG and Wülfert E (1997) Br J Pharmacol 122; 1146-1150
3 Mackenzie L et al, (2002) Clin Neurophysiol 113(4); 586-596
4 Ono J et al, (1990) Funct Neurol 5(4); 345-352
5 Fuentealba J et al, (2011) Neuropharmacology 60; 453-459
6 Levy LM and Degnan AJ, (2013) Am J Neuroradiol 34(2); 259-265
7 Hwang DF and Noguchi T (2007) Adv Food Nutr Res 52; 141-236
8 Peña F and Tapia R (2000) Neuroscience 101(3); 547-561
9 Pulido OM (2008) Mar Drugs 6(2); 180-219
10 Hankir MK et al, (2012) Neuroimage 59(2); 968-978
11 Kehne JH et al, (1992) Br J Pharmacol 106(4); 910-916

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