Metrion offers a range of peripheral and central nervous system (CNS) neuronal assays utilising native rodent tissue and human iPSC-derived neurons.
Our neuroscience assays are used for two main drug discovery purposes: Confirmation of compound effects in native neuronal systems and Neurotoxicity Screening.
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Introduction to Metrion’s Neuroscience services
We offer a range of peripheral and central nervous system (CNS) neuronal assays utilising native rodent tissue and human iPSC-derived neurons.
Metrion’s scientists offer proven expertise in neuroscience drug discovery and electrophysiology, native neuron cell culture, and use of iPSC cell reagents for:
- Pharmacology screening
- Neurotoxicity testing
- Disease modelling
Screening of client compounds in a neuroscience setting is primarily driven by the fact that they have been designed to modulate CNS or peripheral nervous system targets and signalling pathways, including but not limited to ion channels. Metrion can carry out this screening using heterologous cell lines expressing human or rodent neuronal proteins, as well as offering more integrative assays employing rodent and human neuroblastoma cell lines or in acutely cultured native cells from the rodent peripheral and central nervous system.
A second reason to access Metrion’s neuroscience capabilities is for selectivity and safety profiling of lead compounds and IND candidates, either as part of an existing screening cascade or because in vivo testing revealed a neurological signal. Metrion have developed and validated an industry-standard rat cortical CNS neuron assay that can reliably detect the seizure-inducing liability of a wide range of reference compounds (as well as serve as a model to profile anti-epileptic drugs). We also offer have deep expertise with rodent dorsal root ganglion (DRG) sensory neurons which can be used to assess peripheral neurotoxicity.
Neuroscience screening and toxicity testing reaches the ultimate translational step when it is carried out in human iPSC-derived neurons, and Metrion is actively involved in designing and validating such assays for our clients. The use of differentiated human stem cell-derived neurons also allows the creation of patient-derived and disease-specific translational assays, opening the door onto one of the more exciting aspects of modern drug discovery.
We offer a range of neuroscience screening assays of increasing complexity and translational relevance. Firstly, compounds can be tested against specific neuronal ion channel targets expressed in heterologous cell lines using manual and automated patch clamp electrophysiology. Metrion have a panel of peripheral and CNS ion channel cell lines, and can create custom reagents for new targets, human channelopathy mutations, and species orthologues. Secondly, compounds can be tested against endogenous neuronal targets expressed in more physiological rodent and human neuroblastoma cell lines derived from peripheral and CNS origin. Finally, compounds with known or unknown mechanism can be validated by exploring their effects on the function of native rodent and human iPSC-derived neurons.
Neuronal Ion Channel Assays
Metrion’s talented team of neuroscientists and ion channel electrophysiologists have created a variety of ion channel cell lines and assays relevant to neuroscience drug discovery, for both peripheral and central nervous system targets and diseases.
A neuronal Na+ ion channel used as the primary counter-screening target in a major pharma drug discovery collaboration is notoriously difficult to express in heterologous cells, and under standard conditions the original cell line yielded a low success rate assay (Figure 1a).
We tested a number of different cell culture, cell biology and experimental conditions (Figure 1c) to develop an optimised assay on the Patchliner (Nanion) automated patch clamp platform. Our efforts significantly improved the low expression seen in the original reagent without affecting ‘patchability’, yielding a highly efficient gene family selectivity assay (Figure 1b).
CNS neuron ion channels
Metrion scientists have worked on a wide range of CNS ion channel targets including ligand-gated GluR, nAChR, P2XR and GABA-A receptors and voltage-gated Na+, K+ and Ca2+ channels. In many cases, this work is conducted using heterologous cell lines on manual or automated patch clamp platforms, and we can also offer manual patch and MEA recordings from native rodent CNS neurons.
In this example we show human GluR receptor currents recorded by manual patch (A) and on the automated QPatch platform from Sophion (B). These assays can establish EC50 and IC50 concentrations of agonists and antagonists, and investigate the action of negative and positive allosteric modulators.
Our recent Application Note illustrates another example of an CNS ion channel assay, describing the optimisation and pharmacological validation of a QPatch automated patch clamp assay for the ASIC1a ligand-gated receptor which is implicated in stroke and ischemia.
Neuroblastoma cell lines
Immortalised rodent and human neuroblastoma cell lines provide a useful alternative to native peripheral neurons. They endogenously express many relevant membrane receptors and channels, but remove the need for animal sacrifice, thereby meeting 3Rs objectives. The use of rodent or human neuroblastoma cell lines also offer an opportunity to address translational challenges. They do this by confirming compound efficacy in preclinical species and native human neuronal systems.
Metrion scientists have extensive experience using neuroblastoma cell lines for electrophysiological study of native and expressed ion channels (here). In this study we established the profile of voltage-gated Na+ channels endogenously expressed in rodent ND7-23 neuroblastoma cells using the QPatch automated patch clamp platform.
Native neuron assays
DRG sensory neuron ion channel assays
Peripheral sensory neurons from the DRG are a workhouse of neuroscience drug discovery, and Metrion scientists have proven expertise in using this preparation to create assays to study voltage- and ligand-gated ion channels using manual patch clamp and multi-electrode array (MEA) biophysical techniques.
DRG neurons express a wide variety of voltage-gated ion channels that modulate sensory neuron propagation of peripheral stimuli into the spinal cord across the so-called pain ‘gatekeeper’ synapse in the dorsal horn. Sodium and potassium channels control axonal excitability whilst Ca2+ channels control the release of neurotransmitter across the synaptic cleft, making them all attractive targets for pain drug discovery.
Here we show a recording of Ca2+ currents in single rodent DRG neurons exposed to a test compound. This data is from an 8 year drug discovery collaboration with a global pharma company client that successfully delivered novel chemical matter and an IND candidate.
As nervous system side-effects continue to be one of the major causes of drug attrition, it is critical that new drug candidates are tested in predictive neurotoxicity assays.
Neurotoxicity screening can be carried out efficiently and reliably in plate-based assays such as the multi-electrode array (MEA) using peripheral and CNS neurons from native tissue or human iPSC-derived cells. Spiking activity is closely monitored over extended periods using biophysical techniques able to detect acute and chronic effects of test compounds under physiological conditions.
Multi-electrode array platforms enable
s simultaneous recording of the physiological activity from multiple neuronal cells and complex networks. Extracellular field potentials are recorded in a non-invasive manner to characterise neuronal firing before and after compound addition, detecting changes in single neuron activity as well as higher level network synchronisation. The electrophysiological behaviour of populations of disease modified neurons or cells from different sources can also be compared with relative ease.
CNS Seizure Assay
We have validated an industry-standard rat cortical CNS neuron excitability assay on a MEA platform (here) that can reliably detect inhibitory and excitatory effects of a wide range of agricultural and medical agents. Alongside the FDA’s NeuTox initiative we are also developing translational CNS seizure liability assays using human iPSC-derived CNS neurons.
Rat cortical neurons cultured on MEA plates increase their activity to become more co-ordinated and stable over time to match that of postnatal animals (Fig. 5b), after which time test compounds can be assessed for complex effects on cell firing and network bursting (Fig. 6b).
DRG Peripheral Neurotoxicity Assay
It is also important to test compounds for peripheral neurotoxicity, which, for example, is a debilitating side-effect of many chemotherapy treatments. Dorsal root ganglion (DRG) neurons are the most accessible and efficient source of large numbers of peripheral neurons from both rodents and humans, as well as being a major target of morphological toxicity deficits and functional manifestation of inflammatory and neuropathic pain. Accordingly, we offer a number of different DRG neuron readouts using electrophysiological methods to assay drug effects on peripheral neuron excitability and function.
Translational Neuroscience Assays
One of the major challenges of drug discovery is the successful translation of hits and lead compounds into preclinical animal models and human clinical trials. Traversing this so-called preclinical ’valley of death’ requires the use of reliable and predictive animal and human cell and tissue-based assays. Metrion offers a number of phenotypic neuroscience assay formats to enable its customers to validate the efficacy and mechanisms of their compounds in rodent and human neurons, and help translate them towards preclinical testing and eventually into human clinical trials. Our translational neuroscience assays employ a range of peripheral and CNS neuron cell types and functional readouts, including manual patch clamp and multi-electrode array (MEA) recordings from native tissue and human stem cell-derived iPS neurons, and can be designed to meet your specific translational neuroscience needs.
Our neuronal assays are not limited to testing ion channel modulators, but are also suitable for assessing the efficacy and physiological effects of ligands directed against GPCRs, kinases, enzymes, intracellular signalling and homeostatic pathways. They are well suited to validating drugs designed to treat pain, inflammation, epilepsy and a variety of CNS diseases. At the basic level, these assays can be used to confirm that client compounds previously tested in heterologous cell systems are effective in more intact and physiological rodent and human cells and tissues. Such phenotypic assays include a wide range of additional membrane proteins, scaffolding complexes and intracellular signalling cascades which can affect the efficacy of novel test compounds. Thus, we can confirm that the desired potency and mechanism-of-action are seen in rodent species that are typically used in preclinical animal models, and that there are no major species differences. Similarly, the use of human iPSC-derived neurons can help to confirm the efficacy of test compounds prior to testing in rare human tissue, and give confidence that lead candidates may work in human patients as part of clinical trials.
Central Neuronal Firing Assays
Central neuron phenotypic assay platforms at Metrion include manual patch-clamp and multi-electrode array (MEA) techniques, which can be used to validate compound target efficacy, establish target engagement in native tissue, and explore species selectivity. Access to CNS neurons from different brain regions and developmental stages also allows for a comparison of compound effects on cells with different functional profiles, as well as the potential to test compounds on native and iPSC-derived neurons from different, genetically validated, disease states.
is can be monitored from native CNS neurons (e.g. rodent cortical) by measuring their firing behaviour with manual patch clamp (Fig. 7) and MEA electrophysiology platforms (Fig. 7b), where single cell excitability as well as metascale network bursting can be visualised with heat maps and other sophisticated analysis and visualisation tools (Fig. 8).
Peripheral Neuronal Firing Assays
Metrion utilise rodent and human peripheral neuron phenotypic assays to enable our customers to progress their compounds towards preclinical animal models and eventually onto clinical trials. We use manual patch-clamp and techniques to establish target validation, target engagement and species selectivity assays. Physiological activity can be monitored from native tissue such as rodent dorsal root ganglia (DRG) or human iPSC
DRG neurons typically do not fire spontaneous action potentials unless injured or inflamed, so industry-standard excitability assays measure the response of neurons to graded electrical or chemical stimulation. In this example, we show rodent DRG action potential recordings after increasing current injections, and similar responses are seen in human DRG recordings.
Current Clamp Recording
Current-clamp is an information rich technique which allows recordings from individual neurons to measure changes in membrane potential or firing behaviour in response to compound application or current input. Recordings can be used to compare firing characteristics from different cell types, verify findings from other sources (such as MEA) and to ascertain the mechanism of action (MOA) of compounds.
At Metrion, we are highly experienced at recording from native rodent neurons and are currently developing further translational assays such as recordings from stem cell derived neurons and human dorsal root ganglion neurons (see Fig. 9)
Multi-electrode array (MEA) enables simultaneous recording of the physiological activity in multiple peripheral neuronal cells. Extracellular field potentials are recorded in a non-invasive manner to characterise neuronal firing before and after compound addition. For example, these techniques can be used to examine cells in sensory pathways, such as rodent dorsal root ganglia neurons, and their response to ligand application (example shown). The electrophysiological behaviour of populations of disease modified neurons or cells from different sources can also be compared with relative ease.
Human iPSC-derived neurons
Metrion has growing experience utilising human iPSC-derived neurons in our translational and neurotoxicity assays. We work closely with leading commercial iPSC providers to evaluate and validate their neuronal reagents in our own laboratory, and also collaborate with academics and clients to profile bespoke iPSC neuron assay formats and reagents such as patient-derived neurological disease models.
In this case study, we assessed the functional activity of Peri.4U human peripheral neurons on the MEA platform, validating them pharmacologically with a K+ channel modulator.
Metrion neuroscientists also collaborated with an academic group in Germany as part of a project to determine the role of Na+ channels in the increased excitability of patient-derived human iPSC sensory nociceptors expressing mutations found in rare painful neuropathies (reference).
- Metrion Biosciences: high quality ion channel drug discovery service provider. Milner Therapeutics Symposium, Cambridge, 2019
- Development of Native and Stem Cell-Derived Electrophysiological Assays for Neurotoxicology Screening and Translational Drug Discovery. SPS Berlin 2017 poster 142.
- Profiling endogenous sodium channels in the ND7-23 neuroblastoma cell line: implications for use as a heterologous ion channel expression system and native tissue model suitable for automated patch clamp screening. Royal Society for Chemistry Ion Channel Symposium, March 2016.
- The role of Nav1.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients.
- Recent advances in targeting ion channels to treat chronic pain.
- Voltage-clamp and current-clamp recordings from mammalian DRG neurons.
- Multiple sodium channels and their roles in electrogenesis within dorsal root ganglion neurons.
- The role of sodium channels in neuropathic pain.
- A single sodium channel mutation produces hyper- or hypoexcitability in different types of neurons.
- Marc Rogers (Metrion Director and CSO) outlines the benefits of targeting ion channels for pain and some of the hurdles in developing successful ion channel modulators.
- Marc Rogers (Metrion Director and CSO) takes part in a collaborative webinar with Nanion Technologies entitled “Validation and optimization of automated patch clamp voltage-gated Ca2+ channel assays”.
- QPatch automated electrophysiology
- Patchliner automated electrophysiology
- Conventional manual patch clamp electrophysiology
- Plate-based impedance and multi-electrode array techniques
- FlexStation plate-based imaging
Let’s work together
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