In the past decades, a series of medical and technical advances have allowed researchers not only to localize areas involved in specific functions but also to describe the mechanisms underlying these functions. In this context, the development of technologies will bring neuroscience a golden age. Various assays have become unignorable in neuroscience.
Calcium ions (Ca2+) serve as a ubiquitous second messenger within cells across all domains of life, modulating a wide array of cellular processes. Hundreds of proteins have evolved to modulate intracellular calcium levels and to transduce changes in calcium concentrations into downstream cellular signals. Monitoring intracellular calcium levels is a valuable technique for modern drug discovery, allowing functional measurements of target proteins and calcium-mediated physiological pathways.
Fig.1 Ca2+- and light-gated transcriptional reporters of activated neurons. (Wang, 2019)
The developing brain is uniquely sensitive to environmental insults as it is critically dependent on precisely orchestrated developmental processes that must occur in a particular sequence at the right time and location. The prevalence of diagnosed neurodevelopmental disorders is increasing worldwide and fuels public concern regarding the lack of developmental neurotoxicity (DNT) data for many chemicals. Among the thousands of known chemicals, there is only a small fraction proven to cause DNT in humans. The paucity of DNT data is partial since DNT evaluation is not a mandatory requirement unless triggered by evidence of developmental toxicity involving the nervous system, or neurotoxicity, or endocrine disruption in systemic toxicity studies in adult rodents. Neurotoxicity screening is thus unignored in neuro-drug development.
Fig.2 Example plate layout for screening chemicals. (Shafer, 2019)
High content screening (HCS) has gained tremendous popularity in the past few years in the drug discovery industry from advances in fluorescence microscopy and automated screening technologies. The unique architecture of post-mitotic neurons with their elaborate dendritic trees and far-reaching axons imposes extraordinary demands on the mitochondrial system for satisfying the neuron's need for energy, calcium buffering, neurotransmitter metabolism, and other physiological processes. HCS provides an opportunity to rapidly screen chemical or siRNA libraries by imaging subcellular and molecular events of individual cells with an automated fluorescent microscope. HCS is an assay platform capable of multiplexed, high-throughput, and phenotypic screening of small molecules for modulatory effects on neuronal metastasis.
The direct method of immunostaining allowed a single antibody, which is conjugated to an enzyme, to interact with an antigen present on the cell of interest. A substrate is then added, and after a reaction mediated by the conjugated enzyme, the substrate will fluoresce or form an insoluble color product deposited near the antibody. The indirect, or amplifying, technique has proven to be more efficient than the direct technique, particularly in the setting of frozen section tissue examination.
Substrate-integrated MEAs allow simultaneous extracellular recording of electrical activity from many individual sites in electrically active tissues. MEA systems offer a great deal of flexibility regarding biological tissue and experimental design. A wide variety of electrically excitable biological tissues may be placed onto the MEA. This includes primary cultures of nervous system tissue from many different regions, i.e. mouse frontal cortex cells, tissue slices (e.g. hippocampal slice), or retinas.
Ion channels are a very important target class because of their involvement in many physiological processes. Historically, ion channel targeted drug discovery has been hampered by the unavailability of high-throughput platforms utilizing electrophysiological techniques for the characterization of compound activity. Two major approaches have formed the basis for cell-based ion channel screening: the highest throughput optical assays representing indirect measures of ion channel function, and the introduction of higher-throughput electrophysiological methodologies. In combination with non-electrophysiological but higher throughput methods, the implementation of these technologies provides a highly integrated approach for ion channel targeted drug discovery and will likely have a significant impact on the discovery of new ion channel targeted therapeutics.
A central feature of the nervous system is the presence of an enormous diversity of neurons with different morphologies, molecular profiles, and physiological properties. Neurons are densely packed in the brain, and their intermingled dendritic and axonal processes preclude the visualization of their morphologies. Thus, it often requires a sparse labeling method that randomly labels a small subset of neurons to highlighting their dendritic and axonal processes.
A central challenge in systems neuroscience is linking animal behavior to underlying neuronal circuitry. Only a small percentage of neurons are activated during a specific task or sensory experience, and these can be embedded within a large population of nonactivated neurons that have similar morphology and genetic features. Once these neurons are identified, their functional and molecular properties can provide insight into how the brain performs computation, promotes or inhibits actions, or encodes sensory stimuli. Monitoring neuronal activity will give a chance to reveal the mechanism underlying these challenges.
Phenotypic omics is broadly defined as the acquisition of organism-wide high-dimensional phenotypic data, which will be a natural evolution and supplementation of existing molecular omics paradigms in drug discovery. Phenomic-level data can help to understand genomic variants underlying phenotypes, pleiotropy of responses to pharmaceuticals as well as provide new high-throughput and content-rich screening paradigms in drug discovery.
Fig.3 Phenotypic drug discovery compound screening and validation. (Moffat, 2017)
Focusing on neuroscience over years, Creative Biolabs has established a great industry reputation. Our technology platform has integrated a variety of basic neuroscience assays. With rich experience and excellent expert teams, we are capable of providing mature neuroscience assay services to global customers. If you are interested in any one of the neuroscience assay services on our website or focusing on other content of neuroscience, please don't hesitate to contact us for more information.
Wang, W.; et al. Molecular tools for imaging and recording neuronal activity. Nat Chem Biol. 2019, 15(2): 101-110.
Shafer, T. J. Application of Microelectrode Array Approaches to Neurotoxicity Testing and Screening. Adv Neurobiol. 2019, 22: 275-297.
Moffat, J. G.; et al. Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nat Rev Drug Discov. 2017, 16(8): 531-543.