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KeywordsPersonality Disorder Weizmann Institute Excitatory Amino Acid Transport Neuronal Excitation Personalized Healthcare
- Omics technology
Technology used in the integration of genomics, proteomics, and metabolomics; high throughput isolation, identification and functional characterization of genes, their protein (peptide) products, and associated biochemical pathways and interactions.
Methods that involve the use of optics, genetics, and DNA recombinant technology to target distinct neuronal populations from brain tissue slices (ex vivo) with genes that produce bacterial opsins. These ion-channel membrane-associated proteins can be stimulated selectively by light and visualized through fluorescent tagging. The essential reagents used in optogenetics are light-sensitive proteins. Neuronal control is achieved using optogenetic enhancers, while optical recording of neuronal activities can be made with the help of optogenetic sensors for calcium, vesicular release, neurotransmitter, or membrane voltage (McElligott 2015; Touriño et al. 2013).
A term coined to describe a process where larger molecules are manufactured to interact with smaller previously unrecognized ones. G protein-coupled receptors can be engineered to respond exclusively to synthetic small molecular ligands and not to their natural ligand(s) that permits spatial and temporal control of G protein signaling in vivo. Neuronal excitation or silencing occurs through the expression of G protein-coupled receptors activated by designer drugs called DREADD’s (Designer Receptor Exclusively Activated by Designer Drugs). For both genetic approaches, selective cellular genetic constructs occur as a result of recombinase dependent opsin/DREADD expression in tandem with neuron-specific recombinase expression recognized by specific cell types that allow control over cells at a subcellular level (McElligot 2015; Stachniak et al. 2014).
The focus of this chapter will be on aspects of the advances in the development and uses of neurotransmitter assays for a better understanding of neurotransmission in the context of translational research. Translational research and its outcomes are being used to bring precision medicine into the mainstream for personalized healthcare, from genetics to cognition/behavior – integration of the “omics” and other technologies. Different types of neurotransmitter assays have been and are key in this process. An informatics approach will be used to bring the reader a cross section of databases and resources that can be used for research and educational purposes in the identification and characterization of molecules, processes, and circuitry associated with synaptic transmission.
Traditionally, in both academic and pharmaceutical research, biological assays have attempted to characterize structural/functional aspects of a synapse during neurotransmission using various animal models and cell culture as they apply to normal and pathological conditions. For example, early bioassays used protocols to study the uptake and release of tritiated substrates using intact cells or synaptosomes. In this way one could investigate drug effects on selective transporter-mediated uptake and release of the tritiated substrate. Parallel experiments used high-performance liquid chromatography (HPLC) procedures for electrochemical detection of non-radiolabeled substrates (Janosky et al. 2001). More recently, studies have utilized electrophysiological measurements of receptors that have looked at a specific receptor class, the endocannabinoids (eCBs). These molecules are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. Protocols were developed for measuring cannabinoid and eCB-mediated synaptic signaling in mouse brain slices, including analysis of short-term, long-term, and tonic eCB signaling modes (Báldi et al. 2016 ). Finally, complementary computation studies have also been undertaken to further elucidate aspects of the general synapse. Extracellular neurotransmitter concentrations can vary over a wide range depending on the type of neurotransmitter and location in the brain. A biophysical modeling framework was proposed, based on a cortico-accumbens. The model was used to identify the role of perisynaptic parameters on neurotransmitter homeostasis and to propose glial configurations that could support different levels of extracellular neurotransmitter concentrations (Pendyam et al. 2012).
Basic Biology: Importance of Classification for Identification and Function of Synaptic Molecules
In order to understand neurotransmitter assays, one needs to understand the basic biology of the synapse and neuronal firing. Whether this is chemical or electrical, it involves the following structures and the regulation and formation of multiple nuclear gene products. Generally, one must keep in mind the pre- and postsynaptic neurons (internal secretory vesicles/organelles), their receptors and the synaptic cleft with the regulated release and uptake of neurotransmitter(s) and associated transporters.
Furthermore, proper classification and identification of synaptic molecules and their annotation is essential in experimental design. Some information on classification and function of synaptic molecules follows including that for neurotransmitters and their specific transporters. Classification of the major neurotransmitter families comprise amines (quaternary, e.g., acetylcholine and mono, e.g., dopamine), amino acids (e.g., glutamate), neuropeptides (opioids), peptides (e.g., oxytocin), and gases (e.g., nitric oxide). Functionally, vesicular neurotransmitter transporters can mediate storage inside secretory vesicles in a process that involves the exchange of lumenal H+ for cytoplasmic transmitter. Retrieval of the neurotransmitter from the synaptic cleft catalyzed by sodium-coupled transporters is critical for the termination of the synaptic actions of the released neurotransmitter (Elbaz et al. 2010).
An example of their general classification system and complex biology can be found for sodium neuro-transporter serotonin symporter, and, in this case, specifically its N terminal domain. Most reference sites referred to in the text provide tutorials on how to navigate their databases, and in this example we provide links to the molecule’s biological domains/function/process and its detailed source description (http://www.ebi.ac.uk/interpro/entry/IPR013086).
“Neurotransmitter transport systems are integral to the release, reuptake, and recycling of neurotransmitters at synapses. High affinity transport proteins found in the plasma membrane of presynaptic nerve terminals and glial cells are responsible for the removal from the extracellular space of released-transmitters, thereby terminating their actions (PMID: 15336049). Plasma membrane neurotransmitter transporters fall into two structurally and mechanistically distinct families. The majority of the transporters constitute an extensive family of homologous proteins that derive energy from the cotransport of Na+ and Cl−, in order to transport neurotransmitter molecules into the cell against their concentration gradient. The family has a common structure of 12 presumed transmembrane helices and includes carriers for gamma-aminobutyric acid (GABA), noradrenaline/adrenaline, dopamine, serotonin, proline, glycine, choline, betaine, and taurine. They are structurally distinct from the second more-restricted family of plasma membrane transporters, which are responsible for excitatory amino acid transport. The latter couple glutamate and aspartate uptake to the cotransport of Na+ and the counter-transport of K+, with no apparent dependence on Cl− (PMID: 8811182). In addition, both of these transporter families are distinct from the vesicular neurotransmitter transporters (PMID: 8103691, PMID: 7823024).
Biological process GO:0006836 neurotransmitter transport.
GO:0005335 serotonin/sodium symporter activity.
GO:0005887 integral component of plasma membrane ” (EMBL-EBI 2016).
Current Approaches for Investigating Genetic Variation and Function of Synaptic Molecules
New assays, in contrast to older ones, will put more emphasis on identifying and characterizing genic variation and their pathogenic variants associated with these molecules. Identifying these variants will be important in the design and validation of current synaptic bioassays. This information coupled with pharmacogenomics profiles that identifies drug metabolism variation in individuals will provide leads for drug discovery and more efficacious targeted treatment for personalized healthcare.
One such site that documents omic’s information on synaptic molecules is Genecards (http://www.genecards.org) (Weizmann Institute of Sciences 2016a). It provides information on summaries, genomics including products for regulatory elements and epigenetics, proteins, their attributes, protein products, antibodies and bioassays, domains and protein families of the gene product and function, pathways and interactions, drugs and compounds, expression products, and gene variants. An example for a product inquiry and link follows: http://sabiosciences.com/neuroscience.php (Sabiosciences 2016). This company provides PCR arrays and protocols to profile different neurotransmitters, neurotrophins, ion channels, and neurogenesis processes.
In the context of the subject matter for the encyclopedia, a general link to personality disorders can be found at MalaCards. This site facilitates queries into human disease (http://www.malacards.org/search/results/personality%20disorders) and provides information on genes, tissues, related diseases, publications, pathways, drugs (Weizmann Institute of Sciences 2016). For example, the solute carrier family 6 (neurotransmitter transporter), member 4, is a gene associated with personality disorders along with nine others (http://www.genecards.org/cgi-bin/carddisp.pl?gene=SLC6A4) (Weizmann Institute of Sciences 2016b).
Advances in Monitoring Neurotransmission
The development of optogenetic and chemogenetic tools has provided a more precise way for probing circuit dynamics in the brain. Optogenetic methods involve the use of optics, genetics, and DNA recombinant technology to target distinct neuronal populations from brain tissue slices (ex vivo) with genes that produce bacterial opsins. These ion-channel membrane-associated proteins can be stimulated selectively by light and visualized through fluorescent tagging. The essential reagents used in optogenetics are light-sensitive proteins. Neuronal control is achieved using optogenetic enhancers, while optical recording of neuronal activities can be made with the help of optogenetic sensors for calcium, vesicular release, neurotransmitter, or membrane voltage. This light-based method is in contrast to the analytical method of fast scan analytical voltammetry (FSCV) which utilizes electrical stimulation in vivo to study neurotransmitter release (McElligott 2015; Touriño et al. 2013).
Chemogenetics is a term coined to describe a process where larger molecules are manufactured to interact with smaller previously unrecognized ones. G protein-coupled receptors can be engineered to respond exclusively to synthetic small molecular ligands, like clozapine oxide (CNO), and not to their natural ligand(s) that permits spatial and temporal control of G protein signaling in vivo. Neuronal excitation or silencing can occur through the expression of G protein-coupled receptors activated by designer drugs called DREADD’s (Designer Receptor Exclusively Activated by Designer Drugs). These designer receptors act on endogenous intracellular pathways, whereas optogenetic constructs act on ion channels or pumps. For both genetic approaches, selective cellular genetic constructs occur as a result of recombinase dependent opsin/DREADD expression in tandem with neuron-specific recombinase expression. Newer constructs involve viral encoding of promoter regions that recognize specific cell types and allow control over cells at a subcellular level (Stachniak et al. 2014; McElligott 2015).
The aforementioned approaches are being combined with fast scan cyclic voltammetry (FSCV) to better understand neurotransmission dynamics. FSCV is an analytical method that measures changes in neurotransmitter concentrations over short time intervals through electrical stimulation or behaviourally induced neurotransmitter release. It has been used to characterize the dynamics of dopamine uptake and release in the subregions of the striatum. The application of these technologies should refine spatial neural circuitry mapping of the brain and further our understanding of neurotransmission dynamics by more precisely defining/differentiating neurotransmitter types and their signaling components (McElligot 2015; Stachniak et al. 2014; Touriño et al. 2013).
A similar successful approach that combines optogenetics, microscopy, and electrophysiology to study cellular communication can be found through a webinar link provided by Andor Technology and Lab Roots (http://www.labroots.com/webcast/the-benefits-of-combining-optogenetics-microscopy-and-electrophysiology). Signaling pathway elements can be genetically modified to enable precise and spatially targeted light control of biology down to a single cell level. Targeted light can be used in order to control neuronal excitation and so gain better insight into how nerve cells communicate within the context of a network and their impact on the whole organism (Wilde 2016).
High Throughput Analysis: Complexity of Assaying Variants/Subtypes
Detailed discussion of high throughput approaches/screening/analysis for drug study discovery/selection that can involve many agonist/antagonist synaptic molecules is not in the scope of this chapter. However, a link to such studies is provided through PubChem with an example description of a specific subtype (variant), the protein target 5-hydroxytryptamine (serotonin) receptor 1A (Homo sapiens) (https://pubchem.ncbi.nlm.nih.gooassay/567#section=Topv/bi). “Widely expressed in the human brain, 5-hydroxytryptamine (5-HT, serotonin) receptors have been shown to have an important role in depression as well as other cognitive and metabolic disorders. Agonists to 5-HT1a subtype, a protein-coupled heterotrimeric G receptor that inhibits production of cyclic adenosine mono phosphate (cAMP), have been shown useful as anxiolytics and antidepressants. Discovering novel modulators of the 5-HT1a serotonin receptor may not only help probe the function of this receptor, but also help better understand the complex relationship among the 5-HT receptor subtypes (NCBI PubChem Open Chemistry Database 2016).”
This bioassay record (AID 567) belongs to the assay project for drug development “Summary of the probe development efforts to identify agonists of the 5-Hydroxytryptamine Receptor Subtype 1E (5HT1E).” It can also be associated with the summary AID 1676 and a total of nine additional BioAssay records in PubChem.
Similarly, for an example of high throughput electrophysiological studies, the reader is referred to http://www.labroots.com/webinar/novel-applications-automated-electrophysiology-ion-channel-drug-discovery?
“Voltage-gated ion channels represent important drug targets. This assay allows for robust assessment of state-dependent effects of test agents and enables direct comparison of compound potency across several ion channel subtypes at equivalent levels of inactivation. In addition to determination of state dependency and selectivity, the assay provides valuable information on the kinetics of compound association and disassociation (Cern 2016).”
Complementary imaging information on aspects of structure/function of the human brain and its mapping (Beatty et al. 2015) also provides new insights into brain organization/circuity and may potentially validate neurotransmission findings. A general link to imaging studies can be found at http://www.humanconnectomeproject.org (NIH HumanConnectome project 2016).
An informatics approach was used to inform the reader of progress in translational research to better understand the process of neuronal transmission and to provide some insight into the complexity of identifying and characterising synaptic molecules and their population variants. The use of bioassays that incorporate optogenetic and chemogenetic approaches, electrophysiological methods/tools, and microscopy is contributing to improve our knowledge of synaptic transmission and neural circuitry in normal and disease states.
- Beaty, R., Kaufman, S., Benedek, M., Jung, R., Kenett, Y., Jauk, E., Neubauer, A., & Silvia, P. (2015). Personality and complex brain networks: The role of openness to experience in default network efficiency. Human Brain Mapping, 37, 773–779. doi:10.1002/hbm.23065.CrossRefPubMedPubMedCentralGoogle Scholar
- Cern, R. (2016). Novel high throughput approach to evaluate state dependence and selectivity of voltage gated ion channels. Webinar retrieved from http://www.labroots.com/webinar/novel-applications-automated-electrophysiology-ion-channel-drug-discovery?
- EMBL-EBI. (2016). Interpro sequence analysis and classification. Retrieved from http://www.ebi.ac.uk/interpro/entry/IPR013086
- Janowsky, A., Neve, K., & Eshleman, A. (2001). Uptake and release of neurotransmitters. Current Protocols in Neuroscience, 2, 7.9.1–7.9.22.Google Scholar
- NCBI PubChem Open Chemistry DataBase. (2016). Bioassay Record for AID 567. Retrieved from https://pubchem.ncbi.nlm.nih.gooassay/567#section=Topv/bi
- NIH HumanConnectome Project. (2016). Retrieved from http://www.humanconnectomeproject.org/
- SaBiosciences a Qiagen Company. (2016). Neuroscience. Retrieved from http://sabiosciences.com/neuroscience.php
- Stachniak, T., Ghosh, A., & Sternson, S. (2014). Chemogeentic synaptic silencing of neural circuit localizes a hypothalamus-midbrain pathway for feeding behaviour. Neuron, 82, 797–808. doi: 10.1016/j.neuron.2014.04.008. Epub 2014 Apr 24.Google Scholar
- Weizmann Institute of Science. (2016a). The human disease database. Retrieved from http://www.malacards.org/search/results/personality%20disorders
- Weizmann Institute of Science. (2016b). The human gene database. Retrieved from http://www.genecards.org/cgi-bin/carddisp.pl?gene=SLC6A4
- Wilde, G. (2016). The benefits of combining optogenetics, microscopy and electrophysiology. Webinar. Retrieved from http://www.labroots.com/webcast/the-benefits-of-combining-optogenetics-microscopy-and-electrophysiology