Molecular mechanisms underlying the transmitter release at the serotonergic axon terminals
Lead Research Organisation:
MRC Laboratory of Molecular Biology
Department Name: Neurobiology
Abstract
We are applying for a confocal microscope so that we can characterize the properties and the mechanisms of neurotransmitter release at the serotonergic terminals.
One in four adults experiences at least one diagnosable mental illness in any given year. Mental illnesses represent the largest single cause of disability in the UK and worldwide. The serotonin system is the most widely used target for treating mental disorders, and selective serotonin reuptake inhibitors are the most commonly prescribed antidepressants. Despite its importance, we still know very little about the molecular player involved in serotonin release in the brain. Meanwhile, there are other neurotransmitters, such as glutamate, that can be released together with serotonin from the same nerve. We don't know the mechanism behind the decision made by the nerve, about when and how the individual transmitter is released. In this study, we plan to answer these questions. Recently, scientists find that they can label the neighbouring proteins of an interesting molecule with biotin and then identify these labelled proteins by mass spectrometry. We use this strategy to map the proteins that are involved in the regulation of the release of serotonin and other transmitters localized in the same nerve. To study the functions of these proteins, we have established a 3D model mimicking the projection of the nerves containing serotonin that can be put in a dish. This is achieved by using organoid and assembloid technologies, culturing cells from certain brain regions of mouse embryos to form structures resembling neuronal connections in the brain. We can use molecular tools to express or knock out certain genes from neurons in our 3D model. These tools include optogenetics, genetically encoded fluorescent sensors, and CRISPR-Cas9 gene-editing system. Optogenetic tools are light-sensitive proteins that can modulate neurons' activity when they are triggered by light. The newly developed genetically encoded serotonin sensor will generate a fluorescent signal once it detects serotonin. We will express optogenetic tools in the serotonergic cells and activate several spots in the nerves by shining multiple beams of laser, and monitor serotonin release from these nerves by imaging the surrounding cells that express serotonin sensors. We can also use a glutamate sensor to detect glutamate release from the same nerves. At the same time, we will perform electrophysiological recording from the imaged neurons to examine the effects caused by these transmitters. This experiment requires high resolution in both time and space, as well as coordination among the multi-point stimulation system, the imaging system and the electrophysiological system. We will achieve this by setting up a multi-functional live imaging rig with a high-end laser scanning confocal. This will allow us to characterize the properties of transmitter release at the serotonergic nerve terminals. We will then use CRISPR-Cas9 strategy to knock out the genes encoding the proteins we mapped and use our multi-functional rig to test how the removal of these proteins influences the release properties.
Our results will provide a fundamentally deeper understanding of the molecular mechanism behind co-transmission at the serotonergic terminals. They will also uncover new molecular and physiological signatures of serotonin transmission that could provide entirely novel drug targets for treating mental disorders.
One in four adults experiences at least one diagnosable mental illness in any given year. Mental illnesses represent the largest single cause of disability in the UK and worldwide. The serotonin system is the most widely used target for treating mental disorders, and selective serotonin reuptake inhibitors are the most commonly prescribed antidepressants. Despite its importance, we still know very little about the molecular player involved in serotonin release in the brain. Meanwhile, there are other neurotransmitters, such as glutamate, that can be released together with serotonin from the same nerve. We don't know the mechanism behind the decision made by the nerve, about when and how the individual transmitter is released. In this study, we plan to answer these questions. Recently, scientists find that they can label the neighbouring proteins of an interesting molecule with biotin and then identify these labelled proteins by mass spectrometry. We use this strategy to map the proteins that are involved in the regulation of the release of serotonin and other transmitters localized in the same nerve. To study the functions of these proteins, we have established a 3D model mimicking the projection of the nerves containing serotonin that can be put in a dish. This is achieved by using organoid and assembloid technologies, culturing cells from certain brain regions of mouse embryos to form structures resembling neuronal connections in the brain. We can use molecular tools to express or knock out certain genes from neurons in our 3D model. These tools include optogenetics, genetically encoded fluorescent sensors, and CRISPR-Cas9 gene-editing system. Optogenetic tools are light-sensitive proteins that can modulate neurons' activity when they are triggered by light. The newly developed genetically encoded serotonin sensor will generate a fluorescent signal once it detects serotonin. We will express optogenetic tools in the serotonergic cells and activate several spots in the nerves by shining multiple beams of laser, and monitor serotonin release from these nerves by imaging the surrounding cells that express serotonin sensors. We can also use a glutamate sensor to detect glutamate release from the same nerves. At the same time, we will perform electrophysiological recording from the imaged neurons to examine the effects caused by these transmitters. This experiment requires high resolution in both time and space, as well as coordination among the multi-point stimulation system, the imaging system and the electrophysiological system. We will achieve this by setting up a multi-functional live imaging rig with a high-end laser scanning confocal. This will allow us to characterize the properties of transmitter release at the serotonergic nerve terminals. We will then use CRISPR-Cas9 strategy to knock out the genes encoding the proteins we mapped and use our multi-functional rig to test how the removal of these proteins influences the release properties.
Our results will provide a fundamentally deeper understanding of the molecular mechanism behind co-transmission at the serotonergic terminals. They will also uncover new molecular and physiological signatures of serotonin transmission that could provide entirely novel drug targets for treating mental disorders.
Technical Summary
We are applying for a confocal microscope so that we can characterize the properties and the mechanisms of transmission at the serotonergic terminals. Mental illnesses represent the largest single cause of disability in the UK and worldwide. The serotonin system is the most widely used target for treating mental disorders. The majority of serotonin axons bear varicosities for extrasynaptic release (volume transmission), and our knowledge of the molecular mechanisms behind it is very limited. Meanwhile, serotonin neurons also release other transmitters such as glutamate, and we know very little about how co-transmission is modulated at their axon terminals. Here, we use the proximity labelling method to profile the serotonin release machinery and dissect the release action of individual co-transmitters by combing confocal imaging and electrophysiological recording. We have established an in vitro 3D model by advanced organoid technology to mimic the long-distance projection from midbrain serotonin neurons to the cortical and subcortical regions of the forebrain. The candidate components of the release machinery generated from the proximity biotinylation-facilitated quantitative proteomics will be first examined in our organoid model and then mouse brain slices. To be able to accurately detect the transmission event, we will need a platform to do simultaneous real-time manipulation, high-resolution live imaging, and electrophysiological recording. We will achieve this by setting up a multi-functional live imaging rig with a high-end laser scanning confocal to apply the combination of transmitter sensor imaging, calcium imaging, optogenetics and electrophysiology. Our results will provide a fundamentally deeper understanding of the molecular mechanism behind co-transmission at the serotonergic terminals. They will also uncover new molecular and physiological signatures of serotonin transmission that could provide entirely novel targets for psychiatric drug development.
