Using microfluidics and mass spectrometry to study peptide release from neurons

Microfluidics;Mass spectrometry;Neuropeptide;Neuron;Dorsal root ganglia;Aplysia californica

Croushore, Callie

关键词: Microfluidics;    Mass spectrometry;    Neuropeptide;    Neuron;    Dorsal root ganglia;    Aplysia californica;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46908/Callie_Croushore.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Neurons convey information through trillions of connections to affect animal behavior. Communication within these connections is primarily chemical and these chemical signals can function as modulators, hormones, transmitters, and trophic factors, enacting change at local and distant targets. Understanding and identifying local signals that establish and enrich neuron-neuron interactions can give insight into neuronal network formation and function, and are essential for the implementation of key effectors of repair that restore function to a damaged neural network. Neuropeptides are a class of signaling molecules that are involved in a wide range of physiological functions including sleep, learning and memory, feeding, and pain. Abnormal levels of neuropeptides have been implicated in a number of diseases and disorders such as depression and epilepsy. To gain a better understanding of the role that neuropeptides play, we are interested in sampling and identifying cues within local microenvironments around well-defined neuronal networks and assessing the effects of the identified molecules on neuronal network function. Within this context, we have combined microfluidic devices with mass spectrometry detection to effectively stimulate, sample, and detect neuropeptides from a well-defined culture. Microfluidics allow us to gain key information regarding the conditions which initiate or inhibit release by precisely controlling the extracellular environment. Mass spectrometry provides high sensitivity detection over a large dynamic range without requiring analyte pre-selection, enabling fast and accurate peptide discovery and detection.Here, we demonstrate two separate device designs for culturing neurons, applying selective chemical stimulations, and collecting released peptides. The first design employs micro-valve controlled stimulation channels to selectively apply chemical stimulations to cultured Aplysia californica neurons within the device. By controlling the length of stimulationivadditions, the amount of stimulation necessary to enact release was determined for two different chemical stimulations, elevated K+ and insulin. A robust response was observed as the onset of peptide release was different for each stimulation. The device system was then applied to a more complex system, the dorsal root ganglia (DRG) neurons of Rattus norvegicus.In order to study peptide release from DRG neurons, a healthy and viable culture was first established. Immunohistochemical staining revealed long, viable process growth in the presence of glia. MS profiling was performed on the culture and a large number of signals in the peptide region were observed. Both DRG clusters and cultures were stimulated with elevated K+ and several peaks differed between control and stimulation additions. While numerous peaks were observed, these putative peptides must be identified and confirmed with liquid chromatography (LC)- MS sequencing. To that end, DRG tissue, cell, and release samples were sequenced using two LC-MS platforms. Over 500 peptides were identified from 390 unique proteins within the DRG samples and a broad range of classifications were observed. Future work in this system involves focusing efforts toward neuropeptide detection.The second device system consists of a novel method to quantify release using MS imaging of microchannels on-chip. As peptides flow down PDMS channels, they interact and adsorb to a C18 functionalized silicon substrate. MS imaging is then performed on the channels and map of peptide localization throughout the channels is generated. We have created a novel method to quantify release by relating the length of peptide adsorption down a channel to the amount of peptide present. For each peptide of interest, a calibration curve is created and adsorbed peptide lengths can be compared to the calibration curve to determine peptide amount. We also demonstrate modifications of this device which yield both quantitative and temporal information regarding peptide release by instituting microvalve-controlled collection channels.