Neuropeptides form a large group of transmitter molecules involved in diverse biological functions including sleep, reward, feeding, learning and memory. Investigation into the specifics of neuropeptide release in vivo, such as timing and location, has been a challenge due to lack of in vivo detection methods for neuropeptides. Microdialysis is currently the only available option to monitor neuropeptide levels.
Cell-based neurotransmitter fluorescent reporter (CNiFER) is an alternative method for in vivo detection of neuropeptides with high spatial and temporal resolution. The method relies on human embryonic kidney (HEK) cells that are genetically modified to express (1) a G-protein coupled receptor (GPCR) specific for the neuropeptide of interest and (2) a fluorescent calcium (Ca2+)-sensor. CNiFER converts neuropeptide binding into a fluorescence signal which can be detected by microscopy.
Somatostatin (SST) is a neuropeptide which has been used as a molecular marker to classify a group of interneurons in the cortex. However, studies in other parts of the brain suggested SST can be released into the extracellular medium and function as a neurotransmitter. In the present study, CNiFER clones were developed with physiological level sensitivity to SST. One of these clones was used to show that endogenous SST can be released by optogenetic stimulation of cortical somatostatin-expressing interneurons (SST-INs). Subsequent experiments revealed that the amount of SST released was dependent on the frequency of light delivery. A second CNiFER clone was used to show SST can also be released by cholinergic stimulation of SST-INs.
In conclusion, in vivo application of SSTR2 CNiFER clones demonstrated that SST is not only a molecular marker that define a group of cortical interneurons but can be released into the extracellular medium by direct (optogenetic) or indirect physiological (cholinergic) stimulation of SST-INs. Application of CNiFER methodology to neuropeptides can help study into neuropeptide transmission in vivo.
|Advisor:||Slesinger, Paul A.|
|Commitee:||Devi, Lakshmi, Ellis-Davies, Graham, Morishita, Hirofumi, Rudy, Bernardo|
|School:||Icahn School of Medicine at Mount Sinai|
|School Location:||United States -- New York|
|Source:||DAI-B 81/5(E), Dissertation Abstracts International|
|Keywords:||Acetylocholine, Calcium imaging, Interneuron, Neuropeptide, Somatostatin, Two photon microscopy|
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