B-006 Locus Coeruleus Activation Causes Dynorphin Release in Mouse Brain: Implications for Neural Inflammation

Abstract Background Stress hormones are released in response to neural signals from the brain. It’s been shown in our research group that the electrostimulation of locus coeruleus (LC), a brainstem nucleus (mimicking stimulation of the LC by stress) causes the release of the stress hormone corticosterone. We hypothesize that LC, similar to its stress response, may also cause the release of an peptide dynorphin (DYN), that has been previously shown to upregulate cytokine, chemokine and reactive oxygen species and thus initiate an inflammatory response. The LC projects to different parts of the brain while DYNs are also present in different brain regions and thus the LC may be the pathway by which DYNs are released. To confirm our hypothesis, we compared DYN concentrations in cerebrospinal fluid (CSF) in electro-stimulated versus non-stimulated LC mice, quantifying by using a highly sensitive LC-MS/MS method developed in our lab. Confirmation of this hypothesis is important to understanding the pathway by which inflammation occurs in the brain leading to neuropathology. Methods In this research mice groups were used as non-LC-stimulated control mice and LC-stimulated mice. Each mouse is anesthetized and immobilized on a stereotaxic device, with the skull exposed and electrodes inserted into the LC nucleus. For the electro stimulated group the brain was stimulated for 20 minutes. Brainstem stimulation was created by connecting the digital stimulus isolation amplifier (10V) to the stimulating electrode to depolarize the LC neurons in the brain and the control group received the same surgery with placement of the electrode on the LC without stimulation. CSF was collected. To inhibit protease/peptidase activity and prevent protein degradation, acetic acid is immediately added to the collected CSF. We developed an LC-MS/MS MRM technique (m/z 572.6? m/z 136.1) for detecting DYN B using a novel peptidase enzyme, Lys-N. An AB Sciex 5500 QTRAP triple quadrupole tandem mass spectrometer was coupled to Shimadzu HPLC. DYN B was separated from other CSF components using a Luna Omega 1.6 µm Polar C18 100 Å, LC Column 50 x 1.0 mm. The mobile phases were A, water 0.1% formic acid, and B, acetonitrile 0.1% formic acid, gradient 0 – 80% B in 17 min, flow rate 0.2 ml/min. Results Stimulated samples have a significant increase in DYN B release, with concentrations nearly 5 times higher than the baseline levels for ten runs of each stimulated and non-stimulated, with a p-value < 0.01(1.59E-11). Calibration curves (peak area=0.4158*[DYN B pg/mL] + 0.7392, R2= 0.9661) were 0, 0.5, 1, 2 and 5 pg/mL in CSF matrix. For matrix effect and recovery, the slope of CCF calibration plot is 84% of the working standard calibration plot which meets acceptability. The limit of detection is 0.125 pg/mL. Conclusion A sensitive LC-MS/MS MRM technique for quantification of DYN B in CSF has been developed using an unconventional peptidase enzyme Lys-N to generate a specific peptide fragment. This technique was used to confirm the hypothesis that stimulation of the LC leads to the increase of DYN B in mouse brain as measured in CSF.