The goal of our study was to quantitatively assess the differences on the cerebral metabolites and to identify the factors determining the alterations of endogenous biomolecules on stress-induced sleep disturbance in rats using in vivo MR Spectroscopy (1H MRS) and in vitro liquid chromatography-tandem mass spectrometry (LC-MS/MS).Sprague-Dawley rats (sham-controls [CNTL]: n=9; stress-induced sleep perturbation [SSP] rats: n=11) were used in this study and we exposed the stress-induced, sleep-perturbed rat model to the psychological stressor (cage exchange method).¹ All CNTL rats were also exchanged the clean cage at the same time in order to synchronize the ultradian cycles between the two groups. All in vivo MR assessments were carried out using a horizontal 9.4 T/160 mm magnet. For VOI localization and identification of the anatomical region, multi-slice, T2-weighted MR images were acquired using a fast spin echo (FSE) pulse sequence (repetition-time [TR] = 4000 ms, effective-echo-time [TEeff] = 32.95 ms, echo-spacing [ESP] = 10.98 ms, echo-train-length [ETL] = 32, average = 1, field-of-view = 30 × 30 mm, slice thickness = 1 mm, and matrix size = 256 × 256). The VOI (2.0 × 2.5 × 3.0 = 15.0 µL) position was targeted to the right dorsal hippocampal region. Water suppressed in vivo spectra were acquired using a point-resolved spectroscopy pulse sequence (TR = 5000 ms, TE1/TE2/TEtotal = 7.46/6.01/13.47 ms, spectral width = 5.0 kHz, average = 384, number of data points = 2048). Acquired in vivo raw data were analyzed using a fully blind spectral process, using LCModel software with a simulated basis set containing 18 metabolites, as follows: Alanine (Ala); aspartate (Asp); creatine (Cr); phosphocreatine (PCr); gamma-aminobutyric acid (GABA); glutamine (Gln); glutamate (Glu); glucose (Glc); glycine (Glyc); glycerophosphocholine (GPC); scyllo-inositol (sI); myo-inositol (mIns); lactate (Lac); N-acetylaspartate(NAA); N-acetylaspartylglutamate (NAAG); phosphocholine (PCh); glutathione (GSH); and taurine (Tau). After the in vivo 1H-MRS assessment, all rats were sacrificed using CO2 inhalation. All whole-brain tissue samples were quickly and carefully harvested from the removed skull. The dried sample was reconstituted with 20μL of 50.0% methanol prior to LC-MS/MS analysis. Serotonin (5-HT) and dopamine (DA) were analyzed with LC-MS/MS equipped with 1290 HPLC (Agilent), Qtrap 5500 (ABSciex), and a reverse phase column (Pursuit-5-C18). Multiple-reaction-monitoring was used in the positive ion mode, and the extracted ion chromatogram corresponding to the specific transition for each analyze was used for quantitation. Fig.1 shows representative in vivo 1H MR spectra acquired from the right dorsal hippocampal region. The in vivo 1H MR spectra were assigned the following 17 cerebral neurochemical signals: Ala, Asp, Cr, PCr, GABA, Glc, Gln, Glu, Glyc, GPC, PCh, GSH, mIns, NAA, NAAG, Tau, and tCho. Fig. 2A and B illustrates showed that the independent t test revealed significant differences in the cerebral metabolite concentrations between the two groups, and thus indicating a significant stress-induced effect on quantified metabolite concentrations. The GABA (p=0.040) and Gln (p=0.038) concentrations were significantly higher in the SSP rats than in the CNTL rats. In addition, Fig. 3 indicates that the Gln/Glu (p=0.009), Gln/tCr (p=0.037), and GABA/Glu (p=0.025) ratios were significantly higher in the SSP rats than in the CNTL rats. Fig. 4 shows the 5-HT and DA concentrations that were quantified from the in vitro LC-MS/MS of twenty brain hemispheres. The 5-HT concentrations (p=0.036) were significantly lower in the SSP rats than in the CNTL rats. Our study results revealed that the pairs of biomolecule and metabolite signals that were significantly positively correlated in the hippocampal region were as follows: GABA vs. Gln (R2=0.198); and DA vs. 5-HT (R2=0.412) concentrations (Fig. 5A and B). We suggest that significantly higher Gln concentrations and Gln/Glu, Gln/tCr, and GABA/Glu ratios in the SSP rats than those in the CNTL rats may reflect the hyper-activity of glutamine synthetase and GABAergic receptors while there was a declining glutamatergic activity.^2-4 Moreover, our primary findings suggest that the GABA, Gln, and Glu signals in the hippocampal region are particularly sensitive and vulnerable to stress-induced sleep perturbation.^5-7 Significantly lower 5-HT concentrations in SSP rats than in the CNTL rats might reflect that the decreased producing rate of serotonin is possibly due to the impairment or dysfunctions in L-tryptophane and the 5-HTPlevels.^8-10Our in vivo 1H MRS and in vitro LC-MS/MS results suggest that the GABA, Gln, and 5-HT signals in the hippocampal region of the rats are particularly sensitive and vulnerable to stress-induced sleep perturbation. Our in vivo 1H-MRS and in vitro LC-MS/MS results suggest several novel metabolic markers for the cerebral neurobiological effects of stress-induced sleep perturbation in rat brain.