An optimized approach for in vivo Met- and Leu-Enk measurement.

A. Timeline of in vivo sample collection on day 1 and methionine oxidation reaction overnight, sample processing on day 2, and data acquisition on the LC-MS, days 3-5. The microdialysis probe is implanted via stereotaxic surgery in the Nucleus Accumbens Shell. The mouse is allowed to recover before being connected to the microdialysis lines. Samples are then collected at a rate of 0.8 μL/min for 13 minutes each. After collection is completed, the samples are oxidized overnight. On day 2 the samples undergo solid phase extraction (SPE) and are then dehydrated and reconstituted using formic acid (HCOOH) before being acquired on the LC-MS. B. Custom microdialysis probe specifications including membrane size and inner and outer diameters (ID and OD respectively) compared to fiber photometry probe specifications including OD of optic fiber and numerical aperture (NA). C. Before the methionine oxidation at reaction (Rxn) time 0, Met-Enk exists in three different forms with varying intensities, unoxidized (multi-peak), singly oxidized (multi-peak), and doubly oxidized (single peak). After the reaction completes (Rxn time 1), most of the detected signal is in the doubly oxidized form and shows a single peak (>99% signal intensity). D. (Left) Forward calibration curve of Leu-Enk and Met-Enk showing the peak area ratios as the light standard levels are varied. (Right) Reverse calibration curve of Leu-Enk showing the relationship between the heavy standard concentration applied and the measured concentration based on the instrument. E. Same setup as D but for Met-Enk. F. Violin plots showing that high K+ ringer’s solution increases the release of both Leu-Enk and Met-Enk compared to baseline levels in artificial cerebrospinal fluid (aCSF) (Leu-Enk n=9, Met-Enk n=18). The dashed center line indicates the median. G. The evoked concentrations of Met-Enk to Leu-Enk in the same samples show that Met-Enk is consistently released at a higher level than Leu-Enk (n=9). H. Met-Enk is released at a factor of 2.97 that of Leu-Enk as shown by linear regression analysis of the data in f, suggesting a linear relationship between the two peptides. Data in F, G, and H are transformed to a log scale. In F, 2-way ANOVA analysis shows a main effect of peptide, solution, and interaction. The p-values reported were calculated using a Sidak’s multiple comparisons test.

Experimenter handling evokes the release of Met- and Leu-Enk in the NAcSh.

A. Experimenter handling during microdialysis causes a significant increase in the release of Leu- and Met-Enk in comparison to baseline. B. During experimenter handling, Met-Enk is consistently released at a higher rate than Leu-Enk in the same samples. C. Linear regression analysis of the data in B shows that Met-enk is released at a rate of 3.12 times the rate of Leu-Enk during experimenter handling suggesting a linear relationship between the two peptides. D. A negative correlation (−0.3141) shows that if a high concentration of Met-Enk is released in the first two samples, the concentration released in later samples is affected; such influence suggests that there is regulation of the maximum amount of peptide to be released in NAcSh. E. The negative correlation in panel d is reversed by using high K+ buffer to evoke Met-Enk release, suggesting that the limited release observed in D is due to modulation of peptide release rather than depletion of reserves. Data in A-E are transformed to a log scale and n=24 animals. In panel A, 2-way ANOVA analysis shows a main effect of peptide, treatment, and interaction. The p-values reported were calculated using a Sidak’s multiple comparisons test. F. Schematic describing the viral strategy and probe placement for the fiber photometry experiment in pEnk-Cre mice injected with the calcium sensor GCaMP6s in the NAcSh. G. Average z-score trace following the first, second, and third events involving experimenter handling. H. Heatmaps showing individual mouse z-scored fiber photometry responses before and after experimenter handling. I. Bar graphs showing the averaged z-score responses before experimenter handling and after experimenter handling (n=10 for G-I).

Fox odor exposure activates enkephalinergic neurons and drives the release of Met-Enk in the NAcSh.

A. Viral strategy and probe placement for the fiber photometry experiment in pEnk-Cre mice injected with the calcium sensor GCaMP6s in the NAcSh. B. Averaged z-score traces of the first, second, and third exposure to fox odor. C. Bar graphs showing the averaged z-scored fiber photometry responses before and after exposure to fox odor D. Heatmaps showing individual mouse z-scored fiber photometry responses before and after exposure to fox odor. (n=9, for A-D) E. Experimental timeline describing the fox odor microdialysis experiment. F. There is a significant increase in Met-Enk concentration during fox odor exposure (sample 4), in comparison to sample, 3 before odor exposure. Data is transformed to a log scale and the p-value was calculated using a paired t-test (n=11). G. Pie charts showing the variation across animals in Met-Enk and Leu-Enk release profiles in response to exposure to fox odor, suggesting that such exposure may selectively cause the release of Met-Enk but not Leu-Enk. Increased release showed higher concentration of Met-Enk during exposure to fox odor (sample 4) than the sample before (sample 3), some release showed a comparable level to the sample before exposure, and no release showed no quantifiable concentration during exposure to fox odor.