*2.6. Serum Corticosterone, Luteinizing Hormone and Total Protein Concentration Measurement*

For the measurement of serum corticosterone and protein concentration, the animals were decapitated 30 min after icv. treatment. For the assessment of serum LH, decapitation was performed 15 min after icv. treatment. Trunk blood was collected into test tubes and left at room temperature for 30 min to clot, then it was centrifuged for 10 min at 3500 rpm. The samples were stored at −80 ◦C until the assays were performed. Serum corticosterone concentration was measured using a competitive corticosterone ELISA kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer's instruction. Serum LH concentration was determined using a sandwich LH ELISA kit (Wuhan Xinquidi Biological Technology Co., Wuhan, China), according to the manufacturer's instructions. The Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used, according to the manufacturer's instructions for the measurement of total serum protein concentration. The absorbance was measured at 595 nm with a NanoDrop OneC microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.7. Ex Vivo Superfusion*

Before the ex vivo superfusion, the animals did not undergo icv. cannulation. The rats were rapidly decapitated, and their brains were removed from the skull. Dissection was performed with the help of a brain matrix, a tissue puncher and razor blades, on a filter paper moistened with phosphate-buffered saline, on top of a Petri dish filled with ice. The NAc was removed from both sides, following the method of isolation described by Heffner [54]. The VTA was isolated as described by Salvatore et al. [55]. The tissue was cut to 300 μm slices and incubated for 30 min in 5 mL of Krebs solution (Reanal, Hungary) bubbled with carbogen gas (5% CO2 and 95% O2). Then 5 μL of [3H]GABA (PerkinElmer Inc., Waltham, MA, USA) was added to the NAc and 5 μL of [3H]Dopamine (PerkinElmer Inc., Waltham, MA, USA) was added to the VTA or the NAc. Afterwards the slices were transferred evenly into the four cylindrical chambers of the superfusion system (Experimetria Ltd., Budapest, Hungary), and superfusion with carbogen-bubbled Krebs solution was started at body temperature (37 ◦C). A constant flow rate of 227, 7 μL/min was maintained with a peristaltic pump (Minipuls 2, Gilson, Middleton, WI, USA). After 30 min of superfusion, the collection of superfusates into Eppendorf tubes was started with a multichannel fraction collector (FC 203B, Gilson, Middleton, WI, USA). Fractions were collected every two minutes for 32 min. At 6 min, 1 μg of Kp-8 dissolved in 1 mL of Krebs solution was added directly into the chambers. From the 12th minute of fraction collection, electrical stimulation of square-wave impulses was delivered for two minutes (ST- 02 electrical stimulator, Experimetria Ltd., Budapest, Hungary). Then, the tissue from each chamber was transferred into a beaker containing 600 μL of Krebs solution for ultrasonic homogenization (Branson Sonifier 250, Emerson Electric Co., St. Louis, MO, USA).

Afterwards 3 mL of Ultima Gold scintillation cocktail (Perkin-Elmer Inc., Waltham, MA, USA) was pipetted into 4 rows of 17 scintillation vials. Subsequently, 200 μL of the 16 fractions collected and of the suspension of the tissue from the corresponding chamber were added into each row of vials. The samples were homogenized mechanically for 30 min.

The radioactivity of samples was detected with a liquid scintillation spectrometer (Tri-carb 2100 TR, Hewlett-Packard Inc., Palo Alto, CA, USA). Fractional dopamine or GABA release (FR) was calculated from the counts per minute (CPM), according to the equation below, in which *i* stands for the number of fraction and *n* = 16. *CPM17* refers to the CPM of the homogenized tissue sample corresponding to the fraction:

$$FR\_i = 100 \cdot \frac{CPM\_i}{4 \cdot CPM\_{17} + \sum\_{i+1}^{n} CPM\_i}$$

#### *2.8. Statistical Analysis*

Data are presented as mean + SEM. Statistical analysis and graph editing were performed using the GraphPad Prism 8 software. One-way ANOVA with Holm-Sidak's post-hoc test was applied for the analysis of EPM results. One-way ANOVA with Dunnett's post-hoc test was used for the analysis of cumulative OF results, as well as for the evaluation of serum corticosterone, LH and total protein measurements. Two-way RM ANOVA with Holm–Sidak's post-hoc test was performed for the evaluation of 5-min intervals in the OF test as well as for the interpretation of dopamine and GABA release from the NAc. Mixed-effects analysis with Holm–Sidak's multiple comparison test was performed for the evaluation of fractional dopamine release from the VTA. Kruskal–Wallis test with Dunn's post-hoc test was performed for the analysis of MB results. Curve fitting for ELISA tests was performed according to the manufacturers' instructions.
