Detection of Tac4 mRNA in mouse and human samples with RNAscope technique

RNAscope in situ hybridization (ISH) was performed on 4-month-old male WT mice (n = 4). Animals were deeply anesthetized with an overdose of urethane (2.4 g/kg) and perfused transcardially with ice-cold 0.1 M phosphate-buffered saline (pH = 7.4) followed by 4% paraformaldehyde (PFA) in Millonig’s phosphate buffer. Dissected brains and spinal cords (SC) were postfixed for 72 h at 4 °C in PFA solution. The brains were coronally and the SC horizontally sectioned using a vibrating microtome (VT1000S, Leica Viosystems, Wetzlar, Germany). The 30 mm sections were stored in 1 × PBS with 0.01% Na-azide until further use (Merck KGaA, Darmstadt, Germany).

Perfused human cerebellar sample was obtained from one subject (Table 1) who died due to causes not directly involving any brain disease or damage, and without having medical history of neurological or psychiatric disorder either. All procedures were carried out in compliance with the Declaration of Helsinki and approved by the Regional Committee of Science and Research Ethics of Scientific Council of Health (ETT TUKEB 31443/2011/EKU, renewed: ETT TUKEB 15032/2019/EKU). Subject went through autopsy in the Department of Pathology of St. Borbála Hospital, Tatabánya. Informed consent by relatives was obtained for the use of brain tissue and for access to medical records for research purposes. The brain was removed after death, and the internal carotid and vertebrate arteries were cannulated. First, physiological saline containing 0.33% heparin was perfused through this system (1.5 L in 30 min), after which perfusion continued with Zamboni fixative solution containing 4% paraformaldehyde, and 0.2% picric acid in 0.1 M phosphate buffer (PB, pH = 7.4, 2 h, 4 L). Post-mortem interval (PMI) was determined between the time of death and the start of perfusion by Zamboni fixative. Frozen human somatomotor cortex, basal ganglia and spinal cord tissues were also obtained for RNAscope ISH (n = 4) in short post-mortem delay (1–12 h) without any major neuropathological alterations (Table 1). Tissue samples were microdissected in the Human Brain Tissue Bank, Semmelweis University. The human brain microdissection was approved by the Medical Research Council (ETT TUKEB); 5912–2/2018/EKU (Human Brain Tissue Bank, Semmelweis University, Budapest, Hungary), conducted with European directives and regulations. Informed consent was obtained from all participants and/or legal guardians. Following slow thawing of the frozen samples and 72 h of post-fixation, sectioning was performed [20] and processed as described earlier [23].

Table 1 Patient data (age, sex, time after death, and cause of death) of human brain samples

RNAscope ISH was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostic (ACD), Newark, CA, USA) according to the manufacturer’s protocols. Mouse and human sections were hybridized with probes specific to mouse Tac4 (ACD, Cat. No. 449651) or human TAC4 (ACD, Cat. No. 896671), mouse Vglut1 (ACD, Cat. No. 416631), human VGLUT1 (ACD, Cat. No. 415611), mouse Vglut2 (ACD, Cat. No. 319171), mouse Gad1 (ACD, Cat. No. 400951), human GAD1 (ACD, Cat. No. 404031), and mouse Chat (ACD, Cat. No. 408731) probes and were visualized by fluorescein (1:750 for Chat, Vglut2, VGLUT1, GAD1, and 1:3000 for Vglut1), cyanin3 (Cy3) (1:750 for Tac4 and TAC4), and cyanin 5 (Cy5) (1:3000 for Gad1).

Mouse (ACD, Cat. No. 320881) and human (ACD, Cat. No. 320861) 3-plex positive control probes specific to Polr2a mRNA (fluorescein), Ppib mRNA (Cy3), and Ubc mRNA (Cy5) gave a well-recognizable signal in randomly selected sections of the areas of interest, while no signal was detectable with 3-plex negative (ACD, Cat. No. 320871) control probes upon hybridization and channel development (images not shown).

For expression studies on dopaminergic neurons in mice, some slides were further processed for immunofluorescence using rabbit anti-TH antibody (1:2000, Abcam, Cambridge, UK, Cat. No. Ab112). Superclonal Alexa Fluor 488 goat anti-rabbit (1:1000, Invitrogen Antibodies, Cat. No. A11034) was used as a secondary antibody.

All sections were counterstained with 4′,6-diamino-2-phenylindole (DAPI) and covered with ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) for confocal imaging. Preparations were scanned by a Nikon Eclipse Ti2-E confocal microscope with 40x or 60x objectives. Virtual colors were selected to depict fluorescent signals: blue for DAPI; green for Vglut1, Vglut2, Chat, and TH; red for Tac4/TAC4 and GAD1; and white for Gad1, VGLUT1, and TAC4. Brightness/contrast adjustment with maximum intensity of separate channels was processed using FIJI (version 1.53c, NIH, USA).

RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA isolation was performed using TRI-Reagent (Thermo Fischer Scientific, Waltham, MA, USA) and Direct-zol™ RNA MiniPrep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The concentration and purity of total RNA were determined using the Nanodrop ND-1000 Spectrophotometer V3.5 (NanoDrop Technologies, Inc., Wilmington, DE, USA). Samples were treated with 1 U DNase I enzyme to eliminate remaining genomic DNA. Tissue cDNA was synthesized with the Applied Biosystems™ High-Capacity cDNA Kit High-Capacity RT Kit (Thermo Scientific, Waltham, MA, USA). qPCR measurement of the mouse Tac4 was carried out using the QuantStudio™ 5 system (Life Technologies Magyarország Ltd, Hungary), using the eukaryotic translation elongation factor (Eef2) and glucuronidase beta (Gusb) as reference genes. Measurements were performed in triplicates, in a reaction volume of 10 µl, containing 1x SensiFAST™ Probe Lo-ROX mix (Meridiane Bioscience, Memphis, USA), 400 nM probe primer mix (forward and reverse), and 20 ng cDNA. FAM-conjugated TaqMan™ Gene Expression Assays (Thermo Scientific, Waltham, MA, USA) were used to amplify the target loci, Tac4: Mm00474083_m1; Eef2: Mm00833287_g1; and Gusb: Mm01197698_m1. The geometric means of the Cq values were calculated for both reference genes, and the fold changes of Tac4 were calculated using the ΔΔCt method [24, 25].

Animals

We used young (3–4 months old), middle-aged (12 months old), and old (18 months old) male and female C57BL/6 and HK-1 deficient (Tac4–/–) mice weighing 25–50 g, bred and kept in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of Pécs. Animals were housed at a constant temperature (24–25 °C), 50–60% relative humidity, a 12–12-h light–dark cycle, and free access to water and standard rodent food.

Behavioral tests

All testing was conducted during the animals’ day cycle in an appropriately illuminated (400 lx) room. The animals were allowed to acclimate to the testing room for 30 min before the measurements. The mice were gently carried (e.g., in a strainer or in hand) from the cage to the testing apparatus. Four tests were used, which can be divided into two groups. The first group consisted of the rotarod and static bar tests, which examined dynamic and static motor coordination. The second group of tests, horizontal bar and grid tests, examined muscle strength. There was always a minimum of two days between each test, during which the animals were not exposed to any other influences. In all cases, only one test trial was performed and evaluated.

Rotarod test

Dynamic motor coordination was determined using a Rotarod apparatus (Ugo Basile, Italy). The rod rotates continuously and increasingly faster under the paws of the animals during the test, with a diameter of 3 cm and 30 cm above the base of the apparatus [10]. Two three-minute-long training sessions on the rod, which moves at a constant speed (10 rpm), were performed before the measurement. In the test trial, the starting speed was set to 10 rpm, the maximum rotation speed to 40 rpm, and the acceleration speed to 6 rpm/min. Mice could spend up to 300 s on the machine. The time spent on the rod was plotted and compared between the groups.

Static bar test

Static rod test measured static motor coordination. During the test, five wooden rods, each 60 cm long and of different thicknesses (35 (rod 1), 28, 22, 15, and 9 (rod 5) mm in diameter) were fixed to a laboratory shelf so that the rods extended horizontally into space. The height of the rods above the floor was 60 cm, and a soft cushion was placed underneath them to protect against a fall. We worked from the thickest rod to the thinnest. At the end of the pole, near the shelf, was the finish line, which was reached by turning from the side of the pole facing the space. The cut-off time was 120 s. Orientation time (the time required to turn around) and transit time (the time needed to reach the target line) were determined [26].

Horizontal bar test

The horizontal rod test assessed muscle strength. Two metal rods of different thicknesses were used, held horizontally by a wooden frame. They were 38 cm long and were held 49 cm above the surface of the bench by wooden support posts. The diameters of the poles were 4 and 6 mm. The test involved placing the animals at the midpoint of the pole by their front paws, from where they must climb onto the pole and touch the wooden frame with their nose or paws. They had 30 s to complete the task. Protective element(s) were placed under the pole, similar to the static pole test. The time required to complete the task/time on the bar was converted into scores. Shortly, a fall between 1–5 s meant 1 point, between 6–10 s = 2, 11–20 s = 3, 21–30 s = 4, and after 30 s = 5 points. Touching the bar with the forearm or nose without falling = 5 points [26]. The sum of the scores obtained on the two different bar thicknesses was the composite score.

Grid test

The grid test is a commonly used method for testing muscle strength [27]. In the experiment, animals were placed on a metal grid held by four poles 20 cm above the table. This grid is turned upside down, and the mice are then required to hold on to it using all four limbs for up to 180 s. The time spent on the grid was plotted and compared.

Ethical consideration

Our testing methods and procedures comply with all requirements of Government Decree No. 40/2013 (14.II.) on the performance of animal experiments and the European Parliament's directives (2010/63). The University of Pécs Ethics Committee for Animal Experiments has approved the experimental protocols (Licence No. BA/73/00657-3/2022).

Statistics

For the rotarod test, the static rod test, and the grid test, a two-way ANOVA with Fischer’s post hoc test was used. The results of the horizontal rod test were evaluated using the Kruskal–Wallis and Dunn’s post hoc test. Statistical analysis was performed using GraphPad Prism 8 software. Additionally, the effect sizes were also calculated using Hedges’ g (difference in means divided by the pooled and weighted standard deviation) in all relations. Given that the study was conducted on a small sample size, the statistical significance level (p-value) alone could not provide reliable information. Hence, we used effect size to interpret the results, providing a more direct picture of the magnitude and practical significance of the effect [28]. An effect size > 0.2 was considered small, >0.5 as medium, and >0.8 as large [29]. The results for each group were presented as mean ± SEM. The number of elements per group was 8–25.