Animals and housing
Male (n = 40) and female (n = 40) outbred WIST:RccHan rats were purchased from Envigo (Venray, Netherlands) and arrived at the institute on (PD 21). These rats were used to characterize the behavioral sequelae of ASI in adulthood. A separate cohort of male (n = 24) and female (n = 24) from the same supplier (that also arrived on PD 21) was used for characterizing the molecular of OTRs. Rats were housed individually (Makrolon Type III cages) or in groups of four (Makrolon Type IV cages) under a standard diurnal 12 h light-dark cycle, temperature 23 ± 3, and humidity (40–60%) with free availability of tap water and standard laboratory chow without any enrichment. Male and female rats were housed in separate colony rooms. All experiments were approved by the local animal care committee (Regierungspräsidium Karlsruhe, Referat 35, Karlsruhe, Germany, AZ35-9185.81/G-289/18) following the guidelines of the European Union (2010/63/EU).
Study Design
All rats were weaned on PD 21 and were pseudo-randomly selected for housing into either the early adolescent social isolation (EASI), late adolescent social isolation (LASI) condition or control (CTL) condition. Rats were housed in groups of four rats per cage. Each isolation condition lasted for three weeks. Two cohorts of rats where used in the behavioral study, in order to handle the large amount of rats used in the study. In the first cohort, we used eight control rats, eight EASI and four LASI rats of each sex. In the second cohort, we used eight control rats, four EASI, and eight LASI rats of each sex. In the EASI condition, the rats were socially isolated from PD 21 to 42, and in the LASI condition, the rats were socially isolated from PD 42 to 63 (Fig. 1). For the duration of the social isolation, rats had no somatosensory contact but had olfactory, auditory, and visual stimuli of the other rats in the same colony room from the same and different conditions. At the end of the isolation period rats were rehoused with rats from the same condition. Simultaneously, control rats were rehoused with other control rats to equalize potential rehousing stress among groups. Rats remained group housed for the remainder of the experiment.
Behavioral testing began with the elevated plus maze (EPM) (PD 90), followed by the open field test (OFT) (PD 92), novel object recognition (NOR) (PD 94), social interaction and social recognition memory (SIT/SRM) (PD 96), and Hotplate test (PD 98) (Fig. 2). All behavioral testing was done during the first five hours to the light-ON cycle (inactive). A separate cohort of rats was used for the molecular characterization of OTR alterations in adulthood following ASI. These rats underwent the ASI procedure as in experiment 1 but did not undergo behavioral testing. Instead, the rats were sacrificed on PD90 within the first two hours of the light-ON cycle.
Behavioral Tests
We chose commonly used behavioral tests to assess for anxiety-like, social and memory processes as well as pain sensitivity that had previously been shown to be altered by social isolation and had been validated in our lab. All behavioral tests were performed during the first five hours of the inactive phase (light ON) of the diurnal cycle. Rats were given at least 48 hr rest between tests. All videos were recorded and evaluated offline by an expert blinded to the experimental manipulations. The estrous cycle of females was tracked after the elevated plus-maze (EPM) and hotplate test (HP) because there are indications that anxiety-like behaviors (29) and thermal pain sensitivity (30, 31) are influenced by the estrous cycle. All behavioral apparatuses were cleaned with 70% alcohol solution at the start of each day, between trials, and after each day of testing to prevent the transmission of olfactory cues. Next, the apparatuses were cleaned with water and allowed to dry as evidence suggests that strong scented solutions like alcohol can influence behavioral results (32).
Estrous cycle cytology
Cytological vaginal smears were collected immediately after the elevated plus-maze and hotplate test to monitor the estrous cycle phase, as evidence points to the estrous cycle phase influencing both anxiety-like behavior and pain sensitivity in these two tests (29, 30). The samples were analyzed under a light microscope (V300, Will Wetzlar) and characterized into two categories estrus/diestrus and proestrus/metestrus groups, where pain sensitivity differences appeared.
Elevated plus maze
To measure anxiety-like behaviors, we used the EPM, which is an apparatus shaped like a plus sign made of dark gray PVC. It has two open arms measuring 12 cm × 50 cm each and two enclosed arms measuring 12 cm × 50 cm × 50 cm each that surround a middle platform measuring 12 cm × 12 cm, 50 cm above the floor. At the beginning of each trial, a rat was gently placed on the middle platform facing an open arm and then allowed to explore the EPM (90 lx) for 5 min. The subsequent video analysis assessed the time spent in the open and closed arms, number of entries made into the open or closed arms (where an entry was defined as all four paws in a particular arm), head dips, and risk assessment. Risk assessment was defined as the act of placing only the head or forepaws in the open arm without any accompanying movement of the hind legs, even if the rat subsequently entered the arm. The percentage of time spent in the open arms was calculated using the following formula: open arm time / (center + open arm + closed arm time) × 100. While center time was calculated using: center time / total time (center + open arm + closed arm time) × 100
Open field test
To assess the locomotor activity of the animals, we used the open field test, which measures the movement of test rats (33). The apparatus comprised four uniformly sized arenas, each measuring 50 cm × 50 cm × 50 cm and was constructed from dark gray PVC. One day before testing, the rats were habituated to the experimental room for 15 min. On the test day, the rats were brought into the experimental room and habituated for 5 min before the test started. The rats were gently placed in the center of the arena facing a random side, and locomotor activity was measured during a 30-minute test (50 lx). The distance travelled in the OFT was measured in centimeters.
Novel object recognition test
To assess object recognition memory in rats, we employed a test that comprised two phases, namely the initial 5 min acquisition phase (P1) and the 3 min test phase (P2), separated by an inter-trial interval (ITI) of 15 min. The rats were habituated to the open field for 15 min one day prior to testing. The objects under investigation were made of ceramics or glass. To ensure the accuracy of the test results, all objects and the test arena were thoroughly cleaned and dried with 70% ethanol before and during the test. We have conducted preliminary tests in our laboratory to find equally attractive to the subjects (approximately 50% preference) (data not shown) and used these for the test (see Supplementary materials). During P1, the rat was placed in the center of the open field and exposed to two identical unknown objects (A), after which the rat was returned to its home cage and the objects were cleaned and dried. In P2, the rat was returned to the open field and presented with the familiar object A′ (an identical copy of the object presented in P1) and a novel test object (B). The duration of object exploration (sniffing, touching an object with whiskers, and licking) was recorded for both P1 and P2. The discrimination between the exploration time of the novel object and the familiar object was expressed as a percentage of the total exploration time of both objects during P2 [100/(A′+B) × B], whereas the discrimination index was calculated by subtracting the exploration time of the familiar object A′ from the novel object B in P2 (B − A′).
Social interaction and recognition memory
To evaluate social interactions and social recognition memory (SRM) in rats, we utilized an experimental design, as described previously (34). The test involved exposing the experimental rat to an unfamiliar young adolescent same-sex social partner (5–6 weeks old) for a duration of 5 min in the open field. No habituation was required, as the rats had already been exposed to the open field across the OFT and NOR. The experimental rat was placed in the open field and allowed to explore for 1 min, after which the stimulus rat was placed in the open field, and the SIT test began. The frequency of various social behaviors, including contact behavior such as social exploration including anogenital and non-anogenital investigations, were quantified for only the experimental rat. Additionally, the frequency of rearing and self-grooming was recorded.
In the second part of the test assessing social recognition memory, the initial 5-minute social interaction period with the unfamiliar social partner (A) served as the sample phase (P1) for the social recognition test (P2). In the subsequent test for social recognition memory, a second unfamiliar adolescent of the same sex (B) was introduced during the test (P2) after a 15-minute inter-trial interval. During P2, the familiar (A') and novel social partners (B) were presented to the experimental animal for 3 min, and the time for social investigation (anogenital, non-anogenital exploration, and approach/following) for the test rat was recorded. To calculate the social discrimination percentage, we used a within-subjects design, where we calculated the exploration time of the novel conspecific expressed as a percentage of the total exploration time of both conspecifics during P2 [100/(A'+B) × B].
Thermal pain sensitivity
Thermal pain sensitivity was quantified using a hot plate apparatus (Ugo Basil, New Jersey, USA) with a fixed temperature of 52.5°C ± 0.1°C. This experimental setup was conducted in accordance with the methods established in previous studies (35), and video recording of the behavior was analyzed offline frame-by-frame. The experiment was performed in the colony room of the experimental rats to reduce potential environmental stress-induced analgesia (36). In short, rats were gently placed onto the hotplate platform at the beginning of the experiment when the hotplate was at 52.5°C, and the test was terminated as soon as the rat showed the first heat-provoked reaction or after a cut-off period of 30 s to avoid tissue damage (which no rat reached). The first heat-evoked responses, including foot shake, stamping, paw licking, or jumping off the platform, which were used as a cut-off measure of pain.
Tissue collection and preparation
Rat brains were collected within the first two hours of the start of the inactive cycle. The rats were first dazed and then quickly and painlessly decapitated using a guillotine. The brains were quickly but carefully removed from the skull and flash frozen in 2-Methylbutane (-40°C) until completely frozen (~ 20-40s) and stored at -80°C until further processing.
Brain section preparation
To prepare the flash-frozen brains for sectioning, they were first removed from the freezer (-80°C) and placed in a cryostat-microtome (~ -20°C) (Leica CM 1950, Leica Biosystems) for 1 h for acclimatization prior to sectioning. After acclimatization, frozen brains were embedded in the specimen stage using O.C.T™ (Tissue-Tek) compound consisting of water-soluble glycols and resins. The brains were sectioned into 12 µm slices using a sharp blade, and brain sections were collected from the brain regions of interest using stereotaxic coordinates (Paxinos and Watson 2007). Brain sections from the following Bregma levels were collected; medial prefrontal cortex, Bregma: +3.20 to + 2.20, Nucleus accumbens shell and, Bregma: +1.70 to + 1.00, PVN), Bregma: amygdala, PVT, Bregma: -2.12 to -3.2), and Ventral tegmental area, Bregma: -5.2 to -6.00) (Appendix 7). Slices were collected and embedded onto gelatin-coated SuperFrost Plus slides (Thermo Fisher Scientific) and stored at − 20°C until further analysis.
Saturated oxytocin receptor autoradiography
Receptor autoradiography was performed for OTR using the [125I]-Ornithine Vasotocin Analog (d(CH2)5[Tyr(Me)2,Thr4,Orn8,[125I]Tyr9-NH2]-OVTA; (Perkin Elmer) as the hot ligand, while OT was used as the cold ligand to determine non-specific binding, as previously performed in our lab (Hansson et al. 2018). The specificity of these ligands has been previously reported (37, 38).
Prior to beginning the experiment, the frozen slides were kept at room temperature for 1 h for acclimatization. Slides were then incubated in room temperature pre-incubation buffer (50 mM Tris-HCl, pH 7.4) twice for 5 min before being transferred into cold pre-incubation buffer. Next, the sections were placed in a humidified chamber surrounded by ice, and 800 µL of reaction mix containing50 pM [125I]-OVTA (specific activity:2200 Ci/mmol (PerkinElmer), 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.1% bovine serum albumin, and 0.05% bacitracin was applied to each slide so that all sections were fully covered. Slides were incubated for 60 min at room temperature, and non-specific binding was determined by the addition of 2 µM OT (Tocris) into the incubation mix with [125I]-OVTA. Incubation was stopped by washing the sections three times with ice-cold washing buffer (50 mM Tris-HCl, 10 mM MgCl2) for 5 min, followed by dipping in ice-cold deionized water. Last, the sections were dried overnight under a stream of frigid air and left to dry overnight in the cold room (4–6 Celsius).
To visualize and analyze the data, phosphor imaging plates (FUJI imaging plates, Storage Phosphor BAS-IP SR2025 Screen, GE Healthcare Life Sciences) were exposed for 72 h to the slides with brain sections and scanned in a phosphoimager (Fuji Phosphoimager Typhoon FLA 700, GE Healthcare Life Sciences), as previously described (Hansson et al. 2018). Digital images of the phosphor imaging-generated data were analysed using MCID Image Analysis Software (InterFocus Imaging Ltd). Regions of interest (ROI) were defined based on anatomical landmarks, as illustrated in Fig. 7. The total and non-specific binding (in the presence of the cold ligand) was determined for each ROI on adjacent sections, and the non-specific signal was subtracted from the total signal of each ROI. Similar to our previous work, [125I]-quantitation standard curves (Amersham, GE Healthcare Life Sciences) were used to extrapolate the measured optical densities (photostimulable luminescence per mm2) of the tissue-equivalent OXTR densities from sections into nCi/mg (38). Binding in femtomoles per milligram (fmol/mg) was calculated according to the saturation binding equation (B = Bmax*[R]/(Kd +[R]), where Bmax represents the maximal bound receptor, Kd represents receptor affinity (Kd = 0.1 nM) in rat tissue (39), and [R] represents the concentration of the radioligand with which the specific activity of the radio ligand could be calculated. Data are defined as 0% (CTL) and changes in binding density show increase and decrease from baseline in Fig. 7 and raw data expressed as fmol/mg protein (mean ± SEM) can be found in the Supplementary Materials (Appendix 7 & 8).
Data analysis
Since we collected data from two cohorts of rats (see Methods), we first tested for cohort differences with a student’s t-test. The cohorts did not difference statistically in any of the behavioral tests and therefore proceeded with a combined analysis of both cohorts. The data analysis proceeded using univariate and mixed analysis of variance (ANOVAs) and statistically significant interactions, and main effects were followed up using Bonferroni-corrected pairwise comparisons, except when the interaction involved a within-group factor; paired t-tests were used. An alpha level of p < 0.05 (two-tailed) was set as the level of statistical significance, and we report partial eta squares as estimates of effect sizes or Hedge’s g along with individual data points for clarity. Statistical analyses were conducted using SPSS (29.0), and all graphs were illustrated in GraphPad Prism (8.0).