P11 deficiency increases stress reactivity along with HPA axis and autonomic hyperresponsiveness

Patients suffering from mood disorders and anxiety commonly exhibit hypothalamic–pituitary–adrenocortical (HPA) axis and autonomic hyperresponsiveness. A wealth of data using preclinical animal models and human patient samples indicate that p11 deficiency is implicated in depression-like phenotypes. In the present study, we used p11-deficient (p11KO) mice to study potential roles of p11 in stress responsiveness. We measured stress response using behavioral, endocrine, and physiological readouts across early postnatal and adult life. Our data show that p11KO pups respond more strongly to maternal separation than wild-type pups, even though their mothers show no deficits in maternal behavior. Adult p11KO mice display hyperactivity of the HPA axis, which is paralleled by depression- and anxiety-like behaviors. p11 was found to be highly enriched in vasopressinergic cells of the paraventricular nucleus and regulates HPA hyperactivity in a V1B receptor-dependent manner. Moreover, p11KO mice display sympathetic–adrenal–medullary (SAM) axis hyperactivity, with elevated adrenal norepinephrine and epinephrine levels. Using conditional p11KO mice, we demonstrate that this SAM hyperactivity is partially regulated by the loss of p11 in serotonergic neurons of the raphe nuclei. Telemetric electrocardiogram measurements show delayed heart rate recovery in p11KO mice in response to novelty exposure and during expression of fear following auditory trace fear conditioning. Furthermore, p11KO mice have elevated basal heart rate in fear conditioning tests indicating increased autonomic responsiveness. This set of experiments provide strong and versatile evidence that p11 deficiency leads to HPA and SAM axes hyperresponsiveness along with increased stress reactivity.

3 building measurements, litter nests were carefully removed, and a wad of pure cotton weighing 2 g was placed in the food tray. Since all cotton pulled into the cage by the mice was added to the nest, the percentage of the provided nesting material used in a 24-h period was considered a reliable measure of nest construction 4 . In the pup retrieval test, pups were removed from the home cage for 5 min, and placed in a separate clean cage kept at 33±1°C under an infrared heat lamp. Four pups were scattered in the quadrant opposite to the nest and the time the mother retrieved the first and the last pup was recorded within a 10-min period.
Nesting behaviors and licking/grooming were also manually scored during the observation period.

Adult behavioral phenotyping:
We found no rearing-induced differences in adult behavioral phenotype (data not shown), therefore animals from the MS groups seen in Figure 1a were excluded from the subsequent adult phenotyping results and statistics. Resilience to maternal stress was previously reported in some mouse strains, including C57BL/6, whose dams are characterized as providing better care to their pups following periods of deprivation 5,6 . This suggests that the gene  environment interaction observed in early postnatal age was dissipated by overcompensation of maternal care. Nevertheless, it is also evident that the response of C57BL/6 pups to maternal care and the life-long consequences of p11 deficiency are mutually exclusive.
Mice were tested at one-week intervals, i.e. testing took place over four consecutive weeks and tests took place at identical times of the day, to avoid circadian-dependent changes.

Open field test:
Mice were placed in a 55 cm x 55 cm squared box for measurement of locomotion during a trial duration of 5 minutes. Total distance travelled, average speed and time spent in the 4 centre zone were measured using an automated video tracking system (Noldus Ethovision XT 11, Wageningen, Netherlands).

Post-shock vocalizations and passive avoidance test:
Mice were placed in the light compartment of a step-through passive avoidance apparatus (Ugo Basile, Comerio-Varese, Italy) for 60 s, before a sliding door was opened and the mouse entered a dark compartment (training latency). After the animal stepped into the dark compartment with all four paws, the door automatically closed and a weak electrical stimulus (0.3 mA, 2 s duration, scrambled current) was delivered through the grid floor. Immediately post-shock, mouse vocalizations were recorded for 5 s and the number of vocalizations emitted as well as vocal amplitude were measured using Avisoft Ultra Sound Gate Analyzer (Avisoft Bioacoustics, Glienicke, Germany). After a 24-h delay, a mouse was again placed in the light compartment and the step-through latency to return to the dark compartment was measured (retention latency).

Light-dark exploration test:
The light-dark exploration test was conducted as previously described 7 . The cumulative time spent by mice in a brightly illuminated vs. dark compartment over a 15-min session was detected and analyzed using an automated video tracking system (Noldus EthoVision XT 8, Wageningen, The Netherlands).

Response to acute swim stress, serum collection and quantification of hormones:
Testing took place between 09:00 and 12:00 for all sets of all cohorts, to ensure that no circadian variation in hormones confounded the results. Mice were placed into 50 cm tall tanks filled with water (temperature 24±1°C) to 20 cm depth with 7-min test duration. After removal from the water tank, mice were either gently dried and placed in their home cages 5 lined with dry paper towels for a 24-min (Supplementary Figure 3) rest, or were immediately (within 1 min, all other stress conditions) euthanized by decapitation. Immobility in the forced swim test was analyzed in one of the cohorts from video recordings using Noldus EthoVision.
For pup ACTH analysis, we collected blood samples from a separate set of non-stressed litters than the one used for USV recordings. Pups were taken from homozygous breeding pairs (WT: n=13, p11KO: n=15) at PND3, 6, 9 or 12. Sex was not differentiated given that it is widely accepted that ACTH is not sexually differentiated in this developmental period.
Litters were gently moved into a separate cage (kept at 33±1°C by electrically heated pads) to which some of the litter home cage bedding material was added. Trunk blood was quickly collected by decapitation. To meet the minimum assay volumes, each sample of trunk blood had to be pooled from 6 (PND3), 4 (PND6), or 3 (PND9 &12) pups.
Serum collection, ACTH and corticosterone assays were conducted as previously described 8 . ACTH (MD Biosciences, St. Paul, MN, USA) and corticosterone (Enzo Life Sciences, Farmingdale, NY, USA) were measured using commercially available ELISA kits, following the manufacturer's instructions. Serum samples were used undiluted (ACTH) or diluted 1:10 in assay buffer (corticosterone).
Tissue collection, RNA extraction, reverse transcription and quantitative PCR: Immediately following blood collection, brains were quickly removed, snap frozen in isopentane (Sigma-Aldrich, St. Louis, MO, USA) cooled in dry ice, and subsequently stored at -80°C. Adrenal glands were also dissected, frozen in dry ice and stored at -80°C.
In a cryostat chamber kept at -20°C, one 2 mm thick coronal slice (0.0 to -2.0 mm from bregma) was cut from each brain using a 0.5 mm interval mouse brain matrix (AgnThos, Lidingö, Sweden). A tissue piece of approximately 2 x 2 x 2 mm (width x height x thickness) generously encompassing the paraventricular nucleus (PVN) of the hypothalamus was excised 6 from this slice using a scalpel blade. The tissue pieces were immediately placed on dry ice and then kept at -80°C until RNA extraction.
Hypothalamic tissue samples were disrupted in lysis buffer (RNeasy Mini kit, Qiagen, Hilden, Germany) using an ultrasonic processor (EpiShear Probe Sonicator, Active Motif, La Hulpe, Belgium). Total RNA was subsequently extracted from the lysates (RNeasy Mini kit, Qiagen) and samples were treated with RNase-free DNase I (Thermo Scientific, Stockholm, Sweden).
Per sample, an aliquot of 0.5 µg DNase-treated RNA was primed with random hexamers and International, St. Paul, MN, USA) were implanted intraperitoneally as described before 14 .
ECG was remotely measured via a receiver board (RLA1020, Data Sciences International), digitally recorded (4 kHz sampling rate) and analyzed offline using Chart software (v7.1, ADInstruments, Spechbach, Germany). The intervals between successive R-peaks of the ECG complex served as inter-beat intervals to determine instantaneous HR (given in beats per min; bpm). ECG signals were digitally recorded at 4 kHz sampling rate. HR variability was calculated on the basis of the root-mean-square of subsequent heart beat interval differences (RMSSD, given in ms) 14 . After a recovery period of 2-3 weeks mice were subjected to novelty exposure for a total of 34 min as reported before 14

Experimental design and Statistical analysis
The sample sizes were based on previous reports to ensure adequate power. The experimenter was blinded to group allocation during experiment, and analysis was automated.
Animals were pseudo-randomly allocated to experimental groups, so that each cage would contain balanced group distribution. Outliers in the data were calculated using the Grubb's test calculator tool from GraphPad (GraphPad, San Diego, CA, USA), and removed.
Normality of the data distribution was checked with a Normal probability plot (InVivoStat program 16 ). Statistical analysis was carried out by two-way or three-way analysis of variance (ANOVA) or repeated measures ANOVA followed by Fisher's least significance difference (LSD) post-test, where indicated, using the InVivoStat 16 program. All data were plotted using GraphPad Prism 8 (GraphPad, San Diego, CA, USA) All data is presented as mean ± standard error of the mean (SEM) of the number of animals per group detailed in Supplementary Table   1.

Pup ACTH and Maternal behavior
To check whether the greater number of vocalizations seen in non-stressed p11KO mice reflects the HPA activity, we took trunk blood samples under non-stressed (control) conditions from a separate set of litters and saw that ACTH levels do not reflect the pups USVs ( Supplementary Fig. 1a). This finding is in line with evidence that USVs are negatively correlated with plasma ACTH concentrations under the stress hyporesponsive period 17 , and indicates that the USVs are a behavioral response differentially regulated from HPA axis activity at this stage of development.
Mouse maternal behavior is strongly influenced by pup calls in the ultrasonic range reviewed in 18, 19 . We therefore used an undisturbed set of litters (normal rearing conditions) to investigate maternal behavior of p11KO versus WT mice. We first evaluated nesting behavior  Figure 1e). Overall, we found no evidence for impaired maternal behavior in p11KO mice. In fact, some aspects of maternal behavior appear to be enhanced in p11KO compared to WT mice, perhaps as a consequence of enhanced USVs from p11KO pups (see above). This in turn suggests that exacerbated separation distress in p11KO pups is driven by endogenous factors rather than poor maternal behavior. 13

Mobility in the Open Field (OF) test
Measurements of mobility in the open field show that there is an overall higher mobility in the p11KO mice, as indicated by significantly higher pathlength (Supplementary Figure 2a) and average speed (Supplementary Figure 2b) in p11KO females, compared to wildtypes.
However, we did not see a significant difference in zone transitions (data not shown). Since we also see that p11KO mice have a significantly higher activity when placed in a novel environment (Figure 4f), results suggest that the higher mobility in p11KO may be a noveltyinduced arousal reaction.

Serum corticosterone levels in p11KO
We compared hormone concentrations at baseline (non-stressed, control), 1-min post-stress and 24-min post-stress (Supplementary Figure 3). Female p11KO mice had approximately 3fold higher baseline corticosterone when compared to that of WT mice. There was no effect of genotype on basal corticosterone secretion in male mice.
Swim stress increased circulating levels of corticosterone, at 1 min post-stress, in both genotypes and sexes. Female p11KO mice showed approximately 50% higher corticosterone level than WT mice 1 min post-stress. Corticosterone level at 24 min post-stress was 70% higher in female p11KO mice than WT mice (insert in Supplementary Figure 3).
Interestingly, corticosterone concentrations in male p11KO mice were the same as in WT at 1 min post-stress, but 50% higher than WT after a 24-min post-stress resting period

Norepinephrine and epinephrine levels presented as ng/mg adrenal tissue
Supplementary Figure 6 shows absolute measures of adrenal norepinephrine and epinephrine levels normalized against mg adrenal tissue. In each experimental run, epinephrine levels were higher than the norepinephrine levels, but the magnitude differed. To facilitate comparisons from these distinct experimental runs, we present these data as percent of WT vehicle/control in Figure 3 of the main paper. Figure 3 also indicates relevant statistical analyses.

P11 expression in cardiac tissue
We performed fluorescent in situ hydridization (RNAscope) with a p11 probe to detect its expression in WT mouse cardiac tissue (Supplementary Figure 7). Previous studies showed that p11 regulates cardiomyocyte calcium through 5-HT4 receptors 21 . We detected low amounts of p11 mRNA in cardiac tissue (Supplementary Figure 7).