Serotonin deficiency induced after brain maturation rescues consequences of early life adversity

Brain serotonin (5-HT) system dysfunction is implicated in depressive disorders and acute depletion of 5-HT precursor tryptophan has frequently been used to model the influence of 5-HT deficiency on emotion regulation. Tamoxifen (TAM)-induced Cre/loxP-mediated inactivation of the tryptophan hydroxylase-2 gene (Tph2) was used to investigate the effects of provoked 5-HT deficiency in adult mice (Tph2 icKO) previously subjected to maternal separation (MS). The efficiency of Tph2 inactivation was validated by immunohistochemistry and HPLC. The impact of Tph2 icKO in interaction with MS stress (Tph2 icKO × MS) on physiological parameters, emotional behavior and expression of 5-HT system-related marker genes were assessed. Tph2 icKO mice displayed a significant reduction in 5-HT immunoreactive cells and 5-HT concentrations in the rostral raphe region within four weeks following TAM treatment. Tph2 icKO and MS differentially affected food and water intake, locomotor activity as well as panic-like escape behavior. Tph2 icKO prevented the adverse effects of MS stress and altered the expression of the genes previously linked to stress and emotionality. In conclusion, an experimental model was established to study the behavioral and neurobiological consequences of 5-HT deficiency in adulthood in interaction with early-life adversity potentially affecting brain development and the pathogenesis of depressive disorders.

Brain serotonin (5-HT) system dysfunction is implicated in disorders of emotion regulation, such as anxiety and depression, viewed as multifactorial conditions influenced by multiple gene-by-gene and gene-by-environment interactions 1 . Previous research identified genetic variants regulating the expression of the gene encoding the rate-limiting enzyme of neuronal serotonin (5-HT) synthesis, tryptophan hydroxylase-2 (TPH2) 2,3 , which converts the essential amino acid tryptophan (TRP) into 5-OH-TRP, the direct precursor of 5-HT 4,5 .
Depletion of TRP by dietary intervention (acute TRP depletion, ATD) has traditionally been used in assessing the influence of 5-HT deficiency on emotion dysregulation and the pathogenesis of depressive disorders. TRPfree diet produces an acute and profound reduction in brain TRP and consequently brain 5-HT synthesis [6][7][8] . ATD decreases anxiety in patients with anorexia nervosa 9 and also lowered mood in healthy participants and individuals with bulimia nervosa 10,11 . However, the mechanisms of ATD and the impact of reduced brain 5-HT on behavior are still not well understood 12 . Animal model studies provide evidence for alterations in brain 5-HT release following ATD, resulting in altered anxiety-and depression-related behavior 13 .
Genetic manipulation in mice, which results in embryonic inactivation of Tph2 or other 5-HT neuron development-related genes (e.g. Pet1, Lmx1b), provided insight into the behavioral and physiological consequences of 5-HT deficiency in the brain 14,15 . Several studies showed that neonates with a constitutive Tph2 inactivation present viability and growth-related problems compared to wildtype counterparts 16,17 . While these mice retain normal 5-HT neuron morphology and physiology, and are indistinguishable from controls in adulthood 18,19 , several behavioral phenotypes have been reported [20][21][22] . These phenotypes arising from lifelong Tph2 inactivation may be the consequence of impaired functioning of other relevant genes, the expression of which is, at least Induction of Tph2 inactivation. For temporal and spatial control of Tph2 recombination, mice homozygous for Tph2 exon five flanked by loxP-sites (Tph2 fl/fl ), the palindromic recognition sites of Cre recombinase 26 backcrossed onto a C57Bl/6N background were crossed with Tph2 null mutant mice (Tph2 −/− )to generate Tph2 fl/− hemizygous mice, which lack one Tph2 allele from the beginning of their life similar to heterozygous Tph2 knockout mice (Tph2 +/− ). These mouse lines were crossed with C57Bl/6N.Tg Tph2creERT2 transgenic mice, which express CreERT2 under the control of the murine Tph2 promoter exclusively in the raphe nuclei , to generate Tg Tph2creERT2 /Tph2 fl/fl and Tg Tph2creERT2 /Tph2 fl/− mice respectively. Tg Tph2creERT2 /Tph2 fl/fl or Tg Tph2creERT2 /Tph2 fl/− males were crossed with non-transgenic females in order to control the number of Tph2creERT2 transgenes (n = 1) in the animals. In order to initiate Cre/loxP-mediated recombination to generate an induced raphe nuclei specific Tph2 knockout (Tph2icKO), male Tg Tph2creERT2 /Tph2 fl/fl or Tg Tph2creERT2 /Tph2 fl/− mice aged 10-12 weeks were injected twice a day for 5 consecutive days with 1 mg of Tamoxifen (TAM; Sigma Aldrich, St. Louis, USA) 27 resulting in Tph2 Δfl/− mice or Tph2 Δfl/fl . In this respectTph2 Δfl/− mice model an Tph2icKO based on a hemizygous background, while Tph2 Δfl/fl mice model an Tph2icKO based on a wildtype like genetic background during development. Tg Tph2creERT /Tph2 +/+ mice injected with TAM (Tph2 Δ+/+ ) were used as controls. Vehicle injected mice of same genotype were used as controls in the maternal separation experiments (Tph2 CON).
Immunohistochemistry. The efficacy of TAM-induced time-specific Tph2 inactivation in the brainstem raphe region was assessed by fluorescence immunohistochemistry in the 4th and 6th week after treatment. Thin sections (30 µm) of brain of mice (n = 7-9/group) were cut on cryostat and stored at − 80 °C. Frozen sections were dried for 15-20 min. An antigen retrieval was conducted as previously described 26 . Sections were cooled down to 40 °C and washed 3 × 5 min in Tris-buffered saline (TBS). Unspecific binding sites for the antibodies were blocked for 90 min at room temperature (RT) with blocking solution (5% normal goat serum, 0.25% Triton-X100 in TBS). Sections were incubated overnight with the primary antibody (1:400, goat-anti 5-HT; Immunostar; Hudson; USA) diluted in blocking solution (TBS-T) at 4 °C in a humid chamber. Following three 5-min washing steps in TBS, sections were incubated in the dark with the respective fluorescent secondary antibodies (1:400 Cy3) diluted in blocking solution, lasting 90 min. Sections were washed 3 × 5 min in TBS. For staining of the cell nuclei, sections were treated with 300 µm DAPI diluted 1:1000 in TBS for 5 min. The number raphe neurons that were immunoreactive (ir) for 5-HT in dorsal (DRN: B6, B7) and median raphe nuclei (MRN: B5, B8, B9) 28,29 based on cell nuclei surrounded by anti-Tph2 signal using Fiji software 30 on four consecutive pictures of anterior raphe (DRN and MRN) corresponding to Bregma-4.95 mm to-4.47 mm spaced 180 µm 31 . Neurochemistry. For high performance liquid chromatography (HPLC), three brain regions (hippocampus, the dorsal raphe and the amygdaloid complex) were quickly dissected under a stereo microscope. For this, the brain (n = 4-7) was sliced with the aid of a metallic matrix, which allows sectioning at equal intervals on a cold plate. The identified regions were dissected out with a preparation spatula and kept frozen at − 80 °C until use. The brain homogenates were prepared and analyzed to standard protocols 19,25 . Body weight and food intake. Body weight of mice (n = 18-25) used for the baseline behavioral study was measured weekly for 7 weeks starting from the first week of injection. Body-weight measurements were conducted from 10 to 11 a.m. Two weeks after the last injection, mice were single-housed and their food and water-intake was measured weekly.
Behavioral assessment. Mice were subjected to behavioral testing starting from 4 weeks after TAM injections. In the baseline study, one group of mice (n = 13-15/group) was first tested for anxiety-like behavior in the light-dark transition test (LDT), followed by an open-field test (OF) to assess locomotor activity in a novel inescapable environment 32 . A second group of mice (n = 7-11/group) was used to assess anxiety-and depressionlike behaviour including an elevated-plus maze test (EPM), sucrose preference test and lastly Porsolt swim test (PST) 33 . Observations were recorded with VideoMot2 (TSE Systems, Bad Homburg, Germany) and later analyzed with EthoVision XT 11.5 (Noldus, Wageningen, The Netherlands). For details see supplementary methods. www.nature.com/scientificreports/ Maternal separation. In the second study, pups of the Tg Tph2creERT2 /Tph2 fl/fl genotype were separated on the second postnatal day (P2) from their mothers and kept in fresh cages for 3 h daily (between 10.00 and 13.00), for 14 consecutive days (P2-16). Ambient temperature was ensured by infrared light, positioned 70 cm above the cage. Non-MS mice were not separated from the dams but were handled during routine cage changes. Mice were weaned at 25 days after birth and kept in groups of 2 to 5 mice per cage and injected with either TAM (Tph2 Δfl/fl ) or vehicle (CON) (n = 8-10/group). After 4 weeks, mice were tested for anxiety-related behavior in the EPM, LBD and OF followed by SPT and FST for depression related behavior with 3 days inter-trial time, all tests were done as described in supplementary methods.
Quantitative real-time PCR. Quantification of relative gene expression was performed by quantitative real-time PCR (qRT-PCR). cDNA was generated as previously described 34 . The reaction was run in triplicates using SYBR green dye according to manufacturer instructions. Reaction mixture comprised 6 µl (SYBR green + Primer (F + R)) mix and 4 µl cDNA making 10 µl reaction volume each. Mean efficiencies were calculated by LinReg 35 . Relative expression data were calculated by qBase + (Biogazelle, Zwijnaarde, Belgium), with the normalization factors obtained from geNorm (geNorm M < 0.5) 36  Data analysis. Data obtained from this study were analyzed and displayed using GraphPad Prism version 6.07 for Windows (GraphPad Software, La Jolla California USA, www. graph pad. com). The total numbers of 5-HT ir cells counted in the anterior raphe of Tph2 icKO were compared with that of Tph2 CON mice using Kruskal-Wallis statistic (H), while Dunn's multiple comparison test was used to compare means between the groups of mice. Behavioral outcomes on baseline anxiety-and depression-like behavior were analyzed by one-way ANOVA. The course of 5-HT depletion from week 2, 4 and 6 were analyzed by two-way ANOVA as well as MS and gene expression data, with Tukey's multiple comparison post hoc test used to compare means. Students t-test was used for comparisons between Tph2 Δ+/+ and Tph2 fl/fl mice (Figs. S1 and S2). An α < 0.05 was set as level of significance.

Efficiency of induced Tph2 inactivation.
The half-life of Tph2 is approximately 2.5 days 37 . Thus, we first evaluated the time required after induced Tph2 recombination to reduce the amount of Tph2 synthesis to a level similar to constitutive Tph2 KO mice by immunohistochemistry (Fig. 1a). Four weeks after TAM treatment, effective recombination of Tph2 was indicated by a significant drop of 5-HT ir cells in the DRN ( Neurochemistry. In order to relate the absence of 5-HT ir cells to concentrations of monoamines in the raphe and target brain regions, we measured the concentrations of 5-HT and its metabolite, 5-hydroxyindolacetic acid (5-HIAA), as well as norepinephrine (NE) and dopamine (DA) in the raphe, hippocampus and amygdala at all three time points after TAM induction. Two-way ANOVA revealed a tendency towards significance in the raphe for genotype × time interaction (F (4,40) = 2.299; p = 0.0756). Indeed, from week 2 to 6 Tph2 Δ+/+ presented increased 5-HT concentrations (p = 0.0032) in the brainstem at a comparable level to wildtype (Tph2 +/+ ) controls 19 , and showed significantly higher concentrations compared to Tph2 Δfl/− (p = 0.016) and Tph2 Δfl/fl (p = 0.0783) mice. The concentration of 5-HT in Tph2 Δfl/− and Tph2 Δfl/fl mice was relatively stable at all time points, remaining at low concentrations (67.0 ± 13.6 ng/ml), similar to those reported in constitutive Tph2 −/− mice 19 (Fig. 2a, left panel). Moreover, the concentrations of the 5-HT metabolite 5-HIAA remained consistently low in both genotypes of Tph2 icKO mice at all time points examined. However, two-way ANOVA revealed a significant genotype × time interaction (F (4,40) = 8.157; p < 0.0001; Fig. 2b, left panel), showing higher 5-HIAA in Tph2 Δ+l+ compared to Tph2 Δfl/fl and Tph2 Δfl/− (both p < 0.0001) only two weeks after injections.

Anxiety-and depression-like behavior in MS-exposed mice.
Several studies suggested that the functionality of 5-HT in adulthood may be primed by early-life adversity to render an individual susceptible to emotion related disorders [38][39][40] . Therefore, Tg Tph2creERT2 /Tph2 fl/fl mice, were subjected to MS stress from P2-P16, injected with TAM in adulthood and tested for anxiety-and depression-like behavior (Fig. 4a).
In the OF test, no MS × treatment interaction in the number of visits to the center (F (1,32) = 2.275; p = 0.1413; Fig. 4h) was detected. Considering the total time spent in the center, a significant MS × treatment interaction effect (F (1,32) = 6.562; p = 0.015; Fig. 4i) was observed. In the Non-MS group, Tph2 Δfl/fl mice showed a tendency towards less time in the center (p = 0.077) compared with Tph2 CON. Inter-group comparison revealed that Non-MS Tph2 Δfl/fl mice were more anxious than MS-exposed Tph2 Δfl/fl mice (p = 0.035). Furthermore, MS exposure potentially increased total distance travelled in the aversive center (F (1,32) = 6.562; p = 0.09; Fig. 4j).
Furthermore the SPT showed no significant effects on sucrose preference (F (1,31) = 0.3531; p = 0.5566; Fig. 4m). With respect to total fluid consumed, a significant main effect of treatment occurred (F (1,31) = 20.9; p < 0.0001; Fig. 4n). Tph2 Δfl/fl mice consumed more fluid than Tph2 CON mice independent of aversive early-life stress experience. These outcomes indicate that reduction in adult brain 5-HT concentrations may not predispose to lack of pleasure that characterizes anhedonia but rather increases energy metabolism similar to constitutive Tph2 KOmice 20,71 .
Gene expression. The effect of MS-induced anxiety-related behavior on expression of genes, that are viewed as indicators of 5-HT system functionality in the raphe region, hippocampus and amygdala was also examined. Tph2 expression indicated no significant MS × treatment interaction in the raphe region (F (1,31) = 0.1104; p = 0.7419). However, a significant main effect of treatment (F (1,31) = 30.05; p < 0.0001) was apparent (Fig. 5a). Thus, expression of Tph2 was significantly reduced both in Non-MS and MS-exposed Tph2icKO mice which confirms the efficiency of conditional Tph2 inactivation.
Avpr1a was reported to influence anxiety in humans and rodents 34 . In this study a significant MS × treatment interaction (F (1, 31) = 5.44; p = 0.0263; Fig. 5e) was observed in Avpr1a expression in raphe. Post-hoc analysis revealed Avpr1a expression in MS exposed Tph2 CON mice was higher than MS-exposed Tph2 icKO (p = 0.017).
No differences were detected in the Non-MS cohort in both hippocampus and amygdala (Fig. S4d).

Discussion
The first part of this study capitalized on the TAM induced Cre-mediated inactivation of Tph2 to significantly reduce 5-HT synthesis in adult mouse brain. A transgenic Tph2-CreERT2 mouse line and the induction protocol described by (Weber et al. 2011) was used to induce a Tph2 knockout in adulthood. Although few 5-HT positive cells in the raphe region remained, which may point to ineffective nuclear translocation of Tph2-CreERT2 probably due to reduced expression of Tph2 in certain 5-HT immunoreactive neurons, we achieved similar recombination efficiencies as reported for other induced Tph2 KO mice using CMV-CreERT2 transgenics as Cre driver line 41 or using AAV-Cre viral injection into the rostral raphe nuclei 42 . Furthermore, measurement of 5-HT concentrations four weeks after TAM induction in the raphe, hippocampus and amygdala revealed efficiently reduced 5-HT concentrations in Tph2 icKO comparable to constitutive Tph2 −/− null mutant mice 19 . Efficient reduction of brain 5-HT and 5-HIAA within 4 weeks of TAM treatment has previously been reported 43 , which indicates that the TAM-mediated induction approach requires a relatively long time period before a significant deficiency in brain 5-HT concentration is established. However, the HPLC results showing low concentrations of 5-HT in Tph2 Δ+/+ combined with highly increased 5HIAA levels at week 2 differed to previous results of non-injected Tph2 +/+ mice in previous studies 19 . The increased 5-HT turnover in wildtype controls due to the stressful TAM injection protocol may represent a short time adaptive mechanism of the 5-HT system probably similar to restraint stress models 44 . Although, 5-HT concentrations in Tph2 Δfl/− and Tph2 Δfl/fl mice did not differ in comparison with Tph2 Δ+/+ , the low 5-HIAA levels in both Tph2 Δfl/− and Tph2 Δfl/fl groups reflect reduced 5-HT turnover similar to Tph2 −/− null mutant mice in the DRN 19 . Nevertheless, it seems that it takes at least four weeks in Tph2 Δ+l+ mice to recover from the injection stress indicated by increasing 5-HT concentrations in the DRN similar to that of Tph2 +l+ wildtype mice without any injection 19 . Interestingly, in the hippocampus and amygdala this effect on Tph2 Δ+/+ is detected as well but it seems that it takes longer after TAM injections that 5-HT levels become degraded as compared to the raphe nuclei, which might be due to differentially edited or spliced Tph2 mRNA isoforms with enhanced stability itself and or its coding proteins 45 within serotonergic projections targeting these regions. Still, remaining 5-HT in induced Tph2 Δfl/− and Tph2 Δfl/fl mice may arise from blood platelet 5-HT due to inefficient perfusion of the brain and/or from blood-borne 5-HTP derived from Tph1 enzymatic activity, which may cross the blood brain barrier and be converted into 5-HT by AADC 16,19 .
In line with the above explanation is an early increased concentration of NE in the brain coupled with expected reduction in 5-HT concentrations, which was probably caused by the injection procedure 44 . This shows that acute stress coupled with reduced 5-HT metabolism has an immediate influence on the NE and dopamine www.nature.com/scientificreports/ systems 42 . An effect, which normalized in the 4th week after TAM injections. Thus, the observed alterations in brain DA and NE concentrations add on the results from other studies 42,43 . Interestingly, unlike Tph2 −/− mice, which exhibit reduced anxiety-like behavior 21,22,46 , Tph2 Δfl/− and Tph2 Δfl/fl mice showed no differences in anxiety-and depression-like behavior. However, locomotor hyperactivity or panic-like responses in aversive inescapable novel environment in Tph2 −/− mice 32 and locomotor hyperactivity as described for Tph2 icko mice 42 were specifically observed in Tph2 Δfl/− , which resemble genetic heterozygous knockout mice with a 20-30% reduction in raphe 5-HT levels 19 . This is in line with reports of involvement of DR and MnR 5-HT in responses to future threat 47,48 . In contrast, Tph2 Δfl/fl did not differ in any behavioral assessment compared to Tph2 Δ+/+ mice. Indeed, it points towards a two-hit effect 49 . Based upon a primed heterozygous genetic background during development, an induced or acquired 5-HT deficiency may provoke more pronounced panic-like flight responses in inescapable aversive conditions, while a development without changes in the 5-HT system like in Tph2 Δfl/fl is able to compensate further environmental or genetic impacts in adulthood despite the near complete ablation of 5-HT synthesis.
Interaction of early life MS stress and aberrant adult brain 5-HT on behavior. In order to further investigate this effect during early development, we used early life MS, which has been commonly used to study G × E interaction on anxiety and depression in mice 50,51 and has been shown to impact anxiety-like behaviors in rodents via the 5-HT system 52,53 . Exposure of wildtype mice, on a mixed c57BL6/J-129S6/Sv background, to MS significantly decreased distance travelled and time spent in the center of the OFT, while MS had no effect in Tph2 KI mice 54 . Here, we could show that MS produced a similar effect in vehicle injected Tph2 fl/fl control mice and this effect was rescued by inducing a Tph2 deficiency. This shows that MS exposure and reduced brain 5-HT differentially influence anxiety-like behavior and may compensate each other.
Thus, this study complements numerous studies, which focused specifically on manipulation of Tph2 expression in adult mouse brain and its effect on emotion-related behavior 42 . Aberrant 5-HT neurotransmission in the adult brain either via pharmacological interventions 55,56 or induced gene inactivation 42,43 was unable to destabilize behavioral adaptive mechanisms that are established during early brain maturation.
Notably, neither MS nor induced adult 5-HT deficiency altered preferences for sucrose solution indicating no differences in hedonic-like behaviors. Furthermore, no alterations in depression-like behavior in the PST were observed in this study. One explanation may be that maternal care on reunion with pups increased and this may have dissolved the MS effect on depression-like behaviors. Thus, it may well be that the MS protocol used was not robust enough to impact depression-like behavior in the mice 57 and requires further studies including investigation of maternal behaviour. MS exposure neither affected hedonic behavior in rats 58 , nor did it impact anxiety-and depression-related behavior in C57BL/6 mice 59 . Even in TPH2 KI mice, which have reduced brain 5-HT concentrations throughout life, MS exposure did not alter anxiety-and depressive-like behaviors 60 . By contrast, some studies have associated MS exposure with abnormal behavior and stress induced alteration in neurotransmitter concentration 61,62 . Noteworthy, most rodent studies, which found an association between MS with behavioral alterations, were largely done in rats [62][63][64] . Indeed, some C57BL/6 mouse strains appear to be resilient to neonatal MS stress 57,59,[65][66][67][68][69][70] , which may explain the weak MS effects observed in this study.
However, Tph2 icKO mice consumed more fluid than Tph2 CON mice, which points towards an acute metabolic effect of 5-HT deficiency in Tph2 KO mice 71 . The observed increase in food, water and percentage sucrose consumption by Tph2 Δfl/− and Tph2 Δfl/fl mice has also been reported in Tph2 −l− mice 20,71 , which reflects an increased energy need, rather than altered anhedonia as a symptom of depressive-like behavior. This supports the assertion that strong reductions in 5-HT metabolism in adulthood are implicated in the pathophysiology of eating disorders through various hormonal and receptor systems 72,73 , independent of 5-HT functions during development.
Gene expression in raphe, hippocampus and amygdala. Early-life stress does not alter the expression of Tph2 in mice 60 and rats 52 . In contrast, an association between MS exposure and raphe region-specific reduction in Tph2 expression of C56BL/6 J mice has been reported 74 . Here, the relative expression of Tph2 in Tph2 icKO was significantly lower than Tph2 CON mice in raphe, while MS exposure alone did not alter Tph2 expression.
We detected no differences in expression of Htr1a, Htr2a and Avpr1a in target regions of MS-naive mice. This confirms our earlier work, which also reporting no change in expression of Htr1a and Htr2a genes in non-stressed Tph2 conditional KO mice 75 . A slightly altered expression of Maoa in amygdala and hippocampus may point to specific compensatory mechanisms due to MS 53 . Furthermore, MS exposed mice showed altered expression of Htr1a and Htr2a in the raphe independent of Tph2 icKO, which is in line with previous MS studies 76,77 . Alterations in 5-HT receptors in target regions of 5-HT neurons have previously been associated with altered anxiety and exploratory behavior 78 . Here, this may explain the dampening effect of MS on total locomotor activity in OF and EPM.
However, a Tph2 icKO reduced expression of Maoa in the raphe region, pointing towards a direct effect of strong 5-HT depletion on MaoA dependent 5-HT turnover processes. Thus, effects mediated by adverse life experience and associated with altered Maoa expression 34 may be prohibited in Tph2 icKO mice through a dysfunctional 5-HT system in adulthood. Interestingly, Avpr1a expression was only affected in the raphe after the mice were maternally separated. We did not find altered Avpr1a expression in the hippocampus, which was correlated after late adverse life experiences with reduced anxiety-like behavior 34 . Nevertheless, Avpr1a is present in the dorsal raphe, the mesencephalic central gray and the caudal linear raphe 79 . Additionally, an Avpr1a knockout as well as pharmacological blockade of Avpr1a function in rodents reduced aggression and resulted in anxiolytic and anti-depressive-like effects 80 www.nature.com/scientificreports/ Tph2 icKO mice may be directly attributable to altered Avpr1a expression. This highlights the interaction of arginine vasopressin-dependent signaling with the 5-HT system in the brainstem following MS as a potential therapeutic target for treating emotional dysregulation. In conclusion. Our findings establish a "double-hit" experimental model to study the behavioral and neurobiological consequences of 5-HT deficiency in adulthood in interaction with early-life stress experience potentially affecting emotion regulation and the pathogenesis of depressive disorders.