6.1 Lack of brain serotonin affects postnatal
development and serotonergic neuronal circuitry
formation
Sara Migliarini1, PhD, Giulia Pacini1,3, PhD, Barbara Pelosi1,3, Gianluigi Lunardi2, PhD, Massimo Pasqualetti1,4, PhD
1Department of Biology, Unit of Cellular and Developmental Biology,
University of Pisa, Pisa, Italy
2Department of Oncology, Sacro Cuore Hospital of Negrar, Verona, Italy
3G.P. and B.P. contributed equally to this study 4To whom correspondence should be addressed:
Massimo Pasqualetti
Unit of Cellular and Developmental Biology University of Pisa
S.S.12 Abetone e Brennero 4, 56127 Pisa - Italy Tel +39-050-2211496 Fax +39-050-2211495 e-mail: [email protected]
Running title: Serotonin depletion affects serotonergic innervation
Grants supporting this work were Prin 2008 (200894SYW2) from Italian Ministry of Education, University and Research (MIUR), and Orphan_0108 program from Toscana Life Sciences Foundation to M.P. BayGenomics and MMRRC provided E14Tg2a.4 ES cells; Dr. D. Court provided DY380 cells; Dr. S. Dymecki provided ACTB::FLPe mouse line; Dr. Y. Bozzi provided BDNF probe.
ABSTRACT
Despite increasing evidence suggests that serotonin (5-HT) can influence neurogenesis, neuronal migration and circuitry formation, the precise role of 5-HT on central nervous system (CNS) development is only beginning to be elucidated. Moreover, how changes in serotonin homeostasis during critical developmental periods may have etiological relevance to human mental disorders, remains an unsolved question. In this study we address the consequences of 5-HT synthesis abrogation on CNS development using a knock-in mouse line in which the tryptophan hydroxylase 2 (Tph2) gene is replaced by the eGFP reporter. We report that lack of brain 5-HT results in a dramatic reduction of body growth rate and in 60% lethality within the first 3 weeks after birth, with no gross anatomical changes in the brain. Thanks to the specific expression of the eGFP, we could highlight the serotonergic system independently of 5-HT immunoreactivity. We found that lack of central serotonin produces severe abnormalities in the serotonergic circuitry formation with a brain region-specific effect. Indeed, we observed a striking reduction of serotonergic innervation to the suprachiasmatic and thalamic paraventricular nuclei, while a marked serotonergic hyperinnervation was found in the nucleus accumbens and hippocampus of Tph2 mutants. Finally, we demonstrated that BDNF expression is significantly up-regulated in the hippocampus of mice lacking brain 5-HT, unmasking a possible regulatory feedback mechanism tuning the serotonergic neuronal circuitry formation. On the whole, these findings reveal that alterations of serotonin levels during CNS development affect the proper wiring of the brain that may produce long-lasting changes leading to neurodevelopmental disorders.
Key words: serotonin, Tph2; serotonergic innervation; BDNF; neural
development; neurodevelopmental disorders.
INTRODUCTION
Serotonin (5-HT) is a neurotransmitter implicated in the modulation of numerous physiological processes including mood, sleep, aggressivity and sexual behaviour 1-4. Serotonergic neurons represent one of the most early-born and widely distributed neuronal systems in the mammalian brain. These neurons collectively form the raphe system and cluster in distinct nuclei (B1-B9) providing a widespread serotonergic innervation to the whole central nervous system (CNS) 5, 6. The synthesis of 5-HT and the expression of its receptors early in embryonic development, as well as its maternal and placental source to the foetus, has led to the hypothesis that serotonin could act as a growth regulator in specific developmental events 7-10. Support for a role of 5-HT in brain development comes from the idea that dysregulation of serotonergic signalling is at the origin of disorders thought to have developmental bases, such as schizophrenia, affective disorders, anxiety, autism and mental retardation 11-13. Genetic models in mice indicated that
excess of brain 5-HT levels obtained by knocking-out the serotonin transporter (Slc6a4) or monoamine oxydase-A (MAO-A), genes involved in 5-HT re-uptake and degradation, respectively, prevents the development of topographically organized whisker-barrel fields in the mouse somatosensory cortex 14, 15. Further, recent findings showed that increased 5-HT transmission could also alter normal cortical development causing incorrect interneuron migration 16. Taken together these observations indicated that normal 5-HT neurotransmission is required for a correct brain development. On the other hand, analysis of mouse models lacking most serotonergic neurons, obtained inactivating genes involved in the specification of 5-HT identity, such as Lmx1b and Pet1, failed to show brain malformations 17, 18. Recently, animal models with targeted inactivation of tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme for the synthesis of central 5-HT, have been generated 19-23. In different genetic models, serotonin depletion consistently resulted in physiological and behavioural disturbances 19, 22-24. However, lack of serotonin in Tph2 knockout mouse lines did not produce any gross cellular or morphological alteration in the CNS. How can these results be reconciled with the developmental role ascribed to serotonin? On one side, maternal availability of serotonin during early foetal life to the developing brain may explain the absence of overt CNS morphological defects. On the other, it can be hypothesized that lack of brain serotonin may selectively affect fine tuning modulation of developmental events, whose alterations would require subtle analysis at cellular level.
In this regard, the present work aims to investigate whether constitutive depletion of brain serotonin might affect development of the CNS and/or the serotonergic system itself. To this purpose we genetically introduced the enhanced green fluorescent protein (eGFP) into the Tph2 locus in a way to highlight the serotonergic neurons independently of serotonin immunoreactivity. Overall, we report that Tph2 -/- mice show a reduced body growth rate and a high mortality level and, most importantly, we found for the first time that lack of serotonin produces severe abnormalities in the serotonergic circuitry formation with a brain region-specific effect.
MATERIALS AND METHODS Generation of the Tph2::eGFP knock-in mouse alleles
Tph2::eGFP(FRT-neo-FRT) knock-in recombinant allele was generated by
homologous recombination in E14Tg2a.4 embryonic stem (ES) cell line 25. The targeting vector was prepared replacing 105 nucleotides of Tph2 gene downstream the first ATG codon with the cDNA coding for the vital reporter gene eGFP starting with its second codon. A selection marker cassette, flanked by FRT sites, was placed 3’ to the eGFP cDNA in the opposite transcriptional orientation. We obtained the BamHI - ClaI genomic fragment (pBKS-Tph2-BC) containing 3.3 kb and 3.9 kb upstream and downstream
the first ATG codon of the Tph2 gene, respectively. To generate the Tph2::eGFP(FRT-neo-FRT) targeting vector we used a recombination-based strategy carried out in DY380 bacteria 26. Two 500 bp long homology arms (HAs) were generated by PCR amplification of mouse pBKS-Tph2-BC plasmid DNA using the following primer sets: forward 5’-ATTATTGGATCCGAATTCATAATGTAACGTCATGCCAGCAGA-3’ and reverse 5’-ATTATTCCATGGCTCCCGATGTAATTTTCTTT-3’ for the 5’
homology arm, and forward
ATTATTCTCGAGTATCGAGTATCTGGCAGTCTCCG-3’ and reverse 5’-ATTATTGTCGACGAATTCTGCTTCCTTAACTCTTCCATCAATTA-3’ for the 3’ homology arm. To obtain the p-5HA/eGFP/3HA vector, the 5’ and 3’ homology arms were subcloned as BamHI-NcoI and XhoI-SalI fragments into the BglII-NcoI and XhoI sites of the pIRES-eGFP vector (Clontech), respectively. A removable neo selection cassette 27, was cloned into the XhoI site of the 5HA/eGFP/3HA plasmid to generate the p-5HA/eGFP/FRT-neo-FRT/3HA vector. p-p-5HA/eGFP/FRT-neo-FRT/3HA and pBKS-Tph2-BC were co-electroporated in DY380 E. coli following standard protocols previously described 26 to obtain the Tph2::eGFP(FRT-neo-FRT)
targeting vector. The NotI linearized Tph2::eGFP(FRT-neo-FRT) targeting
construct was electroporated into E14Tg2a.4 ES cells (BioRad Gene Pulser Unit, 3 µF/800V) and neomycin selection was maintained for 7 days. Neomycin-resistant cells were screened for the homologous recombination event using Southern blot analysis. Of 96 neomycin-resistant clones, four clones resulted to have the desired integration within the Tph2 locus. Two independent Tph2::eGFP(FRT-neo-FRT) ES cell clones were injected into C57BL/6 host blastocysts and implanted into pseudo-pregnant females to generate chimeric mice. Germline transmission was obtained from two male chimeras and the Tph2::eGFP(FRT-neo-FRT) mouse line was established on
mixed (129/Ola-C57BL/6), 129/Ola, and C57BL/6 (backcrossed onto C57BL/6 wild-types for at least 9 generations) genetic background. Tph2::eGFP(FRT-neo-FRT) heterozygous mice were mated to the ACTB::FLPe
deleter 28 to excise the neo selection cassette for the generation of the Tph2::eGFP(FRT) reporter mouse line. Genomic DNA from Tph2::eGFP(FRT)
mice was digested with HindIII and analyzed by Southern blot to confirm flp-mediated excision of FRT-flanked neo cassette. Mice were routinely genotyped for the identification of Tph2::eGFP(FRT-neo-FRT) and/or Tph2::eGFP(FRT) alleles by PCR in standard conditions using the following
primers: forward ACTGCTCTTCAGCACCAGGGTT-3’ and reverse 5’-CCAGCTGCTTGTACTTTGTGGA-3’ for the wild-type Tph2 allele; forward
5’-TGCTAAAGCGCATGCTCCAGAC-3’ and reverse
5’-GATAGGGAACTTCACTTTCTATTT-3’ for the Tph2::eGFP(FRT-neo-FRT) allele; forward 5’-GTAGGTGTCATTCTATTCTGGG-3’ and reverse 5’-GATAGGGAACTTCACTTTCTATTT-3’ for the Tph2::eGFP(FRT) allele.
Mice were housed in standard plexiglas cages at constant temperature (22±1°C) and maintained on a 12/12 h light/dark cycle, with food and water ad libitum. Experimental protocols were conducted in
accordance with the Ethic Committee of the University of Pisa and approved by the Veterinary Department of the Italian Ministry of Health.
HPLC measurements
Animals were sacrificed by decapitation and brain tissue samples were rapidly dissected out and frozen at -80°C until analysis. For the determination of serotonin content, frozen tissue was homogenized in lysis buffer containing 10 mM ascorbic acid and 2.4% perchloric acid. Brain tissue levels of serotonin were measured using HPLC with electrochemical detection (HPLC-ECD).
Evans blue dye assay
Evans blue dye method was used to calculate the circulating cerebral blood volume (CBV) in Tph2 mutant animals. Briefly, 1% Evans blue (Sigma Aldrich, St Louis, MO, USA) in phosphate-buffered saline (PBS, pH 7.5) was injected into the tail vein of adult mice. 5 minutes after injection, forebrain and hindbrain were dissected out and processed as described in Uyama et al, 1988. Volume calculations were based on Evans blue dye standards dissolved in whole blood (0.01–1 µg/µl) and processed in parallel to tissue samples. Circulating blood volume was calculated per milligram of forebrain and hindbrain tissue sample and used to assess blood-derived 5-HT content in brain tissue of Tph2 mutant animals (Supplementary figure 3).
Histological, immunohistochemical and in situ hybridisation analyses
For histological and immunohistochemical analyses animals were perfused transcardially with 4% paraformaldehyde, brains were dissected, post-fixed o/n at 4°C and sections (50µm thick) were obtained with a vibratome (Leica Microsystems). Histological Nissl-staining and immunohistochemistry were performed following standard protocols. Briefly, immunohistochemical procedure was carried out on free-floating sections using primary rabbit anti-5-HT antibody (1:500, Sigma-Aldrich), primary rabbit anti-eGFP antibody (1:2000, Molecular Probes) or primary chicken polyclonal anti-eGFP antibody (1:200, Aves Labs). Secondary antibody dilutions were: 1:500 Rhodamine Red-X goat anti-rabbit IgG (Molecular Probes); 1:500 Oregon Green 488 goat anti-rabbit IgG (Molecular Probes); 1:200 Fluorescein (FITC) labelled anti-chicken IgY (Aves Labs).
For in situ hybridization analysis, fresh brain tissue was embedded in Tissue Tek (Sakura), and frozen at -80°C. 14 µm cryostat sections were cut in the coronal plane and in situ hybridization was performed according to protocols using either digoxigenin-labelled (Tph2, 1.5 Kb; eGFP, 0.7 Kb) or
35S-labelled (BDNF, 0.5 Kb) antisense RNA probes as previously described
29, 30. In digoxigenin-labelled in situ hybridization experiments, NBT/BCIP
(Roche) was used as substrate for alkaline phosphatase, while in radioactive in situ hybridization, sections were exposed to Biomax MR X-ray films (Kodak) for two to four days. For each probe or antibody used, sections from different genotypes were processed in parallel in order to minimize the
experimental variability. Images were taken with an Eclipse E600 microscope (Nikon) equipped with a DS-SMc digital camera (Nikon), with a MacroFluo microscope (Leica) or with a SP5 confocal microscope (Leica). Confocal images two-dimensional reconstructions on stacks were performed using ImageJ software (http://rsb.info.nih.gov/ij).
Image analysis and quantification
eGFP-positive serotonergic neurons were counted on consecutive coronal sections throughout the rostro-caudal extent of both dorsal and median raphe of Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. Count of eGFP-positive cells was performed blind and slide identity was not revealed until correlations were completed. Reported value represents average cell body number from 3 different animals for each genotype.
Levels of eGFP fluorescence and BDNF expression were quantified by relative optical density (ROD) measurements. Sections from Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice were immunoreacted for eGFP and pictures were taken using a low-power objective lens (4x) setting a constant measuring window. For each experiment, control adjacent sections were processed omitting the primary antibody. After conversion to grayscale 8-bit, OD was quantified using ImageJ software and ROD value for specific brain regions was determined after subtraction of background OD.
eGFP-positive neurons were resolved on 20 consecutive sections using the analyze particle function of the ImageJ software. Subsequently, ROD of fluorescence of selected neurons was measured for both Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- animals. ROD of the
eGFP-immunoreactive fiber fluorescence was quantified on consecutive coronal sections of dorsal (AP -1.06/-2.46 relative to Bregma) and ventral (AP -2.70/-3.64 relative to Bregma) hippocampus, and throughout the cortical layers of the somoatosensory cortex (AP -0.10/-1.82 relative to Bregma) 31.
Images of BDNF expression on autoradiography film resulting from radioactive in situ experiments, were scanned at a resolution of 3200 dpi and the OD was evaluated in the pyramidal cell layer of Ammon’s horn (CA) and in the granule cell layer of the dentate gyrus (DG) of the hippocampus, and in cortex as described above. Background OD value was determined in structures of the same section devoid of specific signal and subtracted for correction to obtain the ROD value for both wild-type and Tph2 mutant mice.
Statistical analysis
Data are expressed as mean ± SEM and tests of significance were conducted using Wilcoxon signed-rank test for unpaired data. Growth rate results were analysed using analysis of variance (ANOVA) followed by Tukey’s post hoc test. Kaplan–Meier survival plots were analysed using Logrank test. P values resulting from statistical tests or post-hoc comparisons are indicated.
RESULTS
Generation of the Tph2::eGFP(FRT-neo-FRT) knock-in mouse allele expressing eGFP in serotonergic neurons
A Tph2::eGFP(FRT-neo-FRT) knock-in mutant allele was generated to disrupt the Tph2 gene and to allow faithful Tph2-like expression of the eGFP reporter. To compare eGFP levels between heterozygotes and homozygotes, the targeting vector was prepared with a FRT-flanked neo cassette cloned in the opposite transcriptional orientation than the eGFP gene to repress the reporter expression within the targeted locus (Figure 1a)32.
We first evaluated the capability of the neo cassette to repress eGFP expression. Starting from mid-gestation to late post-natal stage, we could not detect any eGFP expression in the brainstem of heterozygous Tph2::eGFP(FRT-neo-FRT) knock-in mice (Figure 1b, Supplementary Figure
1a-b). To excise the neo cassette and to generate the recombined Tph2::eGFP(FRT) allele (Fig 1a), Tph2::eGFP(FRT-neo-FRT) mice were mated to
the ACTB::FLPe deleter 28. Interestingly, presence of eGFP was readily observed in Tph2::eGFP(FRT) heterozygous mice starting from 11.5 days post coitum (dpc) embryos to adulthood with a distribution mirroring that of Tph2 mRNA (Figure 1c, Supplementary Figure 1a,c). Immunostaining confirmed the presence of eGFP in all 5-HT immunoreactive neurons (Figure 1d-f). Notably, serotonergic neurons of the Tph2::eGFP(FRT) mouse line express eGFP in their neurites, thus permitting the direct visualization of the entire projection pattern in the whole CNS (Figure 1g-h). Tph2::eGFP(FRT-neo-FRT) +/- animals were intercrossed to obtain Tph2-deficient (-/-) mice and effective disruption of Tph2 was confirmed using in situ hybridization and immunohistochemistry (Supplementary Figure 2a-d). In line, high-performance liquid chromatography (HPLC) analysis showed a dramatic depletion of 5-HT content in the CNS of mutants (Supplementary Figure 2e). Conversely, 5-HT amount in pineal gland, duodenum and whole blood was comparable between mutants and controls (Supplementary Figure 2e). To address the origin of residual brain 5-HT found in Tph2-null mice, we used the Evans blue dye method (Supplementary Figure 3) 33, 34. Importantly, these results demonstrated that in Tph2 mutants neuronal 5-HT synthesis is abrogated and, in turn, unveil that the remnant amount of this neurotransmitter derived from circulating cerebral blood.
Lack of central 5-HT affects survival and growth rate of Tph2-null mice.
Mutants were born at normal Mendelian ratios and did not show at birth any noticeable difference from wild-type (wt) and heterozygotes. Strikingly, few days after birth, evidence of a growth rate defect was apparent and about 60% of Tph2-null mice from both the pure (C57BL/6-F9) and mixed (129/Ola-C57BL/6) genetic background died within the first 3 weeks after birth (Figure 2 a-d). Although mutants exhibited no obvious deficits in suckling ability showing milk in their stomachs, already at postnatal day 3
(P3) they were significantly smaller than wild-types and heterozygotes and the body weight difference between genotypes continued to increase up to the third week after birth (p<0.001) (Figure 2d). After weaning the surviving Tph2-null mice slowly approached the weight of their siblings (Figure 2d), they are fertile and show a normal life span.
Subsequently, we analyzed the effect of lack of 5-HT on the development of highly stereotyped anatomical and histological brain structures using Nissl staining on brain coronal sections and no obvious abnormalities were found in Tph2-null mice as compared to wild-types (Supplementary Figure 4a-b).
We then asked whether development of serotonergic neurons was affected in mice missing the foreseen trophic autocrine action of 5-HT 35-37. To address this question we compared the neuroanatomical distribution and the total number of eGFP-expressing neurons in the rhombencephalon of Tph2::eGFP(FRT) +/- and compound heterozygote Tph2::eGFP(FRT)/(FRT-neo-FRT)
-/- mice. First, to exclude that lack of 5-HT affects Tph2 transcriptional activity, thus making levels of eGFP fluorescence not comparable between heterozygotes and homozygotes, we measured relative optical density (ROD) in the somata of eGFP-positive neurons and we found no difference between genotypes (Supplementary Figure 4c). An undistinguishable staining pattern was observed in the two genotypes demonstrating that eGFP-expressing neurons can differentiate and maintain both comparable distribution and normal neuronal shape up to adulthood, despite the absence of 5-HT (Supplementary Figure 4d-g). Similarly, the number of eGFP-expressing cells in the dorsal and median raphe was unchanged (Supplementary Figure 4h).
Effects of 5-HT depletion on serotonergic neurite outgrowth in the brain of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice
To unveil a potential role of 5-HT on the development of serotonergic system, we explored the hodological organization of the raphe nuclei. Serotonergic innervation was highlighted by immunofluorescence on brain coronal sections of Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/-
animals and evaluation of fiber density was performed using densitometric measurements and confocal microscopy analysis. Surprisingly, in the Tph2-null mice, we were able to identify three distinct scenarios in which the density of serotonergic fibers appeared reduced (i.e. suprachiasmatic nucleus of the hypothalamus and thalamic paraventricular nucleus), increased (i.e. nucleus accumbens and hippocampus), or unaffected (i.e. cerebral cortex).
The cerebral cortex of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice appeared
normally innervated by serotonergic fibers, as highlighted by eGFP-immunoreactivity, from layer I to VI, with a little fluctuant density in distinct layers of specific cortical regions. Accordingly, densitometric analysis of eGFP-immunoreactivity in the parietal cortex (primary somatosensory, S1) resulted in a comparable ROD measured in both Tph2::eGFP(FRT) +/- and
Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice (Supplementary Figure 5a). Moreover, confocal analysis showed a similar path arrangement of serotonergic innervation in the two genotypes (Supplementary Figure 5 b-c), further confirming unaltered serotonergic projections to the cerebral cortex of 5-HT depleted mice.
In contrast, serotonergic fibers failed to properly innervate the suprachiasmatic nucleus (SChN) of the hypothalamus and the thalamic paraventricular nucleus (PVN) of Tph2 mutants. Serotonergic fibers innervating the SChN form a highly stereotyped topographic arrangement as highlighted by eGFP fluorescence in Tph2::eGFP(FRT) +/- mice (Figure 3a-b; Supplementary Figure 6a). In the SChN of both P14 and adult Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice, eGFP-immunofluorescence was dramatically reduced in intensity as compared to heterozygotes (Figure 3 a-f; Supplementary Figure 6 a-b). Although barely visible, eGFP-positive fibers left in the SChN region depicted a pattern resembling the distribution of serotonergic innervation observed in normal mice (Figure 3 b,e; Supplementary Figure 6 a-b). Furthermore, high magnification confocal images in the medial region of the SChN revealed that the reduction of fluorescence signal was due to a remarkable reduction in the number of varicosities of the residual serotonergic fibers (Figure 3 c,f). Similarly, in Tph2-null mice the clustered serotonergic fibers reaching the PVN appeared consistently reduced in their density in the centre of the nucleus, while they resulted trimmed off at the edges (Figure 3 g-l; Supplementary Figure 6 c-d).
An opposite phenotype was found in the nucleus accumbens and hippocampus of mutant brains. Density of serotonergic fibers to the nucleus accumbens shell (NAs) in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice appeared
remarkably increased and the crescent-shaped fluorescent territory resulted enlarged on its medio-lateral extent as compared to Tph2::eGFP(FRT) +/-
animals (Figure 4 a-f). Confocal analysis further confirmed the presence of highly packed axons forming a dense tangle of eGFP-positive fibers (Figure 4 b-c,e-f). Moreover, supernumerary serotonergic fibers found in the NAs of Tph2-null mice appeared to be mainly represented by fibers with thick axonal diameter and characterized by the presence of large varicosities (Figure 4 c,f).
In the hippocampus of adult Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice, Tph2
enzymatic activity disruption resulted in a marked hyperinnervation that was already detectable at P14 (Figure 4 g-r; Supplementary Figure 7 a-h). The serotonergic hyperinnervation extent was quantitatively evaluated in adults for each of the four layers in both dorsal and ventral hippocampal regions. Comparison of eGFP-immunoreactivity between the two genotypes revealed an overall significant increase of the density of 5-HT axons in all the hippocampal layers analysed (p<0,001; Supplementary Figure 8). Interestingly, we reported a higher hyperinnervation in the ventral hippocampal region of each layer as compared to the dorsal counterpart. Specifically, the density of the serotonergic fibers in the stratum oriens of the dorsal and the ventral hippocampus of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice
was +25% and +47% of that measured in heterozygotes, respectively (Supplementary Figure 8). Similarly, the ROD in the dorsal and the ventral hippocampus of 5-HT depleted mice was +40% and +41% in the stratum radiatum, +39% and +44% in the stratum lacunosum-moleculare, and +32% and +49% in the molecular layer of dentate gyrus (Supplementary Figure 8).
Up-regulated BDNF expression in the hippocampus of Tph2 mutant mice
A number of experimental evidences suggests brain derived neurotrophic factor (BDNF) as a crucial molecule in promoting development and establishment of neural circuitry of the serotonergic system 38-40. We assessed BDNF mRNA expression levels by means of ISH using 35
S-radiolabeled antisense BDNF probe on brain coronal sections of both wild-type and Tph2 -/- adult mice. In the cortex of serotonin-lacking brains, mRNA expression levels for BDNF were undistinguishable from that of wild-type animals. In contrast, in the hippocampus of Tph2 -/- mice, the intensity of the autoradiographic signal appeared noticeably higher as compared to controls showing a remarkable and significant increase of BDNF expression in both the pyramidal cell layer of Ammon’s horn (CA) and the granule cell layer of the dentate gyrus (DG) (p<0,001; Figure 5). Interestingly, we observed that the extent of the BDNF over-expression in mutant mice was higher in the ventral hippocampal region (+72% and +42% in the pyramidal and granule cell layer, respectively) as compared to the dorsal region (+26% and +30% in the pyramidal and granule cell layer, respectively) (Figure 5e).
DISCUSSION
Here we report that lack of central serotonin affects postnatal development, and that serotonin is crucial for the correct establishment of the serotonergic neurite projections during critical periods of neuronal wiring. Our finding reveals that areas with the highest susceptibility to serotonin levels for the correct neuronal circuitry formation are those causally associated to the control of behaviours altered in psychiatric disorders including schizophrenia and anxiety.
Genetic disruption of the brain serotonin biosynthetic enzyme Tph2 resulted, in our Tph2 mouse line, in a dramatic growth impairment that is comparable in extent to what reported in other Tph2 knockouts 19, 22. Our results showed that the weight at birth of mutant animals did not differ from that of wild-type and heterozygous littermates, indicating that lack of brain serotonin does not affect foetal growth and development but rather impacts on early postnatal life. In light of the pleiotropic action of serotonin, the origin of growth retardation may be multifactorial and two main non-exclusive reasons might account for the observed deficit, nutritional and/or metabolic. The former hypothesis according to which dysphagia or hypophagia would be at the origin of the growth deficiency appears weakened by the evidence that we found normal amount of milk in the stomach of mutants and the fact
that even the early removal of competing siblings did not improve the phenotype. By contrast, a metabolic cause for the growth retardation is well supported by the marked reduction of insulin-like growth factor-1 (IGF1) levels in serum, as showed by Alenina and co-worker 19. Accordingly, the finding that normal IGF1 levels are restored after two months of age is in agreement with the observation that growth curves are nearly parallel after weaning, with the weight of mutant animals that approaches that of controls around P90 19.
Our results on the reduced survival rate are in line with what reported by Alenina and collaborators but we could neither explain the reasons of early death observed in Tph2 mutant pups nor exclude the growth impairment being at the origin of the premature lethality. Interestingly, recent findings have shown that serotonin deficit alters autonomic circuits causing lethality in animal models displaying some features of sudden infant death syndrome (SIDS) 41. Thus, further studies will be required to determine whether lethality shown by Tph2 mutant animals might be associated to facets of SIDS.
Support to a role for serotonin in brain development comes from data showing that perturbation of serotonergic signalling affects CNS development and that precise serotonin levels are crucial for normal neuroanatomy and cytoarchitecture establishment 9, 12. However, mouse models in which serotonin was depleted via Tph2 inactivation failed to show abnormalities in CNS development 19, 20, 22, 23. In line, our data showed that lack of brain serotonin results neither in detectable alterations of brain neuroanatomy nor in changes of serotonergic neuron development within the raphe nuclei. Therefore, a provocative view is that serotonin is not required for brain development. Support to this paradoxical hypothesis comes from the absence of overt CNS developmental abnormalities in serotonin receptor knock-out mice 9. Nevertheless, studies from both vertebrate and invertebrate organisms have supported the idea that serotonin regulates neurite outgrowth and establishment of neuronal connectivity during development 42-46. The availability of the Tph2::eGFP knock-in mice allowed for the first time to highlight serotonergic fibers irespective of serotonin immunoreactivity and made possible to visualize directly the development and the formation of the serotonergic neuronal circuitry. Thanks to the presence of strong eGFP fluorescence, our mouse model made possible for the first time to perform accurate analyses of axonal projections of serotonergic neurons in animals lacking brain serotonin. Absence of brain serotonin resulted in overt alterations of serotonergic innervation in several regions of the rostral brain. Intriguingly, besides the apparently normal distribution observed in cortex and striatum (not shown), development of serotonin axon terminal arborisation exhibited, as a consequence of Tph2 inactivation, a diametrically opposite response in distinct brain areas. Specifically, we reported a dramatic reduction of serotonergic innervation in the suprachiasmatic nucleus and in the thalamic
paraventricular nucleus. Conversely, an extensive axon terminal arborisation resulting in a serotonergic hyperinnervation was observed in the nucleus accumbens and in the hippocampus of Tph2 mutant mice. These results demonstrate that brain serotonin has an important role in regulating the wiring of the serotonergic system during brain development and that serotonin can act either promoting or inhibiting serotonergic axon terminal arborisation relying on specific target brain districts.
An intriguing question is whether serotonin acts throughout the development of the serotonergic innervation or whether specific developmental phases 5
,
particularly sensitive to serotonin modulation, exist. In Tph2 mutants, serotonergic projections reach all brain districts weakening the possibility of a specific serotonergic pathway rerouting. This evidence, rather, strongly suggests that serotonin exerts its role during the period of terminal field development either promoting or inhibiting sprouting of serotonin axon terminals. Support to this hypothesis comes from the fact that the abnormal sprouting of serotonin axon terminals matches the temporal progression of terminal field development. In fact, suprachiasmatic and thalamic paraventricular nuclei, in which the establishment of serotonergic innervation is completed by P10 5, showed already at P14 the fiber density reduction seen in adult mutants; whereas, in the hippocampus, in which serotonergic terminal field matures over a more protracted time span 5, we observed a progressive increasing hyperinnervation that peaks in adult mice. Although the precise mechanism through which serotonin acts to modulate the development of axon terminal arborisation is unknown, activation of specific serotonin receptors is likely to play a pivotal role. Both pharmacological and genetic results provided evidences that specific serotonin receptors mediate the trophic effects of serotonin 46-48. Therefore, the complex and vast operational heterogeneity of the serotonergic system signalling 49,
together with the dynamic spatio-temporal expression of serotonin receptors during brain development 50-52,
may account for the opposite effects of serotonin on serotonergic fiber formation. This view suggests that serotonin has an autoregulatory role on the serotonergic axon terminal arborisation with selective, brain region-dependent, trophic effects. Similarly, in several invertebrate systems, serotonin depletion altered serotonergic fiber development without precluding normal differentiation of serotonergic neurons 42, 53, 54. Overall, these data disclose the presence of an autoregulatory control mediated by serotonin that is conserved throughout animal evolution.The evidence that Tph2 -/- mice showed an altered arborisation of serotonergic terminals raised the question of how the lack of serotonin induces sprouting defects. In the hippocampus, but not in the cortex, we demonstrated a clear up-regulation of the endogenous BDNF expression that parallels with the marked serotonergic hyperinnervation. These findings are in good agreement with a large body of evidences showing a neurotrophic effect of BDNF upon serotonergic axons. Cerebral infusion of
BDNF promotes sprouting of serotonergic axons at the site of injection 39, 40. Conversely, BDNF downregulation observed in BDNF +/- mice causes a decrement in the serotonergic fiber density in the hippocampus 38. Together with the well-known action of BDNF on the serotonergic system, our results about the effects of serotonin depletion on BDNF expression unmask a possible regulatory feedback mechanism through which serotonin itself might regulate the formation of the serotonergic neuronal circuitry in the hippocampus. Reduction of serotonin levels may trigger increased endogenous BDNF signalling that in turn exerts a neurotrophic effect upon serotonergic axons. A higher density of serotonergic axon terminals would result in additional serotonin release sites and, likely, in an augmented availability of serotonin, thus restoring the original BDNF levels that, consequently, would turn down the serotonergic axon sprouting. A recent report by Hill and collaborators provides strong support to this hypothesis and suggests that serotonin would modulate BDNF levels via the activation of the 5-HT2C receptor 55.
In conclusion, our results demonstrate that serotonin, besides its role as a neurotransmitter, represents an important signal required during CNS development involved in the formation of the highly precise architecture of the nervous system and in particular in the fine tuning for patterning the correct serotonergic branching at selected regions of the brain. We also found that depletion of serotonin resulted in an up-regulation of BDNF expression in the hippocampus, thus matching the observed hyperinnervation. These findings indicate that homeostasis of serotonin is required to establish the complex network of serotonin release sites. The evidence that serotonin levels are crucial for the proper wiring of the brain contributes to confirm that exposure to genetic, pharmacological or environmental insults altering the serotonergic signalling produces long-lasting changes that may prompt to neuropsychiatric disorders thought to have developmental origins such as schizophrenia, autism and mental retardation.
ACKNOWLEDGEMENTS
We thank Dr. M. Carta, Dr. R. Nisticò and Dr. A. Usiello for valuable discussions and comments on the manuscript. We also thank the following for kind gifts of reagents: BayGenomics and MMRRC (E14Tg2a.4 ES cells), Dr. D. Court (DY380 cells), Dr. S. Dymecki (ACTB::FLPe mouse line), Dr. Y. Bozzi (BDNF probe). This work was supported by Italian Ministry of Education, University and Research (MIUR) (Prin 2008, 200894SYW2) and Toscana Life Sciences Foundation (Orphan_0108 program) to M.P.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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FIGURE LEGENDS
Figure 1 Generation of the Tph2::eGFP mouse line expressing eGFP in
serotonergic neurons. (a) Diagram showing the targeting strategy for knock-in replacement of Tph2 and representative Southern blot analysis confirmknock-ing homologous recombination in ES cells (BglI and StuI digests) and genotyping of mice upon flp-mediated recombination (HindIII digest). (b,c) Ventral view of adult brain rhombencefalic region of Tph2::eGFP(FRT-neo-FRT) +/- and Tph2::eGFP(FRT) +/- mice which showed live eGFP fluorescence of
the boxed area in the inset (b) upon flp-mediated recombination. (d-f) Representative confocal images of adult Tph2::eGFP(FRT) +/- dorsal raphe nuclei showing the presence of eGFP in all serotonin positive neurons. (g,h) Sagittal section of a 12.5 dpc Tph2::eGFP(FRT) +/- mouse immunostained for
eGFP showing that eGFP expression highlights serotonergic system (g), and higher magnification of serotonergic fibers projecting rostrally (h). B: BglI, H: HindIII, S: StuI, RPa: raphe pallidus nucleus, RMg: raphe magnus nucleus, LPGi: lateral paragigantocellular nucleus, MnR: median raphe nucleus, DR: dorsal raphe nucleus. Scale bars:300 µm (b-c), 200 µm (d-h).
Figure 2 Postnatal lethality and growth rate impairment in Tph2-null mice.
(a) Graph showing the survival of Tph2 +/+, +/- and -/- mice from the C57BL/6 and mixed genetic backgrounds (n=333 and n=234, respectively). (b) Kaplan–Meier analysis showing the survival curve within the first month after birth of Tph2 mutants (n=60) vs wild-type (n=56) and heterozygotes (n=118). (c) Representative picture of wild-type and Tph2 knockout animals at postnatal day 25. (d) Growth curve of Tph2 -/- mice displaying significant
weight reduction as compared to wild-type (n=56) and heterozygous (n=118) littermates. Growth rate curve of mutants dead by P25 is shown separately (open circle, n=35). Mutants that survive to adulthood (open triangle, n=25) reach near normal weight at 3 months of age. Data are presented as mean ± SEM. ***p<0,001, *p<0,05, ns: not significant.
Figure 3 Altered serotonergic innervation to the suprachiasmatic (SChN)
and thalamic paraventricular nuclei (PVN) in Tph2::eGFP(FRT)/(FRT-neo-FRT) mutant mice. (a-f) Representative confocal images of coronal sections immunostained for eGFP through the SChN of Tph2::eGFP(FRT) +/- (a-c) and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- (d-f) adult mice. (a,d) DAPI staining showing
normal SChN morphology. (b,e) Distribution of eGFP+ serotonin fibers around the pear-like shaped SChN in Tph2::eGFP(FRT) heterozygous and Tph2::eGFP(FRT)/(FRT-neo-FRT) mutant animals. (c,f) High magnification images showing the dramatic reduction of the eGFP+ fiber density in mutant mice. In the insets, a digital magnification (5x) showing a remarkable reduction of varicosities in Tph2::eGFP(FRT)/(FRT-neo-FRT) mutants as compared to the Tph2::eGFP(FRT) controls. (g-l) Representative images of coronal sections
through the PVN of Tph2::eGFP(FRT) +/- (g-i) and Tph2::eGFP(FRT)/(FRT-neo-FRT)
-/- (j-l) adult mice. (g,j) Nissl staining showing the characteristic morphology of PVN, which appear normally developed in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/-
mice as compared to Tph2::eGFP(FRT-neo-FRT) +/- mice. A dashed line is drawn
around the PVN. (h,k) Confocal immunostaining analysis showing eGFP+ serotonergic innervation to the PVN. Fibers in Tph2::eGFP(FRT)/(FRT-neo-FRT)
mutants are present in a more restricted area with a significantly reduced density. (i,l) The higher magnification further illustrates the reduction in axonal arborizations in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. Scale bars: 200
µm (a-b,d-e,g-h,j-k), 15 µm (c,f,i,l).
Figure 4 Serotonergic hyperinnervation in the nucleus accumbens (NA) and
in the hippocampus of Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. (a-r) Fluorescent
microscopy images of coronal sections immunostained for eGFP showing serotonergic fiber distribution through the nucleus accumbens (a-f) and hippocampus (g-r) of adult animals. (a) eGFP-positive serotonergic fibers in the NA are mainly distributed in the shell (dashed line). (d) In Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice the density of eGFP+ fibers results
remarkably increased and the stereotyped crescent-shaped structure in the NA shell appears enlarged. (b-c,e-f) Confocal high magnification images confirm the hyperinnervation of serotonergic fibers in Tph2::eGFP
(FRT)/(FRT-neo-FRT) -/- mice and show an increase of the number of serotonergic varicosities
containing strong eGFP fluorescence. (g,m) Epifluorescence images of coronal sections showing the laminar arrangement of eGFP+ serotonergic fibers in dorsal and ventral hippocampus in Tph2::eGFP(FRT) +/- mice. (j,p) In Tph2::eGFP(FRT)/(FRT-neo-FRT) mutants an overall increase in the density of
serotonergic fibers is observed with a higher distribution of eGFP-positive axons within the stratum lacunosum-moleculare. (h-i,k-l,n-o,q-r) Confocal
higher magnification of the boxed region of stratum lacunosum-moleculare. Images further demonstrate the hyperinnervation of serotonergic fibers and show the presence of supernumerary eGFP-positive axons in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. Co: nucleus accumbens core, Sh:
nucleus accumbens shell, Or: stratum oriens, Rad: stratum radiatum, LMol: stratum lacunosum-moleculare, Mol: stratum moleculare. Scale bars: 300 µm (a,d,g,j,m,p), 50 µm (b,e,h,k,n,q), 15 µm (c,f,i,l,o,r).
Figure 5 Up-regulation of BDNF expression in the hippocampus of
Tph2-mutant mice. (a-d) Representative autoradiogram of coronal sections showing BDNF expression in dorsal (a,b) and ventral (c,d) hippocampus of wild-type (a,c) and Tph2-null (b,d) mice. BDNF mRNA expression level results to be more intense in Tph2-mutants as compared to wild-type mice, both in dorsal and in ventral hippocampal formation. (e) Quantification of BDNF expression levels in the hippocampus and cortex of wild-type (n=4) and Tph2-null (n=5) mice. Densitometric analysis of autoradiograms confirms a significant increase in BDNF expression in the CA and DG areas, both in the dorsal and ventral hippocampus of Tph2-mutants, while BDNF level results to be unaltered in the cortex of mutants as compared to wild-types. Data are presented as mean ± SEM. ***p<0,001; ns: not significant. Scale bars:
500 µm.
SUPPLEMENTARY FIGURE LEGENDS
Figure S1 Analysis of eGFP expression in the Tph2::eGFP(FRT) +/- mice
upon Flp-mediated recombination. (a) Coronal section showing Tph2 mRNA expression in the dorsal and median raphe nuclei of wild-type adult mice. (b) Absence of eGFP expression in the raphe region of Tph2::eGFP(FRT-neo-FRT) +/- mice. (c) Presence of eGFP mRNA in dorsal and median raphe nuclei of Tph2::eGFP(FRT) +/- mice. MnR: median raphe nucleus, DR: dorsal raphe nucleus. Scale bars: 300 µm.
Figure S2 Disruption of Tph2 results in loss of serotonin synthesis in
Tph2::eGFP -/- mice. (a,b) In situ hybridization on coronal sections of wild-type (a) and Tph2 mutants (b) showing loss of Tph2 gene expression. (c,d) Immunohistochemistry on coronal sections of wild-type and mutant confirming abrogation of 5-HT synthesis in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/-
mice. (e) Serotonin levels measured by HPLC in the hindbrain, forebrain, pineal gland, blood and duodenum of wild-type and Tph2::eGFP
(FRT)/(FRT-neo-FRT) -/- animals (n=5 per group). Data are presented as mean ± SEM.
***p<0,001. DR: dorsal raphe nucleus. Scale bars: 300 µm (a-b), 250 µm
(c-d).
Figure S3 Evaluation of blood serotonin content in the brain. (a-b)
Representative pictures of an animal before and after Evans blue dye injection into the tail vein. (c) Calculation of the whole blood-derived serotonin present into the forebrain and hindbrain of Tph2 -/- mice (n=6). Data are presented as mean ± SEM.
Figure S4 Brain neuroanatomy and cytoerchitecture appeared unaltered in
Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. (a,b) Representative Nissl-stained
coronal sections obtained from wild-type and Tph2 mutant adult brains. (c) Relative optical density (ROD) measured in eGFP-positive neurons from both dorsal and median raphe of Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice (n=3 animals per genotype). (d-g)
Representative confocal images showing the distribution and morphology of serotonergic neurons in heterozygotes (g,e) and mutants (f,g), as highlighted by the presence of eGFP. (h) Graph showing the count of eGFP immunoreactive cell bodies in the raphe of Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice (n=3 per genotype). Data are presented as mean ± SEM. ns: not significant. DR: dorsal raphe nucleus. Scale bars: 750 µm (a-b), 200 µm (d,f), 15 µm (e,g).
Figure S5 Normal development of serotonergic fibers in the cerebral cortex
of Tph2::eGFP(FRT)/(FRT-neo-FRT) null mice. (a) Comparison of relative optical density of eGFP+ serotonergic axons measured through the primary somatosensory cortex of Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice (n=3 per genotype). (b,c) Representative composit confocal analysis
on coronal section of Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- primary somatosensory cortex displaying the distribution of serotonergic fibers throughout the cortical layers (I-VI). Data are presented as mean ± SEM. Scale bars: 100 µm.
Figure S6 Impaired serotonergic innervation to the suprachiasmatic (SChN)
and thalamic paraventricular nuclei (PVN) is already present in P14 Tph2::eGFP(FRT)/(FRT-neo-FRT) mutant mice. (a,b) eGFP-positive serotonergic fibers show dense innervation in the SChN already at P14 in coronal sections of Tph2::eGFP(FRT) +/- brains. eGFP immunofluorescence shows a marked reduction of the number of fibers innervating the SChN in Tph2::eGFP(FRT)/(FRT-neo-FRT) mutants. (c,d) Coronal sections of PVN showing less eGFP+ serotonin fibers in Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice as compared to control Tph2::eGFP(FRT) +/- animals. Scale bars: 150 µm.
Figure S7 Hyperinnervation of serotonergic fibers in the hippocampus of
Tph2::eGFP(FRT)/(FRT-neo-FRT) mutant animals progressively increases during postnatal development. (a-d) Low magnification images showing the eGFP-positive serotonergic fibers in the hippocampus of P14 animals. In Tph2::eGFP(FRT) +/- mice few fibers are present both in dorsal (a) and ventral
(b) hippocampus with a uniform distribution. In Tph2::eGFP(FRT)/(FRT-neo-FRT)
mutants a higher number of eGFP-positive fibers can be detected both in dorsal (c) and ventral (d) hippocampal regions. (e-h) Microphotographs showing the dorsal and ventral hippocampal regions at P21. (e,f) At P21 the number of serotonergic fibers in the hippocampus is progressively augmented as compared to P14 and their typical laminar arrangement start to be recognizable. (g,h) In P21 Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice a higher
density of eGFP+ fibers highlights a hyperinnervation of serotonergic fibers. LMol: stratum lacunosum-moleculare. Scale bars: 300 µm.
Figure S8 Quantification of eGFP-positive fibers in the hippocampus of
Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice. (a) Diagram
showing the regions in the dorsal (left) and ventral (right) hippocampal formation in which relative optical density of serotonergic fibers has been quantified. (b) Graph showing the quantification of serotonergic fiber density in Tph2::eGFP(FRT) +/- and Tph2::eGFP(FRT)/(FRT-neo-FRT) -/- mice (n=3 per
genotype). Data are presented as mean ± SEM. ns: not significant. ***p<0,001. Or: stratum oriens, Rad: stratum radiatum, LMol: stratum lacunosum-moleculare, Mol: stratum moleculare.
Figure S1
Figure S3
Figure S5