Silvina Laura Diaz

Nicolas Narboux-Nême

Sara Trowbridge

Sophie Scotto- Lomassese

Felix Kleine Borgemann

Sebastian Jessberger

Bruno Giros

Luc Maroteaux

Evan Deneris

Patricia Gaspar
email: patricia.gaspar@inserm.fr

Increased survival of newborn neurons in the dentate gyrus of mice with constitutive depletion of serotonin

Keywords: genetic mouse models, adult neurogenesis, hippocampus, 5-HT depletion, 5-HT1A, 8-OH-DPAT, PCPA

Increased adult neurogenesis is a major neurobiological correlate of the beneficial effects of antidepressants. Indeed, selective serotonin (5-HT) re-uptake inhibitors, which increase 5-HT transmission, enhance adult neurogenesis in the dentate gyrus (DG) of the hippocampus.

However, the consequences of 5-HT depletion are still unclear since studies using neurotoxins that target serotonergic neurons, reached contradictory conclusions on the role of 5-HT on DG cells proliferation. Here, we analyzed two genetic models of 5-HT depletion, the Pet1 -/-and the VMAT2 sert-cre conditional knockout mice, that have 80% and 95% reductions of hippocampal 5-HT, respectively. In both models, we found unchanged cell proliferation of the neural precursors in the DG subgranular zone, whereas a significant increase in the survival of newborn neurons was noted 1 and 4 weeks after BrdU injections. This pro-survival trait was phenocopied pharmacologically with 5-HT synthesis inhibitor PCPA treatment in adults, indicating that this effect was not developmental. Furthermore, one week administration of the 5-HT 1A receptor agonist, 8-OH-DPAT, in Pet1 -/-mice normalized hippocampal cell survival. Overall, our results indicate that constitutive 5-HT depletion does not alter the proliferation of neural precursor in the DG but promotes the survival of newborn cells, an effect which involved activation of the post-synaptic 5-HT 1A receptor. The role of 5-HT in selective neuronal elimination points to a new facet in its multiple effects in controlling neural circuit maturation.

Introduction

The production of new neurons in the subgranular zone of the dentate gyrus (DG) of adult mammalian brain is a unique form of structural plasticity which enables hippocampal circuits to adapt to changes in the environment [START_REF] Koehl | A new chapter in the field of memory: adult hippocampal neurogenesis[END_REF]. The dentate gyrus subgranular zone continuously generates cells, but a large proportion (70-90%) of these adult newborn cells is cleared in the first four weeks after their generation [START_REF] Snyder | Adult-born hippocampal neurons are more numerous, faster maturing, and more involved in behavior in rats than in mice[END_REF].

Numerous molecular signals and processes have been involved in regulating the neurogenesis and survival of newborn cells and their integration in neural circuits [START_REF] Fabel | Physical activity and the regulation of neurogenesis in the adult and aging brain[END_REF][START_REF] Kempermann | Seven principles in the regulation of adult neurogenesis[END_REF][START_REF] Malberg | Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus[END_REF][START_REF] Van Praag | Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus[END_REF][START_REF] Warner-Schmidt | Hippocampal neurogenesis: opposing effects of stress and antidepressant treatment[END_REF]. Among these factors, neural activity has been shown to promote cell survival, namely glutamatergic and GABAergic afferents were found to play a role in the modulation of neurogenesis [START_REF] Toni | Synapse formation on adult-born hippocampal neurons[END_REF].

Serotoninergic axons originating from the raphe nuclei innervate the DG [START_REF] Jacobs | Structure and function of the brain serotonin system[END_REF] and have also been involved in the control of adult neurogenesis. This is largely based on studies showing that chronic exposure to the selective 5-HT reuptake inhibitors (SSRIs), a widely used class of antidepressants, increased proliferation of neural progenitors [START_REF] Banasr | Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone[END_REF][START_REF] Diaz | 5-HT(2B) receptors are required for serotonin-selective antidepressant actions[END_REF][START_REF] Malberg | Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus[END_REF][START_REF] Petrik | The neurogenesis hypothesis of affective and anxiety disorders: are we mistaking the scaffolding for the building?[END_REF] and survival of the newborn cells in the DG [START_REF] Diaz | 5-HT(2B) receptors are required for serotonin-selective antidepressant actions[END_REF][START_REF] Encinas | Fluoxetine targets early progenitor cells in the adult brain[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF].

However, the physiological effect of constitutive 5-HT transmission on proliferation and survival remains a matter of debate since pharmacological depletion of 5-HT by the use of the selective 5-HT toxin (5-7 dihydroxytryptamine) led either to a reduction (Brezun andDaszuta, 1999, 2000), or to no change [START_REF] Huang | The role of 5-HT1A receptors in the proliferation and survival of progenitor cells in the dentate gyrus of the adult hippocampus and their regulation by corticoids[END_REF][START_REF] Jha | Selective serotonin depletion does not regulate hippocampal neurogenesis in the adult rat brain: differential effects of pchlorophenylalanine and 5,7-dihydroxytryptamine[END_REF] of newborn cell number.

To evaluate the effects of a constitutive 5-HT depletion, we analyzed two genetic models: the Pet1 -/-mice, in which a majority of 5-HT neurons in the raphe fail to differentiate [START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF], and the VMAT2 sert-cre mice, which lack vesicular monoamine storage mechanisms in 5-HT neurons [START_REF] Narboux-Neme | Severe serotonin depletion after conditional deletion of the vesicular monoamine transporter 2 gene in serotonin neurons: neural and behavioral consequences[END_REF]. We report that basal proliferation is undisturbed in these two mouse strains, whereas survival of newborn cells is increased. In addition, similar results were obtained using a pharmacological approach for depleting 5-HT in adult WT mice, indicating that this effect is not due to developmental alterations. A normalization of the increased survival phenotype with 5-HT1A agonists in both, Pet1 -/-and pharmacologically 5-HT depleted mice, revealed an unsuspected role of this receptor in controlling the number of surviving newborn cells in the hippocampus.

Because it has been recently proposed that the immature DG cells are critical for pattern separation [START_REF] Nakashiba | Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion[END_REF][START_REF] Sahay | Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation[END_REF], a form of memory that enables to encode somewhat similar contexts as distinct events, we examined this form of memory in the Pet1 -/-mice. However, no change in memory performance in this test could be observed.

Material and Methods

Animals

Procedures involving animals were conducted in accordance with the directives of the European Community (council directive 86/609) and the French Agriculture Ministry (council directive 87-848, 19 October 1987, permissions 00782 to PG). The Pet1 -/-mouse line has been described previously [START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF]. Heterozygote Pet1 +/-females were mated with Pet1 +/-or Pet1 -/-males to produce mixed litters. Both Pet1 +/+ and Pet1 +/-served as controls, since no differences of 5-HT levels or 5-HT innervation between the two genotypes has been reported [START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF] or noted in the present study.

The VMAT2 sert-cre mouse line has been described previously [START_REF] Narboux-Neme | Severe serotonin depletion after conditional deletion of the vesicular monoamine transporter 2 gene in serotonin neurons: neural and behavioral consequences[END_REF].

Briefly, these mice result from crossing a floxed VMAT2 mouse line (Bruno Giros, to whom correspondence regarding the mouse line should be addressed) to a SERT cre mouse line [START_REF] Zhuang | Targeted gene expression in dopamine and serotonin neurons of the mouse brain[END_REF]. VMAT2 lox/lox :: SERT cre/+ mice were mated with female VMAT2 lox/lox to generate recombinant and control littermates as previously described (Narboux-Neme et al,

2011).

The two mouse lines were maintained on a C57BL/6J background and littermates were used in all experiments. After weaning, males and females were housed separately (5 animals per cage) and maintained under standard laboratory conditions (22±1°C, 60% relative humidity, 12-12 hrs light-dark cycle, food and water ad libitum).

Pharmacological studies were performed on wild type (WT) C57BL/6J mice (purchased from Janvier, France).

Proliferation and survival assays.

Dividing cells were labelled with the thymidine analogue 5-Bromo-2'deoxyuridine (BrdU; Sigma, B9285) as previously described [START_REF] Diaz | 5-HT(2B) receptors are required for serotonin-selective antidepressant actions[END_REF]. To study cell survival at 4 and 16 weeks, it is necessary to use relatively high doses of BrdU to ensure that it is not diluted in the process of re-division of daughter cells [START_REF] Wojtowicz | BrdU assay for neurogenesis in rodents[END_REF]. Animals received 2 injections of BrdU 150 mg/kg at 2 hrs interval, a dose that has previously been proved to be non toxic for proliferating and immature neurons [START_REF] Hancock | Does 5-bromo-2'-deoxyuridine (BrdU) disrupt cell proliferation and neuronal maturation in the adult rat hippocampus in vivo?[END_REF]. BrdU was dissolved in 0.9% NaCl at 50°C, and the pH was set at 7.4 with NaOH 10M. 7-week-old VMAT2 sert-cre , Pet1 -/-and their respective controls (n=3-6/experimental group) were sacrificed either 24 hrs, 1 week, 4 weeks or 16 weeks after BrdU administration and brains were recovered for cell proliferation and survival studies.

Chronic treatment with PCPA

To study the effect of chronic 5-HT depletion in adults, 4 week-old WT mice received a daily intraperitoneal (i.p.) injection of PCPA at 300 mg/kg during the first 3 days, and at 100 mg/kg for the rest of the treatment (Suppl Fig. 2) [START_REF] Vitalis | Embryonic depletion of serotonin affects cortical development[END_REF]. After 4 weeks of PCPA treatment, BrdU was administered as described above. A group of mice was sacrificed 24 hrs later for proliferation assays and a second group continued receiving PCPA injections during 1 or 4 extra weeks for survival assays.

8-OH-DPAT treatment

A group of 4 weeks PCPA-treated and control Pet1 -/-mice (7-8 week-old) received 2 injections of BrdU as described above. 24 hrs later, animals received 2 daily i.p. injections of the 5-HT 1A agonist 8-OH-DPAT at 0.5 mg/kg or vehicle (NaCl 0.9%) during 7 days and sacrificed the day of the last injection.

Retrovirus-mediated labeling of new hippocampal granule cells 300 nl of retroviral vector CAG-GFP [START_REF] Zhao | Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity[END_REF] were injected into the dentate gyrus of Pet1 -/-and Pet1 +/-mice at the following coordinates: anteroposterior, -1.7; lateral, -/+1.7; ventral, -1.75 by using the bregma as reference. Mice were perfused one week later and 100 µm sections were made with a vibratome (VT1000S, Leica).

Histological methods

Mice were deeply anesthetized (xylazine, 20 mg/kg; pentobarbital, 50 mg/kg) and transcardially perfused with 5 ml of NaCl 0.9% and 50 ml of 4% paraformaldehyde in 0.1M phosphate-buffered saline (1X PBS, pH 7.4) for 15 min. Brains were recovered, postfixed for 24 hrs at 4ºC and sliced to 50 µm thick coronal sections with a vibratome. Sections were stored at -20ºC in cryoprotectant (30% ethylene glycol, 30% glycerol in 0.12M phosphate buffer) if not used immediately. Newborn cells were revealed by peroxidase immunostaining of BrdU. Free-floating sections were incubated overnight in 0.1% H 2 O 2 , and exposed to 2 N HCl for 1 hr. Sections were incubated overnight at 4°C with a rat anti-BrdU (1:400; AbDserotec OBT0030; clone BU1/75-ICR1), which was then detected with a biotinylated goat anti-rat antibody during 2 hrs (1:400; Vector). Revelation steps included 1 hr incubation in a streptavidin horseradish peroxidase complex (1:400 Amersham) followed by an exposure to 3,3'-diaminobenzidine (DAB) used as substrate.

To identify the type of newborn cells produced in the DG, double-labeling of BrdU and neuronal or astroglial markers were carried out. Rat anti-BrdU (1:400; AbDserotec OBT0030) was combined with a goat anti-doublecortin (1:500; Santa Cruz, immature neuronal marker), an IgM mouse anti-calbindin (1:12000; AbCys: CP331, mature neuronal marker), a rabbit anti-calretinin (1:500; Swant, immature marker) or a rabbit anti-GFAP (1:800; Dako Cytomation, astroglial marker) (overnight, 4°C). Sections were then incubated for 2 hrs at room temperature in the corresponding secondary fluorescent antibodies (1:500; Molecular Probes).

To evaluate morphology of retrovirus-infected new neurons, GFP expression was intensified by permeabilizing 100 µm-thick sections with proteinase K (10 µg/ml; 2 min), which was subsequently blocked with 2 mg/ml glycine. GFP was then detected with a rabbit anti-GFP at 4°C overnight (1:5000; Molecular Probes) and revealed at room temperature for 2 hrs with a donkey anti-rabbit (1:500; Molecular probes).

Coronal sections from the hippocampus of WT mice were employed for in situ hybridization as described previously [START_REF] Narboux-Neme | Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs)[END_REF]. The following cDNA were used: 5-HT 1A (IMAGE:8861702), 5-HT 2B (IMAGE:5344328) 5-HT 3A (IMAGE:9053347), 5-HT 4 (IMAGE:9053710), 5-HT 6 (IMAGE:40104889), 5-HT 7 (IMAGE:40126305) dig-UTP-labeled probes were used and detected using an antibody coupled to alkaline phosphatase (1/2000; Roche) and NBT/BCIP was used as blue substrate for in situ revelation.

Analysis and Counting Procedure

The number of BrdU-labeled cells was quantified with a bright-field microscope with a 40X objective, on serial sections through the entire hippocampus. One out of 6 sections of the series was counted (300 µm intervals; 6 sections per series). BrdU-labeled cells were counted in the subgranular zone (SGZ). Cells were considered to be BrdU + when their nuclei were completely filled with DAB product or showed clear patches of labeling. To facilitate the comparison between the three different mouse models, across different experiments, the number of BrdU + cells was normalized to the control values and expressed as %.

The surface of the DG was measured in 2-and 7-week old VMAT2 sert-cre and control mice.

Briefly, images from Nissl stained-sections through the entire hippocampus (3 mice/experimental condition) were acquired with a 10X objective and the granular cell layer was outlined using Image J software.

In proliferation and survival experiments, double-labeled cells (BrdU/ doublecortincalbindin -calretinin -GFAP) in the granular cell layer of the dentate gyrus were imaged with a confocal laser scanning microscope (SP2, Leica, Mannheim, Germany). 45 to 85 BrdU + cells per mouse (n = 5-6 mice/experimental group) were analyzed in their entire z-axis with a 1-µm step to exclude false double-labeling. Double-labeled cells were expressed as percentage of the total BrdU + cells.

In viral-labeling experiments, images were acquired with a 40X objective, with lateral and zaxis resolutions of 385 and 700nm respectively. Dendritic trees of GFP-expressing new neurons were visualized in 3-D reconstructions using the NeuronStudio software [START_REF] Rodriguez | Automated threedimensional detection and shape classification of dendritic spines from fluorescence microscopy images[END_REF] to quantify the number of dendritic nodes (n = 3-4 mice/experimental group; a total of 193 and 181 GFP + cells was analyzed in Pet1 -/-and Pet1 +/-mice, respectively).

Pattern separation

This behavioral test was carried out as previously published [START_REF] Sahay | Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation[END_REF] using contextual fear-discrimination learning, a type of hippocampal-dependent learning test that captures the animal's ability to discriminate two similar contexts. Briefly, adult (8 to 10 weekold) Pet1 -/-male mice were exposed to two similar contexts, with only one of them associated to a foot-shock. The shock-associated training context A, and the similar (no-shock) context B, shared many features, such as a stainless steel grid floor and a plastic roof. The differences between contexts were defined in a pilot study. The context B differed from the context A in that two colored paper inserts were used to cover the lateral walls and a mild limonene scent was used as an olfactory cue. An alcoholic solution was used to clean the grids between runs.

The same experimenter manipulated all the animals during all the experiments. Mice were individually brought into the testing room. For discrimination learning, mice were exposed to the training context in which they received a single 2 s foot shock of 0.8 mA at 180 s after placement in the chamber. Mice were taken out of the chamber 15 s after termination of the foot shock and returned to their home cage. After 1 hr, each mouse was placed in the similar B context, in which they were left for 180 s and were never shocked. On the test-days 1, 4, 7,

Statistical analysis

Differences between experimental groups for neurogenic responses were analyzed by the Mann Whitney test. Behavioral studies were analyzed by the One-way ANOVA for repeated measures test. Two-way analysis of variance (ANOVA) was employed in the case of the 8-OH-DPAT chronic treatment with genotype and treatment as main factors. Bonferroni's test was used for post hoc comparisons. In all cases, p < 0.05 was considered statistically significant.

Results

We examined the consequences of constitutive 5-HT depletion on adult DG neurogenesis in two previously characterized genetic mouse models of 5-HT depletion, the Pet1 -/-and the VMAT2 sert-cre mice [START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF][START_REF] Narboux-Neme | Severe serotonin depletion after conditional deletion of the vesicular monoamine transporter 2 gene in serotonin neurons: neural and behavioral consequences[END_REF].

In the Pet1 -/-, an 80% depletion of brain 5-HT levels is documented [START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF]. Basal cell proliferation was assessed in the subgranular zone of the DG in young adults 24 hrs after BrdU injections and we found that the total number of BrdU labeled cells was unchanged in Pet1 -/-mice (p=0.69; death between the first and fourth week after their generation [START_REF] Dayer | Short-term and longterm survival of new neurons in the rat dentate gyrus[END_REF][START_REF] Snyder | Adult-born hippocampal neurons are more numerous, faster maturing, and more involved in behavior in rats than in mice[END_REF]. To determine when a difference in survival rate becomes visible in the Pet1 -/-mice, we analyzed newborn cell survival 1 week after BrdU injections and found that Pet1 -/-mice already presented an increase of BrdU-labeled cells as compared to control (p <0.029; Fig 1 c). These results suggest that 5-HT may participate in the new neuron elimination process shortly after their production in the DG, since we calculated that in control mice, 50% of the newborn cells are lost as early as 1 week after generation confirming that cell death occurs very rapidly after proliferation (Suppl. Given these unexpected results and considering that a few residual 5-HT axons still innervate the subgranular layer of the DG in the Pet1 -/-mice [START_REF] Fernandez | Investigating anxiety and depressive-like phenotypes in genetic mouse models of serotonin depletion[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF], we performed the same experiments in another mouse model of 5-HT depletion: the VMAT2 sert-cre mice, in which a complete depletion of 5-HT-innervation has been reported [START_REF] Narboux-Neme | Severe serotonin depletion after conditional deletion of the vesicular monoamine transporter 2 gene in serotonin neurons: neural and behavioral consequences[END_REF]. Again, in the proliferation assay, the total number of BrdU labeled cells was unchanged in VMAT2 sert-cre mice (p=0.96) 24 hrs after BrdU injections (Suppl. Fig 2a). In addition, the ratio of BrdU + cells co-expressing the cell cycle marker Ki67 was not different between VMAT2 sert-cre and control mice (Suppl.

Fig 2e),

suggesting that, at least during the first 24 hr, the rate of rounds of division is similar in both genotypes. We next evaluated new neuron survival and found that similarly to observations in Pet1 -/-mice, a significant increase in the number of BrdU-labeled cells was observed in VMAT2 sert-cre mice at 4 weeks (p=0.0016) and 1 week (p=0.0152) after BrdU injection as compared to controls (Suppl. Fig 2 b -d). The surface of the DG was measured in 2-and 7week old VMAT2 sert-cre and no significant difference was detected compared to control mice (Suppl. Fig 2f ), suggesting an absolute increase in the number of immature cells in VMAT2 sert-cre mice. Altogether, these results indicated that in these two constitutive hyposerotonergic mouse models, relying on different mechanisms of 5-HT depletion, newborn cells are generated in normal numbers but survive in greater numbers.

To determine whether excess of surviving neurons are maintained or eventually cleared later when neural networks become consolidated, we counted BrdU-labeled cells 16 weeks after the initial BrdU injection in VMAT2 sert-cre mice. This experiment showed a 4-fold increase in new neuron survival (1335 ± 501; vs. control 342 ± 66, n=2) similar to the one observed after 4 weeks. Altogether these results indicate that constitutive lack of 5-HT neurotransmission results in an increased survival of newborn cells in the DG, with a cumulative effect between 1 and 4 weeks. These supernumerary cells remain integrated in the hippocampal circuits 4 months after their genesis. Both Pet1 -/-and VMAT2 sert-cre mice have decreased 5-HT during development that could alter the normal maturation of hippocampal circuits [START_REF] Trowbridge | Genetic models of serotonin (5-HT) depletion: what do they tell us about the developmental role of 5-HT?[END_REF]. Indeed, postnatal growth retardation is observed in hyposerotonergic mouse models (Narboux-Neme et al, 2013). To determine whether the observed effects on cell survival may result from defects during development, we analyzed mice in which 5-HT depletion was induced in adulthood. PCPA (the inhibitor of TPH, the enzyme responsible of 5-HT synthesis) was administered in 7-8 week-old wild type mice during 4 weeks, to induce a pharmacological depletion of 5-HT. By the end of this treatment, BrdU was administered to evaluate both cell proliferation and survival as in the others mouse models (Suppl Fig. 3). The chronic 4 week treatment with PCPA in adult WT mice mimicked the effects previously seen in Pet1 -/-and VMAT2 sert-cre mice: no change in cell proliferation (p=0.63; One possible cause of the increased survival rate of the newborn cells in the DG of hyposerotoninergic mouse models could be a change in the maturation rate of newly produced neurons, as demonstrated after SSRI administration [START_REF] Wang | Chronic fluoxetine stimulates maturation and synaptic plasticity of adult-born hippocampal granule cells[END_REF]. We analyzed colocalization of BrdU with calretinin and doublecortin, which are markers of immature neurons, and with calbindin, a marker of more mature neurons. Experiments were performed in the Pet1 -/-and control mice, 1 and 2 weeks after BrdU injections. These experiments showed an identical proportion of co-labeled cells in both genotypes (Fig 2a-a', b-b'), indicating that the surplus surviving newborn cells matured normally. The cell fate of the 4 week-old hippocampal newborn neurons was examined by using double labeling of BrdU and the mature neuronal marker NeuN. The proportion of newborn cells that became neurons (80-90%) was similar in all genotypes. Finally, we analyzed the morphological maturation of the newborn neurons, using retroviral vectors that integrate only in the dividing cells. Newborn GFP-labeled cells showed a comparable morphological maturation of dendritic trees in Pet1 -/- and control mice (Fig 2 c-c').

Immature DG cells have been recently proposed as playing a role in the pattern separation process [START_REF] Nakashiba | Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion[END_REF][START_REF] Sahay | Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation[END_REF]. To evaluate the functionality of these supernumerary immature DG cells found in hyposerotonergic mice, Pet1 -/-mice were tested in a contextual fear discrimination learning. In a pilot study, we evaluated the level of freezing in the shock-associated context on day 0 and found negligible levels of freezing for both genotypes (p=0.09; suggesting that Pet1 -/-and control mice acquired and retained contextual fear conditioning at a similar rate in our experimental conditions. As expected, control mice showed comparable levels of freezing in both contexts during the first 6 days (Fig 3 b), after which, the discrimination index increased progressively, showing their capacity to discriminate between the shock-associated and no-shock contexts. The level of discrimination was unchanged in Pet1 -/-mice compared to controls (p=0.60), suggesting that the increased number of immature DG cells in the Pet1 -/-mice does not modify performance in pattern separation.

The consistency in the increased survival rate of newborn neurons observed in the 3 different chronic hyposerotonergic models analyzed here suggests that a lack of 5-HT during the first weeks after cell generation in the hippocampus hinders the regulatory mechanisms that normally operate to restrict their number before integrating into functional circuits. We hypothesized that 5-HT may act through specific 5-HT receptors to help selected surviving cells and/or to remove surplus neurons. We examined 5-HT receptor expression in the DG using in situ hybridizations with a large panel of 5-HT receptor probes. We found expression of 6 5-HT receptors in the DG of the hippocampus: the 5-HT 1A , 5-HT 2B , 5-HT 3A 5-HT 4, 5-HT 6 , and 5-HT 7 receptors (Fig 4 a). The 5-HT 1B and the 5-HT 2A-C receptors were expressed in the hippocampus but not in the DG cells. The 5-HT 5 probe did not provide an interpretable signal. Among the 5-HT receptors expressed in the granular cells of the DG, the 5-HT 1A receptor matched best the localization of newborn neurons in the subgranular zone, as also suggested in a recently published work [START_REF] Klempin | Oppositional effects of serotonin receptors 5-HT1a, 2, and 2c in the regulation of adult hippocampal neurogenesis[END_REF]. To determine whether the lack of 5-HT 1A receptor stimulation in the DG of hyposerotonergic mice may be responsible for the enhanced survival phenotype, we evaluated cell survival in both Pet1 -/-and PCPA-treated mice following a 1-week treatment with the 5-HT 1A receptor agonist 8-OH-DPAT. Since the 5-HT raphe neurons projecting to the hippocampus do not develop in the Pet1 -/-mice [START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF], the effects of 8-OH-DPAT in these mutants are essentially postsynaptic. As shown above, the number of 1 week-old neurons was significantly higher in Pet1 -/-mice (p<0.05) and in PCPA-treated mice (p<0.01) compared with their respective control groups (Fig 5 a,b). In control mice, a 1-week treatment with 8-OH-DPAT did not change cell survival. However, the number of newborn cells reestablished the control levels after the 1-week treatment with 8-OH-DPAT in the Pet1 -/-(p<0.05) and the PCPA-treated mice (p<0.01), suggesting a cell-autonomous regulation of cell survival via the post-synaptic 5-HT 1A receptor. Thus, 5-HT 1A post-synaptic receptor stimulation can reverse the enhanced survival observed in genetic and pharmacological models of constitutive 5-HT depletion. no effect on neural stem cells or progenitor cell cycle as demonstrated in VMAT sert-cre mice in which the ratio BrdU + -Ki67 + / BrdU + -Ki67 -is similar to that in control mice.

Majority of the cells generated in the DG of WT mice die during the cell differentiation process [START_REF] Snyder | Adult-born hippocampal neurons are more numerous, faster maturing, and more involved in behavior in rats than in mice[END_REF], and it was even recently demonstrated that this cell death occurs between 1 and 4 days after cell birth [START_REF] Sierra | Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis[END_REF]. In our study, we also found in WT animals, that most of the newborn neurons are eliminated within the first week of maturation.

By contrast, all our three 5-HT depletion models show a robust increase in newborn neuron survival, an effect that is observed as early as one week after their birth and that seems to last at least for 16 weeks, with no change in the DG surface. Interestingly, the magnitude of increase in the cell survival was inversely correlated with the 5-HT amount: increase in DG cell survival was stronger in VMAT2 sert-cre mice that show the most severe 5-HT depletion [START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF][START_REF] Narboux-Neme | Severe serotonin depletion after conditional deletion of the vesicular monoamine transporter 2 gene in serotonin neurons: neural and behavioral consequences[END_REF], compared to the Pet1 -/-mice and PCPA treated mice in which biosynthesis 5-HT level is not totally abolished [START_REF] Vitalis | Embryonic depletion of serotonin affects cortical development[END_REF], These observations indicate that 5-HT is required in the process of eliminating newborn neurons.

Several 5-HT receptors have been implicated in the neurogenic effects of antidepressants [START_REF] Banasr | Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone[END_REF][START_REF] Diaz | 5-HT(2B) receptors are required for serotonin-selective antidepressant actions[END_REF][START_REF] Klempin | Oppositional effects of serotonin receptors 5-HT1a, 2, and 2c in the regulation of adult hippocampal neurogenesis[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF][START_REF] Soumier | Region-and phase-dependent effects of 5-HT(1A) and 5-HT(2C) receptor activation on adult neurogenesis[END_REF]. Particularly, the downregulation of presynaptic 5-HT 1A receptors induced by chronic SSRI treatment has been proposed to be required for the expression of some behavioural effects (Popa et al., 2010). To determine which 5-HT receptors is involved in the effects of 5-HT depletion on neuron survival, we performed an in situ hybridization screen and found that 5-HT 1A receptor displays an interesting distribution because it is expressed in the subgranular zone as previously shown [START_REF] Klempin | Oppositional effects of serotonin receptors 5-HT1a, 2, and 2c in the regulation of adult hippocampal neurogenesis[END_REF], where stem cells and progenitor cells are located [START_REF] Encinas | Fluoxetine targets early progenitor cells in the adult brain[END_REF]. However, 5-HT 1A -/-mice show no change in basal levels of BrdU incorporation [START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF] and studies using various 5-HT receptor agonists or antagonists have all concluded that 5-HT 1A agonist stimulation from one to two weeks had no effect in DG BrdU + cell numbers in WT animals [START_REF] Banasr | Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone[END_REF][START_REF] Huang | The role of 5-HT1A receptors in the proliferation and survival of progenitor cells in the dentate gyrus of the adult hippocampus and their regulation by corticoids[END_REF][START_REF] Klempin | Oppositional effects of serotonin receptors 5-HT1a, 2, and 2c in the regulation of adult hippocampal neurogenesis[END_REF][START_REF] Soumier | Region-and phase-dependent effects of 5-HT(1A) and 5-HT(2C) receptor activation on adult neurogenesis[END_REF]. Indeed, we confirmed that a one-week treatment with 8-OH-DPAT did not alter BrdU + cell number in control mice. In contrast, the same treatment normalized the number of newborn cells to control levels in both Pet1 -/-mice and PCPA-treated WT mice. The strong reduction of the 5-HT innervation in the hippocampus of Pet1 -/-mice allows us to conclude that the results obtained herein reflect mostly the involvement of post-synaptic 5-HT 1A receptor stimulation in the regulation of cell survival and suggest a role for 5-HT, via the 5-HT 1A receptor, in the differentiation-or-death decision of newborn cells. Interestingly, the vast majority of DG newborn cells undergoes death by apoptosis in the first 4 days of their life, during the transition from amplifying neural progenitors to early post-proliferative neuroblasts [START_REF] Sierra | Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis[END_REF]. The 5-HT survivalapoptosis decision, 5-HT 1A receptor-dependant, may correspond to this transition from late dividing neural progenitors to early postmitotic neuroblasts.

Increased survival of newborn neurons may be expected to have impacts on memory and learning (rev. in [START_REF] Koehl | A new chapter in the field of memory: adult hippocampal neurogenesis[END_REF] and depressive-like phenotypes (rev. in [START_REF] Petrik | The neurogenesis hypothesis of affective and anxiety disorders: are we mistaking the scaffolding for the building?[END_REF], although recent reviews underlined the difficulty to establish a causal role of increased neurogenesis for the control of these behaviours. Cytostatic drugs, irradiation, and genetic manipulation demonstrated that the ablation of newborn neurons impairs several forms of hippocampus-depending learning and memory. However, controlled elimination of newborn cells by cell death is also crucial, since mice with disruption of the pro-apoptotic factor Bax performed worse than control mice in spatial memory tests, whereas pattern separation was improved [START_REF] Dupret | Spatial learning depends on both the addition and removal of new hippocampal neurons[END_REF][START_REF] Sahay | Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation[END_REF]. Likewise, another genetically modified mouse model with an increased population of young DG cells exhibited enhanced pattern separation between similar contexts [START_REF] Nakashiba | Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion[END_REF]. In contrast, we report here that Pet1 -/-mice have a normal performance in pattern separation, although they have an increased number of immature DG cells. However it is possible that in the hyposerotoninergic mouse lines, the functional deficits caused by globally decreased 5-HT neurotransmission prevents the potential beneficial effect of an increased number of immature neurons in the DG.

Moreover, despite the apparently normal maturation of newborn neurons at early stages (1 week) it is possible that late maturation or integration of these neurons in functional circuits is suboptimal in conditions of hyposerotoninergia. It remains to be determined whether the increase in the number of adult born neurons is responsible for some of the behavioral traits observed in the Pet1 -/-mice such as enhanced conditioned fear responses [START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF]. Moreover it should be noted that our results are consistent with a possible relationship between enhanced neurogenesis and reduced depression-like behaviour [START_REF] Pittenger | Stress, depression, and neuroplasticity: a convergence of mechanisms[END_REF], since VMAT2 sert-cre mice displayed enhanced struggling in the tail suspension test (Narboux-Neme et al, 2011) while Pet1 -/-showed no changes floating time in the forced swim test [START_REF] Diaz | 5-HT(2B) receptors are required for serotonin-selective antidepressant actions[END_REF], and both lines showed anxiolytic-like response in the noveltysuppressed feeding test [START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF], Narboux-Neme et al, 2011).

The present findings add new insights in the mechanisms and pathways that finely regulate early stages of neurogenesis and pave the way for future studies to better comprehend the factors influencing the fate of immature newborn DG cells.

  8 and 10, mice were first exposed to context A before context B, whereas on the test-days 2, 3, 5, 6 and 9, they were first exposed to context B. Measurement of the freezing levels in both the A training context (3 min pre-shock) and the similar B context (3 min) each day allowed the assessment of discrimination between the two contexts and was computed as a discrimination index: (Freezing A context -Freezing B context )/ (Freezing A context + Freezing B context ). A score of 0 indicates complete lack of discrimination (equal freezing levels in both contexts), whereas a score of 1 indicates perfect discrimination (no freezing in context B).

  Fig 1 a). However when BrdU labeled cells in the DG were counted 4 weeks after the BrdU injections, a significant increase was observed in Pet1 -/- mice as compared to controls (p <0.046; Fig 1 b), indicating an increase in the survival rate of newborn cells. It is known that 50 to 75% of the newly produced cells in the DG undergo cell

Fig

  Fig 1 d), but a significant increase in cell survival at 4 weeks (p<0.02; Fig 1 e) and at 1 week (p=0.05; Fig 1 f) after BrdU administration. These results demonstrated that inducing a 5-HT depletion in adult life is enough to improve survival of the newborn neurons in the DG.

  Fig 3 a). Similar increased freezing times were found for both genotypes on day 1, i.e. 24 hrs after the first exposure to the context A (p=0.24; Fig 3 a),

FiguresFigure 1 .

 1 Figures

Figure 2 .Figure 3 .

 23 Figure 2. Normal differentiation of newborn neurons in Pet1 -/-mice. (a-b') Multiple immunohistochemistry were performed on control of Pet1 -/-mice one week after BrdU injection. The proportion of cells expressing a differentiation marker within BrdU positive cells was measured and compared between genotypes. We checked the expression of the immature neuron markers Calretinin (CalR a, a') and Doublecortin (Dcx, b, b'), as well as the expression of the marker of mature DG neurons Calbindin (Cabp, a, a'). No difference was found in the expression of these markers between genotypes. (c-c') Analysis of the morphology of newborn neurons was performed one week after the injection of GFPexpressing retrovirus. The number of nodes was quantified for each labeled cell as a marker of differentiation. No significant difference was found between controls and Pet1 -/-mice. Scale bar = 20µm

Acknowledgments:

This research was funded by Institut National de la Recherche et de la Santé (Inserm), the Université Pierre et Marie Curie (Paris 6), the European Commission (FP7-health-2007-A-201714) and the Agence Nationale pour la recherche (ANR-MNP-neur-032). SD and NN were supported by fellowships from the Région Ile de France, ST was supported by the Henry Russell Shaw travelling fellowship from Harvard University. The laboratory of PG is part of the Ecole des Neurosciences de Paris, training network.

Discussion

The present observations demonstrate that 5-HT is: (i) dispensable for neural stem cell and progenitor proliferation in the DG and (ii) necessary for the removal of surplus immature newborn neurons via post-synaptic 5-HT 1A receptor stimulation.

A number of studies report that enhanced hippocampal neurogenesis is required for the therapeutic response to the increased 5-HT levels induced by SSRIs treatments [START_REF] Malberg | Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF] rev. in [START_REF] Samuels | Neurogenesis and affective disorders[END_REF][START_REF] Samuels | Neurogenesis and affective disorders[END_REF][START_REF] Samuels | Neurogenesis and affective disorders[END_REF].

Conversely this observation would predict that, reduced neurogenesis should be observed in hyposerotonergic mice, although there is as yet little evidence that impaired neurogenesis has a causal role in the etiology of depression [START_REF] Pittenger | Stress, depression, and neuroplasticity: a convergence of mechanisms[END_REF]see the meta-analysis in [START_REF] Petrik | The neurogenesis hypothesis of affective and anxiety disorders: are we mistaking the scaffolding for the building?[END_REF]). Initial studies in rats by Daszuta et al. showed a strong reduction in hippocampal cell proliferation after serotonergic 5,7-DHT lesions in the raphe [START_REF] Brezun | Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats[END_REF][START_REF] Brezun | Serotonergic reinnervation reverses lesion-induced decreases in PSA-NCAM labeling and proliferation of hippocampal cells in adult rats[END_REF]. However, subsequent studies using 5,7-DHT lesions by ICV injections, reported a change neither on cell proliferation [START_REF] Huang | The role of 5-HT1A receptors in the proliferation and survival of progenitor cells in the dentate gyrus of the adult hippocampus and their regulation by corticoids[END_REF] nor on cell survival [START_REF] Jha | Selective serotonin depletion does not regulate hippocampal neurogenesis in the adult rat brain: differential effects of pchlorophenylalanine and 5,7-dihydroxytryptamine[END_REF]. These conflicting reports suggest that 5-HT could play multiple and perhaps opposing roles, via different 5-HT receptors, in the regulation of the different neurogenic stages.

Nevertheless, experimental factors, such as mode of 5-HT depletion/up-regulation, duration of treatment and timing of observations, could probably also account for these discrepancies [START_REF] Huang | The role of 5-HT1A receptors in the proliferation and survival of progenitor cells in the dentate gyrus of the adult hippocampus and their regulation by corticoids[END_REF][START_REF] Jha | Selective serotonin depletion does not regulate hippocampal neurogenesis in the adult rat brain: differential effects of pchlorophenylalanine and 5,7-dihydroxytryptamine[END_REF]. Here, we found that there was no change in cell proliferation in the DG of three independent models of 5-HT depletion: complete loss of 5-HT-storage in the VMAT2 sert-cre axons (Narboux-Neme et al, 2011), failure to differentiate raphe neurons in Pet1 -/-mice [START_REF] Hendricks | Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior[END_REF][START_REF] Kiyasova | A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei[END_REF], and pharmacological inhibition of 5-HT synthesis. These results indicate that 5-HT depletion had

Disclosure:

The authors have no financial interests or conflict of interest to disclose, (a) On day 0, Pet1 -/-and control mice showed negligible levels of freezing in context A before receiving the foot shock (n= 4-6). On day 1, both groups showed comparable levels of conditioning to training context A (n= 11-13). (b) Freezing levels as indicated by the discrimination ratio, i.e. (Freezing A context -Freezing B context )/ (Freezing A context + Freezing B context ) were similar in Pet1 -/-and control mice (n= 6-13). Data are expressed as mean + SEM. Representative 5-HT 1A , 5-HT 2B , 5-HT 3A , 5-HT 4 , 5-HT 6 , and 5-HT 7 mRNA labeling in the DG of WT mice determined by in situ hybridization . Scale bar = 100µm After a 1 week-treatment, 8-OH-DPAT (0.5 mg/kg; twice a day) did not affect cell survival in control mice, but hindered the increased basal cell survival phenotype described in PET1 -/- mice (a) as well as in 4-week-treated PCPA WT mice (b). Data are expressed as mean + SEM (* , + p <0.05; ** , ++ p <0.01; n=3-4).

Supplementary Figure 1.

The comparison of the number of BrdU + cells in WT mice 24h and 1 week after BrdU injection indicates that 50% of the cells labeled one day after BrdU injection are missing one week later. Data are expressed as mean + SEM (*** p <0.001; n = 4-5 mice for each group).

Supplementary Figure 2.

(a) Cell proliferation in the subgranular zone 24 h after BrdU administration was similar in control mice and VMAT2 sert-cre (n=7-8). On the contrary, cell survival in the cell granular The surface of the DG measured in 7-week old VMAT2 sert-cre mice was similar in both groups (n=5). Scale bar = 20µm Supplementary Figure 3. Experimental protocol employed for 5-HT depletion during adulthood. PCPA was administered daily during 4 weeks (300 mg/kg for the first 3 days; 100 mg/kg for the rest of the treatment) to WT animals. BrdU (2 injections at 150 mg/kg injected 2 hr apart) was administered i.p. after the first 4 weeks of PCPA treatment. A group of animals was sacrificed 24 hr after BrdU treatment for evaluating cell proliferation whereas another group went on receiving PCPA 100mg/kg and sacrificed 1 week or 4 weeks after BrdU treatment for evaluating cell survival.


Thomas Degueule
email: degueule@cwi.nl

Tanja Mayerhofer
email: mayerhofer@big.tuwien.ac.at

Andreas Wortmann
email: wortmann@se-rwth.de

Engineering a ROVER Language in GEMOC STUDIO & MONTICORE: A Comparison of Language Reuse Support

Keywords: Software Language Engineering, Language Reuse, Language Workbenches, Rover, GEMOC Studio, Monti-Core

Domain-specific languages (DSLs) improve engineering productivity through powerful abstractions and automation. To support the development of DSLs, the software language engineering (SLE) community has produced various solutions for the systematic engineering of DSLs that manifest in language workbenches. In this paper, we investigate the applicability of the language workbenches GEMOC STUDIO and MONTICORE to the MDETools'17 ROVER challenge. To this effect, we refine the challenge's requirements and show how GEMOC STUDIO and MONTICORE can be leveraged to engineer a Rover-specific DSL by reusing existing DSLs and tooling of GEMOC STUDIO and MONTICORE. Through this, we reflect on the SLE state of the art, detail capabilities of the two workbenches focusing particularly on language reuse support, and sketch how modelers can approach ROVER programming with modern modeling tools.

I. INTRODUCTION

Domain-specific languages (DSL) improve software engineering productivity and system quality through powerful domain abstractions and increased automation. To support DSL development, the software language engineering (SLE) [START_REF] Kleppe | Software Language Engineering: Creating Domain-Specific Languages Using Metamodels[END_REF] community has proposed various solutions for the systematic development, use, deployment, and maintenance of DSLs and related tools. These solutions manifest in language workbenches [START_REF] Erdweg | The State of the Art in Language Workbenches[END_REF] that assist language engineers in all DSL engineering phases.

In this paper, we present the language workbenches GEMOC STUDIO [START_REF]The GEMOC Studio[END_REF] and MONTICORE [START_REF] Krahn | MontiCore: a framework for compositional development of domain specific languages[END_REF] to demonstrate and compare their language engineering support on the MDETools'17 ROVER challenge [START_REF]Model-Driven Engineering Tools Challenge (MDETools'17)[END_REF]. GEMOC STUDIO is based on the Eclipse Modeling Framework (EMF) [START_REF] Steinberg | EMF: Eclipse Modeling Framework[END_REF] and provides support for implementing executable DSLs and supporting tooling. This includes meta-programming approaches for defining DSL interpreters and generic components for efficiently developing DSL tools including model animators and debuggers. MONTICORE offers support for implementing textual DSLs with context-free grammars and provides a powerful infrastructure for developing analyzers, transformations, and code generators.

We present implementations of the RASPIROVER DSL, a DSL for defining the architecture and behavior of Raspberry Pi operated rovers, with both language workbenches. In these implementations, we aimed at reusing as many DSLs as possible to ease and speed up the DSL engineering process for the RASPIROVER DSL. We show (i) how to develop DSLs for software-controlled rovers with both language workbenches by reusing existing DSLs, (ii) the tool support that both workbenches offer to engineers for using the developed DSLs, and (iii) the individual strengths of GEMOC STUDIO and MONTICORE with regard to their support for engineering DSLs and supporting tools. We put a particular emphasis on language reuse [START_REF] Erdweg | Language Composition Untangled[END_REF] techniques offered by both language workbenches to facilitate DSL engineering.

In the following, Section II details the ROVER challenge studied in this paper. Section III and Section IV describe the solutions developed with GEMOC STUDIO and MONTICORE, respectively. Section V compares the language engineering support provided by the two studied language workbenches focusing particularly on language reuse. Finally, Section VI concludes.

II. ROVER LANGUAGE ENGINEERING CHALLENGE

Both GEMOC STUDIO and MONTICORE have been used in the past for implementing DSLs facilitating the development of software-controlled rovers. The ARDUINOML language [START_REF]Arduino Modeling GitHub Project[END_REF] has been implemented using GEMOC STUDIO for modeling the architecture and behavior of Arduino-based systems, such as Arduino-operated rovers. The MONTIARCAUTOMATON (MAA) [START_REF] Ringert | Language and Code Generator Composition for Model-Driven Engineering of Robotics Component & Connector Systems[END_REF] architecture description language has been implemented using MONTICORE for modeling the architecture and behavior of robotics applications, such as the architecture and behavior of LEGO NXT operated rovers as well as powerful service robots [START_REF] Ringert | Language and Code Generator Composition for Model-Driven Engineering of Robotics Component & Connector Systems[END_REF]. While MAA is designed to be a platformindependent DSL for developing robotics applications running on any kind of platform, ARDUINOML is specific to the Arduino platform.

In this paper, we present a case study on reusing these and other existing DSLs to develop a new RASPIROVER DSL customized for the development of Raspberry Pi operated rovers, such as the Eclipse PolarSys Rover [START_REF] Polarsys | PolarSys Rover[END_REF]. Our goal is to showcase the language reuse support offered by MONTICORE Before we present the implementations of RASPIROVER DSL with GEMOC STUDIO and MONTICORE, we first describe the requirements imposed on the development of RASPIROVER DSL regarding desired language features, reuse of existing DSLs, and DSL tooling.

A. Language Requirements

To set the language requirements for RASPIROVER DSL, we investigated several DSLs that have been used in the past to model rover systems. In particular, we investigated ARDUINOML and MAA mentioned before, as well as the UML-RT implementation of PAPYRUS-RT [START_REF]The Eclipse Foundation[END_REF]. What the investigated modeling languages have in common is that they provide concepts for modeling both the hardware architecture of a rover and the behavior of the rover's control software. There are however considerable differences, in particular in how behavioral aspects are described. For instance, ARDUINOML provides an action language for defining the behavior, while PAPYRUS-RT relies on UML state machines with embedded C++ code. Based on our investigations, we decide to include in RASPIROVER DSL a hardware architecture description language and an action language as detailed in the following.

Rover Hardware Architecture Description Language: RASPIROVER DSL should enable the definition of the hardware setup of a Raspberry Pi operated rover. This includes defining hardware parts of the rover, such as the different actuators and sensors, as well as their connection to the rover's control unit through describing the mapping of the rover's board's pins to hardware modules. The primary rationale for including the hardware mapping into the DSL is to make this mapping explicit in a rover model, such that it can be leveraged in code generation and for interacting with the rover when executing a rover model.

Rover Control Language (RCL): For defining the behavior of rovers, RASPIROVER DSL should provide an imperative rover control language. We choose an action language because even if other behavioral languages (such as UML state machines) are used, an action language is still required to define finegrained behaviors. Furthermore, the action language should abstract from a concrete programming language used to implement rover control software by providing high-level actions commonly supported by rovers. Therefore, RCL should comprise [START_REF] Kleppe | Software Language Engineering: Creating Domain-Specific Languages Using Metamodels[END_REF] and conditional statements as well as variables, primitive values, and variable assignments; (2) rover-specific queries and actions, in particular, dedicated queries for retrieving the temperature and humidity of a rover's environment, for obstacles in front of and behind the rover, and for messages posted to a central message board (e.g., messages remotely sent to a rover), as well as actions for moving forward and backward, turning, stopping, logging, and sending messages; (3) units for defining distances to travel forward or backward, as well as the rotation angle for turning.

Fig. 1 shows an exemplary rover hardware description model that should be definable with RASPIROVER DSL. It features a control unit, two motors allowing the rover to drive forward, backward, and turn, an obstacle sensor on the front of the rover, as well as a message subscriber component allowing the rover to receive input commands. Fig. 2 shows an example of a rover control program written with RASPIROVER DSL defining that the rover should travel between two obstacles. In particular, it specifies that the rover should travel forward for 3 cm as long as no obstacle is detected in front of the rover (ll. [START_REF] Krahn | MontiCore: a framework for compositional development of domain specific languages[END_REF][START_REF]Model-Driven Engineering Tools Challenge (MDETools'17)[END_REF][START_REF] Steinberg | EMF: Eclipse Modeling Framework[END_REF]. When an obstacle is detected, the rover should turn in the opposite direction (l. 7). This is repeated until a "stop" command is received via the message board (l. 3).

B. Reuse Requirements

Since we focus in this study on a comparison of language reuse support offered by the investigated language workbenches, we require that existing DSLs previously engineered with GEMOC STUDIO and MONTICORE should be reused wherever possible.

For implementing RASPIROVER DSL as specified above, we identify several DSLs that can be reused in GEMOC STUDIO and MONTICORE as shown in Table I. In particular, we aim to reuse ARDUINOML for the GEMOC STUDIO solution, as well as MAA and Java/P for the MONTICORE solution.

The GEMOC STUDIO solution relies on a revised version of the unit domain model proposed in [START_REF] Mayerhofer | Adding Uncertainty and Units to Quantity Types in Software Models[END_REF], while the units are implemented by hand in the MONTICORE solution. We manually implement the rover-specific queries and actions from scratch in both cases.

C. Tool Requirements

Regarding tooling support, we want to reuse any existing tool of the reused languages for the resulting RASPIROVER General Action Language ARDUINOML Java/P [START_REF] Schindler | Eine Werkzeuginfrastruktur zur agilen Entwicklung mit der UML/P, ser[END_REF] Units Reused Newly from [START_REF] Mayerhofer | Adding Uncertainty and Units to Quantity Types in Software Models[END_REF] implemented Rover Actions Newly implemented DSL language. This comprises, for instance, editors, model animators, model debuggers and code generators available for ARDUINOML and MAA. Other than that, we impose no further requirements, as the set of tools that can be developed with GEMOC STUDIO and MONTICORE is quite diverse. For instance, GEMOC STUDIO mostly supports graphical languages, while MONTICORE focuses on textual languages.

As another example, GEMOC STUDIO specializes in language interpreters while MONTICORE specializes in code generators.

III. THE RASPIROVER DSL IN GEMOC STUDIO

The GEMOC STUDIO [START_REF]The GEMOC Studio[END_REF] is a language workbench for designing and using executable DSLs. It is built atop various modeling technologies of the Eclipse ecosystem (e.g., EMF, Xtext, Sirius) and contributes new components, such as a model animator and a model debugger [START_REF] Bousse | Supporting Efficient and Advanced Omniscient Debugging for xDSMLs[END_REF], along with customizable execution engines [START_REF] Bousse | Execution Framework of the GEMOC Studio (Tool Demo)[END_REF]. Following the GEMOC approach, the abstract syntax of a DSL is specified in Ecore [START_REF] Steinberg | EMF: Eclipse Modeling Framework[END_REF], its concrete syntax in either Xtext or Sirius, and its execution semantics using various meta-languages (e.g., K3 [START_REF] Diverse | K3 -Executable Metamodeling[END_REF], ALE [START_REF] Leduc | Revisiting Visitors for Modular Extension of Executable DSMLs[END_REF], or xMOF [START_REF] Mayerhofer | xMOF: Executable DSMLs based on fUML[END_REF]). By specifying these artifacts, users of GEMOC STUDIO then enjoy various facilities, e.g., language-specific debuggers and animators [START_REF] Bousse | Execution Framework of the GEMOC Studio (Tool Demo)[END_REF].

As one of the showcases of the GEMOC STUDIO, the authors designed ARDUINOML, a DSL with simulation and animation capabilities for designing programs that can be deployed on a given configuration of an Arduino board. The metamodel describing the abstract syntax of ARDUINOML consists of two main parts: a description of the hardware deployed on a given Arduino board (the set of modules and their associated pins), and a description of the scenario putting these modules into play with a simple action language providing basic control structures, variables, and expressions. The ARDUINOML language also comes with an associated interpreter.

Rather than starting the development of a new DSL for the PolarSys rover from scratch, one could reuse some parts of ARDUINOML and customize them to the specificities of the PolarSys rover, thereby obtaining a customized RASPIROVER DSL. To achieve this, GEMOC STUDIO includes MELANGE, a meta-language that allows language designers to reuse and compose various DSLs in the creation of new ones [START_REF] Degueule | Melange: A Meta-language for Modular and Reusable Development of DSLs[END_REF]. Using MELANGE, it is possible to prune the Arduino-specific parts of the ARDUINOML language, merge new concepts specific to the PolarSys rover (i.e., rover actions and units as described in Section II-A), and customize the existing execution semantics (i.e., override certain methods of the interpreter) to make the different parts fit together. Fig. 3 depicts the definitions of the various languages reused for implementing RASPIROVER DSL in MELANGE, along with the definition of the RASPIROVER DSL itself. The language implementations are available online. 1First, the rover control language RCL is defined (ll. 3-6). It consists of its abstract syntax, defined in Ecore (l. 4), along with its execution semantics defined in K3 (l. 5). The Units and ArduinoML languages are defined in a similar way (ll. 7-10 and 11-14). Finally, the RasPiRover language composes the reused language components in a meaningful way. First, it slices [START_REF] Degueule | Melange: A Meta-language for Modular and Reusable Development of DSLs[END_REF] the ArduinoML language (l. 17) to extract the hardware-definition part and remove the Arduino-specific parts that should not be reused in the new language. Then, it merges the two other languages RCL and Units into the RasPiRover DSL (ll. [START_REF] Degueule | Safe model polymorphism for flexible modeling[END_REF][START_REF] Haber | Composition of Heterogeneous Modeling Languages[END_REF]. Finally, additional K3 aspects are woven to glue together the different components both syntactically and semantically (l. 22). As an example, Fig. 4 depicts the ProjectToProgramGlue and OverridenProjectInterpreter aspects that, respectively, insert a new containment reference from ArduinoML's Project (l. 1) to RCL's RoverProgram (l. 3), and override the execution semantics of Project to instead delegate to the interpreter of RCL (ll. 7-9). The renaming clauses (Fig. 3, ll. 18-21) simply ensure that all concepts of the three languages end up in the same logical package.

Overall, the MELANGE meta-language allows us to compose the three languages and reuse (parts of) their syntax and semantics (interpreter). As a result, the model execution capabilities of GEMOC STUDIO can be employed for executing RASPIROVER DSL models. The main limitation of the reuse support offered by MELANGE is that the composition operators do not cope with concrete syntax, i.e., the concrete syntax of the resulting language must be defined by hand to benefit from the animation and debugging facilities of GEMOC STUDIO. For more information on the reuse support offered by MELANGE, we refer the interested reader to [START_REF] Degueule | Melange: A Meta-language for Modular and Reusable Development of DSLs[END_REF], [START_REF] Degueule | Safe model polymorphism for flexible modeling[END_REF].

IV. THE RASPIROVER DSL IN MONTICORE

MONTICORE [START_REF] Krahn | MontiCore: a framework for compositional development of domain specific languages[END_REF] is a workbench for the development of modular, textual languages based on extended context-free grammars, Java context conditions and translational realizations of semantics using template-based code generation. It enables reusing (parts of) languages via inheritance, embedding, and aggregation [START_REF] Haber | Composition of Heterogeneous Modeling Languages[END_REF]. Inheritance enables reusing productions from the inherited grammar for which MONTICORE generates parsers, abstract syntax classes, context condition checking infrastructures, and code generation infrastructures. This supports specializing or extending languages while reusing existing tooling from the inherited language. With embedding, extension points in the host grammar are filled with productions from embedded grammars. This enables, for instance, reusing languages for well-defined concerns, such as expression languages. Aggregation loosely combines languages for joint analysis. To this effect, elements used in models of one language that reference elements of models of another language (such as references to data types in an architecture language) are interpreted specific to the integration. This integration is external to both languages and, hence, does not require participating languages to be aware of the integration.

A. Reusable MONTICORE Languages

Many of the languages required for realizing RASPIROVER DSL are already available in MONTICORE: MAA [START_REF] Ringert | Language and Code Generator Composition for Model-Driven Engineering of Robotics Component & Connector Systems[END_REF] enables describing software components and can thus be reused for the hardware architecture description language of RASPIROVER DSL through language inheritance. For realizing the rover control language RCL of RASPIROVER DSL, JAVA/P, the action language of UML/P [START_REF] Rumpe | Modeling with UML: Language, Concepts, Methods[END_REF] can be refined and extended as required. For this, we can first restrict JAVA/P to feature only variable assignments, statements, conditionals, and whileloops. This is achieved by inheritance, i.e., RCL inherits from JAVA/P, and an adds additional context condition that prevents instances of unsupported JAVA/P abstract syntax classes from being used in RASPIROVER DSL models. Then we add new primitives dedicated to movement, sensing, and communication to RCL. Furthermore, the UNITS language RCLEmbedding implements Element = "behavior" Name "{" Statement+"}"; } from RCL from MontiArcAutomaton Fig. 6. Grammars RCL and RasPiRover realizing the definition and integration of RASPIROVER DSL into MAA is embedded into RCL. Consequently, the overall language composition of RASPIROVER DSL is as depicted in Fig. 5. MONTICORE combines the parsers and abstract syntax classes of the languages accordingly.

B. Combining Syntaxes for the RASPIROVER DSL

Leveraging JAVA/P through inheritance enables the integration of new primitives for rover actions and queries via its various interfaces. To this end, rover actions, which resemble statements in JAVA/P, are designed to implement JAVA/P's Statement interface. This enables the usage of the new actions wherever statements are supported, e.g., loops and conditionals. Queries, which resemble literals, are analogously designed to implement MONTICORE's Literal interface, enabling their use wherever literals are supported. With this, the complete RCL grammar is only 17 lines of code (15 new productions) as illustrated in Fig. 6 (top). In MONTICORE, embedding is a specific usage of grammar inheritance that links productions of the grammar to be embedded into extension points of the host grammar. For embedding RCL into RASPIROVER DSL, we leverage MONTICORE's multiple inheritance to realize embedding as linking one interface from the host grammar MAA to productions from the embedded grammar RCL. This is depicted in Fig. 6 (bottom). With this, we obtain a textual concrete syntax as illustrated in Fig. 7.

Well-formedness of RCL programs is checked by new context conditions that ensure, for instance, that results of receive? queries (which return strings) are not compared to numbers. To this end, adapters between the abstract syntax classes generated from MAA's ports and RCL's primitives enable interpreting the latter as method calls of the appropriate return types, i.e., whenever MONTICORE looks up what receive? in the context of JAVA/P is, the adapters return

C. Combining Code Generators for the RASPIROVER DSL

Integrating the behavior of MAA (scheduling, message passing) and RCL (commanding the rover to perform actions) requires ( 1) the integration of their template-based code generators, and (2) a mapping from rover primitives to sending/receiving messages in the architecture (e.g., mapping the primitive forward to sending a messages to a motor).

The MAA code generator requires that for embedded behavior models (in our case RCL programs), a Java class is generated that implements a specific interface of MAA's common Java run-time environment [START_REF] Ringert | Language and Code Generator Composition for Model-Driven Engineering of Robotics Component & Connector Systems[END_REF]. This interface prescribes a compute() method that takes a set of named values (read from incoming ports) as input and returns another set of named values (that are assigned to outgoing ports) as output. Every time a component (e.g., a rover's control unit) should execute its behavior, this method is invoked by MAA. The body of the compute() method is generated by the code generator of RCL. For this, the RCL generator can reuse the JAVA/P generator templates for translating control structure, conditionals, etc. The translation of RCL primitives into sending and receiving messages is specific to the RCL generator and must be implemented accordingly. To take into account the connection among rover components, the generator takes as additional input a map from RCL primitives to port names. Based on this, the generator translates actions to sending messages, and queries to receiving messages.

D. Summary

Overall, MONTICORE facilitates reusing the syntax and semantics of independently developed DSLs. The central artifacts for this are MONTICORE grammars, Java context conditions, the symbol table, and template-based code generators, out of which only the grammars require learning a specific meta-language while the rest is implemented in Java. Hence, MONTICORE focuses on programmatic language engineering. This, however, confronts language engineers with the complexities of Java. Moreover, there is no support for producing graphical editors for MONTICORE languages. The language implementations are available online.2

V. DISCUSSION

As illustrated, the language reuse capabilities of GEMOC STUDIO and MONTICORE differ in the language constituents that can be reused as shown in Table II. For both workbenches, reusing abstract syntax, semantics, and transformations3 is supported. MONTICORE also supports the reuse of concrete syntax as the concrete syntax integrated into the grammars defining the abstract syntax as well. However, the reuse mechanisms of GEMOC STUDIO and MONTICORE differ in their expressiveness (see Table II). While GEMOC STUDIO supports removing abstract syntax elements through slicing, MONTICORE only supports removing abstract syntax elements through well-formedness rules that actively prevent their instantiation. Adding new abstract syntax elements is supported through inheritance by both language workbenches. GEMOC STUDIO also supports merging two metamodels on joint classes. MONTICORE also supports inheriting concrete syntax. Through inheritance, both workbenches also support to change language concepts, whereas embedding, i.e., specification and binding of dedicated language extension points is specific to MONTICORE.

We also investigated the impact of reuse in GEMOC STUDIO and MONTICORE with respect to the reuse that could be achieved in the implementations of RASPIROVER DSL. For GEMOC STUDIO, we measured the numbers of reused metamodel elements (classes, features, operations) and lines of code of the existing interpreters. For the new artifacts, we considered the size of the new RCL language (metamodel and interpreter), the glue code, and the MELANGE file. For MONTICORE, we measured the lines of code in the new grammars and in the reused grammars, as well as their size in terms of new and reused productions. For MONTICORE's generators, we also measured the lines of code of new and reused code. The results are depicted in Table III. As can be seen from these results, we could reuse a large portion of RASPIROVER DSL from existing languages and only needed to implement RCL and some glue code from scratch in both language workbenches. VI. CONCLUSION We presented the quintessential constituents and activities required for implementing RASPIROVER DSL, a DSL for defining the hardware architecture and control software behavior of rovers, with GEMOC STUDIO and MONTICORE. For both language workbenches, we could rely on existing DSLs to build the new rover-specific DSL. Through both case studies, we showed how the different language reuse mechanisms are applied and highlighted how they differ. We hope that this supports practitioners in creating custom DSLs with minimal effort.

Fig. 1 .

 1 Fig. 1. Example rover hardware architecture defined with RASPIROVER DSL

Fig. 3 .

 3 Fig. 3. Using MELANGE to compose the ARDUINOML, RCL and UNITS languages to form a new customized RASPIROVER DSL

Fig. 4 .

 4 Fig. 4. Syntactically and semantically gluing ARDUINOML's Project with RCL's RoverProgram using K3 aspects

Fig. 5 .grammargrammar

 5 Fig. 5. Relationships of the languages reused to implement RASPIROVER DSL in MONTICORE

Fig. 7 .

 7 Fig. 7. Example RASPIROVER DSL model defined in MONTICORE

  general control structures, such as blocks, loops,

	variable
	01	program TravelBetweenObstacles() {
	02	var step = 3 cm;	number with unit
	03	while (not message? = "stop") {
	04		while (not obstacle-front?) {
	05 06	loop	}	forward step primitive rover action	comparison of strings
	07 08	}	turn 180 deg	primitive rover query
	09	}
	Fig. 2. Example rover control program defined with RASPIROVER DSL

TABLE I LANGUAGES

 I REUSED FOR DEVELOPING RASPIROVER DSL

	Sub-language	GEMOC STUDIO	MONTICORE
	Architecture Description	ARDUINOML	MAA [9]

TABLE II COMPARISON

 II OF REUSE SUPPORT IN GEMOC STUDIO AND MONTICORE

	Reuse Support	GEMOC STUDIO	MONTICORE
	Language Components:
	Abstract Syntax
	Concrete Syntax
	Semantics	(interpreters)	(generators)
	Transformations
	Mechanisms:
	Removing Concepts	(slicing)	(context condition)
	Adding Concepts	(merging/inheritance)	(inheritance)
	Changing Concepts	(weaving/inheritance)	(inheritance)
	Language Embedding

TABLE III COMPARISON

 III OF REUSE ACHIEVED WITH GEMOC STUDIO AND MONTICORE (LOC: LINES OF CODE; PROD: PRODUCTIONS; ELEM: ELEMENTS)

	Artifact	GEMOC STUDIO Size of New Artifacts Size of Reused Artifacts	Size of New Artifacts	MONTICORE Size of Reused Artifacts
	Syntax	141 elem. (RCL metamodel) 24 LoC (K3 glue)	361 elem. (metamodels)	21 LoC / 18 Prod. (grammars) 1,267 LoC / 246 Prod. (grammars) 4 LoC / 2 Prod. (glue)
	Semantics	281 LoC (RCL interpreter) 50 LoC (K3 glue)	643 LoC (interpreters)	439 LoC (generators) 1 LoC (Java glue)	1,266 LoC (generators)
	Reuse-Specific	19 LoC (Melange)	-	-	-

https://github.com/tdegueul/gemoc-pirover/

http://www.se-rwth.de/materials/rcl/

Please note that we did not explicitly discuss the reuse of transformations in this paper.