L J Wrobel
email: wrobel@inaf.cnrs-gif.fr

M Ogier

S Autran

V Mézières

Thoby - Brisson

Wrobel Ludovic

Abnormal inspiratory depth in Phox2a Haploinsufficient Mice

Keywords: Breathing, neonate, petrosal ganglion, brainstem

Mutations of genes encoding Phox2a or Phox2b transcription factor induce modifications of different brainstem neuronal networks. Such modifications are associated with defects in the breathing behaviour at birth. In particular, an abnormal breathing frequency is observed in Phox2a -/-mutant mice, resulting from abnormal development of the locus coeruleus nucleus. However, the role of Phox2a proteins in the establishment of cardiorespiratory neuronal pathways is unknown, largely because mutants die shortly after birth. In the present study, we examined the effects of a haploinsufficiency of the Phox2a gene. Phox2a heterozygotes survive and exhibit a significantly larger inspiratory volume and a delayed inspiratory duration compared to wild-type animals during both normoxic breathing and in response to hypoxia. This phenotype accompanied by an unaltered frequency is evident at birth and persists until at least postnatal day 10. Morphological analysis of Phox2a +/-animals reveal no anomaly in the locus coeruleus region, but highlighted an increase in the number of cells expressing the catecholamine-synthesizing enzyme tyrosine hydroxylase, a marker of chemoafferent neurons, in the petrosal sensory ganglion. These data indicate that Phox2a plays a critical role in the ontogeny of the reflex control of inspiration.

Pulmonary ventilation adequately adapts to physiological and environmental changes to meet the organism's needs in oxygen by the combined activity of brainstem central pattern generators (CPG) and peripheral reflexes controlling lung inflation and blood gases level. The neural output of the CPG intermittently activates the inspiratory muscles at a defined frequency [START_REF] Smith | Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals[END_REF][START_REF] Bianchi | Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters[END_REF], Hilaire and Duron, 1999[START_REF] Thoby-Brisson | Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro[END_REF][START_REF] Richter | Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models[END_REF], Onimaru and Homma, 2003;Feldman and Del Negro, 2006). Lung inflation is monitored by stretch receptors that activate the Hering-Breuer reflex to immediately adjust breathing amplitude (tidal volume: VT) and inspiratory duration (Ti) (Duara et al., 1991, 1993[START_REF] Bianchi | Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters[END_REF]. The adequacy of pulmonary ventilation is monitored by chemoceptors sensing levels of oxygen and carbon dioxide allowing the proper matching of ventilation to metabolism and for responses to environmental changes such as hypoxia (Neubauer et al., 1990, Dillon et al., 1991[START_REF] Rigatto | Ventilatory response to hypoxia in unanesthetized newborn kittens[END_REF][START_REF] Roux | Ventilatory and central neurochemical reorganisation of O2 chemoreflex after carotid sinus nerve transection in rat[END_REF][START_REF] Pascual | Progesterone reverses the neuronal responses to hypoxia in rat nucleus tractus solitarius in vitro[END_REF]. Investigations on human breathing dysfunctions show a particular role of peripheral reflexes in controlling breathing activity [START_REF] Dauger | Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways[END_REF]. However, in vivo breathing patterns resulting from the impaired development of peripheral reflexes remain quite unknown. To this end we investigated mutations affecting afferent neurons in the distal sensory ganglia of the ninth and tenth cranial nerves (petrosal and nodose ganglia respectively) and particularly the subpopulation of chemoafferent cells expressing Tyrosine Hydroxylase (TH) in the petrosal ganglia. It appears that developmental genes such as Phox2 paralogs may be responsible for reflex arches set up and for unconscious breathing control [START_REF] Amiel | Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome[END_REF][START_REF] Amiel | Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome[END_REF]. Phox2 is a family of homeodomain transcription factors that includes two paralogs, Phox2a and Phox2b, which are considered to be master regulators of neuronal phenotype and survival in specific subsets of central and peripheral neurons [START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF]. Lossof-function mutations indicate that Phox2b and Phox2a are not completely genetic redundant [START_REF] Brunet | Phox2 genes -from patterning to connectivity[END_REF] and exert a complex reciprocal control on their abilities to promote gene transcription. The current hypothesis concerning these two paralogs is that during development, Phox2a and Phox2b genes are expressed to different degrees, and the balance between Phox2a and Phox2b proteins determines which genetic pattern is expressed in a given cell, and as such its fate [START_REF] Brunet | Phox2 genes -from patterning to connectivity[END_REF]. This seems to be particularly true in catecholaminergic neurons, where Phox2 proteins are competing for expression of the gene encoding dopamine-β-hydroxylase [START_REF] Yang | Paired-like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine beta-hydroxylase gene[END_REF][START_REF] Yang | Paired-like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine beta-hydroxylase gene[END_REF]. Phox2a participates in the differentiation of catecholaminergic neurons in restricted areas. Gene inactivation studies show that Phox2a controls development of the nucleus locus coeruleusand the distal sensory ganglia of the ninth and tenth cranial nerves in mice. These structures mediate major regulations of breathing parameters (Katz and Black, 1986, Erickson et al., 1996, Mortola 2001), control breathing frequency (Hilaire et al., 2004, Li andNattie, 2006) and may control breathing amplitude in rodents. While a homozygous suppression of Phox2a affects both central control of breathing and peripheral reflexes, this mutation is lethal at birth and as such little is known about respiratory insufficiencies resulting from such modifications. In this study, we investigated the breathing behaviour and developmental defects induced by a Phox2a haploinsufficiency in mice, a non-lethal mutation. We observed that Phox2a +/- animals show an increased inspiratory volume during normoxia and in response to hypoxic challenges and a delayed maturation of inspiratory duration. We suspect that this respiratory anomaly involves altered petrosal reflexes rather than central deficits, because the mutation is correlated with specific enlargement of the population of chemoafferent cells in this sensory peripheral structure.

Methods

All experiments were performed in accordance with ethical guidelines defined by the French Agricultural Ministry and the EU Council Directive for the Care and Use of Laboratory Animals (#2889).

Mouse genotyping

Phox2a +/-deficient mice [START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF] were obtained from JF Brunet and C. Goridis (ENS, Paris). The insertion of a neo cassette, a genetic sequence coding for the expression of neomycin, into the transgene allowed us to distinguish wild type locus from the mutant one. To this end, we performed two sets of PCR, one using a pair of probes specific for Phox2a sequence (sigma proligo) and the other using a pair of probes specific for the neo cassette sequence (sigma proligo). To verify the genetic status of mice, we extracted DNA from tail samples of the animals. The PCR products corresponding to each animal were visualized by gel electrophoresis using a 1.5% agarose gel in a Tris Acetate EDTA buffer (TAE, chemicon).

Plethysmographic recordings

Breathing patterns were studied in newborn mice from postnatal day 0 (P0) up to postnatal day 10 (P10). For P0 experiments we recorded ventilation in the first hour after birth 6, 12 and 18 hours later. P0 results are an averaged ventilation of these 4 measures for each animal. At P10, all mice were sacrificed for histological examination. Respiratory activity was measured by a barometric method, whole-body plethysmography (Schweitzer et al, 1990) using one recording chamber where the animals were placed in, a reference chamber and a differential pressure transducer. The plethysmograph chambers were of 20mL and the pressure difference between the two chambers was measured with the differential pressure transducer (Validyne DP 103-14) connected to a sine wave carrier demodulator (Validyne, CD15). After demodulation, the analog signal from the transducer was amplified, filtered, passed through an analog-to-digital converter (Labmaster TL-1, sampling frequency 1 kHz) and then captured and stored on disk. Plethysmograms were calibrated during each recording session by injecting 2.5-5 µl of air in the chamber with a Hamilton syringe. During quiet breathing, a computer-assisted method was used to measure breathing amplitude (tidal volume, V T ) the durations of inspiration (Ti) and expiration (Te) from which respiratory frequency (f) is derived (Jacquin et al., 1996, Dominguez et al., 2001[START_REF] Chatonnet | Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice[END_REF], 2003). Temperature was maintained constant (mean ± SE = 31.1 ± 0.25°C). As the tidal volume depends of body weight, we divided V T by body mass to normalize our results. The total volume of air breathed (Vm) is given by the relation: Vm = V T x f. Breathing pauses lasting more than 1.5 seconds were considered as apneas and were not included in frequency evaluation. Barometric pressure was measured before each recording session to normalize our results.

In all neonates we measured oral temperature before and after each recording session, the time required to right themselves after being layed on their backs, motility corresponding to the duration of limb, neck or body movements in the plethsmographic chamber and the number of jaw openings following peribuccal contact.

Hypoxic tests:

We also studied chemoafferent reflexs in Phox2a +/-mice at P10 using direct plethysmography. Here, the animal's head is isolated from the rest of the body by a thin membrane which divides the recording chamber into two compartments of 20mL each. The head of the animal receives a continuous flow of a normoxic air (500mL/min). This flow rate had no visible effect on the animal's behaviour. Given the flow rate and the volume of the chamber, the calculated time constant for filling the chamber was 2.4 s. The second compartment, containing the body, is the recording chamber. To start the hypoxic challenge, we switched from the normoxic air to a hypoxic mixture (12%O 2 ) without modifying the flow rate. This technique allowed us to flush the normoxic mixture out of the first compartment in less than 4s. . The hypoxic exposition lasted 90 seconds and then we switched back to normoxic air. These short-term hypoxic challenges have been shown to specifically recruit chemoafferent stimuli conveyed by glossopharyngeal neurons (Neubauer et al., 1990).

Recording of respiratory rhythm in vitro:

Experiments were performed on brainstem transverse slices obtained from wild type and heterozygous Phox2a mutant animals using an experimental procedure that will only be briefly described here (for details see [START_REF] Thoby-Brisson | Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro[END_REF]. Animals were decapitated, the brainstem was rapidly removed from the skull and placed in ice-cold artificial cerebro spinal fluid (aCSF composed of in mM: 128 NaCl, 8 KCl, 1.5 CaCl 2 , 1 MgSO 4 , 24 NaHCO, 0.5 Na 2 HPO 4 , 30 glucose, pH = 7.4) equilibrated with carbogen (95% O 2 , 5% CO 2 ). The isolated brainstem was then glued to an agar block, mounted in a vibratome and serially sectionned from rostral to caudal until specific landmarks were reached (inferior olive and nucleus ambiguus). A 450µm-thick slice containing the Pre-Bötzinger complex region was then isolated and placed in the recording chamber maintained at 30 °C and continuously superfused with oxygenated aCSF. Experiments were executed blindly since the genotype of the mouse was not known at the time of the recording session.

Neuronal population activity was recorded via an extracellular electrode positioned under visual control using an IR-DIC microscope at the surface of the slice in the pre-Bötzinger complex region in which the population of respiratory neurons lies. The signals were amplified (Grass, 7P511), filtered (3KHz, 3Hz), integrated (Neurolog System), and recorded using the software program Axoclamp 9 (Axon Instruments, Foster city, CA) and a digitizing interface Digidata1322A (Axon Instruments). Integrated population activity from the pre-Bötzinger complex was in phase with integrated inspiratory activity recorded from the hypoglossal nerve [START_REF] Thoby-Brisson | Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro[END_REF] and was therefore used as a marker for inspiratory population activity.

Immunohistochemistry

P10 mice were deeply anaesthetized with pentobarbital and intracardially perfused with a 0.15M phosphate-buffered saline solution (PBS, pH 7.4), followed by a 4% paraformaldehyde solution prepared in PBS. After craniotomy, the brain was removed and the remaining of the head was hemisected along the midline, to isolate lateral parts of the head containing the right or left inner ear and petrosal ganglia. The three samples were then placed overnight at 4 °C in the 4% paraformaldehyde solution. They were then rinsed in PBS, infiltrated with PBS containing 30% sucrose and stored at 4 °C until used. Serial 20 µm-thick sections were cut throughout each structure studied using a cryostat microtome. Parasagittal or coronal sections for A6, parasagittal section for petrosal ganglia were used to evaluate the number of tyrosine hydroxylase positive neurons. Sets of adjacent sections were stained with cresyl violet and for the tyrosine hydroxylase protein (TH). Sections processed for immunochemistry were incubated overnight at room temperature with an anti-TH polyclonal antibody (antibody purchased from Jacques Boy, France; 1:1000 in dilution buffer (DB): PBS containing 4% bovine serum albumine), rinsed and then incubated for 2 hours at room temperature with an anti-rabbit antibody coupled either to biotin (Vector; 1:200 in DB) or to rhodamine (Jackson Immunoresearch; 1:200 in DB). Staining using the biotinconjugated antibody was revealed with the elite peroxydas kit (vector) following manufacturer's instructions.

Cell counts and morphometric analysis:

Cell number was estimated from 20µm thick serial brain or ganglion sections stained with the appropriate coloration. In every slice of brain or ganglion, cells were counted visually using spot camera visualisation system (spot32, RT Slider, diagnostic instruments). In each slice, only cells exhibiting a visible nucleus were counted. No significant differences could be observed when comparing the mean nucleus diameter. The Abercrombie's correction method was then employed to avoid counting any given cell more than once (Abercrombie, 1946).

Statistical analysis

All results obtained from age-matched wild type and mutant animal groups, not necessarily from the same litter, were compared using an ANOVA test and a Bonferroni/Dunn post hoc of ANOVA in each experiment. Significance is given in the figures as follow: * means p<0.05, ** means p<0.01 and *** means p<0.005. Results are given as mean +/-SEM.

Results

Breathing pattern of Phox2a +/-mice

We employed plesthymographic techniques to analyse ventilatory parameters in WT and mutant mice. These results are presented in table 1. Minute ventilation (VM) in Phox 2a+/-mice was significantly higher that in wild type animals at all ages. This was due to an increased breathing amplitude (tidal volume, VT) with no change in frequency (f) (figure 1, table 1). This breathing pattern was visible from birth (postnatal day 0, P0) and persisted until P10 (table 1) and even into adulthood (data not shown) suggesting that no complete compensation occurs over time. In wild type animals, inspiratory duration (Ti) decreased by 20% over the first 5 days of life. Mutant animals also showed this same reduction in Ti with maturation, but this reduction was delayed by 5 days. As such, at P5, Ti in mutant mice was significantly larger (by 17%) than in wild type animals. We conclude that the Phox2a+/mutation induces an altered inspiratory phenotype characterised by a large VT and delayed maturation of Ti.

We also examined other physiological parameters such as body weight, oral temperature, righting reflexes and suction behaviour in all animals studied. We found no differences between wild type and mutant groups.

Central generation of breathing

Plethysmogram analyses revealed that Phox2a haploinsufficiency induces no modification in breathing frequency (Fig. 1D). Individual measurements showed that in contrast to VT and Ti, no Phox2a +/-phenotype can be derived from the analysis of respiratory and suction rhythms suggesting that central pattern generators activity are not affected (Fig. 2). We also analysed the duration of expiration (Te, table 1) because low frequency, lethal apneas and irregularities in breathing rhythm could be indicative of altered rythmogenesis. In neonates we did not observe any modification of Te (table 1). Finally, we isolated the respiratory rhythm generator in a transverse brainstem slice [START_REF] Smith | Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals[END_REF] and recorded the spontaneous rhythmic activity from respiratory neuronal populations (Koshiya et al., 1999;[START_REF] Thoby-Brisson | Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro[END_REF]. In accordance with in vivo measurements, the respiratory frequency measured in vitro was normal in mutants (14.2 ±1.4 burst/min in wild type versus 15.6 ± 0.6 in Phox2a +/-mutants n=5, Fig. 2A).

Increased chemoreflex control

Plethysmographic recording of breathing under hypoxic conditions were performed on young neonates at postnatal day P10. During a 90 s exposure to a hypoxic mixture containing 12% oxygen (Fig. 3A), the tidal volume and frequency were first increased within 10s of hypoxia, and decreased afterward. At the end of the hypoxic challenge, the ventilatory depression persisted during 60s, after which the animals recovered control respiratory parameters (Table 2, Fig. 3B). Short-term (10 s) responses to hypoxic challenge indicated that chemoafferent inputs to the rhythm generator were active in Phox2a +/-mice and caused an increase in VT induced by hypoxia were larger than normal in Phox2a +/-mice (Fig 3B,upper part). WT and Phox2a +/-mice had the same chemosensitivity, measured by reporting VT during hypoxia to VT in normoxia (expressed in percentage of increase in Table 2). . During long term hypoxic depression where chemoafferences are progressively inhibited, Phox2a +/-and wild-type VT were not significantly different because of a higher depression occurring in Phox2a +/-mice, suggesting that central structures controlling breathing are not affected by the mutation (table2, Fig. 3B upper part). The two types of animals were indistinguishable from their frequency (Table2, Fig. 3B lower part).

Anatomical studies of Phox2a +/-mice

We analysed the anatomical properties of neural structures that were found to be affected by the homozygous mutation of the Phox2a gene and in particular the petrosal sensory ganglion and the locus coeruleus.

The petrosal ganglion contains about 1400 cell bodies among which 10% are dopaminergic chemoafferent neurons expressing tyrosine-hydroxylase (Katz andBlack, 1986, Hellard et al., 2004) which are eliminated in Phox2a -/-mutants [START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF]. We compared the number of TH positive PG neurons in newborn wild-type and mutant animals to see the effect of phox2a haploinsufficiency on the specification of chemoafferent neurons (Fig. 4A). In Phox2a +/-mice the number of TH positive neurons per ganglion was dramatically increased compared to wild-type controls (Fig. 4C, 144+17 in wild-type, n=16 vs 313+7 in Phox2a +/-mice, n=16). However, the total number of PG neurons (Fig. 4B, 1141+106 neurons in wild-type, n=16 vs 1070+48 in Phox2a +/-, n=16) was not significantly different in Phox2a +/-mutants and in wild-type controls, indicating that Phox2a haploinsufficiency resulted in a specific increase in the number of cells expressing TH. By expressing the number of TH+ cells as a function of tidal volume (Fig 4E ), we can see that mice exhibiting the higher values of V T also have the highest number of chemoafferent cells.

To determine whether or not Phox2a haploinsufficiency also increased TH expression in central catecholaminergic cell groups, we examined TH cell number in the pontine noradrenergic group A6, the nucleus locus coeruleus. This structure contains between1200 and 1600 TH positive neurons grouped into three subdivisions, the locus proper, the rostral and ventral subcoeruleus extensions [START_REF] Swanson | The locus coeruleus: a cytoarchitectonic, Golgi and immunohistochemical study in the albino rat[END_REF][START_REF] Bezin | Postna tal development of the tyrosine hydroxylase-containing cell population within the rat locus coeruleus: topological organization and phenotypic plasticity[END_REF][START_REF] Coelln | Loss of locus coeruleus neurons and reduced startle in parkin null mice[END_REF]. Using immunohistochemistry, the general cell morphology, the location and the size of the subdivisions were found normal in mutants' brains examined in coronal (n=4) or parasagittal (n=5) planes (Fig. 5A). Unlike the petrosal ganglion, number of TH positive cells was not significantly different in the locus coeruleus of Phox2a +/-mutants (1141±106 neurons in wildtype, n=9 vs 1070±48 in Phox2a +/-, n=9; Fig. 5B). The location and the shape of locus coeruleus cells were not affected by the mutation and the average soma diameter was not significantly different from control animals (15.9±0.6 µm in wild type versus 16.0±0.7 µm in mutant group). Taken together, anatomical data support the existence of anomalies in the reflex control of inspiration, rather than a central effect mediated by pontine catecholaminergic nuclei in Phox2a +/-mice.

Discussion

The present study describes a Phox2a +/-breathing phenotype characterized by a large V T and a delayed postnatal maturation of Ti. These breathing alterations are associated with an increase in the number of chemoafferent neurons in the petrosal ganglia, while THexpressing neurons in the central nucleus locus coeruleus are unaffected. Interestingly breathing frequency was not modulated by the mutation suggesting that central structures controlling breathing rhythm are not affected. As such, because in Phox2a +/-mice the V T is increased more than normally during the early phase of hypoxia and is correlated with the numbre of chemoafferent neurons, we conclude that this mutation affects the reflex control of inspiration.

Phox2a haploinsufficiency does not affect central respiratory-related structures

Phox2a +/-mice were found to hyperventilate, exhibiting a larger-than-normal average volume of air breathed (Vm) at all ages studied. Hyperventilation results from an abnormality in the inspiratory phase. Genetically-transmitted low respiratory frequency, neonatal apneas and rhythm irregularity are induced by other mutations and are associated with an abnormal development of the brainstem rythm generators (Jacquin et al., 1996[START_REF] Shirasawa | Rnx deficiency results in congenital central hypoventilation[END_REF], Dominguez et al., 2001[START_REF] Chatonnet | Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice[END_REF], 2003[START_REF] Blanchi | MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth[END_REF][START_REF] Viemari | Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice[END_REF], 2005, Durand et al., 2005). These animals die shortly after birth. In some cases such mutations are associated with abnormal suction behaviour (Jacquin et al., 1996). Lethal apneas and other phenotypic traits pointing to dysfunction of the central pattern generators are not observed in Phox2a haploinsufficient animals, as such it is unlikely that the central rhythm generation is significantly affected. Furthermore, in contrast to Phox2a +/-mutation, mutations affecting the maturation of the bulbar respiratory generator abnormal breathing frequency observable after isolation in vitro (Jacquin et al., 1996[START_REF] Shirasawa | Rnx deficiency results in congenital central hypoventilation[END_REF], Dominguez et al., 2001[START_REF] Chatonnet | Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice[END_REF], 2003[START_REF] Blanchi | MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth[END_REF][START_REF] Viemari | Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice[END_REF], 2005, Durand et al., 2005). Breathing deficits described in Phox2a +/-mutants are distinct from those so far identified after the inactivation of genes controlling development of brainstem central pattern generators in the brainstem. In addition, breathing during the delayed responses to hypoxia is mediated by central chemosensory mechanisms as chemoafferences are progressively inhibited (Neubauer et al., 1990, Dillon et al., 1991[START_REF] Oyama | Respiration-modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem-spinal cord of the neonatal rat[END_REF][START_REF] Chatonnet | Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice[END_REF], Li and Nattie, 2006). Those mechanisms appear normal and functional in these mutants although it would be interesting to further compare the central chemosensitivity using in vitro preparations.

The central control of rhythm generation was normal, as well as other behavioural and physiological parameters studied, including neonatal and long-term survival, body weight, righting and jaw opening reflexes, mobility and oral temperature (data not shown). Thus, the abnormal breathing pattern exhibited by Phox2a +/-mice is compatible with normal behavioural development and survival and does not appear to be indicative of acute respiratory distress. The Phox2a haploinsufficiency syndrome may therefore target respiratory controls operating outside the central control of breathing, in particular the reflex control of breathing.

TH expression in the Phox2a +/-locus coeruleus

Previous results on homozygous mutants demonstrate that Phox2a is required for the correct establishment and functional development of the nucleus locus coeruleus [START_REF] Viemari | Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice[END_REF]. This pontine noradrenergic structure provides a tonic excitatory drive that contributes to a normal breathing rate in rats (Guyenet et al., 1993;Jodkowski et al., 1997;[START_REF] Oyama | Respiration-modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem-spinal cord of the neonatal rat[END_REF][START_REF] Dawid-Milner | Respiratory effects of stimulation of cell bodies of the A5 region in anesthetized rats[END_REF]Li and Nattie, 2006) and mice [START_REF] Shirasawa | Rnx deficiency results in congenital central hypoventilation[END_REF][START_REF] Viemari | Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice[END_REF]. Elimination of the locus coeruleus is linked to a severe decrease in the respiratory frequency of homozygous Phox2a -/-mutants (Viemari, et al., 2004, Li andNattie, 2006). Here we show that despite the low expression of the Phox2a protein, locus coeruleus neurons can build a functional pontine catecholaminergic phenotype, since A6 cells are normal in shape, size and anatomical location, and since breathing rhythm is normal.

Phox2a dose-dependent regulation of the sensory neuronal fate

The present study shows that Phox2a has a dose-dependent influence on the development of TH positive cells in the petrosal ganglion. During embryonic development (E10.5-13.5), many petrosal and nodose sensory neurons transiently express TH and dopamine-β-hydroxylase (DBH) and then switch to a non-catecholaminergic phenotype before a subset of ganglion cells begins to accumulate TH (Jonakait et al, 1984). Both the early, transient phase of noradrenergic expression and the late, stable phase of dopaminergic expression are associated with co-expression of Phox2a and Phox2b transcription factors [START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF]Brosenitsch andKatz, 2005, 2002). Transient expression of DBH by petrosal and nodose neurons requires Phox2a expression and is absent in Phox2a null mutant mice [START_REF] Pattyn | Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis[END_REF]). However, homozygous mutants provide no information on later stages because the ganglia degenerate soon after. We presently show that the proportion of TH positive petrosal neurons increases in Phox2a +/- mutants withno change in ganglion cell number. This indicates a specific role of Phox2a in the phenotypic specification of catecholaminergic neuron progenitors.

Phox2a dose-dependent involvement during development of inspiratory controls

Increased tidal volume in Phox2a +/-mutants suggests that Phox2a controls specific neuronal circuits controlling breathing. Tidal volume is protected during changing behavioural demands by reflex compensation against mechanical loads (Mortola, 2001). Vagal reflexes, mediated by pulmonary stretch receptors controlling Ti, are the core mechanisms for immediate load compensation, particularly in newborns, so that vagotomy results in deeper breathing in the newborn than in the adult. When mechanical loads are sustained, the maintain of tidal volume and ventilation also involves chemoreflexes (LaFramboise et al. 1983, Rigatto et al., 1988, Duara et al., 1991, 1993). We hypothesize that the exaggeration of VT and the delayed maturation of Ti observed in Phox2a +/-mice results from the elevated number of dopaminergic chemoafferent neurons. Current hypothesis propose that chemoafferent fibres may act presynaptically on vagal afferent fibres (underlying the Hering Breuer reflex) coming from stretch receptors (Kline et al.,2002) and may modulate mechanical load compensation. Therefore, the possibility that this phenotype might result from yet undetected dysfunction of stretch receptor afferents in the nodose ganglion cannot be excluded. Furthermore Phox2a is also essential for development of the superior cervical ganglion. The superior cervical ganglion provides a major sympathetic input to the carotid body and the noradrenergic input is able to modify the chemosensory afferent pathway. We cannot exclude that the changes observed here in hypoxic ventilatory response are in part dependent on defects in sympathetic nerve activity.

Our finding that the Phox2a +/-mutation affects both the number of TH positive PG neurons and tidal volume supports the hypothesis that Phox2a signalling influences development of load compensation by regulating the number of chemoafferent cells. The fact that V T is increased in newborn Phox2a +/-mice under all conditions examined, i.e. during room air breathing and during the hypoxic response, indicates that synaptic mechanisms that normally restrict V T are diminished in these animals while chemosensitivity itself is not affected. There are now several mutants or experimental models that testify to a relationship between chemoafferent cell number in the petrosal ganglion and tidal volume. For example, in Phox2b heterozygote mutants, a reduced number of chemoafferent neurons is associated with a blunting of the normal increase in VT in response to hypoxic stimuli [START_REF] Dauger | Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways[END_REF]. Similarly, a reduction in chemoafferent neurons is associated with a decreased tidal volume in heterozygous and homozygous BDNF mutants (Erickson, et al., 1996). These results are in accordance with the hypothesis that chemoafferent signalling is a regulatory pathway of breathing amplitude.

Conclusion

The present study shows that the number of Phox2a alleles influences the breathing behaviour and the fate of petrosal neurons. By interrupting several genetic signalling pathways, complete Phox2a inactivation produces various defects including depression of the central respiratory generator and elimination of locus coeruleus and sensory afferent neurons. Nevertheless, the heterozygous mutation of Phox2a is linked to a more specific phenotype, that of a delayed maturation of inspiratory duration and an increased tidal volume during normoxic and hypoxic breathing, correlated with an increase in the number of chemoafferent neurons. These results identify a higly selective dose-dependent function of Phox2a in specifying petrosal neuronal types during development. This study also provides new insight into understanding how tidal volume and maturation of inspiratory duration can be affected in human syndromes presenting a deregulation of breathing amplitude.

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Figure 4 :

 4 Figure 4: Petrosal catecholaminergic cells in wild type and Phox2a +/-mice.

Acknowledgements

This work was supported by grants coming from the « Centre National pour la Recherche Scientifique » (CNRS) and the European Community (QLG2/CT/2001-01467). Ludovic J Wrobel was awarded by the French « Ministère de la Recherche ». Jean-François Brunet, Christo Goridis and Cyril Ribeyre are gratefully acknowledged for discussions around the article. Christelle Thaeron is acknowledged for her participation in preliminary experiments.

De Pontual L, Nepote V, Attie-Bitach T, Al Halabiah H, Trang H, Elghouzzi V, Levacher B, Benihoud K, Auge J, Faure C, Laudier B, Vekemans M, Munnich A, Perricaudet M, Guillemot F, Gaultier C, Lyonnet S, Simonneau M, Amiel J. (2003). Noradrenergic neuronal development is impaired by mutation of the proneural MASH-1 gene in congenital central hypoventilation syndrome (Ondine's curse). Hum Mol Genet. 12(23),3173-3180 Dillon, G.H., Welsh, D.E., and Waldrop, T.G. (1991). Ventrolateral pons mediates short-term depression of respiratory frequency after brief hypoxia. Respir. Physiol. 121, 87-100. Dominguez del Toro E, Borday V, Davenne M, Neun R, Rijli FM, Champagnat J (2001) Generation of a novel functional neuronal circuit in Hoxa1 mutant mice. J Neurosci 21: 5637-5642. Duara S, Neto GS, Gerhardt T, Suguihara C, Bancalari E (1991) Metabolic and respiratory effects of flow-resistive loading in preterm infants. J Appl Physiol 70: 895-899. Duara S, Suguihara C, Gerhardt T, Bancalari E (1993) Metabolic, hemodynamic, and ventilatory responses to respiratory load in sedated neonatal piglets. J. Appl. Physiol 75: 181-184. Durand E, Dauger S, Pattyn A, Gaultier C, Goridis C, Gallego J (2005) Sleep-disordered breathing in newborn mice heterozygous for the transcription factor Phox2b. Am J Respir Crit Care Med 172: 238-243. Erickson JT, Conover JC, Borday V, Champagnat J, Barbacid M, Yancopoulos G, Katz DM (1996) Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing. J Neurosci 16: 5361-5371. Feldman JL, Del Negro CA. (2006) Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci. 7(3):232-42. Guyenet PG, Koshiya N, Huangfu D, Verberne AJ, Riley TA (1993) Central respiratory control of A5 and A6 pontine noradrenergic neurons. Am. J. Physiol. Regul. Integrative Comp. Physiol. 264: R1035-R1044. Hellard D, Brosenitsch T, Fritzsch B, Katz DM (2004) Cranial sensory neuron development in the absence of brain-derived neurotrophic factor in BDNF/Bax double null mice. Dev. Biol. 275: 34-43. Hilaire G, Duron B (1999) Maturation of the mammalian respiratory system. Physiol Rev. Apr;79(2):325-60 Hilaire G, Viemari JC, Coulon P, Simonneau M, Bevengut M. ( 2004) Modulation of the respiratory rhythm generator by the pontine noradrenergic A5 and A6 groups in rodents. Respir Physiol Neurobiol. 143(2-3):187-97. Jacquin TD, Borday V, Schneider-Maunoury S, Topilko P, Ghilini G, Kato F, Charnay P, Champagnat J (1996) Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice. Neuron 17: 747-758. Jodkowski JS, Cole SK, Dick TE (1997) Prolongation in expiration evoked from ventrolateral pons of adult rats. J. Appl. Physiol. 82: 377-381. Jonakait GM, Markey KA, Goldstein M, Black IB (1984) Transient expression of selected catecholaminergic traits in cranial sensory and dorsal root ganglia of the embryonic rat. Dev Biol 101: 51-60. Katz DM, Black IB(1986) Expression and regulation of catecholaminergic traits in primary sensory neurons: relationship to target innervation in vivo. J Neurosci 6:983-989. Kim HS, Seo H, Yang C, Brunet JF, Kim KS (1998) Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2a-binding sites. J Neurosci 18: 8247-8260. Kline DD, Takacs KN, Ficker E, Kunze DL (2002) Dopamine modulates synaptic transmission in the nucleus of the solitary tract. Jneurophysiol 88(5): 2736-44. Koshiya N, Smith JC (1999) Neuronal pacemaker for breathing visualized in vitro. Nature 400: 360-363. LaFramboise WA, Tuck RE, Standaert TA, Woodrum DE (1983) An elastic loading system for ventilatory studies in small animals. J Appl Physiol 54: 314-317 Li A, Nattie E (2006) Catecholamine neurones in rats modulate sleep, breathing, central chemoreception and breathing variability. J. Physiol. Lond. 570: 385-396. Mortola, J.P (2001) Respiratory physiology of newborn mammals, a comparative perspective. The John Hopkins University Press Neubauer, J.A., Melton, J.E., Edelman, N.H. (1990) Modulation of respiration during brain hypoxia. J. Appl. Physiol. 68 : 441-451. Onimaru H, Homma I. (2003) A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci. 23(4):1478-86.


O Polach

A Bottcher

D Vannuci

J Sima

H Schelle

Hugues Chollet

G Gotz

Garcia Prada

D Nicklish

L Mazzola

A Böttcher

D Vannucci

G Götz

M Garcia Prada

D Nicklisch

M Berg

M Osman

Validation of multi-body models for simulation in authorisation of rail vehicles

Keywords: Model validation, multi-body simulation, railway vehicle, running dynamics, running characteristics, acceptance, authorisation, certification, homologation

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INTRODUCTION

Cost and duration of vehicle testing for the acceptance of running characteristics according to EN 14363:2005 [START_REF]Railway applications-Testing for the acceptance of running characteristics of railway vehicles-Testing of running behaviour and stationary tests[END_REF] could be reduced by using multi-body simulations which have already been employed in rolling stock design and development for several years. This, however, is only possible if there is confidence that the simulation results have been produced by using a validated railway vehicle model [START_REF] Evans | Challenges in simulation of rail vehicle dynamics[END_REF], [START_REF] Bruni | Modelling of suspension components in rail vehicle dynamics context[END_REF]. The recent revision prEN 14363:2013 [START_REF]Railway applications-Testing and simulation for the acceptance of running characteristics of railway vehicles-Running behaviour and stationary tests[END_REF] considers application of multi-body simulations, but without specifying quantitative limits for a successful model validation; an assessment by an independent reviewer is required instead. A development of method, criteria and limits for validation of vehicle models intended for simulations for the acceptance of running characteristics of railway vehicles was a topic investigated in Work Package 5 of the research project DynoTRAIN.

These investigations were carried out by comparing simulations with measurements from a testing campaign carried out in four European countries in October 2010 and accompanied with continuous measurement of track irregularities and rail profiles. The following vehicle models were assessed:

• 2 models of locomotive DB BR 120 (in simulation tools Simpack and VOCO)

• 2 models of DB passenger coach Bim (in simulation tools Simpack and VOCO)

• 2 models of empty freight wagon Sgns with Y25 bogies (in simulation tools Simpack and VOCO) • Model of laden freight wagon Sgns with Y25 bogies in Simpack

• Laas freight unit consisting of two 2-axle flatbed freight wagons with UIC link suspension modelled in Simpack. Furthermore, other models of two recently developed vehicle types were assessed using measurement results provided by the suppliers of vehicles.

VALIDATION EXERCISES

The comparisons between the simulation and the measurement were carried out for all vehicle models and model configurations under the same conditions and in the same manner for selected sections of test runs, called validation exercises. One validation exercise consists of one curve passing scenario including both transitions and parts of straight track; in this context the word "section" means a part of track; it does not mean section according to the definition in EN 14363:2005 [START_REF]Railway applications-Testing for the acceptance of running characteristics of railway vehicles-Testing of running behaviour and stationary tests[END_REF]. A total of 17 validation exercises were selected from DynoTRAIN measurements, representing all 4 track zones from straight track down to very small radius curves (250 m ≤ R ≤ 400 m). They were from 3 countries: Germany (11 sections), Italy (4) and Switzerland [START_REF] Evans | Challenges in simulation of rail vehicle dynamics[END_REF].

The assessment by comparisons between the simulation and the measurement results contained (see an example in Fig. 1):

• Assessment based on measured quantities, filtered and processed by analogy with EN 14363:2005 [START_REF]Railway applications-Testing for the acceptance of running characteristics of railway vehicles-Testing of running behaviour and stationary tests[END_REF]. • Subjective engineering assessment using simple "Yes/No" method by project partners as well as during a workshop with invited experts, where a set of selected plots were assessed by 26 workshop attendees. • Validation metrics, i.e. quantitative measures comparing simulation and measurement in the time histories with the aim to maintain agreement with engineering judgement [START_REF] Schwer | Validation metrics for response histories: perspectives and case studies[END_REF]. The analyses of about 1 000 simulation runs considered about 50 000 pairs of values evaluated by analogy with EN 14363, together with more than 6 000 plots assessed subjectively by the project partners as well as 120 selected plots during the workshop. Each of the plots comparing the time or distance histories was assessed by calculating the validation metrics values by Sprague and Geers and by Russell: Magnitude error factor M i , phase error factor P i and comprehensive error factor C i , where indices i = SG for validation metrics by Sprague and Geers and i = R for validation metrics by Russell; see [START_REF] Schwer | Validation metrics for response histories: perspectives and case studies[END_REF] for error factors definitions.

EVALUATION OF VALIDATION CRITERIA AND LIMITS

Correlations between the quantities evaluated by analogy with EN 14363, the assessments by validation metrics and the subjective assessments of plots were analysed. The relationship between the assessments and the simulation results achieved was investigated in order to specify criteria and limits ensuring reliable model validation and at the same time allowing a successful validation applying the state of the art modelling and simulation, see Fig. 2. The effects of using the actual track data (measured track irregularities, rail profiles) as opposed to random track data and the use of stationary tests for the model validation in regards to simulation of the on-track tests were also investigated.

Final agreement on limits

The model validation criteria and limits should take into account errors in the measurement of running dynamics quantities, measurement of track layout and track irregularities, measurement of rail profiles and wheel profiles, scatter of test conditions as e.g. friction coefficient between wheel and rail as well as generally stochastic character of the test results. The investigations showed that the quantities based on EN 14363:2005 so far provide the best potential for quantitative criteria and objective validation assessment. An application of these criteria on a few single pairs of compared simulation -measurement values, however, does not provide sufficient information about an overall model performance considering the stochastic character of the test results. It is therefore proposed to assess a whole set of simulationmeasurement pairs for each quantity. The proposed model validation process considers an assessment of 12 quantities based on measured forces between wheel and rail and vehicle body accelerations, evaluated on a minimum of 12 test sections. The validation is assessed comparing the mean and standard deviation of differences between simulation and measurement for each particular quantity with the validation limits.

PROPOSED VEHICLE MODEL VALIDATION

The proposed validation assessment is based on quasi-static values and maximum values of wheel/rail contact quantities (Y, Q, Y/Q and ΣY) and rms and maximum values of vertical and lateral car body accelerations. The simulation and measurement results of these quantities should be compared on at least 12 test sections, called validation exercises. The selected validation exercises shall contain sections from all 4 curvature zones according to EN 14363, at least 3 sections from each zone.

Each quantity should be evaluated using at least two signals, e.g. vertical acceleration above the leading and trailing bogie, thus, at least 24 simulated values S v compared to the corresponding measured values M v of each quantity. Each compared simulated as well as measured quantity is filtered and processed according to the requirements in Table 1, whereby the frequency values (%-values) are calculated from the cumulative curve. For the maximum value calculated as 0.15% or 99.85%-value, the higher magnitude of the 0.15%-and 99.85%-values (absolute value) is used. The 50%-values (medians) are applied with their sign to approve the agreement of both magnitude and direction of those quantities.

The difference D v between the simulated value S v and the corresponding measured value M v is to be evaluated for each value and each quantity, whereby this difference is transformed so that, if the magnitude of the simulation value is higher than the magnitude of the measurement (simulation overestimating the measurement), the difference is positive, and vice versa:

0 for ) ( ¹ - = v v v v v v M M M M S D
(1)

0 for = = v v v M S D
The following values are calculated for the whole set of differences D v between the simulation and measurement for each quantity (e.g. for all Y qst values):

• Mean of differences between simulation value S v and measurement value M v • Standard deviation of the same set of differences. The standard deviation of the set of differences between simulation value S v and the measurement value M v for each quantity should be below their validation limit shown in Table 1. For each quantity the mean of the set of differences between the simulation value S v and the measurement value M v should be lower than a validation limit equal to 2/3 of the related limit for the standard deviation. The validation limits for accelerations (standard deviation as well as mean of differences) for freight vehicles and vehicles without bogies or without secondary secondary suspension are twice the relevant limit values for other vehicles.

DICSUSSION OF VALIDATION RESULTS

An advantage of the proposed validation procedure is that this assessment represents an overall assessment of a large number of data. It is not practical to carry out such an assessment by using engineering judgement of the plots, as it would involve having to display, check and document the approval of such a large number of diagrams. The calculation of characteristic parameters of mean and standard deviation of differences between the simulation values S v and the measurement values M v , and their comparison with the validation limits, however, allows a fast identification of quantities with large deviation.

The specified set of 12 quantities covers the quasi-static as well as dynamic behaviour of the vehicle in regard to the vehicle acceptance, which is the intended range of the application for a validated model. The vehicle's safety relevant behaviour and the track loading results are validated by comparing quantities measured using force measuring wheelsets. The representation of vehicle ride is validated by comparing the rms values and maximum values of car body accelerations. The signal processing is carried out by analogy with EN 14363 for both the measurement and simulation, thus allowing direct use of the acceptance tests data. The only additional requirement on the measurement evaluation is the calculation of quasi-static values of the sum of guiding forces and Y/Q ratios.

The weakness of the model in question can be identified by displaying normalised validation criteria, as it can be seen in Fig. 456789. The mean and standard deviation of differences between simulation and measurement are normalised by the proposed validation limits (see Table 1). A vehicle model is thus validated if magnitudes of all values are lower than 1.

Fig. 4 shows examples of model validation results for vehicles tested in DynoTRAIN project. These initial models were developed using available parameters including measured profiles of wheels and rails and measured track irregularities, however without any model adjustments. From the four vehicle models compared, only the initial model of the Bim coach fulfils the validation limits. The other vehicle models must be adjusted to be validated. The model adjusted by comparison with stationary tests was further improved by adjustment of the estimated height of the car body centre of gravity identified from the comparisons with the on-track tests results. This adjustment of the uncertain parameter of the car body centre of gravity could possibly be identified from the stationary sway test; however, this test was not available. The validation results of the same vehicle modelled by IFSTTAR (Fig. 8) exceed the validation limits even after the model adjustment; thus, this model failed the proposed validation criteria. 9 shows the effect of the vehicle model adjustments by comparisons with stationary tests on the validation results of the High Speed EMU for TCDD (Turkey) conducted by CAF. The stationary tests were divided in to 2 groups. The validation results are shown for the model before the comparisons and adjustments using stationary tests, but considering the measured static wheel loads, for the model after adjustments using the twist test (wheel unloading test) and for the model further improved by comparisons with the remaining available stationary tests (bogie rotational resistance test, measurement of roll coefficient and sway test). As can be seen, the model improvements by comparisons with the stationary tests are rather marginal; occasionally, the results for some quantities are even worse than before the adjustment. It is believed that this rather small model improvement by stationary tests is due to well known model parameters of this recently developed vehicle compared to older vehicles which were tested in the DynoTRAIN project. Nevertheless, the High Speed EMU model failed the validation. This can be explained by the missing data of track irregularities and rail profiles, which were replaced by estimated data. The presented examples demonstrate the importance of actual data, both infrastructure data as well as vehicle parameters, for a successful model validation.

CONCLUSIONS

Investigations in the DynoTRAIN project compared simulations of several vehicle models with measurements from a testing campaign using a test train consisting of 4 types of vehicles and a total of 10 force measuring wheelsets, accompanied with continuous measurement of track irregularities and rail profiles. The analyses of these comparisons were used to develop criteria and limits proposed for the validation of multi-body vehicle models intended for simulations of on-track tests in the railway vehicle authorisation process.

The proposed model validation criteria and limits take into account errors in the measurement of running dynamics quantities, measurement of track layout and track irregularities, measurement of rail profiles and wheel profiles, scatter of test conditions as e.g. friction coefficient between wheel and rail as well as generally stochastic character of the test results. They are based on 12 quantities covering the quasi-static and dynamic wheel/rail force measurements and vertical as well as lateral vehicle body accelerations. For each quantity, a set of at least 24 comparisons simulationmeasurement is evaluated using values based on EN 14363 from at least 12 sections which represent all 4 test zones according to EN 14363 from straight track to curves with very small radius. The agreement between simulation and measurement is assessed comparing the mean value and standard deviation for a set of differences between simulated and measured values of each quantity with the proposed validation limit.

The proposed validation method allows identification of model weaknesses and a model improvement by adjusting uncertain model parameters. The investigations of the importance of comparisons with stationary tests showed, that the model adjustments using stationary tests results may be useful to identify unknown model parameters. The model improvement using stationary tests in regard to simulation of the on-track tests is often marginal if reliable vehicle model data is available; moreover, the model results can be even worse than before the model adjustment. A comprehensive comparison with the on-track tests is indispensable for reliable validation of a vehicle model intended for the simulation of on-track test. This validation may require not only measured track irregularity data and measured wheel and rail profiles, but possibly also additional measurements. For example, if the tested vehicles are used as propelling vehicles during the on-track test, a measurement of the longitudinal creep forces or the driving torque and an appropriate modelling of the tractive effort in simulations may be necessary for a successful model validation.

Fig. 2

 2 Fig. 2 Evaluation of validation criteria and limits carried out in DynoTRAIN

Fig. 3

 3 illustrates the calculation of differences between the simulation values S v and the measurement values M v for the example quantity rms-value of vertical acceleration at the vehicle body. The left diagram displays the simulation values S v and the measurement values M v . The right diagram shows the differences D v , and their mean value and standard deviation, which are used for comparisons with the validation limits specified

Fig. 3

 3 Fig. 3 Example of differences between simulated and measurement values (left diagram) and calculation of their mean and standard deviation (right diagram)

Fig. 4 Fig. 5 Fig. 6

 456 Fig.[START_REF]Railway applications-Testing and simulation for the acceptance of running characteristics of railway vehicles-Running behaviour and stationary tests[END_REF] Normalised values of mean and standard deviation of differences between simulation and measurement for the initial models of locomotive (Siemens), Bim coach (Bombardier Transportation), laden freight wagon Sgns (TU Berlin) and freight wagon unit Laas (Alstom)

Fig. 7

 7 Fig. 7 Example of Bim passenger coach by Bombardier Transportation: F1 -initial model, T1a -model after adjustments by comparisons with stationary tests, T2amodel configuration T1a further adjusted by comparisons with on-track tests

Fig. 9

 9 Fig. 9 Effect of model adjustments by comparisons with stationary tests on the validation results of the High speed EMU modelled by CAF: A1 -initial model before adjustments by stationary tests, G1 -model after adjustment by comparison with the twist test (wheel unloading test), O1 -model G1 further adjusted by comparison with bogie rotational resistance test, roll coefficient measurement and sway test

Table 1

 1 in Table1. Quantities compared, their filtering and processing and the proposed limits for model validation in regard to simulation of on-track test

	Quantity	Notation Unit	Filtering	Processing	Validation limit for standard deviation * )
	Quasi-static guiding force	Y qst	kN	Low-pass filter 20 Hz	50%-value (median)	5
	Quasi-static wheel load	Q qst	kN	Low-pass filter 20 Hz	50%-value (median)	4 (1+0.01 Q 0 ) Q 0 -static wheel load [kN]
	Quasi-static quotient Y/Q	(Y/Q) qst	-	Low-pass filter 20 Hz	50%-value (median)	0.07
	Quasi-static sum of guiding forces	ΣY qst	kN	Low-pass filter 20 Hz	50%-value (median)	6
	Guiding force, maximum	Y max	kN	Low-pass filter 20 Hz	0.15%/99.85%-value *** )	9
	Wheel load, maximum	Q max	kN	Low-pass filter 20 Hz	99.85%-value *** )	6 (1+0.01 Q 0 ) Q 0 -static wheel load [kN]
	Quotient Y/Q, maximum	(Y/Q) max	-	Sliding mean (2 m window)	0.15%/99.85%-value *** )	0.10
	Sum of guiding forces, maximum	ΣY max	kN	Sliding mean (2 m window)	0.15%/99.85%-value *** )	9
	Car body lateral acceleration, rms	* rms y& &	m/s 2	Band-pass filter 0.4 to 10 Hz	rms-value	0.15 k a ** )
	Car body vertical acceleration, rms	* rms z& &	m/s 2	Band-pass filter 0.4 to 10 Hz	rms-value	0.15 k a ** )
	Car body lateral acceleration, max.	* max y& &	m/s 2	Band-pass filter 0.4 to 10 Hz	0.15%/99.85%-value *** )	0.40 k a ** )
	Car body vertical acceleration, max.	* max z& &	m/s 2	Band-pass filter 0.4 to 10 Hz	0.15%/99.85%-value *** )	0.40 k a ** )

*) Validation limit for mean of differences simulation-measurement is 2/3 of the limit for standard deviation **) k a -coefficient in regard to vehicle design; freight vehicles and vehicles without bogies or without secondary suspension, respectively: k a = 2, other vehicles: k a = 1 ***) Absolute values of simulated value S v and measured value M v

ACKNOWLEDGEMENTS

This abstract describes work undertaken in the context of the DynoTRAIN project, Railway Vehicle Dynamics and Track Interactions: Total Regulatory Acceptance for the Interoperable Network (www.triotrain.eu). DynoTRAIN is a collaborative projectmedium-scale focused research project supported by the European 7th Framework Programme, contract number: 234079 and is led by UNIFE.