Tamás Freund

Didier Le Ray

Frédéric Brocard

Céline Bourcier-Lucas

François Auclair

Philippe Lafaille

Dr Réjean Dubuc
email: dubuc.rejean@uqam.ca

Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys by

Keywords: Brainstem, Acetylcholine, Nicotine, Locomotor control, MLR

In lampreys as in other vertebrates, brainstem centers play a key role in the initiation and control of locomotion. One such center, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is done remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR-controlled locomotion.

The local application of either acetylcholine or nicotine exerted a direct dose-dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase-immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with D-tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi-intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.

INTRODUCTION

Locomotor behaviors include both sensory-evoked locomotion organized at the rhombencephalic level and goal-directed locomotion that requires higher brain structures [START_REF] Grillner | Neurobiological bases of rhythmic motor acts in vertebrates[END_REF][START_REF] Mcclellan | Command systems for initiating locomotion in fish and amphibians: parallels to initiation systems in mammals[END_REF]. The neural elements responsible for these locomotor behaviors are not fully identified, nor are the underlying cellular mechanisms involved. In both cases, however, the supraspinal locomotor command is likely to be channeled via the reticulospinal (RS) system, which constitutes the main descending system involved in the initiation and control of locomotion in lampreys [START_REF] Brodin | Reticulospinal neurons in lamprey: transmitters, synaptic interactions and their role during locomotion[END_REF][START_REF] Ohta | Monosynaptic excitatory amino acid transmission from the posterior rhombencephalic reticular nucleus to spinal neurons involved in the control of locomotion in lamprey[END_REF]. Reticulospinal neurons directly activate spinal locomotor networks [START_REF] Rovainen | Synaptic interactions of reticulospinal neurons and nerve cells in the spinal cord of the sea lamprey[END_REF][START_REF] Buchanan | Activities of identified interneurons, motoneurons, and muscle fibers during fictive swimming in the lamprey and effects of reticulospinal and dorsal cell stimulation[END_REF] and receive sensory information from various sources [START_REF] Rovainen | The Biology of Lampreys[END_REF][START_REF] Bussières | Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys[END_REF]Dubuc et al., 1993a,b;Viana Di Prisco et al., 1995;[START_REF] Deliagina | Initiation of locomotion by lateral line photoreceptors in lamprey: behavioural and neurophysiological studies[END_REF], as well as feedback from the spinal locomotor networks [START_REF] Dubuc | The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey[END_REF]. Cells with such input/output characteristics have been referred to as "command neurons" (see [START_REF] Frost | Single neuron control over a complex motor program[END_REF], and this role has been proposed for lamprey RS neurons [START_REF] Mcclellan | Activation of 'fictive swimming' by electrical microstimulation of brainstem locomotor regions in an in vitro preparation of the lamprey central nervous system[END_REF][START_REF] Mcclellan | In vitro CNS preparations: unique approaches to the study of command and pattern generation systems in motor control[END_REF][START_REF] Mcclellan | Brainstem command systems for locomotion in the lamprey: localization of descending pathways in the spinal cord[END_REF].

Recently, we described a cellular mechanism by which cutaneous inputs are transformed into an escape locomotor command through intrinsic properties of RS neurons in lampreys [START_REF] Di Prisco | Role of sensoryevoked NMDA plateau potentials in the initiation of locomotion[END_REF], 2000). Moreover, we established that brainstem regions rostral to the rhombencephalon are not essential for escape locomotion [START_REF] Cardin | Motor responses elicited by skin stimulation in lampreys[END_REF]. Goal-directed locomotion on the other hand, has only recently attracted significant interest in lampreys (see El [START_REF] El Manira | Diencephalic projection to reticulospinal neurons involved in the initiation of locomotion in adult lampreys Lampetra fluviatilis[END_REF], although exploratory, food seeking, and attack behaviors are all important for the survival of these animals. We have recently shown that lamprey RS cells are controlled by a brainstem structure comparable to the mesencephalic locomotor region (MLR; [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF] originally described in mammals (for reviews see: [START_REF] Shik | Neurophysiology of locomotor automatism[END_REF][START_REF] Jordan | Initiation of locomotion in mammals[END_REF].

Little is known about the mechanisms by which the MLR acts onto RS neurons to generate locomotion. The pedunculopontine nucleus (PPN), traditionally associated to the physiologically defined MLR (for reviews see: [START_REF] Garcia-Rill | The basal ganglia and the mesencephalic locomotor region[END_REF][START_REF] Jordan | Initiation of locomotion in mammals[END_REF], contains cholinergic neurons that project to the reticular formation of cats and rats [START_REF] Mesulam | Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1-Ch6)[END_REF]Garcia-Rill & Skinner, 1987a;[START_REF] Skinner | Locomotor projections from the pedunculopontine nucleus to the medioventral medulla[END_REF][START_REF] Lai | Brainstem projections to the ventromedial medulla in cat: retrograde transport horseradish peroxidase and immunohistochemical studies[END_REF]. Interestingly in mammals and birds, the focal injection of acetylcholine agonists within the reticular formation elicits locomotion (Garcia-Rill & Skinner, 1987a;[START_REF] Sholomenko | Avian locomotion activated by brainstem infusion of neurotransmitter agonists and antagonists. I. Acetylcholine, excitatory amino acids and substance P[END_REF]. Moreover, cholinergic cells were recently seen in the isthmic region of lampreys [START_REF] Pombal | Distribution of choline acetyltransferase-immunoreactive structures in the lamprey brain[END_REF]. However, no direct link between a cholinergic command originating from the MLR and locomotion was ever reported in any vertebrate species.

We investigated in lampreys, the effects of cholinergic agonists on RS neurons and on locomotor behavior. Here, we describe that acetylcholine induces a nicotinic receptormediated depolarization of RS cells and elicits locomotion. We also provide evidence supporting our hypothesis that cholinergic inputs play a substantial role in the MLR command to RS cells in lampreys.

MATERIALS AND METHODS

Animal preparation

Experiments were performed on larval (n=59) and adult (n=28 newly transformed, and 3 spawning) lampreys, Petromyzon marinus. All procedures conformed to the Canadian Medical Research Council guidelines and were approved by the University Committee for Animal Care and Use. Under tricaine methanesulphonate (MS 222, 100 mg/l; Sigma-Aldrich, Oakville, ON) anesthesia, the animals were opened along the ventral midline and eviscerated. The dissection and experiments were performed in cold oxygenated Ringer's (9 o C, pH 7.4) with the following composition (in mM):, 2.1 KCl, 2.6 CaCl 2 , 1.8 MgCl 2 , 4 Hepes, 4 Dextrose, 1 NaHCO 3 . Some experiments were performed in saline with higher divalent cation concentrations, the sodium chloride concentration reduced accordingly (in mM): 10.8 CaCl 2 , 7.2 MgCl 2 , and 103 NaCl. The rostral end of the body was dissected up to the last gill, and the brain and spinal cord were isolated in vitro with the underlying cranium and notochord kept for support. The spinal cord was cut either at the first spinal segment out of 100 or between segmental levels 10 and 20. The responses to the local application of cholinergic agonists were similar in either case.

A semi-intact preparation (n=42) was also used, where the rostral end of the animal was dissected out as described above and pinned down to the Sylgard bottom of the experimental chamber perfused with Ringer's. The caudal two thirds of the body and the tail were left intact to swim freely behind, and insulated bipolar EMG electrodes were inserted into the myotomes with an inter-electrode distance of 3 mm (Teflon coated stainless steel wire, diameter 50 m; California Fine Wire Company, Grover Beach, CA).

Two pairs of EMG electrodes were usually placed between segmental levels 20 and 25, one on each side of the body. For decerebration purposes, a complete transection was made between the mesencephalon and the diencephalon. However, in experiments where locomotion was initiated by local application of a cholinergic agonist, the transection was usually performed lower, between the mesencephalon and the rhombencephalon. The local application of cholinergic agonists provided similar results whether the mesencephalon was kept or not.

Electrophysiology and drug application

The MLR of semi-intact lampreys was electrically stimulated (1-10 A, 1 ms, 5-10 Hz) with glass-coated tungsten electrodes (5 M with 10 m tip exposure) using a pulse generator S88 (Grass Instrument Co, Quincy, MA) connected to a stimulus isolation unit (Grass Instrument Co). For later histological identification, an electrolytic lesion (n=12) was made at the stimulation site using positive DC current (5 A) applied for 10 s at the end of experiments.

Reticulospinal neurons in the middle (MRRN, n=113) and posterior (PRRN, n=5) rhombencephalic reticular nuclei were impaled under visual guidance with sharp glass micropipettes (4M K-acetate, ~100 M). In all experiments, the impaled neurons were the largest reticular neurons described by Müller (see [START_REF] Rovainen | The Biology of Lampreys[END_REF] in the brainstem of the lamprey, and only cells with a resting membrane potential consistent throughout the experiment were considered in this study. The intracellular signals were recorded using an Axoclamp 2A (Axon Instruments Inc., Foster City, CA) and directed to a computer via a TL-1 DMA interface (Axon Instruments Inc., sampling rate: 1-5 kHz). Suction electrodes were used to monitor the activity of ventral roots on each side between segmental levels 10 and 20. Acquisition and analysis of RS neuron responses (i.e., peak amplitudes, areas, 10-90% slopes) were performed using the pClamp9 pack program (Axon Instruments, Inc.).

Small doses (0.4-10.6 nL) of either acetylcholine (1-5 mM) or nicotine (1-5 mM) were pressure-applied locally onto RS neurons through a glass micropipette using a Picospritzer (General Valve Corporation, Fairfield, NJ). Dose-response curves were built by randomly applying short-duration ejections of the drugs with a minimal time interval of 1 min: the random application was used to prevent possible cumulative effects , and the 1 min interval was tested by repeating three times the ejection of the same drug quantity as the minimal delay required to avoid any possible desensitization of the receptors. Fast Green was added in the pipette to monitor the extent of the drug diffusion.

When only Fast Green and Ringer's were ejected onto RS cells, no cellular responses were ever observed. To elicit locomotion, larger volumes (30-50 nL) of either cholinergic agonist solutions were ejected over the entire MRRN on both sides. A higher concentration of the cholinergic agonists (5 mM) was sometimes used because the ejected solution tended to diffuse in the bathing Ringer's before reaching the RS cells. All other drugs were dissolved in fresh Ringer's and bath applied. These included: 6-cyano-7nitroquinoxaline-2,3-dione (CNQX, 10 M), 2-amino-5-phosphonopentanoate (AP-5, 100 M), dizocilpine (MK-801, 100 M), atropine (10 M), physostigmine (eserine, 100 M), D-tubocurarine (30-100 M), -bungarotoxin (0.1 M), tetrodotoxin (TTX, 3 M) and N-methyl-D-aspartate (NMDA, 100 M). In such experiments, a Vaseline wall was built just below the rhombencephalon delimiting two distinct, watertight perfusion compartments. NMDA was applied in the spinal cord bath, whereas all other drugs were applied into the brainstem bath. After each bath application of a drug, a wash out period of 15 minutes to more than one hour depending on the drug ejected, was allowed for recovery. All drugs were purchased from Sigma-Aldrich.

Statistical analyses

Results are expressed as means ± standard errors. The correlation between the duration of drug ejection and the size of the induced responses was examined with regression analysis. In all cases, linear regressions fitted best the data. The statistical significance for differences between means was established with either a paired Student's t-test (comparisons of 2 groups) or an ANOVA test (comparisons of more than 2 groups).

All statistical analyses were carried out using Origin software (OriginLab Corporation, Northampton, MA).

Anatomical studies

Procedures: Immunohistochemistry for choline acetyltransferase (ChAT), the last enzyme in the synthesis of acetylcholine, was used to study in details the distribution of cholinergic neurons in the isthmic region of adult lampreys Petromyzon marinus (n=26).

The brains were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight. After agarose embedding, transverse sections were cut at 50µm thickness on a vibratome (TPI, St Louis, MO) and collected in 0.1 M phosphate buffer (PB). Free-floating sections were treated with 1% H 2 O 2 in phosphate buffered saline (PBS) for 30 minutes to reduce endogenous peroxidase activity. After rinsing in 0.1 M PBS, the presence of ChAT was revealed according to a procedure adapted from [START_REF] Marin | Distribution of choline acetyltransferase immunoreactivity in the brain of anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians[END_REF], replacing the peroxidase-antiperoxidase technique by the avidin-biotin one.

Briefly, sections were incubated with the following solutions: i)-Purified goat anti-ChAT serum (Chemicon, Temecula, CA) diluted 1:100 in 0.1 M PBS with 0.5% Triton X-100, 15% normal horse serum (Gibco, Burlington, ON) and 2% bovine serum albumin (Vector, Burlingame, CA) for 40 hours at 4°C; ii)-0.1 M PBS (3 x 10 minutes); iii)-Biotinylated horse anti-goat serum (Vector) diluted 1:50 in the same buffer for 1 hour at room temperature; iv)-0.1 M PBS (3 x 10 minutes); v)-Avidin-horseradish peroxidase reagent (Vectastain, Elite ABC KIT, Vector) diluted as directed by the manufacturer in PBS for 1 hour at room temperature in darkness; vi)-0.1 M PBS (3 x 10 minutes).

Finally, immunostaining was visualized with the glucose oxidase method of [START_REF] Shu | The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system[END_REF]. However, in order to achieve a better control on the duration of reaction, all the reagent concentrations were halved with respect to the initial procedure described by these authors. As a control, the primary antiserum was omitted from a series of sections in each experiment, and this resulted in no specific labeling of somata or fibers.

In some experiments, immunohistochemistry for ChAT was performed on brainstems where an electrolytic lesion had previously been made in the MLR at the end of electrophysiological experiments (n=12). The fixed brains were frozen and cut at 20 m thickness on a cryostat (American Optical, Buffalo, NY), and the sections were mounted on gelatinized slides and left to dry overnight. Immunocytochemistry for ChAT was then performed as described above, but on the mounted sections.

Analysis: Counts and cell size measurements of labeled neurons were made from transverse sections on one side of the brainstem in the rostral rhombencephalon and the mesencephalic tegmentum. All ChAT-immunoreactive cells were counted each time they disappeared as the focus was changed using an optical dissector method [START_REF] West | Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator[END_REF]; see also [START_REF] Coggeshall | Methods for determining numbers of cells and synapses: a case for more uniform standards of review[END_REF]. In one specimen, the soma diameter was measured along the longest axis as seen on transverse sections. Photomicrographs were acquired with a digital camera (Coolpix 995, Nikon, Japan) custom-fitted on a microscope (Optiphot-2, Nikon). All the figures were designed using CorelDraw 9 software (Corel Corp., Ottawa, ON) and 3-dimensional reconstructions of the distribution of ChAT-immunoreactive neurons were made using the Autocad 12 software (Autodesk Inc., Montreal, QC).

RESULTS

Cholinergic responses in RS neurons

The local application of acetylcholine (1 mM) onto RS neurons elicited depolarizing responses, the amplitude of which increased linearly with the duration of the drug ejection, until spiking threshold was reached (n=34; Fig. 1A1). The responses, although reduced, persisted under bath application of 3 µM TTX (n=3), indicating that acetylcholine exerted direct excitation of RS neurons. There was a linear increase in the responses as the ejection duration increased (Fig. 1B1). The response area, which takes into account both the amplitude and the duration of the response, displayed a strong positive correlation with the duration of the ejection in normal saline (r=0.97; p<0.001; Fig. 1A2) as well as under TTX bath perfusion (r=0.97; p<0.001; Fig. 1B2). With a higher concentration (5 mM) of acetylcholine in the ejection pipette, a focalized ejection onto a given RS neuron generally produced a complete desensitization of the depolarizing response, whereby a second ejection was ineffective, and several minutes were required for recovery (n=5; not shown). Most of the RS neurons tested responded similarly to the local ejection of acetylcholine.

The bath perfusion of the nicotinic antagonist D-tubocurarine completely blocked the depolarizing response to acetylcholine (n=3; Fig. 1C1), suggesting that the latter resulted exclusively from the activation of nicotinic receptors. In contrast, bathapplication of either atropine (n=5; Fig. 1C2), or a combination of both NMDA and non-NMDA glutamatergic antagonists, AP5 (n=4) or MK-801 (n=3) and CNQX (n=7) respectively (not shown), did not alter the subthreshold response of RS neurons to acetylcholine. This indicated that the small amplitude depolarizations induced by acetylcholine, in the absence of any network influences, were mediated exclusively by nicotinic receptors.

Properties of the nicotinic responses

When nicotine (1-2 mM) was substituted for acetylcholine in the ejection pipette, RS neurons from either the MRRN or the PRRN displayed dose-dependent depolarizations similar to those elicited by acetylcholine (n=14; Fig. 2A). Short-duration ejection evoked a small depolarization, and the area of the responses increased with the duration of the ejection. The best fit of this relation was obtained with a linear regression (r=0.97, p<0.001; Fig. 2B). Similarly to acetylcholine, nicotine induced depolarizing responses, the largest of which reached spiking threshold (Fig. 2A). Here again, repeated local applications of a high concentration (5 mM) of nicotine induced a complete desensitization of the response, requiring several minutes for recovery (n=3; not shown).

In contrast, repeated pressure-applications of a low concentration (1 mM) of nicotine unmasked interesting properties of the RS neuron responses: when a subsequent nicotine ejection was performed before the membrane potential had returned to its resting value, summation occurred in all of the tested RS cells (Fig. 2C; n=5). The membrane potential of the RS neurons depolarized until spiking threshold was reached and sustained firing of action potentials occurred. Thereafter, additional applications of nicotine did not depolarize the cell membrane further. The temporal summation of responses may be an important factor in the recruitment of RS cell activity by the MLR (see below).

Initiation of swimming by cholinergic agonists

Because acetylcholine and nicotine evoked sustained firing in the RS neurons of both MRRN and PRRN, we investigated the possibility that the drugs could also induce swimming in a semi-intact preparation (n=17). Repeated pressure applications of acetylcholine (1 or 5 mM) into the 4th ventricle over most of the MRRN on both sides evoked a long-lasting depolarization in RS neurons accompanied by an increase in EMG activity recorded bilaterally from the body and followed by the onset of swimming (Fig. 3A). Swimming (EMG bursts) developed after spiking occurred in the recorded RS cell and lasted for as long as the cell remained depolarized. The quality of swimming was variable from one experiment to another, and the excitability of the preparation appeared to be critical. Indeed, about half of the acetylcholine applications resulted in only a tonic increase in EMG activity on both sides (not shown). We found no clear relation between the occurrence of this tonic activity and the state of the preparation, suggesting that some yet unknown and uncontrolled factors intrinsic to the RS system might be involved. In contrast, in the other half of the trials, swimming that could last for several minutes was elicited and displayed alternate activity in the right and left side EMGs (Fig. 3B,3C). The body movements were then comparable to those occurring in intact animals with a wave of lateral displacement propagating smoothly from the rostral part of the body to the tail region (not illustrated). Similar results were obtained when nicotine was substituted for acetylcholine in the ejection pipette (n=4; not shown), or when either drugs were ejected over RS cells in the PRRN (n=2; not shown).

Cholinergic control of the swimming activity

Similar experiments were carried out in the in vitro isolated brainstem-spinal cord preparation where acetylcholine (1 or 5 mM) was locally applied onto the whole MRRN in the absence of prior locomotor activity (n=12). Fictive locomotion that could last for several minutes (Fig. 4A) was then elicited and ventral root discharges alternated on both sides (Fig. 4B). Nicotine (1 mM; n=6), ejected either on the MRRN or on the PRRN under the same conditions, also triggered fictive locomotion (not shown).

To study the effects of cholinergic agonists on ongoing fictive locomotion, a double bath paradigm was used where the spinal cord was perfused with Ringer's containing 100 µM NMDA to activate the spinal locomotor networks, whereas the brainstem was perfused with normal Ringer's (Fig. 5A). In these experiments (n=9), the ventral root discharges (Fig. 5B left part) initially showed an unstable rhythmic activity immediately after adding NMDA to the spinal bath. This progressively evolved into a fast and stable locomotor rhythm, some 7-10 min later (diamonds in Fig. 5C). The effects of a single local ejection of acetylcholine (1 or 5 mM) onto the whole MRRN (n=12) differed whether studied on the early unstable or the later stable fictive locomotor rhythm. When ejected early after the NMDA application, acetylcholine produced, with a delay of 30-60 s, a significant increase in the frequency of the ventral root bursts. This is illustrated in figure 5B where there is a 86.7±0.6% (mean over 8 experiments: 38.4  8.7%) reduction in period and a 89.1±0.6% (mean: 38.6  9.9%) reduction in burst duration. Cumulative data from 12 experiments (Fig. 5C) indicate that acetylcholine consistently speeded up the early locomotor rhythm as compared to when NMDA was applied alone to the spinal cord bath (compare squares with diamonds in Fig. 5C). The inset shows the early part of the graph with an expanded time base. Figure 5D shows that the cycle duration for the first 30 cycles after application of acetylcholine (black bars) was significantly shorter (p<0.001) than under control conditions (gray bars). The reduction was less pronounced for the following 30 cycles, with no significant difference afterwards. In contrast, when the fictive locomotor rhythm had already stabilized after NMDA perfusion (n=7), pressure-ejection of acetylcholine onto RS cells did not elicit any changes in the period nor the burst duration (empty bars in Fig. 5D). This was also true for the five experiments in which acetylcholine was ejected after a previous ejection of the drug had already accelerated the fictive locomotor rhythm (included in the empty bars of Fig. 5D).

ChAT-immunoreactive neurons

The results described above suggest that acetylcholine receptors are present on lamprey RS neurons and that their activation elicits locomotion. In order to locate the brainstem cholinergic cells that may possibly innervate RS cells, immunohistochemistry directed against ChAT was used (n=26). Amongst the brainstem populations of ChATimmunoreactive neurons, which included also various groups of motoneurons (see also [START_REF] Pombal | Distribution of choline acetyltransferase-immunoreactive structures in the lamprey brain[END_REF], two distinct groups of ChAT-immunoreactive neurons were observed in the tegmentum at the mesopontine border, in an area that included the isthmus and the caudal mesencephalon. The first group was located at the rostrocaudal level of the trochlear nucleus, and ChAT-immunoreactive cells were clustered medially in the periaqueductal gray (Fig. 6A,B). Cell counts varied from 545 to 781 with a median of 628 (n=6). Some of these strongly ChAT-immunoreactive cells were in close proximity with the conspicuous isthmic Müller cell I1 (nomenclature of [START_REF] Rovainen | The Biology of Lampreys[END_REF] in the anterior rhombencephalic reticular nucleus (Fig. 6B). Another group of ChATimmunoreactive cells, i.e. rostral and lateral to the trochlear nucleus, consisted of small ovoid cells that were sparsely distributed in the tegmentum (Fig. 6C,D). Cell counts ranged from 253 to 403 with a median of 352 (n=5). The cells were significantly less numerous than in the caudal group (p<0.01). The mean size of cell body in the rostral group of ChAT-reactive neurons (10.7±0.2 µm) was also significantly smaller (p<0.01) than in the caudal group (11.8±0.2 µm). A 3-D representation illustrating the location of ChAT-immunoreactive neurons within this area of the brainstem (Fig. 7) revealed how the caudal group of ChAT-immunoreactive neurons (Yellow spheres) tightly surrounded the isthmic Müller cell I1 (Green sphere; Fig. 7B,C). More rostrally, the cells from this group tended to be less compact and were more ventral. There was then a transition between this group of cells and the rostro-lateral group of ChAT-labeled neurons (Red spheres), which occupied a more dorsal location further rostrally (Fig. 7C).

Effects of nicotinic antagonists on the RS neuron response to MLR electrical stimulation

The electrophysiological observations above indicated a role of acetylcholine in the initiation and the control of locomotion in lampreys. The immunohistochemical experiments confirmed the presence of cholinergic cells at the mesopontine border in the lamprey MLR defined by [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. We investigated whether these cells could be a source of cholinergic input to rhombencephalic RS neurons by performing electrical stimulation of the MLR and recording intracellularly from RS neurons. The neurotransmitters involved in the MLR-RS synapses were first investigated: the effects of the nicotinic antagonists D-tubocurarine (30-100 M) or -bungarotoxin (0.1 M) were tested on the EPSPs evoked by single electrical stimulation of the MLR (Fig. 8A1). There was a significant decrease in the EPSP amplitude (~25 %; n=23; p<0.001; Fig. 8A2) and slope (~23 %; n=23; p<0.05; not shown). Conversely, a bath application of the acetylcholinesterase inhibitor physostigmine (100 M), which prevents the degradation of acetylcholine in the synaptic cleft, dramatically increased both the amplitude (~62 %; n=3; p<0.05) and the slope (~56 %; n=3; p<0.05) of the response evoked by single MLR electrical stimulation (Fig. 8B1-2). Because D-tubocurarine did not produce a complete block of the response to MLR stimulation, we investigated the possibility that glutamate might also mediate a part of the MLR input to RS neurons in the lamprey (Fig. 8C). A mixture of NMDA and non-NMDA receptor antagonists (200 µM AP5 and 25 µM CNQX, respectively) was bath applied, and the MLR was stimulated electrically. Single EPSPs were dramatically reduced (~39%; n=2; p<0.05; Fig. 8C1) but a substantial depolarizing response persisted. This AP5-and CNQX-insensitive component remained constant in high calcium-high magnesium saline and was able to follow a frequency of stimulation of 25 Hz with no changes in latency and amplitude (Fig. 8C2). This clearly demonstrated that the non-glutamate component was monosynaptic. Interestingly, the addition of D-tubocurarine (30 µM) to the glutamate antagonists mixture did not completely suppress the MLR-evoked response, and a small EPSP remained (Fig. 8C1).

Although our pharmacological experiments indicated the involvement of various neurotransmitters in the MLR-evoked EPSP in lamprey RS neurons, they nevertheless confirmed that cholinergic inputs are present. Furthermore, electrolytic lesions were performed at the stimulation site within the MLR and ChAT-immunohistochemistry was carried out on the brainstem sections (Fig. 9). ChAT-labeled neurons were found around the coagulation site close to the ventricular surface (Fig. 9A,C), corresponding to the more caudal group of cholinergic cells (Fig. 9B).

The implication of the cholinergic component from the MLR to RS neurons was thereafter investigated on locomotor activity in a semi-intact preparation (n=8). The MLR was stimulated at 5 Hz and EPSPs were evoked in RS cells which displayed a buildup of depolarization until the threshold for spiking was reached and swimming activity followed (Fig. 10A1; see also [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. Consistently, after selectively adding the nicotinic antagonist D-tubocurarine (30-50 M; n=5) to the brainstem bath, the membrane potential of RS neurons remained mostly below spiking threshold and swimming did not occur (compare Fig. 10A1 andA2). There was a significant decline of both the peak and the area of the MLR-evoked compound EPSPs (p<0.001; Fig. 10B1-2).

The depolarization resulting from the summation of EPSPs induced by repetitive MLR stimulation was decreased (compare traces in control, black, and under D-tubocurarine bath perfusion, gray, in Fig. 10B2; compare also gray circles from control, black circle in Fig. 10B3). Opposite effects were observed when the selective cholinesterase inhibitor physostigmine (100 µM) was applied to the brainstem bath: the EPSPs evoked by single electrical stimulation of the MLR were increased in both amplitude and duration (p<0.05, n=3; not shown, but see Fig. 8B for single MLR stimulation), and the depolarization resulting from the 5 Hz stimulation of the MLR was substantially increased (Fig. 10B3, empty circles) when compared to control (Fig. 10B3, black circle). This resulted in the occurrence of swimming (not shown). It is noteworthy that swimming could still be evoked under D-tubocurarine, but only with higher stimulation intensities (generally, a 30-40% increase was required; not illustrated). These results indicate that the locomotor input from the MLR to RS cells of lampreys is, at least, partly cholinergic.

DISCUSSION

The present study is in accordance with an important role of cholinergic inputs onto rhombencephalic RS neurons in the initiation and control of MLR-evoked locomotion in a vertebrate. Acetylcholine elicited a nicotinic receptor-mediated depolarization in RS cells, accompanied by action potential firing that induced swimming in a semi-intact preparation. In addition, the cholinergic stimulation of RS neurons accelerated ongoing locomotor activity induced pharmacologically by NMDA. Anatomical experiments confirmed the existence of two populations of isthmic cholinergic neurons located in the MLR, the electrical stimulation of which induced locomotion. Furthermore, the perfusion of a nicotinic antagonist dramatically depressed the MLR-evoked locomotor command, preventing the occurrence of swimming at the same stimulation strength.

Cholinergic inputs from the MLR to RS neurons in lamprey

Two distinct groups of ChAT-immunoreactive cell bodies were present in the mesopontine tegmentum of lampreys, as previously observed by [START_REF] Pombal | Distribution of choline acetyltransferase-immunoreactive structures in the lamprey brain[END_REF].

The first group consisted of densely clustered cells in the periaqueductal gray, and the second, at a more rostral level, comprised fewer cells that were loosely distributed more laterally within the tegmentum. Comparison with immunohistochemical data from other species (cats: [START_REF] Mitani | Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat[END_REF]rats: Jones, 1990;and amphibians: Marin et al., 1997) suggests that the medially clustered cells corresponds to the laterodorsal tegmental nucleus (LDT), and the more laterally located group of cells to the PPN.

The MLR is a functionally defined brain structure that does not correspond to any single nucleus. Several lines of evidence suggest that cholinergic neurons of the PPN may be part of the MLR in mammals [START_REF] Garcia-Rill | Activity in the mesencephalic locomotor region during locomotion[END_REF]Garcia-Rill & Skinner, 1987a;Garcia-Rill et al., 1987;[START_REF] Rye | Pedunculopontine tegmental nucleus of the rat: cytoarchitecture, cytochemistry, and some extrapyramidal connections of the mesopontine tegmentum[END_REF][START_REF] Coles | The mesencephalic centre controlling locomotion in the rat[END_REF][START_REF] Skinner | Locomotor projections from the pedunculopontine nucleus to the medioventral medulla[END_REF].

However, no direct relationship has yet been provided. Here, we demonstrate that the electrical stimulation of the mesopontine region containing cholinergic cells, defined as the MLR of lampreys [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF] evokes swimming in a semi-intact preparation, the occurrence of which is depressed by exposing RS neurons to the nicotinic antagonist D-tubocurarine. Moreover, previous studies in mammals indicate that some of the mesopontine cholinergic neurons project to the reticular formation (Garcia-Rill & Skinner, 1987a;[START_REF] Rye | Medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum in the rat[END_REF]. For example, cholinergic neurons of the PPN and the LDT project specifically to the ventromedial medulla in rats [START_REF] Skinner | Locomotor projections from the pedunculopontine nucleus to the medioventral medulla[END_REF]. In our study, the slope of the responses to MLR stimulation was affected by the nicotinic drugs D-tubocurarine, -bungarotoxin and physostigmine, supporting the hypothesis that the cholinergic input acts directly on RS neurons. Moreover, we found that the non-glutamate component of the MLR-evoked EPSP followed without any changes in amplitude nor delay a 25 Hz frequency of stimulation. Furthermore, we have previously shown that cells in the MLR region are retrogradely labeled from injections of cobalt-lysine in the rhombencephalic reticular formation [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. Hence, although we cannot exclude the possibility of another synapse intercalated between the MLR and RS cells, altogether our findings strongly support a monosynaptic cholinergic input from the MLR to RS cells in lampreys. Double labeling techniques (retrograde tracing with dextranamines and ChAT immunofluorescence) will thus be useful to confirm a projection from the MLR cholinergic cells to the rhombencephalic reticular formation.

In addition to the cholinergic component of MLR input to RS neurons, pharmacological studies showed the existence of a large glutamatergic component. This raised the possibility that both glutamate and acetylcholine cooperate to the MLR command. Indeed, we did not investigate this hypothesis further, and experiments are now performed to analyze both the source of glutamate (co-release or release by two distinct MLR neuronal populations) and possible cooperative effects of glutamate and acetylcholine.

Cholinergic responses in RS neurons

In vertebrates, RS neurons respond to cholinergic agonists. Acetylcholine produces either depolarizing, hyperpolarizing, or biphasic responses in RS cells of the cat medial pontine reticular formation, affecting the cell firing frequency in response to glutamate bath perfusion [START_REF] Greene | Biphasic responses to acetylcholine in mammalian reticulospinal neurons[END_REF][START_REF] Grillner | Neurobiological bases of rhythmic motor acts in vertebrates[END_REF]. [START_REF] Kungel | Substance P and other putative transmitters modulate the activity of reticular pontine neurons: an electrophysiological and immunohistochemical study[END_REF] reported mixed effects of cholinergic agonists onto the discharge pattern of neurons in the caudal pontine reticular nucleus of rats. An injection of atropine within the nucleus reticularis pontis oralis produced a decrease in the rate of discharge in RS cells [START_REF] Takakusaki | Discharge properties of medullary reticulospinal neurons during postural changes induced by intrapontine injections of carbachol, atropine and serotonin, and their functional linkages to hindlimb motoneurons in cats[END_REF].

In birds, an injection of carbachol in the ventral division of the central nucleus medulla, an area containing RS cells, induces atropine-sensitive walking, running or flight [START_REF] Sholomenko | Avian locomotion activated by brainstem infusion of neurotransmitter agonists and antagonists. I. Acetylcholine, excitatory amino acids and substance P[END_REF][START_REF] Webster | Origins of brainstem-spinal projections in the duck and goose[END_REF]. However, the drug is not effective when injected in the pontine reticular formation. The present study in lamprey RS neurons revealed dose-dependent nicotinic receptor-mediated depolarizations in response to a focal ejection of acetylcholine. These responses persisted under TTX (although reduced), suggesting a direct effect, but possibly associated with some network amplification. Surprisingly, [START_REF] Matthews | Glycine, GABA and synaptic inhibition of reticulospinal neurones of lamprey[END_REF] reported that acetylcholine had no effects on RS cells of the MRRN. However, because the drug was bath-applied in their study, a desensitization of the responses may have occurred as reported here. We also found that the local ejection of either nicotinic agonists did not always elicit consistent responses in RS neurons. In some cases, the local application of a nicotinic agonist evoked no response or only subthreshold depolarizations from the RS neuron (not illustrated). However, this was temporary, and a longer delay or changing the agonist solution was usually sufficient to observe a cholinergic response. It is possible that some receptor desensitization occurs due to uncontrolled spontaneous cholinergic activity. Supporting this hypothesis is the observation that adding the selective cholinesterase inhibitor physostigmine alone was sufficient to unmask spontaneous acetylcholinemediated depolarizations (n=5; not illustrated).

Acetylcholine and MLR-controlled swimming

As in mammals (Garcia-Rill & Skinner, 1987b;[START_REF] Iwakiri | Stimulus effects of the medial pontine reticular formation and the mesencephalic locomotor region upon medullary reticulospinal neurons in acute decerebrate cats[END_REF], MLR inputs in lampreys are relayed to the spinal cord by the rhombencephalic RS neurons [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. Swimming evoked in response to MLR stimulation generally needs several seconds of repeated stimulation to develop, due to a slow buildup of depolarization in the RS cells; the onset delay of swimming decreases as the intensity or frequency of stimulation increases [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. Here, we report that MLR inputs to RS neurons are partly mediated by acetylcholine, and that nicotinic responses show summation properties. The latter may participate to the slow buildup of depolarization and the delayed swimming onset that occur at low stimulation intensities. According to this assumption, the buildup of the response to a 5 Hz stimulation of the MLR was dramatically reduced in the presence of a nicotinic antagonist and largely increased in the presence of physostigmine, which resulted in the blockade or the facilitation of the initiation of swimming activity, respectively. In such a view, higher stimulation frequencies would result in a faster depolarization in RS cells and a shorter onset delay for swimming. Of course, other neurotransmitters may be involved (see Fig. 8C1) and increasing the intensity of stimulation reversed the blocking of swimming induced with a nicotinic antagonist, presumably through the activation of other non-cholinergic neurons in the MLR. Nevertheless, our results emphasize an important role of acetylcholine in the initiation of locomotion by MLR stimulation.

The local application of nicotinic agonists onto rhombencephalic RS neurons generally produced an increase of the locomotor output. In the absence of prior activity, nicotinic agonists triggered swimming in a semi-intact preparation. When fictive locomotion was induced by perfusion of NMDA onto the spinal cord, the nicotinic activation of RS neurons accelerated the ongoing locomotor rhythm. This acceleration is likely to result from an increase in the excitatory amino acid drive onto the spinal locomotor networks as RS neurons are excited by acetylcholine. This accelerating effect was nevertheless limited, perhaps due to gating of descending inputs during NMDAinduced swimming or to intrinsic limits of the spinal locomotor networks themselves. It was shown previously that vestibular inputs to RS neurons are depressed by adding NMDA to the Ringer's [START_REF] Bussières | Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys[END_REF][START_REF] Alford | Glutamate metabotropic receptor mediated depression of synaptic inputs to lamprey reticulospinal neurones[END_REF]. Wannier and collaborators (1998) demonstrated that lamprey RS neurons have differential effects on locomotion, including both excitatory and inhibitory influences. In our hands, nicotinic stimulation of the rhombencephalic RS neurons only induced an acceleration of the locomotor rhythm, suggesting that excitatory RS neurons are the main cholinergic targets.

Functional considerations

The MLR-evoked locomotor behavior is established on a slow, graded depolarization of the RS neurons [START_REF] Sirota | Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys[END_REF]. The present study indicates that this graded command seems to be mediated, at least in part, by acetylcholine. In contrast, sensory-evoked escape swimming relies on the occurrence of a fast, large and longlasting depolarizing plateau in RS cells in response to a single high intensity stimulus [START_REF] Di Prisco | Role of sensoryevoked NMDA plateau potentials in the initiation of locomotion[END_REF], 2000). The plateaus require the activation of NMDA receptors. This could result from a different function of either command to RS neurons.

The latter produces a fast behavioral response that needs to last long enough (the plateau) to guarantee the safety of the animal, such as for example in the case of a predator attack.

In contrast, the MLR command generates a locomotor behavior that has to be fully adapted (gradual depolarization) and goal-directed compatible with exploratory and appetitive behaviors, which have been proposed to be channeled through the MLR in mammals [START_REF] Sinnamon | Preoptic and hypothalamic neurons and the initiation of locomotion in the anesthetized rat[END_REF]; for a review see [START_REF] Jordan | Initiation of locomotion in mammals[END_REF].

In this study, we focused on the nicotinic component of the cholinergic inputs to RS cells. Muscarinic effects could also be present, although atropine had no effects on acetylcholine-evoked small amplitude depolarizations of RS neurons. We have recently reported that acetylcholine, through muscarinic receptors, modulates the RS cell responses to glutamate, suggesting a role in the control of locomotion (Le Ray et al., in prep). Similarly, anatomical studies showed that numerous neurons in the MLR remained unstained after ChAT-immunohistochemistry, and the MLR-evoked EPSPs in RS neurons were affected only partly by nicotinic antagonists and partly by glutamate antagonists. Taken together, these results demonstrate that the initiation and the control of locomotion require complex mechanisms and that acetylcholine is not the only neurotransmitter involved in the MLR command in lampreys. Rather, goal-directed locomotion may result from the co-activation (cooperation?) of several excitatory commands originating in the MLR including cholinergic and glutamatergic inputs.

Experiments are underway to test this hypothesis.

ABBREVIATIONS

Figure 1 :

 1 FIGURE LEGENDS

Figure 2 :

 2 Figure 2: Nicotine-evoked responses in reticulospinal neurons. A: Local ejections of nicotine (1 mM) of increasing duration (50, 150, 250, 300 and 350 ms, Nico) elicit dosedependent depolarizations in an intracellularly recorded RS neuron. B: Linear relationship between the duration of the nicotine ejection and the area of the RS response. C: A single local ejection of nicotine (150 ms; Nico) evokes a transient depolarization in an RS neuron (left). The depolarizations sum up when the ejections are repeated within a short time interval (right). A and B: same cell.

Figure 3 :

 3 Figure 3: Acetylcholine induced active swimming in a semi-intact preparation. A: Four repeated local ejections (300 ms) of acetylcholine (1 mM, ACh) were made onto the MRRN covering the entire nucleus on both sides without spreading to other rhombencephalic reticular nuclei. A slow depolarization with spiking occurs in a MRRN neuron and is followed by swimming (rEMG and lEMG on the right and the left side, respectively). B, C: The EMG activity can last several minutes (in B, trace cut after 30 s) and shows clear left and right alternation (C). C shows on a faster time base the traces in the gray area in B.

Figure 4 :

 4 Figure 4: Acetylcholine induces fictive locomotion. Three repeated local ejections (300 ms) of acetylcholine (1 mM, ACh) trigger a fictive locomotor bout in ventral roots on the right and left sides of the spinal cord (rVR and lVR, respectively). Fictive locomotion lasts several minutes (A) and shows clear right and left alternation (B). B shows on a faster time base the traces in the gray area in A.

Figure 5 :

 5 Figure 5: Acetylcholine accelerates the NMDA-induced locomotor rhythm. A: Experimental design. A Vaseline wall was built to separate the brainstem from the spinal cord allowing the selective perfusion of the latter with NMDA (100 µM) to elicit fictive swimming. B: Three local ejections (200 ms) of 1 mM acetylcholine onto the MRRN accelerate the ongoing rhythm previously induced by selective bath perfusion of the spinal cord with NMDA (trace discontinued during 30 s). C: Mean period of NMDAinduced bursts as a function of time with (arrow, black squares) and without (gray diamonds) a local application of acetylcholine (n=12). Inset displays an expanded representation of the first 200 s. D: Changes in burst mean period as a function of the number of cycles of fictive swimming in the absence (gray bars) or in the presence (black bars) of acetylcholine local ejection (n=12). Empty bars include examples where acetylcholine was ejected after the NMDA rhythm had already stabilized (i.e., >90 min after NMDA superfusion on the spinal cord) or after a previous injection of acetylcholine had already been made. Note the absence of change in the cycle duration. ***: p<0.001, *: p<0.05, ns: non significant.

Figure 6 :

 6 Figure 6: ChAT-immunoreactive neurons at the mesopontine border. A: Photomicrograph from a transverse section showing clustered ChAT-immunoreactive neurons in the caudal group. B: Magnification of the boxed area in A illustrating ChAT-immunoreactive cells surrounding the large isthmic Müller cell (I1) in the anterior rhombencephalic reticular nucleus. C: Photomicrograph from a transverse section showing ChAT-immunoreactive neurons sparsely distributed in the rostral lateral group. D: Magnification of the boxed area in C. Scale bars: 200 µm in A and C, 50 µm in B and D. mlf: medial longitudinal fasciculus; I1: first isthmic Müller cell of the anterior rhombencephalic reticular nucleus; MT: mesencephalic tectum; IV: trochlear nucleus.

Figure 7 :

 7 Figure 7: Three-dimensional representation of the position of ChAT-immunoreactive cells in the isthmic region. Each sphere represents a ChAT-immunoreactive neuron. For clarity, the diameter of the spheres was doubled as compared to the true diameter of the cells. A: 3-D reconstruction from 29 transverse sections (20m-thickness) showing the clustered periventricular group (yellow spheres) surrounding the large isthmic Müller cell I1 (green sphere) and the more lateral and rostral group in the mesencephalic tegmentum (red spheres). The two neuronal populations are shown at high magnification in transverse view (B) and sagittal view (C). Two orthogonal planes were added for a perspective view. In B, caudal is toward the observer and in C, lateral is toward the observer. Scale bars: 400 m in A and 200 m in B and C. V: ventral; R: rostral; C: caudal; L: lateral.

Figure 8 :

 8 Figure 8: Effects of cholinergic drugs on synaptic responses evoked in RS neurons by single stimuli delivered to the MLR. A1: Depressed MLR-evoked EPSP in RS neurons under D-tubocurarine (D-tubo, 30 µM) perfusion. A2: Histograms of the single EPSP mean peak amplitude and slope in control (filled bars) and under bath perfusion of D-tubocurarine (30-50 µM, empty bars). B1: Enhanced MLR-evoked EPSP under physostigmine (Physo, 100 µM) perfusion. B2: Histograms of single EPSP mean peak amplitude and slope in control (filled bars) and under physostigmine bath perfusion (empty bars). C1: MLR-evoked EPSPs are reduced but not completely blocked by CNQX (25 µM) and AP5 (200 µM). The EPSP component insensitive to glutamate antagonists was further reduced by D-tubocurarine (30 µM), but not abolished. C2: The glutamate antagonists-resistant EPSP followed with a constant latency the MLR high frequency stimulation in a solution of high divalent cations (superposition of 10 consecutive traces). In each condition, mean traces depicted in this figure are averages of three to five responses. ***: p<0.001, *: p<0.05.

Figure 9 :

 9 Figure 9: ChAT-immunoreactive cells in and around the MLR-coagulation site in one larval lamprey. A: Photomicrograph from a transverse section at the isthmic level showing the intact side (on the left) and the caudal part of the coagulation site (on the right). B: Schematic representation from the transverse section showing the location of ChAT-immunoreactive cells and the extent of the coagulation site (gray area) drawn from

Figure 10 :

 10 Figure 10: D-tubocurarine effects on MLR-evoked locomotion. A1: Control response elicited by a 5 Hz stimulation of the MLR (MLR St). The spiking activity induced in the RS neuron by the stimulation of the MLR precedes the onset of swimming characterized by alternate EMG activities on both right and left sides of the body (rEMG and lEMG, respectively). A2: The bath perfusion of D-tubocurarine (30-50 M) dramatically reduces spiking in RS neurons and swimming does not occur, even when the stimulation is maintained for a longer duration. B1: Histograms illustrating the changes in the mean peak and mean area of the compound EPSPs under bath perfusion of D-tubocurarine as compared to control (normalized values). ***: p<0.001. B2: Superimposition of the EPSPs evoked by the 5 Hz stimulation of the MLR in control (black) and under the bath perfusion of D-tubocurarine (gray). B3: Plot of the relative cell depolarization as a function of the relative mean area of the MLR-evoked EPSP. The depolarization corresponding to a mean area in control (black circle) is centered on 100%-area and 100%-depolarization. The perfusion of D-tubocurarine (gray circles) reduces both the EPSP area and the depolarization, whereas perfusion of physostigmine (empty circles) increases both the EPSP area and cell depolarization.

ACKNOWLEDGEMENTS

This work was supported by the Canadian Institutes for Health Research (CIHR) and the Fonds pour la Formation des Chercheurs et l'Aide à la Recherche (FCAR, Québec). D.L.R. was a fellow from the Institut National de la Santé et de la Recherche Médicale (France), the Ministère de l'Éducation du Québec, and the Fondation de l'Université du Québec à Montréal. F.B. was a fellow from the Fondation pour la Recherche Médicale (France) and Jasper Fellowship (Québec). C.B.L. and P.L. received a studentship from FCAR and CIHR grants, respectively. The authors wish to express their gratitude to D. Veilleux for her assistance with the experiments and to Y. Breton for his expertise in producing the 3D representations of cell groups. We thank Mr. W.D. Swink and Mrs. M.K. Jones from the Lake Huron Biological Station as well as Mr. J.E. Gersmehl, E. Howe, and W. Boufard from the U.S. Fish and Wildlife Service for their kind supply of lampreys.


Michel Belliard

Sophie Bajard

Thierry Cadiou
email: thierry.cadiou@cea.fr

Modelling of a Passive Hydraulic Lock concept for the Mitigation Transfer Tubes in a SFR core

Keywords: Sodium-cooled Fast Reactor, thermal-hydraulics in sub-assembly, PHYLOCK, multi-scale, CFD, component scale, system scale

In the context of the safety increase of the integrated Sodium-cooled Fast Reactor, we present an original safety complementary disposal to prevent or mitigate the consequences of a hypothetical severe accident consecutive to a loss of reactor cooling sequence. This disposal is set by ad-hoc empty transfer tubes, in place of some fuel sub-assemblies present in the reactor core, using an innovative passive hydraulic lock. It is based on the concept of hydraulic diode controlled by the flowrate of primary pumps. In case of the loss of the primary flow, it provides a direct flow path between the hot pool, equipped with decay heat removal exchangers, and the cold pool, leading to an easier passage of colder sodium towards the cold pool to promote core cooling by natural convection. In addition, it provides an easy way to discharge corium towards the core catcher in the hypothetical case of severe accident. In this paper, we focus on the assessment of the hydraulic lock function and of the small magnitudes of the perturbations induced by our disposal on the thermal-hydraulic behaviour of the reactor during normal operations. Through an analytical analysis and a multi-scale numerical approach, ranging from the local scale (CFD) to the system one, we claim that the lock function is kept during the normal situations at various power regimes. The leak flow represents no more than 1% of the primary pump flow. In addition, no important reactor thermal-hydraulic perturbation is brought by adding transfer tubes in the core. This is true for normal steady states, whatever the power is, but also for a full-power loss of forced flow accidental transient leading to the natural convection cooling.

Introduction

In a typical Sodium-cooled Fast Reactor (SFR) with an integrated concept [START_REF]Some thermal-hydraulic challenges in sodium cooled fast reactors[END_REF], the primary circuit is entirely inside the main vessel (i.e. no loop), cf. Fig. 1. The integrated SFR design has some intrinsic advantages from the point of view of the safety. In particular, the natural convection of the primary sodium fluid is facilitated thanks to the elevation differences between the heat source (core) and the cold sources (Intermediate Heat eXchangers: IHX). However, this advantage can be broken if the IHX are unavailable. In order to cool down the primary sodium fluid heated in the core, safety exchangers are therefore needed as the Decay Heat Removal (DHR) ones. In this paper, we only focus on the DHR located in the hot pool. The sodium cooled by these safety exchangers is flowing down in the hot pool and faces the difficulty to go back to the bottom of the core, through the IHX inlet being at a higher elevation than the DHR outlet. In order to promote the reactor core cooling by natural convection, it is proposed to substitute ad-hoc empty tubes, open at both ends, to some fuel sub-assemblies present in the reactor core. This is motivated by providing a direct flow path for sodium towards the cold pool. In this situation, the passage of sodium after being cooled by the decay heat removal system is made easier from the hot pool to the cold pool. Moreover, in the hypothetic case of severe accident core fusion due to the loss of DHR exchangers, these transfer tubes must favor the passage of the hypothetical corium from neighboring fuel sub-assemblies to the core catcher. These tubes, denoted M-TT for Mitigation Transfer Tubes, are part of a complementary safety disposal to mitigate the consequences of this kind of accident [START_REF] Bachrata | Fuel discharge toward the core catcher under severe accident conditions with mitigation devices[END_REF]. Their efficiency has been proved in a recent study [START_REF] Bertrand | Status of severe accident studies at the end of the conceptual design of ASTRID: Feedback on mitigation features[END_REF] in which it has been evaluated that thanks to a fast draining of a part of the fuel from the core region through the M-TT, the mechanical energy release is one order of magnitude lower with tubes than without.

Fig. 1 Scheme of the primary circuit of a SFR with M-TT

In case of an eventual pollution from the reactor cover non-reactive gas, empty tubes offer the advantage to avoid or minimize gas retention. Moreover, it let the possibility to wash the M-TT subassemblies. Obviously, the M-TT disposal must work as a lock and induce only small perturbations on the hydraulic behavior of the reactor during normal operations from stand-by to full power state. Hence, no fluid must be allowed to flow between the hot and cold pools, unless a small by-pass flow. For this goal, these empty tubes are equipped with an innovative device, designated under the name of PHYLOCK [4], and based on the concept of hydraulic diode.

The paper is organized in a multi-scale approach as follows. First, the M-TT PHYLOCK concept is described and analytically analysed in Sections 2 and 3. This analysis is supported in Section 4 by a numerical study based on computational fluid dynamics at the local scale. It gives up-scaling elements to construct a stand-alone 3D-model at the component scale using a thermal-hydraulic system code in Section 5. In particular, the hypothetical gas retention issue in the M-TT is numerically analyzed. Then, Section 6 presents the integration of this M-TT 3D-model into a full reactor model at the system scale using the same code. The impact of the M-TT on eventual reactor thermal-hydraulic perturbations is studied for various steady-states regimes as well as for a loss of primary flow transient. Finally, some conclusions and perspectives are given.

M-TT hydraulic lock concept

As presented in the introduction, there are four functional requirements of the transfer tubes:

-prevent the severe accidents by providing a direct path between hot and cold pools and an easier natural-convection core cooling, -mitigate a hypothetical severe accident ensuring the rapid flow of the corium to the core catcher, -ensure compatibility with the core thermal-hydraulics anytime in normal operation, -ensure compatibility with the assembly handling system.

It is important to note that there is a difference of between the hot and cold pools free levels for the nominal steady state (cf. Fig. 2). This elevation difference is related to the pressure loss at the IHX depending on the primary mass flow. Consequently, for a given axial elevation, the hot pool is over pressurized relatively to the cold pool. Then, at steady state: Then, the difference of total pressure , between the two extremities of the tube, is equal to:

(2)

Alternatively, the difference of reduced pressure (with gravity terms) is equal to:

(3)

Fig. 2 Reactor vessel with the transfer tube in normal operation

Due to this pressure difference P, sodium should flow downwards. To avoid this, the transfer tubes are equipped with one or more injectors, fed by the primary pumps, ensuring the tightness between the hot and cold pools in operating situation (cf. Fig. 2 and Fig. 3). Conversely, in case of a primary pump failure, the sodium supply of the injectors is instantaneously stopped. Then this seal disappears allowing the direct passage of sodium between the hot pool and the cold pool. In general, an injector system is used to transfer fluid from a low-pressure region to a higher one. In this case, the injector system of the tube is designed to stop the sodium flow coming from the hot pool to the cold pool.

Core IHX DHR

Transfer Tube

Fig. 3 Principle of the Transfer Tube

The presence of injectors induces some untreated issues for the moment such as:

-the cross-section in the tube is locally reduced by the presence of the injectors (potentially up to 30%), which could possibly limit the passage of the corium, -a possible risk of cavitation in the injector nozzles remains to be studied, -the hypothetical accumulation and retention of sodium dissolved gas in the M-TT, -an eventual thermal cracking of the hexagonal tube could appear at the equilibrium plane if no thermal diffusion is existing between hot and cold sodium.

Analytical approach

The injector is said to be in equilibrium when the flowrate in the upper part of the through tube is zero. In non equilibrium, there is a residual flow going upwards or downwards, depending on the injector flowrate.

Equilibrium state

The domain of study chosen for the analytical approach is shown in Fig. 4. The injector, schematically represented by the red arrow, is positioned in the lower part of the M-TT. Due to the pressure difference P between the hot pool above and the cold pool below, sodium should flow downwards. Thanks to the flowrate injected to counteract it, the downward flow of hot sodium is totally stopped whereas the injected sodium flow is reversed.

Fig. 4 Principle of the injector

Mass conservation equation

At the equilibrium, the upward hot sodium is stopped and the injected flow is totally transferred into the lower part of the tube. In terms of flowrate, the injected sodium will totally go downwards at equilibrium, whereas the hot sodium from the hot pool will be stopped. : sodium density (kg/m 3 )

Momentum balance equation

The momentum balance is written for the M-TT flow reversal region (cf. Fig. 4):

(

: height of the flow reversal region (m)

: upper pressure of the flow reversal region (Pa)

: lower pressure of the flow reversal region (Pa)

: pressure at the injector nozzle (Pa)

If we assume that the pressures at the lower and upper sides of the flow reversal region can be approximated by the hydrostatic pressures and , and that , the momentum balance can be rewritten: This balance is rewritten in the following form, if is negligible.

: pressure drop between the upper and lower part of the tube (Pa)

: injected flowrate at the equilibrium (kg/s) Then using , the equilibrium injector flowrate is expressed as follows:

As a result, the injector flowrate is entirely defined from the tube section , the injector nozzle section , the sodium density , and the reduced pressure difference on either side of the tube.

In view of these results, it appears that the dimensioning of the injectors results from a compromise between the injector velocity and the section of the injector nozzle.

Non-equilibrium state

Considering that the actual flow of the injector is slightly different from the equilibrium flow obtained above, a residual flow appears in the upper part of the tube.

Mass conservation equation

Taking into account this residual flow, the mass balance is written: with the sodium flowrate at the bottom outlet of the transfer tube, the sodium flowrate at the upper part of the tube and the injected flowrate.

Momentum balance equation

The addition of the residual flowrate modifies the momentum equation as follows:

We look for the expression of versus . By derivation, this equation becomes: [START_REF] Tenchine | Status of CATHARE code for sodium cooled fast reactors[END_REF] As is independent of , Hence:

(11)

Or (if ) (12)
After solving this differential equation, the residual flowrate in the upper part is expressed as a function of the injected flowrate:

(13)
With the injected flowrate at equilibrium (see Equation ( 8)).

Local scale: CFD numerical approach

To complete the analytical approach, the numerical simulation based on a CFD approach with STAR-CCM+ software is selected [5] [6]. The STAR-CCM+ approach, by discretising the domain with many cells, can model the fluid flow once the boundary conditions have been imposed.

The physical models retained in this approach are as follows:

-axisymmetric 2D modelling, -stationary calculation, -resolution of Navier-Stokes equations, -a RANS approach.

The turbulence is taken into account by the k- model. It consists in adding a turbulent viscosity to the Navier-Stokes equations, calculated as a function of the turbulent energy k per mass unit, and the dissipation ε per mass unit. Each of those two terms is the solution of a transport equation.

Domain of study

The tube with injector is represented in a simplified manner in Fig. 5. The injector is positioned in the lower part of the domain by a blue arrow. Hot sodium (550°C) descends from the hot pool under overpressure. The injection of cold sodium (400°C) is intended to counteract this descent.

Fig. 5 2D-axisymmetric CFD model of M-TT

Sodium thermophysical properties

Constant thermophysical properties for sodium are considered at a temperature of 475°C, average value between the lower and the upper sodium temperature in the transfer tube, respectively of 400°C and 550°C [START_REF]Physical properties of sodium: a contribution to the estimation of critical coordinates[END_REF].

Boundary conditions

The boundary conditions imposed on the domain composed of the injector and the vertical tube are the following:

-The upper and lower boundaries of the domain are considered, each as open wall of uniform reduced pressure, -the vertical wall is adiabatic, -the sodium flowrate is imposed at the injector nozzle.

Meshing

The 2D-axisymmetric domain is meshed with the STAR-CCM+ software taking into account the injector nozzle. The meshing for this 2D-axisymmetric modelling is shown on Fig. 6. The number of cells is nearly 18,000. The domain extends over the 4.5 m of the height of the transfer tube. The lower part of 80 cm long, called tube nozzle in which is positioned the injector, has a diameter of 0.09 m while the upper part diameter is 0.14 m.

Fig. 6 Meshing of the transfer tube with a zoom on the nozzle

CFD results vs analytical results

The CFD results are firstly presented at the equilibrium state assuming a reduced pressure drop of 0.16 bar between the hot and the cold pools and an injector diameter of 1 cm. They are then compared to the analytical results. Lastly, the influence of this diameter and the pressure drop on the results are evaluated.

Pressure distribution

For the equilibrium injector flowrate value, the reduced pressure distribution (cf. Fig. 7) shows two distinct pressure zones: high pressure in the upper part and low pressure in the lower part delimitated by a sharp transition. This evolution makes it clear the effect of the injector on sodium that elevates it up to a certain height. On the contrary, sodium coming from upwards is stopped at this height. In addition, the high velocity at the injector nozzle leads to a locally even lower pressure than in the lower part of the tube. The axial sodium temperature linearly evolves from the cold pool temperature of 400°C to the hot pool temperature of 550°C (cf. Fig. 8). In contrast to the two separated regions obtained with the pressure distribution, the thermal flux smoothly diffuses throughout the sub-assembly, which leads to the absence of any thermal stratification. This regular evolution corresponds to a situation of a static fluid submitted to a temperature gradient between each side. Consequently, this thermal diffusion existence in the M-TT seems to be pushing the risk of an eventual thermal cracking of the hexagonal tube. However, it needs to be consolidated with CFD computations taking into account the sub-assemblies and the interassembly bypass.

Velocity distribution

The velocity distribution (cf. Fig. 9) is very high at the injector nozzle. The higher the fluid rises, the more its velocity decreases until it reverses.

Fig. 9 Velocity distribution with a zoom on the nozzle

Comparison of analytical and numerical approaches

Table 1 shows the good agreement for the equilibrium injector flowrate, between CFD modelling and the analytical approach. The difference is less than 7.5%. The analysis of this table suggests that doubling the pressure variation only increases the injected flow by a factor of 1.5. In addition, this flowrate is directly proportional to the diameter of the injector.

These results are in agreement with the relation (8) established above, which is equivalent to: with the injector diameter and the reduced pressure difference in the transfer tube. So, the flow increases as the square root of the pressure difference, while it evolves linearly with the injector diameter.

Sensitivity to the equilibrium injector flowrate

The sensitivity to the injector flowrate around the equilibrium flowrate has been established above, cf. (12). Again, Table 2 shows the good agreement for the sensitivity to the injector flowrate between CFD modelling and the analytical approach. Fig. 10 represents the residual flowrate versus the injector flowrate near the equilibrium flowrate for both CFD and analytical approaches, in the case where the injector diameter is equal to 2 cm and the reduced pressure drop in the tube is 0.16 bar. The equilibrium injector flowrate is of 5.2 kg/s but the high value of the slope with an order of magnitude of 15 (19 with the analytical approach and 12 with the CFD one), makes the equilibrium quite sensitive to the injection flowrate.

Fig. 10 Sensitivity of the residual flowrate to the injector flowrate

The analytical approach has led to a simple formulation of the injector flowrate that allows a first dimensioning of the characteristics of the injector. These preliminary results have been refined by the CFD approach.

In the following part, we will consider the component scale that will be then integrated in the system scale.

Component scale: stand-alone 3D-modelling numerical approach

In this section, we present the stand-alone 3D-modelling of the M-TT at the component scale. As our goal is to include this component-scale model in a system-scale model of the SFR, we use the CATHARE code that allows this two-scale description. CATHARE is the French reference thermalhydraulic system safety code [START_REF] Barré | The CATHARE code strategy and assessment[END_REF] [9] originally devoted to the study of water-cooled reactor and extended to other designs including SFR [START_REF] Tenchine | Status of CATHARE code for sodium cooled fast reactors[END_REF]. It is used to model the compressible two-phase flow transients (normal-operation or accidental transients from any kind of failure or size and location of breaks) using a six-equation model (mass, momentum and energy) on a staggered space discretization. As a whole, the reactor system elements as pipes, primary pumps, intermediate heat exchangers and secondary circuits are generally modelled with 0D and 1D elements. However, it is possible to model components (core, steam generators …) with 3D elements. It is the case in this section where we use a 3D element to model the M-TT device. In particular, this approach is motivated by the fact that it is not possible to fully describe the presence of one-phase counter-current flows with 0D or 1D elements.

The 3D mesh is constituted by 5x3x21 cells (radial x azimuthal x z-axis) with a cylindrical geometry; cf. Fig. 11 (left). The tube lower part (nozzle; Ø = 9 cm) and the upper part (Ø = 14 cm) are 0.85 m and 3.65 m in length, respectively. The eventual variations in volume are set using a porosity field. The porosity is null in the peripheral radial meshes of the nozzle part because the diameter is lower than in the upper part: 9 cm versus 14 cm, cf. Fig. 11 (center). Three injectors, modelled by 1D elements, cf. Fig. 11 (right), are connected to the M-TT 3D mesh. The equivalent diameter of the three injectors is 2 cm. Boundary conditions are prescribed at the injectors inlets (imposed mass flow or pressure) and at the top and bottom of the M-TT inlets (imposed pressures).

In an upscaling-like process based on the previous CFD results, we define global quantities to be conserved at the component scale, under a given reduced pressure difference between the hot and cold pools:

 the bypass flowrate that would exist in the M-TT without injector,  the injected flowrate at equilibrium, meaning that there is no flowrate between the tube and the hot pool (negligible value in practise).

The singular head loss values in the 3D element and in the injectors are chosen in order to respect these CFD-prescribed global values. These head losses have been regularly applied all along the tube height. Moreover, the M-TT design envisaged for our typical SFR usually included the presence of an upper neutron shielding in the upper part of the tube. This has been taken into account by the use of a higher head loss value. Following this upscaling-like process, we approximatively recover the CFD value for the injected flowrate at equilibrium ( ~ 5 kg/s and ~ 0 kg/s) under the reduced pressure difference of 0.16 bar, cf. Fig. 12.

The rest of this section addresses simulations of the M-TT at the component scale in order to investigate the model answer to variations of the injector liquid flowrate and to the consequences of a two-phase injection (gas retention issue), as well as the cavitation risk.

Porosity of cells

Risk of gas retention in the M-TT

Here, we face the issue of the reactor cover non-reactive gas (argon), trained in the primary circuit and injected in the M-TT by the injectors, as well as its behaviour in the tube (trapped or released). The main risk should be the trapping of the gas in the M-TT and the neutron consequences on the neighbour assemblies (potential increase of the reactivity due to a void apparition).

In this study, a liquid sodium and argon gas mixture is considered with a mass concentration of the argon in the continuous gas phase of 0.999. We start from a steady state with an injector mass flowrate slightly greater than 5 kg/s. In this case, the flowrate is mainly directed toward the cold pool (4.25 kg/s) with a small flowrate toward the hot pool (0.75 kg/s) that is penalizing considering gas trapping. When the steady state is reached, the injector flow turns linearly in time into two-phase flow during 3 seconds. When the desired void fraction is reached, it is maintained during another 3 seconds, before suddenly stopping the gas injection. Two input void fractions α were used: one low void fraction value (α=10 -2 ) and one high void fraction value (α=0.6). These void fractions are typical of a low gas entrainment and of a bubble entrainment, respectively.

For the both cases, the gas quickly flows out the M-TT in typically one or two minutes. For the low gas entrainment case, the gas leaves the tube by the top and the bottom exits. While, in the bubble entrainment case, the gas mainly flows by the top exit, cf. Fig. 13. After this time delay, the M-TT recovers its initial inventory in liquid, cf. Fig. 14. Then, the issue of gas retention in the M-TT seems excluded.

Risk of cavitation in the injector nozzle

In addition, this study gives an insight in the cavitation risk in the injector nozzle. The velocity increase at the nozzle induces a pressure decrease. even for the highest velocities (here, about 25 m/s), the pressure decrease does not lead to pressure levels lower than the saturation pressure at the temperature of the liquid sodium (about 10 5 Pa for the computed pressure, to compare to the saturation pressure of 50 Pa at 420°C).

Obviously, this result, depending on the nozzle geometry, would be to consolidate in the future. Now, disposing of a M-TT component model, we face the integration of this one into a full system-scale model in the following of this paper.

System scale: system-code numerical approach

The objective of this part is to observe the impact of the M-TT implementation in the core of a typical SFR on the main thermal-hydraulic parameters, in normal operations and during an example of transient situation, using the same system-code than above.

Integration of the M-TT in the reactor modelling

The position of the M-TT in the reactor core is illustrated on Fig. 1. The reference thermal-hydraulic input deck of such a reactor is built with the CATHARE code, and represents the primary system with boundary conditions for the secondary circuit at the IHX. It is composed of 0D and 1D elements of the CATHARE code. Fig. 15 presents this CATHARE data desk, focusing on the central region of the vessel. The core sub-assemblies are grouped in derivations 1 : the inner derivations (fuel sub-assemblies, control rods, inter-assembly bypass …) are connected to a core downstream 0D element representing the central part of the hot pool entry area. The further derivations (in-vessel fuel storage and debugging, steel reflectors …) are connected to a 0D element representing the peripheral part of the hot pool entry area.

The M-TT model, composed of a 3D element as described in Section 5, has been integrated in the reactor input deck, as illustrated on Fig. 15. Compared to the previous section, the nozzle length is extended in order to reach the cold pool: the tube nozzle is 1.7 m height and the body is 3.65 m. The difference of total pressure between the two extremities of the tube is equal to 0.32 bar, corresponding to a reduced pressure (with gravity terms) of 0.135 bar. The head losses values have been calibrated similarly to the Section 5, with respect to the new total length of the tube. There are 3 injectors per tube, which aspire cold sodium from the diagrid. The upper extremity of the tube communicates with the hot pool, whereas the lower extremity communicates with the cold pool. We associate a weight of 21 to the 3D element, in order to represent the 21 transfer tubes supposed to be present in the reactor core.

Fig. 15 M-TT connections with the volumes of the SFR input deck

The M-TT is at equilibrium (see Section 3.1) at nominal power, meaning that there is no flowrate going from the tube to the hot pool (negligible value in practise).

Steady-state in normal operations

1 A derivation corresponds for CATHARE to a 1D element gathering several sub-assemblies, which have identical geometry, materials and neutronic properties.

Primary pump

As previously explained, we want to investigate the thermal-hydraulic behaviour of the primary system including the M-TT in normal operations. In this part, a computation simulating the SFR primary system with 21 M-TT is compared with a reference computation corresponding to the same system without M-TT. This is done for three steady-state power conditions: 100%, 80% and 60% of the Nominal Power (NP) cf. Table 3.

The CPU time corresponding to steady state computation for the whole model including the 21 M-TT is about 2.5 days on a standard desktop station (24 cores, 2 GHz and 32 Gb of RAM).

The primary flowrate going into the IHX is the sum of the flowrates going out of the top part of the core (red numbers in Table 3). This downstream core flow is divided in two parts (depending on the M-TT presence):

(14)
with .

The modelling of the 21 M-TT slightly reduces the flowrate through the core. Indeed, at full power nominal conditions, the 21 M-TT suck a small part of the primary flowrate entering in the diagrid ( ≈ 0.96% of the primary pumps flowrate , see green cells related to M-TT in Table 3), which does not go anymore in the core sub-assemblies. This flowrate reduction is balanced over all derivations.

The main part of the M-TT injector flowrate is discharged in the cold pool ( 0.93% of ) and a small amount is discharged in the hot pool ( 0.03% of ). Consequently, the flow feeding the primary IHX is slightly reduced (-0.93% of ). The tubes are close to equilibrium (cf. Section 3.1).

Table 3 Main thermal-hydraulic parameters for 100%, 80% and 60% NP steady state without / with M-TT These insights at full power nominal conditions are also true for partial-power conditions: the equilibrium state of the transfer tubes is preserved, as shown on Table 3. The main tendencies related to the power reduction are:

-The flowrate ratio sucked by the transfer tubes ( 0.96% of ) is independent of the power; -The flowrate fraction of injected fluid going to the cold pool ( ) slightly increases when the power decreases.

Finally, as a whole, the main effect of the integration of transfer tubes with injectors in a SFR core at steady state (100%, 80% and 60% of NP) is the reduction of flowrate feeding the core sub-assemblies. This lack of flowrate for the core and IHX (primary side) does not exceed 1% of the reference core feeding flowrate, which is negligible.

On one side, the tube functioning is therefore not affected by the steady state power level and remains at equilibrium, meaning that nearly no sodium is rejected into the hot pool by the M-TT. On the other side, no important reactor thermal-hydraulic perturbation is induced by adding transfer tubes in the core whatever the power is.

A loss of flow transient

The correct functioning at equilibrium state of the transfer tubes has been studied for fixed in time boundary conditions in the previous Section. At present, the objective of this part is essentially devoted to the characterization of M-TT behaviour during a transient, with time dependent boundary conditions at both ends of the tube.

The studied transient is a Loss Of Flow (LOF): the primary pumps stop unintentionally and the flowrate in the primary circuit decreases until the establishment of a natural convection loop. The initial state corresponds to nominal conditions at 100% NP. We suppose that the IHX still operate with a nominal secondary flowrate, fostering the core heat removal. Consequently, the DHR exchanger is not activated.

After a quick presentation of the main thermal-hydraulic features, we will stress on the differences of the transient simulations with and without M-TT.

Analysis of the main thermal-hydraulic parameters of the reactor during the transient

The simulation time lasts for 305 seconds, including 5 seconds of steady state at the beginning. Firstly, the pump rotation speed rapidly decreases jointly with the diagrid inflow (cf. Fig. 16), as well as the pressure in the diagrid (cf. Fig. 18). The primary flowrate decreases from a nominal value of 8500 kg/s to a stabilized value of 451 kg/s. It represents the natural convection flowrate.

As the cooling primary flowrate decreases, the core outlet sodium temperature increases from 550°C (nominal value) to 679°C in 20 sec. (cf. Fig. 17). It causes a core power drop by Doppler effect, which finally results in the core outlet temperature decrease once the control rods fall down (~ 19 sec.). After about 100 seconds, the reactor power and outlet core temperature continue to regularly decrease thanks to the IHX heat removal. ), the momentum injected by the injectors is no more sufficient to counter-balance the pressure difference between the two pools. At that moment, a reverse flow takes place at the top of the tubes: the sodium flows down the M-TT. Shortly after, the cold pool pressure increases until the cancellation of the reduced overpressure between the hot and cold pools that exists at steady state. It induces in the tubes  at the upper extremity, another flow direction change around 18 seconds: the sodium again goes out of the M-TT into the hot pool with a stronger flowrate than the residual one at steady state,  at the lower extremity, a reversal of the initial downward flow after 35 seconds.

Finally after 170 sec., a new stabilized state is established: the 21 M-TT are fed with sodium by the cold pool (23.6 kg/s) and discharge the flow mainly to the hot pool (21.8 kg/s) and in a small amount to the diagrid (1.8 kg/s), through the injectors.

Therefore, during this particular LOF transient computed with CATHARE, the 21 M-TT of the core behave on the long term (after 200 sec. of transient) like a core bypass and transfer the sodium from the cold to the hot pool. The bypass flowrate through the 21 M-TT represents less than 6% of the natural circulation loop total flowrate entering through the core admission ( 450 kg/s). It can be noted that this bypass has a very low impact on the reactor transient.

Conclusion and prospects

In the context of the integrated Sodium-cooled Fast Reactor, we have presented an innovative version of the safety complementary disposal M-TT to prevent or mitigate the consequences of a hypothetical severe accident. It provides a passage to the core catcher for a hypothetical corium coming from molten fuel sub-assemblies (mitigation). But, in addition, it induces an improvement of the decay heat removal capacity by natural convection between the core and active hot-pool DHR in case of loss of primary flow and of unavailable IHX (prevention). This disposal is set by ad-hoc empty transfer tubes, in place of some fuel sub-assemblies present in the reactor core, using the innovative passive device PHYLOCK, based on the concept of hydraulic diode. One or more injectors fed by the primary pumps, ensure the tightness between the hot and cold pools in normal operations. But, in case of loss of the primary pumps, it provides a direct flow path between the hot pool, equipped with DHR exchangers, and the cold one leading to an easier passage of cold sodium towards the cold pool.

This paper has focused on the assessment of the lock function and of the slight impact induced by our disposal on the thermal-hydraulic behaviour of the reactor during normal operations. Multi-scale investigations were done through the local scale to the system scale. At the local scale, we have developed an analytical analysis confirmed by CFD numerical simulations. A 3D model, developed at the component scale with a thermal-hydraulic system code and based on the up-scaling of the CDF results, has allowed us to investigate the eventual gas retention and the cavitation issues in the M-TT. Finally, this component model has been integrated in a full reactor model simulation at the system scale. Thanks to these computations, we claim that the lock function is kept during the normal-operation steady states at various power regimes (here, 100%, 80% and 60% of the nominal power). The residual leak flowrate represents no more than 1% of the primary pump flow. In addition, we conclude that no important modification of the reactor thermal-hydraulic is brought by adding transfer tubes in the core. It is also true for a typical full-power loss of forced flow transient leading to a natural convection cooling. In this case, the transfer tubes act as extra bypasses leading to lower the fraction of the natural-convection flow (about -6%) passing through the core with only slight modifications of the core temperature.

Moreover, exploratory 3D-component computations show no gas retention in the device: bubbles from an argon-polluted sodium flowing through the M-TT are quickly ejected into cold and hot pools in a few minutes, whatever the void fraction is (here, 10 -2 or 0.6). Moreover, the saturation pressure for the local sodium temperature, that is a necessary condition for cavitation issues, is never reached at the injector nozzle.

As perspectives, we envisage on one hand, to consolidate these results by other studies concerning normal operations and design basis accident transients. Also, we plan to study the severe-accident prevention capacity of the M-TT through natural-convection simulations including a DHR exchanger model at the system scale coupled with a CFD hot pool model at the local scale. On the other hand, experimental assessment of the M-TT thermal-hydraulics and its sensibility to reactor parameters should be carried out to validate the present numerical modelling.

Glossary

Abbreviation/variable Definition 0D, 1D, 3D Geometry in zero dimension, one dimension, three dimensions

( 1 )

 1 : total height of the tube (m) : upper pressure of the tube (Pa) : lower pressure of the tube (Pa) : sodium density (kg/m 3 )

:

  injector velocity (m/s) : section of the injector nozzle (m 2 ) : injector flowrate (kg/s) : downwards sodium velocity (m/s) : section of the tube (m 2 )

( 6 )

 6 : length of the M-TT above the flow reversal region (m) : length of the M-TT below the flow reversal region (m) with : upper pressure of the tube (Pa) : lower pressure of the tube (Pa)

Fig. 7

 7 Fig. 7 Reduced pressure distribution with a zoom on the nozzle

Fig. 8

 8 Fig. 8 Temperature distribution on the total height of the M-TT

Fig. 11 5 . 1

 1151 Fig. 11 Component scale: 3D meshing (left)zoom on the porosity field at the junction between the nozzle and upper part (center) -1D meshing of the 3 injectors (right)

Fig. 13

 13 Fig. 13 Gas injection in the M-TT: gas mass flowrate at the inlet and outletslow void fraction case (left)high void fraction case (right)

  Without M-TT With 21 M-TT Without M-TT With 21 M-TT Without M-TT With 21 M-TT Q inj (%Q prim )

Fig. 16

 16 Fig. 16 Flowrates evolution in the diagrid and pump rotation speed evolution during a LOF transient without/with M-TT modelling

Fig. 17

 17 Fig. 17 Power and inlet/outlet core temperature evolution during a LOF transient without/with M-TT modelling

  . The pressure, temperatures and reactor power are not impacted by the M-TT introduction in the CATHARE data desk.Let us discuss the M-TT behaviour during the LOF transient, cf. Fig.18. Starting from the steady state, where the transfer tubes suck 81.6 kg/s of sodium from the diagrid volume thanks to the injectors, mainly transferred to the cold pool (79.3 kg/s), the sodium pumped by the injectors decreases towards zero with the primary flowrate and the diagrid pressure.

Fig. 18

 18 Fig. 18 Flowrate and pressure evolution at the both ends of the 21 M-TT during a LOF transient

ε

  Turbulent kinetic energy dissipation rate (J/kg/s) Injector diameter (m) Average sodium density (kg/m 3 ) g Gravity acceleration (m/s 2 ) Height of the flow reversal region (m) Height below the flow reversal region (m) Height above the flow reversal region (m) Total height of the transfer tube (m) Difference of free levels between the hot and the cold pools (m) k Turbulent kinetic energy (J/kg) Pressure at the injector nozzle (Pa) Pressure at the lower part of transfer tube (Pa) Pressure at the upper part of transfer tube (Pa) Pressure drop in the transfer tube (Pa) Reduced pressure drop in the transfer tube (Pa) Core flowrate (kg/s) Flowrate in the peripheral part of the core (kg/s)

Table 1 Comparison between analytical and CFD equilibrium flowrates for different injector diameters and reduced pressure drops Flowrate 0.16 bar 0.36 bar

 1

	 injector	Analytical	CFD	Analytical	CFD
	1 cm	2.6 kg/s	2.5 kg/s	3.91 kg/s	3.8 kg/s
	2 cm	5.2 kg/s	5.2 kg/s	7.8 kg/s	7.6 kg/s
	4 cm	10 kg/s	9.5 kg/s	15 kg/s	14 kg/s

Table 2 Sensitivity to the injector flowrate for different conditions

 2

			0.16 bar		0.36 bar
	 injector	Analytical	CFD	Analytical	CFD
	1cm	32	24	33.5	35
	2cm	19	11.5	19	14
	4cm	4	3	4.2	4

Flowrate in the central part of the core (kg/s) Flowrate delivered by the primary pumps (kg/s) Primary flowrate going into the IHX (kg/s) Injector flowrate (kg/s) Injector flowrate at equilibrium (kg/s) Flowrate at the bottom outlet of the transfer tube (kg/s) Residual flowrate at the top outlet of the transfer (kg/s) Injection nozzle section (m 2 ) Cross-section of the transfer tube (m 2 ) Injection velocity (m/s) Sodium velocity in the lower part of the transfer tube (m/s)