Roux Le
email: leroux@nbcm.cnrs-gif.fr

Amar Nicolas

Muriel

Alexandre Moreau

Philippe Fossier

L E Roux

Involvement of NR2A or NR2B-containing NMDA receptors in the potentiation of cortical layer 5 pyramidal neurons inputs depends on the developmental stage Running title: NR2A or NR2B-containing NMDA receptors in LTP

Keywords: NMDA receptors, LTP, NR2A, NR2B, critical period, cortical networks

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Involvement of NR2A-or NR2B-containing N-methyl-D-aspartate receptors in the potentiation of cortical layer 5 pyramidal neurone inputs depends on the developmental stage.

Nicolas Le Roux, Muriel Amar, Alexandre Moreau, Philippe Fossier

To cite this version:

Nicolas Le Roux, Muriel Amar, Alexandre Moreau, Philippe Fossier. Involvement of NR2A-or NR2Bcontaining N-methyl-D-aspartate receptors in the potentiation of cortical layer 5 pyramidal neurone inputs depends on the developmental stage.. European Journal of Neuroscience, 2007Neuroscience, , 26 (2), pp.289-301. 10.1111Neuroscience, /j.1460Neuroscience, -9568.2007.05671..05671.x. hal-00169854

Introduction

N-methyl D-aspartate receptors (NMDARs) are among the most important receptors in the central nervous system. Largely expressed in the brain, they play a major role in glutamatergic transmission due to their unique functional properties, which include a high Ca 2+ permeability, slow activation and deactivation kinetics, and a voltage-sensitive block by external Mg 2+ [START_REF] Jahr | Voltage dependence of NMDA-activated macroscopic conductances predicted by single-channel kinetics[END_REF][START_REF] Lester | Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents[END_REF]. As a result of these properties, NMDARs play key roles in numerous physiological and pathological brain processes such as synaptic plasticity [START_REF] Choi | The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death[END_REF], schizophrenia [START_REF] Coyle | Ionotropic glutamate receptors as therapeutic targets in schizophrenia[END_REF][START_REF] Coyle | Converging evidence of NMDA receptor hypofunction in the pathophysiology of schizophrenia[END_REF] or epilepsy [START_REF] Dingledine | Excitatory amino acid receptors in epilepsy[END_REF].

The role of NMDARs in the two major forms of synaptic plasticity that contribute to phenomena of learning and memory, long-term potentiation (LTP) and long-term depression (LTD) has been largely determined. Modest elevations of intracellular calcium due to NMDARs activation are thought to trigger depression, whereas large increases of intracellular calcium trigger potentiation [START_REF] Bear | Synaptic plasticity: LTP and LTD[END_REF][START_REF] Yang | Selective induction of LTP and LTD by postsynaptic [Ca2+]i elevation[END_REF].

NMDARs are heterotetramers that consist of the NMDA receptor 1 (NR1) subunit which binds glycine or D-serine and one or more of the NR2 subunits: NR2A-NR2D which bind glutamate [START_REF] Forrest | Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death[END_REF][START_REF] Hollmann | Cloned glutamate receptors[END_REF]. While, NR3A [START_REF] Ciabarra | Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family[END_REF][START_REF] Sucher | Developmental and regional expression pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain[END_REF] and NR3B [START_REF] Chatterton | Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits[END_REF][START_REF] Matsuda | Cloning and characterization of a novel NMDA receptor subunit NR3B: a dominant subunit that reduces calcium permeability[END_REF] have also been recently identified, it is reported that NR2A and NR2B subunits dominate in the forebrain [START_REF] Monyer | Developmental and regional expression in the rat brain and functional properties of four NMDA receptors[END_REF].

The functional properties of the receptor strongly depend on the type of NR2 subunit incorporated [START_REF] Cull-Candy | NMDA receptor subunits: diversity, development and disease[END_REF][START_REF] Cull-Candy | Role of distinct NMDA receptor subtypes at central synapses[END_REF] which changes with age. In the cortex, the NR2B subunit is predominantly expressed early in development [START_REF] Babb | Pre-and postnatal expressions of NMDA receptors 1 and 2B subunit proteins in the normal rat cortex[END_REF] whereas expression of the NR2A subunit increases later. According to the NR2 composition, NMDARs will have different roles in synaptic plasticity, and it has been proposed that potentiation and depression are differentially mediated by NR2A and NR2B, respectively (Liu et al., 2004a;[START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF]. This differential effect could be related to the specific localization of NMDARs subtypes; the NR1/NR2A complex, characterized by rapid offset kinetics, would be present at the synapse, while the NR1/NR2B complex, characterized by slow kinetics, would mainly lie in extrasynaptic sites [START_REF] Rumbaugh | Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons[END_REF][START_REF] Tovar | The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro[END_REF]. However, recent studies suggest that NR2A and NR2B each contribute equally to the induction of potentiation and depression [START_REF] Toyoda | Roles of NMDA receptor NR2A and NR2B subtypes for long-term depression in the anterior cingulate cortex[END_REF][START_REF] Weitlauf | Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation[END_REF][START_REF] Zhao | Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory[END_REF].

In the rat visual cortex it has been shown that the NR1 subunit is co-expressed with both the NR2A and NR2B subunits [START_REF] Hawkins | Biochemical evidence for the coassociation of three N-methyl-D-aspartate (NMDA) R2 subunits in recombinant NMDA receptors[END_REF][START_REF] Tongiorgi | Dark-rearing decreases NR2A N-methyl-D-aspartate receptor subunit in all visual cortical layers[END_REF][START_REF] Babb | Pre-and postnatal expressions of NMDA receptors 1 and 2B subunit proteins in the normal rat cortex[END_REF].

The role of NMDARs in the induction of potentiation in the visual cortex is well established [START_REF] Bear | Neocortical long-term potentiation[END_REF][START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF] but which NR2 subunit(s) might be involved in this plasticity process remains controversial. We have previously shown that application of a high frequency stimulation (HFS) protocol in layer 2/3 does not change the excitatory/inhibitory (E/I) ratio due to parallel potentiation of excitatory and inhibitory inputs received by pyramidal neurons [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]. Here, we investigate the role of NMDARs in the induction of the potentiation of excitatory and inhibitory inputs of layer 5 pyramidal neurons after application of an HFS protocol in layer 2/3 in rat visual cortex (between P18 and P23 or between P29 and P32). We find that for young rats (between P18 and P32) the potentiation of excitatory and inhibitory inputs by an HFS protocol mainly depends on the activation of NR2B-containing receptors, whereas for P29-P32 old rats this plasticity becomes dependent on NR2A-containing receptors. We show that the prevention of potentiating effects on excitation and inhibition by blockade of NMDARs did not change the E/I ratio. Our results bring direct evidence of a functional shift in subunit composition of NMDARs during the critical period (P12-P36) which would explain the relative contribution of both NR2B-and NR2A-containing NMDARs in synaptic plasticity processes.

Materials and Methods

Slice preparation

Parasagittal slices containing primary visual cortex were obtained from P18-P23 or P29-P32 day-old Wistar rats. Briefly, in accordance with the guidelines of the American Neuroscience Association, a rat was decapitated, its brain quickly removed and placed in chilled (5°C) artificial cerebrospinal extracellular solution. Slices of 250 µm thickness were made using a vibratome from the primary visual cortex and then incubated for at least 1 hr at 36°C in a solution containing (in mM) : 126 NaCl, 26 NaHCO 3 , 10 Glucose, 2 CaCl 2 , 1.5 KCl, 1.5

MgSO 4 and 1.25 KH 2 PO 4 (pH 7.5,. This solution was bubbled continuously with a mixture of 95 % O 2 and 5 % CO 2 .

Electrophysiological recordings and cell identification

Current-clamp and voltage-clamp recordings were performed using an Axopatch 1D (Axon Instruments, USA); filtered by a low-pass Bessel filter with a cut-off frequency set at 2 kHz, and digitally sampled at 4 kHz. Layer 5 pyramidal neurons, identified from the shape of their soma and primary dendrites and from their current-induced excitability pattern, were studied The stimulating electrodes were positioned in layer 2-3. Electrical stimulations (1-10 μA, 0.2 ms duration) were delivered in these layers using 1 MΩ impedance bipolar tungsten electrodes (TST33A10KT, WPI). The intensity of the stimulation was adjusted in currentclamp conditions to induce a subthreshold postsynaptic response due to coactivation of excitatory and inhibitory circuits. Under voltage-clamp conditions, the frequency of stimulation was 0.05 Hz and five to eight trials were repeated for a given holding potential.

A control recording was made after 15 min of patch-clamp equilibration at 0.05 Hz, and then drugs were continuously superfused during 20 min, a "drug recording" was made in the same conditions. We next applied an HFS (high frequency of stimulation) protocol in order to induce long term modifications of synaptic strength in the recruited circuits. The HFS protocol was elicited with theta burst stimulation known to induce long term potentiation (LTP) at the synaptic level. It consists of 3 trains of 13 bursts applied at a frequency of 5 Hz, each burst containing 4 pulses at 100 Hz [START_REF] Abraham | Induction and reversal of long-term potentiation by repeated high-frequency stimulation in rat hippocampal slices[END_REF]

Synaptic response analysis

Data were analyzed off-line with specialized software (Acquis1 TM and Elphy TM : written by Gérard Sadoc, Biologic UNIC-CNRS, France). The method is based on the continuous measurement of conductance dynamics during stimulus-evoked synaptic responses, as primarily described in vivo on cat cortex [START_REF] Borg-Graham | Visual input evokes transient and strong shunting inhibition in visual cortical neurons[END_REF][START_REF] Monier | Orientation and direction selectivity of synaptic inputs in visual cortical neurons: a diversity of combinations produces spike tuning[END_REF] and recently validated on rat visual cortex [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]. This method has received further validation on other experimental models [START_REF] Shu | Turning on and off recurrent balanced cortical activity[END_REF][START_REF] Wehr | Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex[END_REF][START_REF] Higley | Balanced excitation and inhibition determine spike timing during frequency adaptation[END_REF][START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]. Evoked synaptic currents were measured and averaged at a given holding potential. In I-V curves for each delay (t), the value of the holding potential (Vh) was corrected (Vhc) from the ohmic drop due to the leakage current through Rs, by the equation: Vhc(t) = Vh(t) -I(t) x Rs. An average estimate of the input conductance waveform of the cell was calculated from the best linear fit (mean least square criterion) of the I-V curve for each delay (t) following the stimulation onset. Only cells showing a Pearson correlation coefficient for the I-V linear regression higher than 0.95 between -90 and -40 mV were considered in order to calculate the conductance change of the recorded pyramidal neuron from the slope of the linear regression.

The evoked synaptic conductance term (gT(t)) was derived from the input conductance by subtracting the resting conductance (g rest ) estimated 90 ms before the electrical stimulation.

The apparent reversal potential of the synaptic current at the soma (Esyn(t)) was taken as the voltage abscissa of the intersection point between the I-V curve obtained at a given time (t) and that determined at rest. Assuming that the evoked somatic conductance change reflects the composite synaptic input reaching the soma, Esyn(t) characterizes the stimulation-locked dynamics of the balance between excitation and inhibition.

Decomposition of the synaptic conductance

To the reversal potentials for excitation and inhibition. Values of these reversal potentials were equal to 0 mV for excitation (Eexc) and to -80 mV for inhibition (Einh). The value of -80 mV used in the decomposition method is the reversal potential of GABA A (and not an intermediate value between GABA A and GABA B ) because in the presence of QX314 (which blocks K + efflux), no variation of the synaptic response was observed.

We showed that the I-V curve in the presence of excitatory synaptic transmission blockers (CNQX, D-AP5) is linear between -80 to +10 mV with a reversal potential equal to -80 mV [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]supplementary material). In the presence of bicuculline, which blocks the inhibitory inputs on the layer 5 pyramidal neuron, the I/V curve for excitation is linear between -80 to +10 mV with a reversal potential equal to 0 mV (data not shown) as already

shown by other studies [START_REF] Wehr | Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex[END_REF][START_REF] Higley | Balanced excitation and inhibition determine spike timing during frequency adaptation[END_REF]. Under our experimental conditions of stimulation of cortical layers leading to subthreshold postsynaptic responses, Esyn(t) which was extrapolated from I-V curves took any intermediate values between -80 mV and -40 mV [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]supplementary material) i.e. within the limits of our voltage excursion (-90 to -40 mV) corresponding to the linearity of I-V curves and between the respective values of Einh and Eexc in such a way that the mathematical conditions of the oversimplification used to calculate gI(t) and gE(t) were fulfilled.

Like all somatic recordings, our recordings cannot make rigorous estimates of synaptic events in the distal dendrites and the conductance estimates are ratios of the overall excitatory and inhibitory drive contained in the local stimulated network [START_REF] Haider | Neocortical network activity in vivo is generated through a dynamic balance of excitation and inhibition[END_REF]. However, our measurements give relative changes in conductance magnitude which reflect the cumulative contributions of excitation and inhibition arriving at proximal portions of the neuron. These relative conductance changes at the somatic level is precisely the result of synaptic dendritic integration that we wish to record at the somatic level because it determines the output signal from the neuron [START_REF] Wehr | Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex[END_REF][START_REF] Higley | Balanced excitation and inhibition determine spike timing during frequency adaptation[END_REF].

To quantify the synaptic conductance changes we calculate the integral (int) over a time window of 200 ms. The contribution of each component was expressed by the ratio of its integral value (intgE or intgI) to that of total conductance change (intgT).

Statistical analysis

Data are expressed as the mean ± the standard deviation of the mean (SD) of n cells. Paired samples for IntgT, IntgE, IntgI, %E or %I between the control condition (before HFS) and a given time after HFS (15, 30, 45 or 60 min) were analysed using the parametrical t-test. Data were considered statistically significant for p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***). Application of 50 µM D-AP5, a specific antagonist of NMDA receptors [START_REF] Yoshimura | Two forms of synaptic plasticity with distinct dependence on age, experience, and NMDA receptor subtype in rat visual cortex[END_REF][START_REF] Huemmeke | Lesion-induced enhancement of LTP in rat visual cortex is mediated by NMDA receptors containing the NR2B subunit[END_REF], for 20 min did not induce significant differences in the global, excitatory and inhibitory conductance changes (Fig. 1A2). These results clearly show that the excitatory and inhibitory components recorded in a layer 5 pyramidal neuron in response to a low frequency of stimulation (0.05 Hz) of layer 2/3 does not depend on activation of NMDA receptors but corresponds to the activation of AMPA and GABA A receptors mainly located on the apical dendrite of the layer 5 pyramidal neuron.

Results

Involvement of NMDARs in the potentiation of excitatory and inhibitory inputs on cortical layer 5 pyramidal neurons induced by HFS protocol application in layer 2/3

In the brain, information is encoded as action potential trains of higher frequency than that used in our protocol (0.05 Hz). We applied an HFS protocol with a higher frequency (see methods), usually used to induce LTP in numerous preparations. Application of this HFS protocol in layer 2/3 induced a long term potentiation of both excitatory and inhibitory inputs on layer 5 pyramidal neurons. Figure 1B shows representative current recordings in a voltage clamp pyramidal neuron, under control condition (Fig. 1B1) and 15 minutes after application of an HFS protocol (Fig. 1B2). HFS application elicits enhanced current responses and In order to examine the involvement of NMDARs activation in these potentiating effects of HFS on excitatory and inhibitory inputs, HFS protocol was applied in the presence of 50 µM D-AP5. No significant increase of current responses and conductance changes was measured (Fig. 1C1 and1C2). No variation of the total conductance change and of inhibitory or excitatory conductance changes was observed through the statistical analysis of the total neuronal population (n = 15; Fig. 1E). Under this condition, the HFS protocol failed to induce potentiation of excitatory and inhibitory inputs. No significant changes in excitation (intgE)

and inhibition (intgI) conductance integrals expressed as percent of intgT was measured (Fig. 1E) and the E/I ratio did not change. These results indicate that NMDARs are recruited by high frequency of stimulation applied in layer 2/3.

Involvement of NR2A and/or NR2B-containing receptors in the potentiation of excitatory and inhibitory inputs of cortical layer 5 pyramidal neurons induced by HFS protocol application in layer 2/3

The involvement of NR2A and/or NR2B-containing receptors in synaptic plasticity is presently strongly debated (Liu et al., 2004b;[START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF][START_REF] Berberich | Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation[END_REF][START_REF] Weitlauf | Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation[END_REF]. To further characterise whether NMDA currents in layer 2/3 pyramidal neurons were due to the activation of NR2A-or NR2B-containing receptors, we then used antagonists for each of these receptors.

Role of NR2B-containing receptors in P18-P23 old rats

Ro 25-6981 [START_REF] Yang | Tonic facilitation of glutamate release by presynaptic NR2B-containing NMDA receptors is increased in the entorhinal cortex of chronically epileptic rats[END_REF] or CP101,606 (Traxoprodil, [START_REF] Chenard | 1S,2S)-1-(4hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol: a potent new neuroprotectant which blocks N-methyl-D-aspartate responses[END_REF][START_REF] Menniti | CP-101,606, a potent neuroprotectant selective for forebrain neurons[END_REF] were used to specifically block NR2B-containing receptors. Representative recordings in the presence of 1 μM of Ro 25-6981 before and after application of HFS protocol showed that the HFS effect was blocked by the NR2B antagonist (Fig. 2A). The statistical analysis of the global population (n = 12; Fig. 2B) confirms that no significant variations of the global, excitatory and inhibitory conductance changes were observed after application of HFS protocol in layer 2/3 in the presence of Ro 25-6981. Consequently, the E/I ratio was unchanged (Fig. 2C). Identical results were obtained after application of HFS protocol in the presence of CP101,606 (n = 18; Fig. 2D,2E,2F). These results reproduced those observed in the presence of D-AP5 and provide evidence for an involvement of NR2Bcontaining receptors in the HFS-induced potentiation of both excitatory and inhibitory inputs on layer 5 pyramidal neurons.

Role of NR2A-containing receptors

NVP-AAM077 [START_REF] Izumi | Zinc modulates bidirectional hippocampal plasticity by effects on NMDA receptors[END_REF] or Zn 2+ (Paoletti & Neyton, 2006) ZnCl 2 was also used to block selectively NR2A-containing receptors because low concentrations of Zn 2+ block NR2A receptors more than NR2B receptors [START_REF] Amar | High-affinity Zn block in recombinant Nmethyl-D-aspartate receptors with cysteine substitutions at the Q/R/N site[END_REF][START_REF] Kohr | NMDA receptor function: subunit composition versus spatial distribution[END_REF]Neyton & Paoletti, 2006). As observed for NVP-AAM077, similar results were obtained in the presence of 200 nM of ZnCl 2 , with excitatory and inhibitory conductance changes all increased (Fig. 3D). This result was confirmed by the statistical analysis of the global population (n = 13; Fig. 3E), which showed that HFS application in the presence of Zn 2+ leads to a mean increase by 26 %, 18 % and 30 % for the total, excitatory and inhibitory conductance changes, respectively. In this case, a slight but not significant (p > 0.05) decrease of the E/I ratio was observed (Fig. 3F). In the presence of NR2A-containing receptors blockers, the potentiating effects of the HFS protocol on layer 5 pyramidal neurons still occurred but were reduced.

Involvement of extrasynaptic NR2B-containing receptors in potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons

NR2B receptors are known to be present in both synaptic and extrasynaptic domains at glutamatergic synapses [START_REF] Cull-Candy | NMDA receptor subunits: diversity, development and disease[END_REF]. To check the involvement of synaptic and/or extrasynaptic NR2B-containing receptors in HFS induced-potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons, synaptic NMDARs were blocked according to the protocol described by [START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF] 4B for the statistical analysis of the global population, n = 14) and the E/I ratio slightly decreases (Fig. 4C). However, the increased percentage of intgE was lower than that obtained after a direct HFS protocol. This discrepancy might be due to the prolonged depressive effects (beyond 30 min) of LFS protocols (mainly on intgE) that we have shown [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]).

Nevertheless, it was possible to obtain a potentiating effect with an HFS protocol, and we could thus test the HFS protocol in the presence of MK801. In the presence of MK-801, no potentiation of excitatory and inhibitory inputs was obtained, but a slight decrease of IntgT, IntgE and IntgI was observed (Fig. 4D for representative recordings and Fig. 4E for statistical analysis of the global population, n = 13) and the E/I ratio remains stable (Fig. 4F), indicating that synaptic NMDARs can account for the potentiation of excitatory and inhibitory inputs of layer 5 pyramidal neurons.

Role of NR2A and/or NR2B-containing receptors in the potentiation of excitatory and inhibitory inputs in P29-P32 old rats

Some studies suggest that the activation of NR2A but not of NR2B induces long-term potentiation (Liu et al., 2004b;[START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF] while other studies proposed that both types of NMDARs can contribute to the induction of LTP [START_REF] Berberich | Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation[END_REF][START_REF] Weitlauf | Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation[END_REF][START_REF] Zhao | Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory[END_REF]. These discrepancies may be related to temporal regulation of NR2 subunits [START_REF] Monyer | Developmental and regional expression in the rat brain and functional properties of four NMDA receptors[END_REF]Liu et al., 2004b). The NR2A/NR2B ratio increases with maturation [START_REF] Yoshimura | Two forms of synaptic plasticity with distinct dependence on age, experience, and NMDA receptor subtype in rat visual cortex[END_REF]Liu et al., 2004c). Our results indicate that for rats between P18 and P23, synaptic NR2B-containing receptors are functional while NR2Acontaining receptors may not yet be fully functional. To test this hypothesis, a new series of experiments was done on P29-P32 old rats.

Role of NR2B-containing receptors in P29-P32 rats

Representative recordings in the presence of 1 μM Ro 25-6981, before and after application of HFS protocol showed an increase of the amplitude of current recordings (Fig. 5A significant change of the E/I ratio was observed (Fig. 5C).

Role of NR2A-containing receptors in P29-P32 rats

Representative recordings in the presence of 0.1 μM NVP-AAM077 before and after application of HFS protocol showed no significant variation (Fig. 5D). The statistical analysis of the global population (n = 13; Fig. 5E) confirms the block of the effects of the HFS protocol in the presence of the NR2A-containing receptors blocker for P29-P32 old rats. Consequently, the E/I ratio was unchanged (Fig. 5F).

Discussion

NMDAR activation is required for the potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons after application of an HFS protocol in layer 2/3. Our results provide evidence for a major recruitment of synaptic NR2B-containing receptors in P18-P23 old rats, relative to the involvement of NR2A-receptors. As well as their possible differential role in physiological processes such as learning and memory [START_REF] Collingridge | Receptor trafficking and synaptic plasticity[END_REF], NR2A and NR2B-containing receptors are thought to be differentially involved in certain central disorders such as chronic pain or dementia [START_REF] Kemp | NMDA receptor pathways as drug targets[END_REF][START_REF] Chazot | The NMDA receptor NR2B subunit: a valid therapeutic target for multiple CNS pathologies[END_REF]Paoletti & Neyton, 2006). It therefore becomes important to determine the contribution and the function of distinct NMDAR subtypes. However, this physiological classification is highly dependent on the selectivity of antagonists that can be used. Ro 25-6981 (ifenprodil) and CP,101-606 (traxoprodil) which bind the N-terminal lysine/isoleucine/valine-binding protein (LIVBP)-like domain of NR2B are now considered as selective antagonists of NR2Bcontaining receptors [START_REF] Fischer | Ro 25-6981, a highly potent and selective blocker of N-methyl-Daspartate receptors containing the NR2B subunit. Characterization in vitro[END_REF][START_REF] Berberich | Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation[END_REF][START_REF] Kohr | NMDA receptor function: subunit composition versus spatial distribution[END_REF]. On the contrary, the specific blockade of NR2A-containing receptors is strongly debated [START_REF] Feng | Proton modulation of alpha 1 beta 3 delta GABAA receptor channel gating and desensitization[END_REF]Paoletti & Neyton, 2006). First, Zn 2+ binds the (LIVBP)-like domain of NR2A and displays a better selectivity for NR2A than for NR2B with a maximal selectivity of 70 % [START_REF] Amar | High-affinity Zn block in recombinant Nmethyl-D-aspartate receptors with cysteine substitutions at the Q/R/N site[END_REF][START_REF] Kohr | NMDA receptor function: subunit composition versus spatial distribution[END_REF]Neyton & Paoletti, 2006). Second, NVP-AAM077 was originally described as a highly selective antagonist of NR2A-containing receptors [START_REF] Auberson | 5-Phosphonomethylquinoxalinediones as competitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition[END_REF]Liu et al., 2004b;[START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF] but later studies on rodents indicate that this compound lacks selectivity for NR2A vs NR2B receptors [START_REF] Berberich | Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation[END_REF][START_REF] Weitlauf | Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation[END_REF]Neyton & Paoletti, 2006). Other studies reported that NVP-AAM077

presents some selectivity at concentrations lower than 1 μM [START_REF] Bartlett | Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus[END_REF][START_REF] Izumi | Zinc modulates bidirectional hippocampal plasticity by effects on NMDA receptors[END_REF] and concluded that it was not possible to find a concentration of NVP-AAM077 that fully block the NR2A receptors while sparing NR2B. According to the work by [START_REF] Bartlett | Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus[END_REF], we used 0.1 μM NVP-AAM077 to check the specific blockade of NR2A receptors.

Indeed, whatever the NR2A antagonist used, the potentiation of excitatory and inhibitory inputs of layer 5 pyramidal neurons occurred but was reduced by about 40 % in P18-P23 old rats. This result could be due to the blockade of NR2A, but also to an unspecific blockade of NR2B subunits.

Recent studies suggest that the activation of NR2A but not NR2B induces long-term potentiation (Liu et al., 2004b;[START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF] although other studies proposed that both types of NMDARs can contribute to the induction of LTP [START_REF] Berberich | Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation[END_REF][START_REF] Weitlauf | Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation[END_REF][START_REF] Zhao | Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory[END_REF]. These discrepancies could be related to the differential distribution of NMDARs subtypes. For example, an important role for extrasynaptic receptors exclusively containing the NR2B subunit [START_REF] Cull-Candy | NMDA receptor subunits: diversity, development and disease[END_REF] is reported in synaptic plasticity [START_REF] Massey | Differential roles of NR2A and NR2Bcontaining NMDA receptors in cortical long-term potentiation and long-term depression[END_REF] or in neuronal cell death [START_REF] Hardingham | Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways[END_REF][START_REF] Hardingham | 2B synaptic or extrasynaptic determines signalling from the NMDA receptor[END_REF]. However, both NR2A-or NR2B-NMDAR subtypes can be located either on synaptic or extrasynaptic sites [START_REF] Thomas | Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons[END_REF]. Here, we show that predominantly synaptic NMDARs are implicated in the observed potentiating effects.

Another possibility to explain these discrepancies is to consider changes in the spatial distribution of NMDA receptors during the developmental stages. While NR1 subunit is ubiquitously expressed in the CNS, NR2 subunit expression is temporally regulated [START_REF] Monyer | Developmental and regional expression in the rat brain and functional properties of four NMDA receptors[END_REF]Liu et al., 2004b) with the NR2A/NR2B ratio increasing during maturation [START_REF] Yoshimura | Two forms of synaptic plasticity with distinct dependence on age, experience, and NMDA receptor subtype in rat visual cortex[END_REF]Liu et al., 2004c). In the rat visual cortex, morphological studies showed that NR1 and NR2B subunits are already expressed at birth and reach their highest level of expression during the second and third postnatal weeks [START_REF] Kew | Developmental changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat cortical neurons[END_REF][START_REF] Tongiorgi | Dark-rearing decreases NR2A N-methyl-D-aspartate receptor subunit in all visual cortical layers[END_REF][START_REF] Babb | Pre-and postnatal expressions of NMDA receptors 1 and 2B subunit proteins in the normal rat cortex[END_REF]. In contrast, NR2A subunits are poorly expressed at birth but progressively increase as development proceeds [START_REF] Kew | Developmental changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat cortical neurons[END_REF] and the insertion of NR2A receptors displace NR2B receptors at extrasynaptic sites (Liu et al., 2004c). Our results

clearly show that the potentiation initially depends on NR2B-containing receptors in P18-P23 old rats, but becomes NR2A dependent for P29-P32 old rats. Thus in order to understand the particular role of NR2A or NR2B in the potentiation process, it is crucial to consider the temporal and spatial NMDARs expression. Hence, rather than a differential role for NR2A-or NR2B-containing receptors in the induction of LTP supposed by recent works [START_REF] Berberich | Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation[END_REF][START_REF] Weitlauf | Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation[END_REF], Zhao et al., 2005), we proposed that both types can be involved in the potentiation but their respective involvement depends on the developmental stage of the cortex.

We show that without under estimating the problem of selectivity of the NR2A or NR2Bcontaining receptors antagonists, it is necessary to consider the developmental stages of the animal. In addition, we cannot exclude the possibility that, for P18-P23 old rats, NR2Acontaining receptors were present but not yet fully functional (not antagonist sensitive), whereas for P29-P32 old rats specific blockers were rather efficient.

The blockade of NMDA receptors prevents the potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons to HFS in layer 2/3 resulting in an unchanged E/I ratio. These results suggest that the NMDARs activation is not required for the induction of the homeostatic control of the E/I balance but is necessary for the homeostatic plasticity expression. Only a few studies report a similar observation in which the blockade of NMDARs prevents the potentiation of inhibitory synapses (IPSCs recorded in cerebellar stellate cells [START_REF] Liu | The activation of excitatory glutamate receptors evokes a long-lasting increase in the release of GABA from cerebellar stellate cells[END_REF]). It is obvious that we cannot localize facilitated synapses in the stimulated networks by HFS protocol and we observed that the stimulation of inhibitory interneurons is mainly disynaptic, involving a synapse between a glutamatergic terminal and the interneuron [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]Supplementary materials). Thus the potentiation of inhibitory inputs following HFS protocol in layer 2/3 is likely indirect, but in any case parallel increases of the strength of excitatory and inhibitory inputs on the layer 5 pyramidal neuron were observed resulting in a close control of the E/I ratio. Indeed, normal brain function requires a delicate balance between excitation and inhibition in neuronal networks, an imbalance of excitation and inhibition underlies various neurological diseases such as epilepsy [START_REF] Cossart | Multiple facets of GABAergic neurons and synapses: multiple fates of GABA signalling in epilepsies[END_REF], Parkinson's disease [START_REF] Llinas | Thalamocortical dysrhythmia: A neurological and neuropsychiatric syndrome characterized by magnetoencephalography[END_REF] or schizophrenia [START_REF] Wassef | GABA and schizophrenia: a review of basic science and clinical studies[END_REF]. A correlation between excitatory and inhibitory neuronal responses to sensory stimuli has been found in many cortical neurons in adult mammalian sensory cortices. For example, in cat primary visual cortex, excitation and inhibition exhibit similar tuning preference and tuning width in orientation-selective neurons [START_REF] Anderson | Orientation tuning of input conductance, excitation, and inhibition in cat primary visual cortex[END_REF][START_REF] Martinez | Laminar processing of stimulus orientation in cat visual cortex[END_REF][START_REF] Monier | Orientation and direction selectivity of synaptic inputs in visual cortical neurons: a diversity of combinations produces spike tuning[END_REF][START_REF] Hirsch | Functionally distinct inhibitory neurons at the first stage of visual cortical processing[END_REF][START_REF] Marino | Invariant computations in local cortical networks with balanced excitation and inhibition[END_REF]. In addition, a proportionality of recurrent excitation and inhibition in cortical networks has been demonstrated to be associated with the persistent activity and is thought to underlie the working memory [START_REF] Shu | Turning on and off recurrent balanced cortical activity[END_REF]. So, neuronal networks adapt to environmental changes by learning and memory, in which the strength of excitatory or inhibitory synapses are changed.

We show using a protocol of stimulation classically used in long-term plasticity, that the E/I ratio can remain unchanged while the strength of excitatory and inhibitory inputs altered. The present results provide evidence for the activation of the homeostatic control of the E/I balance previously reported by Le [START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF]. This control is independent of the activation of NMDARs but appears linked to recurrent interactions between excitatory and inhibitory circuits through layer 2/3 and layer 5 pyramidal neurons. This cortical organization can underlie the HFS-induced potentiation of some excitatory and inhibitory synapses (and the imprinting of some information) and may control the main output pathway of the cortex, layer 5 pyramidal neurons, through a homeostatic mechanism avoiding hyper-or hypoexcitability.

  using the patch-clamp technique in whole-cell configuration. Somatic whole-cell recordings were performed at room temperature using borosilicate glass pipettes (of 3-5 MΩ resistance in the bath) filled with a solution containing (in mM) : 140 K-gluconate, 10 HEPES, 4 ATP, 2 MgCl 2 , 0.4 GTP, 0.5 EGTA (pH 7.3 adjusted with KOH, 270-290 mOsm). The membrane potential was corrected off-line by -10 mV to account for the junction potential. Estimation of the access resistance (Rs) is critical to quantitatively evaluate the relative change of input conductance in response to synaptic activation. After capacitance neutralisation, bridge balancing was done on-line in current-clamp conditions, which provided us with an initial estimate of Rs. This value was checked and revised as necessary off-line by fitting the voltage response to a hyperpolarizing current pulse with the sum of two exponentials. Under voltageclamp conditions, the holding potential was corrected off-line using this Rs value. Only cells with a resting membrane potential more negative than -55 mV and recordings with an access resistance lower than 25 MΩ were kept for further analysis. The firing behavior of neurons was determined in response to depolarizing current pulses ranging from -100 to 200 pA under current-clamp conditions.

Figure 1A -

 1A Figure 1A-C presents representative current recordings in a layer 5 voltage-clamped pyramidal neuron to electrical stimulation (0.05 Hz) in layer 2/3. Current responses show astrong inward current followed by an outward component (Fig.1A). Decomposition of these current responses into distinct conductance changes showed that, as already reported[START_REF] Le Roux | Homeostatic control of the excitationinhibition balance in cortical layer 5 pyramidal neurons[END_REF], inhibition (gI) represents roughly 80 % (light grey trace) and excitation (gE) 20 % (dark grey trace) of the global conductance change (black trace, Fig.1A1).

  increases conductance changes. The statistical analysis of the global population (n = 24) is presented on figure 1D and shows that 15, 30, 45 and 60 min after the HFS protocol, the total, excitatory and inhibitory conductance changes are potentiated by about 45, 35 and 48 %, respectively. However, no significant change in excitation (intgE) and inhibition (intgI) conductance integrals expressed as percent of intgT was measured (Fig 1D, right part) in such a way that the E/I balance was unchanged. It is worth noting that the detected potentiation is likely related to HFS protocol since in absence of such protocol, control experiments show no significant change of IntgT, IntgE or IntgI for one hour (n = 17, data not shown).

  were used to block NR2A-containing receptors. Representative recordings in the presence of 0.1 μM of NVP-AAM077, before and after application of HFS protocol showed an increase in the amplitude of current recordings (Fig.3A, upper traces). Decomposition in conductance showed an increase of total, excitatory and inhibitory conductance changes (Fig.3A, middle and lower traces). The statistical analysis of the global population (n = 13; Fig3B) indicated that application of HFS protocol in the presence of NVP-AAM077 still induced a significant potentiation of the global and inhibitory conductance changes for 45 min. IntgT was enhanced by 26 % (p < 0.01), 21 % (p < 0.05) and 34 % (p < 0.05), 15, 30 and 45 min respectively after HFS protocol. IntgI was significantly enhanced in the same proportions but IntgE was not significantly increased (p > 0.05). In any case, no potentiating effect remains 60 min after HFS protocol. Consequently, the E/I ratio was decreased (p < 0.01, Fig.3C) 15 min after HFS protocol but recovered its control value 30, 45 and 60 min after HFS protocol.

  , upper traces). Decomposition in conductance changes showed an increase of total, excitatory and inhibitory conductance changes (Fig. 5A, middle and lower traces). The statistical analysis of the global population (n = 12; Fig 5B) indicated that application of HFS protocol in the presence of Ro 25-6981 still induced a significant potentiation of the global and inhibitory conductance changes for 45 min. IntgT was enhanced by 42 %, intgE by 40 % and intgI by 51 % after HFS protocol application. Due to parallel potentiation of intgE and intgI, no

Figure 1 .

 1 Figure 1. Effects of NMDA receptor blockade on the potentiation of layer 5 pyramidal neurons inputs. (A-C) Upper traces: current responses of a layer 5 pyramidal neuron to electrical stimulation (black arrow) applied in layer 2/3: (A1) under control conditions and (A2) 20 min after perfusion of 50 μM D-AP5; holding potentials scaled from -75 (bottom trace) to -55 mV (top trace, steps equal to 5 mV). (B1) Under control conditions and (B2) 15 min after HFS protocol; holding potentials scaled from -70 (bottom trace) to -50 mV (top trace, steps equal to 5 mV). (C1) 20 min after application of 50 μM D-AP5 and (C2) 15 min after HFS protocol in the presence of D-AP5; holding potentials scaled from -75 (bottom trace) to -55 mV (top trace, steps equal to 5 mV). Medium traces: decomposition of the responses in total conductance change (gT). Lower traces: decomposition of gT in excitatory (gE, dark grey) and inhibitory (gI, light grey) conductance changes. Note that, for stimulation in layer 2/3 in the presence of D-AP5, no significant change of the response was observed. HFS protocol induced an increase of total, excitatory and inhibitory conductance changes which was prevented in the presence of D-AP5. (D) Left part: relative changes (compared to control) of intgT (black), intgE (dark grey) and intgI (light grey), after application of HFS protocol in layer 2/3 under control conditions (n = 26) (*** p < 0.001, ** p < 0.01 and * p < 0.05, t-test). Right part: relative contribution of excitatory and inhibitory conductances to the total conductance change, after HFS (c: control before HFS protocol). (E) Left part: relative changes (compared to control) of intgT (black), intgE (dark grey) and intgI (light grey), after HFS in layer 2/3 (n = 15) in the presence of 50 μM D-AP5. Right part: relative contribution of excitatory and inhibitory conductances to the total conductance change, after HFS in the presence of 50 μM D-AP5 (c: control before HFS protocol).

Figure 2 .

 2 Figure 2. Effects of the blockade of NR2B-containing receptors on the potentiation of layer 5 pyramidal neurons inputs for P18-P23 old rats. (A) The left column shows representative recordings before HFS application and the right column 60 min after application of HFS in layer 2/3 in the presence of 1 μM Ro 25-6981. Upper traces: current responses of a layer 5 pyramidal neuron to electrical stimulation. Imposed membrane potential ranged for -55 to -75 mV. The amplitude of current responses was unchanged after HFS application in the presence of Ro 25-6981. Medium traces: decomposition of the responses in total conductance change (gT). Lower traces:

Figure 3 .

 3 Figure 3. Effects of the blockade of NR2A-containing receptors on the potentiation of layer 5 pyramidal neurons inputs for P18-P23 old rats. (A) The left column shows representative recordings before HFS application and the right column 60 min after application of HFS in layer 2/3 in the presence of 0.1 μM NVP-AAM077. Upper traces: current responses of a layer 5 pyramidal neuron to electrical stimulation. Imposed membrane potential ranged for -55 to -75 mV. The amplitudes of current responses were slightly increased after HFS application. Medium traces: decomposition of the responses in total conductance change (gT). Lower traces: decomposition of gT in excitatory and inhibitory conductance changes (gE, dark grey and gI, light grey). Total, excitatory and inhibitory conductance changes were all increased. (B) Relative changes (compared to control) of intgT (black bars), intgE (dark grey bars) and intgI (light grey bars), after HFS in layer 2/3 (n = 13) in the presence of 0.1 μM NVP-AAM077. (*** p < 0.001, ** p < 0.01 and * p < 0.05, t-test). (C) Relative contribution of excitation and inhibition conductance changes to the total conductance change, after HFS in the presence of 0.1 μM NVP-AAM077 (c:control before HFS protocol). (D-E-F) Application of HFS protocol in the presence of 200 nM ZnCl 2 (n = 13). Legends are identical to those in A-B-C. (D) Imposed membrane potential ranged for -60 to -80 mV. (E-F) Total, excitatory and inhibitory conductance changes were increased after HFS.

Figure 4 .

 4 Figure 4. Effect of synaptic NMDARs blockade on the potentiation of layer 5 pyramidal neurons inputs.

Figure 5 .

 5 Figure 5. Effects of the blockade of NR2B or NR2A-containing receptors on the potentiation of layer 5 pyramidal neurons inputs for P29-P32 old rats. (A) The left column shows representative recordings from the statistical analysis of n = 12 experiments before HFS application and the right column 60 min after application of HFS in layer 2/3 in the presence of 1 μM Ro 25-6981. Upper traces: current responses of a layer 5 pyramidal neuron to electrical stimulation. Imposed membrane potential ranged for -55 to -75 mV. The amplitude of current responses was increased after HFS application. Medium traces: decomposition of the responses in total conductance change (gT). Lower traces: decomposition of gT in excitatory and inhibitory conductance changes (gE, dark grey and gI, light grey). Total, excitatory and inhibitory conductance changes were all increased. (B) Relative changes (compared to control) of intgT (black bars), intgE (dark grey bars) and intgI (light grey bars), after HFS in layer 2/3 (n = 12) in the presence of 1 μM Ro 25-6981. (C) Relative contribution of excitation and inhibition conductance changes to the total conductance change, after HFS in the presence of 1 μM Ro 25-6981 (c:control before HFS protocol).

  using MK-801, an antagonist of NMDARs that acts only on open channels. MK-801 (10 μM) was bath-applied and a low frequency stimulation protocol (LFS) was delivered in order to activate synaptic NMDARs which can then be blocked whereas extrasynaptic receptors are not activated by this protocol of stimulation. MK-801 was then washed out for 30 min and HFS protocol of stimulation was applied in layer 2/3.

To start, a control condition (omitting MK-801) was used in order to check that application of LFS protocol does not prevent the potentiation induced by the HFS protocol. Significant potentiations were observed for IntgT (by 39 %, p < 0.05), for IntgI (by 42 %, p < 0.05) and for IntgE (by 22 %, p < 0.05) (Fig 4A for representative recordings and Fig.

Acknowledgments

The authors are grateful to Dr. Y.P. Auberson (Novartis Institutes for BioMedical Research, Basel) for the kind gift of NVP-AAM077. We are grateful to Dr. D.W. Owens (Pfizer global research and development) for the kind gift of CP101,606. The authors thank Dr. S. O'Regan and Dr. R. Lewis for critical reading of the manuscript.

Abbreviations

E, excitation; GABA, gamma amino butyric acid; HFS, high frequency of stimulation; I, inhibition; intgE, excitatory conductance integral; intgI, inhibitory conductance integral; intgT, total conductance integral; LTP, long-term potentiation; NMDAR, N-methyl Daspartate receptor.


Plant Biology: Unravelling the Transient Physiological Role for PHO1 in the Seed

During seed development, an important transfer of nutrients occurs between the seed coat and the embryo. A new study reveals that, for inorganic phosphate (Pi), this function is transiently performed by PHO1, a protein associated previously with Pi loading into the xylem.

Main Text

Phosphorus is an essential macronutrient for plant development and signalling. It is acquired in the plants exclusively in the form of Pi from the environment by the PHT1 family of Pi/H+ cotransporters [START_REF] Nussaume | Phosphate import in plants: focus on the PHT1 transporters Front[END_REF], which mediates its transport across the plasma membrane. Interestingly, this family of transporters is involved in both the direct uptake of Pi from soils and the exchanges with arbuscular myccorhizal fungi (a trait shared by 80% of vascular flowering plants [2]). All of these transporters are mainly associated with the influx activities of Pi into the cell, and many additional Pi transporters have been identified for the allocation of this crucial resource in the

different plant cell compartments [2].
For the efflux activity, other key components for Pi homeostasis are the members of the PHO1 family. Their physiological activity was difficult to demonstrate, as they exhibit no homology to known transporters [START_REF] Stefanovic | Overexpression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux[END_REF], but show similarities with the yeast Syg1 protein (a component of the mating pheromone signal transduction pathway) or with the Rcm1 mammalian receptor for xenotropic murine leukemia retrovirus. Another problem with studying these proteins is the difficulty to detect the PHO1 protein at the plasma membrane. A GFP-labeled PHO1 can complement the pho1 mutant but is observed only in the Golgi and trans-Golgi network [START_REF] Arpat | Poirier Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate[END_REF]. This may be due to fine control of the level of this protein at the plasma membrane level through important recycling at the trans-Golgi network level.

Until now, PHO1 proteins were only known to support the Pi transfer from root to shoot [START_REF] Stefanovic | Poirier Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways[END_REF]. Indeed the pho1 mutant exhibits a strong reduction of Pi content in the leaves due to a failure in the loading of Pi into the xylem [START_REF] Poirier | Mutant of Arabidopsis deficient in xylem loading of phosphate[END_REF][START_REF] Hamburger | Poirier Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem[END_REF]. Experiments overexpressing this protein in plant tissue where it was normally not detected triggered Pi export, suggesting a direct role in Pi export for the PHO1 protein [START_REF] Stefanovic | Overexpression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux[END_REF][START_REF] Arpat | Poirier Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate[END_REF].

In Arabidopsis, where they were first identified, the PHO1 family comprises 11 members. They share important homologies (47-94% [START_REF] Wang | Poirier Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis[END_REF]) but phylogenetic studies have revealed that PHO1 and PHO1;H1 belong to a distinct clade from all other PHO1 family members, which would suggest a distinct role independent of Pi homeostasis [START_REF] Secco | Poirier Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons[END_REF]. Such analysis is reinforced by genetic studies revealing that mutants affecting these other members of the PHO1 family exhibited abnormal hypocotyl growth [START_REF] Kang | Ni Arabidopsis SHORT HYPOCOTYL UNDER BLUE1 contains SPX and EXS domains and acts in cryptochrome signaling[END_REF], seed size [START_REF] Zhou | SHORT HYPOCOTYL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development[END_REF] or flowering [START_REF] Zhou | Ni SHB1 plays dual roles in photoperiodic and autonomous flowering[END_REF]. Besides, only PHO1;H1 can also complement the pho1 mutant, supporting a similar physiological role for these two proteins [START_REF] Stefanovic | Poirier Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways[END_REF].

Since the pioneering work on the gene expression map of the Arabidopsis root [START_REF] Birnbaum | A gene expression map of the Arabidopsis root[END_REF],

the transcriptomes of many plant tissues have been analysed. They revealed unexpected and specific locations for many genes, offering the opportunity to investigate their physiological relevance in these tissues. So for phosphate, the recent identification of PHT1 proteins in the root cap offered the opportunity to identify the important contribution of this cell layer to Pi uptake [START_REF] Kanno | A novel role for the root cap in phosphate uptake and homeostasis[END_REF]. In this issue of Current Biology, Yves Poirier's team found a new physiological role for PHO1the transfer of Pi between the chalazal seed coat and the embryo [START_REF] Vogiatzaki | Poirier PHO1 exports phosphate from the chalazal seed coat to the embryo in developing Arabidopsis seeds[END_REF]. Using several reporters such as GFP or GUS fused to PHO1 or PHO1;H1 genes under the control of their own promoters, the authors detected strong expression in the chalazal seed coat. It also revealed a novel level of complexity, as in this system, PHO1;H1 cannot complement PHO1 phenotype, suggesting distinct features between these two proteins, which have been unknown so far [START_REF] Vogiatzaki | Poirier PHO1 exports phosphate from the chalazal seed coat to the embryo in developing Arabidopsis seeds[END_REF]. Such results can appear surprising, as PHO1;H1 is also expressed in the same tissues. However, both genetic studies of mutants and specific tissue complementation confirmed this surprising result.

As pho1 mutation impacts Pi distribution in plants (including the seed), the authors circumvented this problem elegantly by grafting wild-type root stock onto pho1(pho1;H1, or double mutant) mutants to produce plants with normal Pi distribution from root to shoot, but with pho1 background for the seed development [START_REF] Vogiatzaki | Poirier PHO1 exports phosphate from the chalazal seed coat to the embryo in developing Arabidopsis seeds[END_REF].

The other difficulty in the study of Pi is the lack of a technique to visualize the cellular resolution of Pi distribution [START_REF] Kanno | Nussaume Performance and limitations of phosphate quantification: guidelines for plant biologists[END_REF] and the velocity of the transport of this ion. Most physical techniques offering access to ion distribution, for example, synchrotron x-ray fluorescencemicrotomography used to localize iron in the seed [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter[END_REF], can only detect total phosphorus and therefore are not accurate for visualizing the phosphate pool. The main tool remains the labeling of the tissues with radioisotope ( 32 P or 33 P), which can be measured either by real-time imaging [START_REF] Kanno | Development of real-time radioisotope imaging systems for plant nutrient uptake studies Phil[END_REF] or microdissection when high resolution is requested. This latter solution has been used here [START_REF] Vogiatzaki | Poirier PHO1 exports phosphate from the chalazal seed coat to the embryo in developing Arabidopsis seeds[END_REF]. I must underline the difficulty of such experiments, as the role of PHO1 protein during seed development appears to be only transient, and the phenotype can be found only at the mature green stage. The absence of a strong seed phenotype may be due to compensatory mechanisms resulting from the activity of other members of the PHO1 family (distinct from PHO1;H1) or to physiological adaptations that remain to be discovered. This role for PHO1 may also require specific conditions, as illustrated by the study of iron allocation in the seed. Indeed, the role of VIT1 for primary allocation of iron in the provascular strands of the embryo was revealed by specific pH conditions, which promoted an impact at the germination level between the mutant and the wild-type [START_REF] Kim | Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter[END_REF]. For phosphate, similar mechanisms may exist, especially since abnormal growth is only observed when Pi absorption is reduced by more than 80% in normal growth conditions [START_REF] Ayadi | Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling[END_REF], highlighting the capacity of the plant to adapt.

Nevertheless, the analysis reported in the present issue provides novel evidence supporting the role of the PHO1 family in Pi export.

Unravelling the molecular mechanisms involved remains an exciting but very difficult challenge, as all attempts to understand PHO1 transport activities in non-plant systems have failed so far.

Nevertheless, the increasing pace of research in the field of Pi homeostasis should help to fill this gap in the near future.


Keywords: Synthetic Biology, Directed Evolution, Recombination, DNA shuffling, Integron, Metabolic Engineering

Shuffling of DNA cassettes in a synthetic integron

Introduction

Over the past 20 years, efficient mutagenesis techniques have been developed allowing the introduction of random point mutations, as well as sequence shuffling through homologous recombination. However the efficient random recombination of heterologuous sequences largely remains a challenge. It has been achieved in vitro through various techniques of randomized assembly ligation and overlap extension PCR [START_REF] Bittker | Directed evolution of protein enzymes using nonhomologous random recombination[END_REF][START_REF] Gertz | Analysis of combinatorial cisregulation in synthetic and genomic promoters[END_REF][START_REF] Guet | Combinatorial synthesis of genetic networks[END_REF][START_REF] Cox | Programming gene expression with combinatorial promoters[END_REF][START_REF] Tsuji | Random multi-recombinant PCR for the construction of combinatorial protein libraries[END_REF]. However these techniques are limited in the size and number of elements they can combine.

Integron are natural recombination platforms involved in the rapid adaptation of bacteria to changing environment. They are present in the genome of roughly 10% of sequenced bacteria, and are largely responsible for the massive spread of multiple resistances to antibiotics. Integrons consists in an integrase gene (intI) able to capture, stockpile and shuffle gene cassettes. Natural cassettes generally consist in a single promoterless ORF associated to an attC recombination site. Cassettes can be excised through attC x attC recombination into a circular intermediate and preferentially integrated at a primary recombination site downstream of the integrase gene called attI. Different classes of integrons have been identified on the base of the integrase sequence identity (for a comprehensive review see [START_REF] Cambray | Integrons. (Translated from eng[END_REF]).

We use here the class 1 integrase isolated from R388 multiresistant integron, the associated attI1 site, and the attCaadA7 site isolated from an aadA7 spectinomycin resistance cassette. These elements have been extensively described and shown to efficiently recombine. We have already shown how this machinery can be used to deliver gene cassettes of the tryptophan operon and shuffle them to generate a wide variety of combination yielding improved phenotypes [START_REF] Bikard | The synthetic integron: an in vivo genetic shuffling device[END_REF]. Here is a detailed procedure of the steps required to construct a strain with a synthetic integron integrated in the chromosome, to deliver new genetic elements in the array and to shuffle them.

Materials

The minimal platform of an integron consists in the integrase gene and the attI recombination site. In all experiments, the IntI1 integrase is expressed in trans from the pBAD::intI1 plasmid. The attI1 site is integrated into the chromosome at the attB site of bacteriophage lambda using a suicide integration vector (procedure described below). Depending on your application you may want to directly clone cassettes in the integron array that you will integrate into the chromosome.

Biological material: DNA

1. Plasmid pSWK carrying an attI site. The pSWK plasmid carries an R6K origin of replication which depends on the Π protein expressed from the pir gene. It also carries the attP site from bacteriophage lambda, allowing its integration at the lambda attB site in the E.coli chromosome. This plasmid complies with biobrick standard RFC10, and can be found on the registry of standard biological parts as BBa_J99000 (7). 2. The integron attCaadA7 site: BBa_J99001 (7). 3. The integron attI1 site: BBa_J99002 (7). 4. Plasmid pBAD::IntI1 (8). 5. Plasmid pTSA29-CXI-AK [START_REF] Valens | Macrodomain organization of the Escherichia coli chromosome[END_REF]. A thermo-sensitive pSC101 vector carrying the lambda integrase under the control of the thermo-inducible CI857/pR promoter.

Biological material: strains

Culture media and reagents

1. Luria Bertani (LB) culture medium 2. LB agar plates supplied with suitable selection agent 3. Antibiotics were used at the following concentrations: ampicillin (Ap), 100 µg/ml, chloramphenicol (Cm), 25 µg/ml. 4. Thymidine (Thy) and diaminopimelic acid (DAP) is supplemented when necessary to a final concentration of 0.3mM. 5. Glucose and L-arabinose are added at respectively 10 and 2mg/ml final concentration for the repression and induction of the pBAD promoter. 6. CaCl2 solution, 50uM 7. EcoRI, XbaI, SpeI, PstI restriction enzymes. Those are the restriction sites of the RFC10 assembly standard. 8. T4 DNA ligase

Methods

The global idea is to first integrate the attI1 site into the chromosome. This will be the landing platform of any integron cassette of your choice. Cassettes present in the array can be shuffled through the expression in trans of the IntI1 integrase from the pBAD::intI1. Finally, new cassettes can be delivered to the array trough a suicide conjugation assay.

Depending on your application an initial cassette array can directly be integrated together with the attI site. Cassettes are simply created through the association of any DNA element to an attCaadA7 recombination site and cloned downstream of the attI site. This can be done using the construction method of your choice. The method we use here is biobrick standard assembly. We constructed vectors carrying attI (BBa_J99002) and attC sites (BBa_J99001) complying with the RFC10 standard.

Several methods have been described for the efficient integration of DNA in the E.coli chromosome [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF][START_REF] Chaveroche | A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans[END_REF]. The method we chose here is to clone the initial integron cassette array on a vector (pSWK) that can then be integrated at the chromosomal attB site of bacteriophage lambda. pSWK carries an attP site and can thus be integrated at the attB site by the lambda integrase. It has an R6K origin of replication and can only replicate in cells expressing the pir gene. This plasmid can thus be integrated into pircell expressing the lambda integrase in trans (from plasmid pTSA29-CXI-AK), and plating on the pSWK resistant marker will select integration events. However, any other integration method and any other neutral chromosomal locus should be suitable.

New cassettes can be delivered to an integron array through a suicide conjugation strategy. The pSWK vector can be used to this end too. pSWK carries an RP4 origin of transfer, and can be delivered to recipient cells by ω7249 cells expressing the RP4 conjugation machinery. This donor strain can be used in a conjugation assay to deliver cassettes in a recipient strain carrying an integron array and expressing the IntI1 integrase in trans from the pBAD::IntI1.

Construction of the initial cassette array

1. The initial integron array can be constructed with the method of your choice.

Here, we use a classic digestion / ligation / transformation protocol. The use of the RFC10 idempotent assembly standard allows the easy association of any biobrick to an attCaadA7 site and its cloning into the integron array on the pSWK vector. For more details on biobrick assembly see partsregistry.org. The pSWK vector can only replicate in cells expressing the pir gene.

Transformation is thus carried out into pi1 cells, and selection on LB agar + Chloramphenicol (25 microg/ml) + Thymidine (70 microg/ml) plates.

Integration of the initial cassette array into the chromosome

1. You first need to transform the pTSA29-CXI-AK plasmid carrying the lambda integrase into your strain. The method of your choice can be used to prepare competent cells. We use here a classical CaCl2 chemo-competent protocol. After heat-shock, cells need to be incubated at 30°C for the expression period and plated on LB agar + Ap at 30°C in order for the plasmid to stably replicate. 2. In order to integrate your pSWK into these cells, you now need to prepare competent cells expressing the lambda integrase. Launch an overnight culture at 30°C in LB + Ap from a single colony 3. Dilute the overnight culture to the 1/100 th in LB and grow at 30°C until OD600=0.2 4. Switch the incubation temperature to 42°C until 0.6< OD600<0.8 . During this period, the lambda integrase will be expressed from the CI857-PR promoter. 5. Prepare CaCl2 competent cells (or any other type of competent cells. The integration event is very efficient, so you don't need to prepare ultracompetent cells for this to work). (see note 1) 6. Transform with at least 20 ng of pSWK DNA, and incubate for the expression time for 1H at 42°C. 7. Plate on LB agar + Cm and grow overnight at 42°C. Doing all this steps at 42°C ensures that the lambda integrase is expressed and that at the same time the pTSA29 is lost. 8. The following day you should have colonies with the pSWK integrated at the attB site of your strain. 9. Check for proper integration by doing a colony PCR using a primer binding on the pSWK and one binding on the chromosome.

Cassette shuffling through the IntI1 integrase expression

1. Transform the pBAD::IntI1 in your cells using the method of your choice.

2. Induce the integrase by diluting an overnight culture to the 1/100 th in LB + Ara. You can prepare a negative control in LB + Glu. Grow overnight (see note 2) 3. Plate cells on the selective media of your choice. Recombinations induced by the integron machinery happen at frequencies below 10 -2 . In order to find recombined clones, you need a powerful screening assay. In our initial study where we shuffle the genes of the tryptophan biosynthesis operon, the selection is performed in tryptophan auxotroph strains and recombinants are selected on minimal media [START_REF] Bikard | The synthetic integron: an in vivo genetic shuffling device[END_REF].

Delivery of new cassettes through conjugation

1. Transform the pSWK carrying the cassettes of interest into ω7249 cells. We use CaCl2 competent cells, but any method is suitable. Remember that ω7249 cells need DAP to grow. (see note 3) 2. This strain will be your donor strain in the conjugation assay. Depending on your application you may want to use several donor strains to combinatorially deliver cassettes of interest in a single recipient strain. The recipient strain needs to carry the pBAD::intI1 plasmid and an attI site in the chromosome. Dilute an overnight culture of the donor strain(s) to the 1/100 th in LB + DAP and of the recipient strain in LB+DAP+Ara (see note 4). 3. Grow donor and recipient cells to 0.4< OD600<0.6 4. Mix 1ml of donor cells with 4ml of recipient cells and filter onto a 0.22um membrane. (see note 5)

5. Place the filter on a fresh LB+DAP+Ara plate, the side with the cells facing up. Incubate 6H at 37°C. 6. Put the filter in a 50 ml tube with 5 ml LB and vortex to remove the cells from the filter. 7. Plate the cells on the selective media of your choice. (If the pSWK vector you use carries a resistance that is not already present in your recipient strain, you can select for the integration of the whole vector in the integron array. It is then possible to get rid of the vector backbone using a counter selection marker, leaving all or some of the cassettes in the chromosome.)

Notes

1. It is important that competent cells are kept on ice throughout the whole process. 2. The longer the cells are growing with the integrase expressed, the higher the recombination frequency will be. To improve recombination frequency, several passages in LB+Ara can be done. Note however that without selection, cassettes are lost at a frequency similar to the one of reordering events (7). 3. The use of the DAP auxotroph donor (ω7249) for the conjugation delivery protocol guaranties that you get rid of the donor by simply selecting on LB + antibiotic marker of the pSWK, as DAP is completely absent in LB. 4. The recipient strain does not actually need DAP to grow, but adding DAP in the medium allows to later mix the cultures without worrying about killing the donor cells 5. If you do not have the equipment to do this, you can mix the cells and centrifuge them gently 4min at 2000g, discard the supernatant, resuspend the cells in 100 microl and pipette onto the filter.)

1 .

 1 pi1 E.coli cells (DH5α DthyA::(erm-pir116) [EmR]), for the replication of the pSWK plasmid (10) 2. MG1655 E.coli cells (or any E.coli strain where you want to integrate the synthetic integron) 3. ω7249 E.coli cells ((F-) RP4-2-Tc::MuΔnic35 ΔdapA::(erm-pir) [KmR ErmR]). ω7249 cells express the pir gene allowing the replication of vectors carrying the R6K replication origin. It also expresses the RP4 conjugation machinery allowing the transfer of plasmids carrying the RP4 origin of transfer[START_REF] Babic | Construction of an improved RP4 (RK2)-based conjugative system[END_REF].