Rim Boubakri

Christian Jelsch

Frederic Lefebvre

Chérif Werner Kaminsky

Ben Nasr

Kamel Kaabi
email: kamel_kaabi@yahoo.fr

Frédéric Lefebvre

Ben Cherif

Nasr

Keywords: Square planar, Square pyramidal, Cu(II) complex, X-ray diffraction, Coordination compound, Hirshfeld surface

  Crystal structure, Hirshfeld surface analysis and physicochemical studies of two new Cu(II) complexes with the ligand 2-amino-6-methylpyrimidin-4-(1H)-one

Introduction

The rational design and synthesis of transition metal complexes have become very attractive research fields owing to their intriguing structural diversity and enormous potential applications such as gas storage [1], gas separation [START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF], ion-exchange [3], molecular electronics [4], luminescence [5], non-linear optical behavior [6] and magnetism [7]. These complexes have also drawn increased attention because of their important role in the field of bio-inorganic chemistry. Among them, copper complexes have been extensively studied because of their widespread use in a range of applications ranging from catalysis [START_REF] Tsipis | [END_REF] to medicinal chemistry [9,10] or electro-optics [11,12]. The properties of complexes are dependent on the nature of coordinating ligands and donor atoms. Copper complexes containing heterocyclic organic ligands, such as pyrimidine, have shown a wide range of biological and pharmaceutical activities that include DNA binding and cleavage, antimicrobial, anticancer and antioxidant behavior [13 -16]. Pyrimidine ring systems in nucleic acids, vitamins and coenzymes offer some effective binding sites for metal ions. The presence of more than one hetero-atom and the higher π acidity of the pyrimidine ring as compared to that of pyridine bases play an important role in coordination chemistry and also serve to improve biological systems [17][18][19].

As part of our continuous involvement in the study of metal complexes of ligands of interesting molecular structures for suitable applications [20], we report here the synthesis and structural characterization of two Cu(II) complexes with the ampymo (2-amino-6methylpyrimidin-4-(1H)-one) ligand (Scheme 1). This work indicates that the solvent used in the synthesis of the compounds has a direct effect on the crystal structure diversity. Indeed, by use of the same reagents in the synthesis but of two different solvents, two crystal structures with a different unit cell content were obtained. Scheme 1. Structural formula of ampymo (2-amino-6-methylpyrimidin-4-(1H)-one). A solution of [Cu2(O2CMe)4(H2O)2] (10 mg, 0.025 mmol) in ethanol (10 mL) was added dropwise to a solution of ampymo (26 mg, 0.2 mmol) in ethanol (10 mL). After stirring for 30 min, the mixture was filtered. Crystals suitable for X-ray analysis were obtained after five days by evaporating the filtrate at room temperature (yield = 71%). Anal. Calc.: C, 42.21; H, 4.98; N, 24.62%. Found: C, 41.92; H, 4.56; N, 24.43 %.

Experimental

Preparation of [Cu(ampymo)2(H2O)3](O2CMe)2 (2)

A solution of [Cu2(O2CMe)4(H2O)2] (10 mg, 0.025 mmol) in water (10 mL) was added dropwise to a solution of ampymo (13 mg, 0.1 mmol) in water (20 mL). After stirring for 30 min, the mixture was filtered. Crystals suitable for X-ray analysis were obtained after five days by evaporating the filtrate at room temperature (yield = 67%). Anal. Calc.: C, 34.60; H, 5. 35; N, 17.29%. Found: C, 34.12; H, 5.72; N, 17.06 %.

X-ray single crystal structural analysis

2.1. X-ray single crystal structural analysis of (1)

A colorless prism, measuring 0.33 x 0.11 x 0.10 mm 3 was mounted on a loop with oil.

Data were collected at 100 K on a Bruker APEX II single crystal X-ray diffractometer using Mo-radiation. The crystal-to-detector distance was 40 mm and the exposure time was 10 seconds per frame for all sets. The scan width was 0.5 o . Pertinent experimental details are listed in Table S1. The diffraction data were integrated and scaled using SAINT, SADABS within the APEX2 software package by Bruker [START_REF]Bruker APEX2 (Version 2.1-4), SAINT (version 7.34A), SADABS (version 2007/4)[END_REF]. Solution by direct methods (SHELXS, SIR97 [22]) produced a complete heavy atom phasing model consistent with the proposed structures. The structures were completed by difference Fourier synthesis with SHELXL [START_REF]Sheldrick SHELXL-97[END_REF]24]. Scattering factors are from Waasmair and Kirfel [25]. Isotropic thermal parameters Ueq of hydrogen atoms were fixed such that they were 1.2Ueq of their parent atom Ueq for CH's, NH's, and NH2's -and 1.5Ueq All non-hydrogen atoms were refined anisotropically by full-matrix least-squares. The drawings were made with Diamond [START_REF] Brandenburg | Diamond Version 2.0 Impact GbR[END_REF] and Mercury [START_REF] Macrae | [END_REF].

X-ray single crystal structural analysis of (2)

Intensity data for (2) were collected at 293 K on a New Xcalibur, EosS2 single crystal diffractometer using graphite monochromated Mo-Ka radiation (k = 0.71073 Å). Unit cell determination, data collection and data reduction were performed using the CrysAlisPro software [START_REF] Crysalispro | Agilent Technologies[END_REF]. A symmetry-related (multiscan) absorption correction had been applied. The structures were solved with SHELXT [START_REF] Sheldrick | [END_REF] and refined by a full-matrix least-squares procedure based on F 2 (Shelxl-2014) [START_REF]Sheldrick SHELXL-97[END_REF]. Four square aligned residual electron density maxima of up to 1.12 eÅ 3 were found, located co-planarly with the ligands at about 1 Å distance from the copper atom. The crystal data are gathered in Table S1. The drawings were made with Diamond [START_REF] Brandenburg | Diamond Version 2.0 Impact GbR[END_REF] and Mercury [START_REF] Macrae | [END_REF].

IR spectroscopy

The spectra were recorded in the range 4000-400 cm -1 by means of a Perkin-Elmer FTIR spectrophotometer 1000 using samples dispersed in spectroscopically pure KBr pressed into a pellet.

DFT calculations

Quantum chemical calculations were performed with the Gaussian 09 program on the two copper complexes. As periodic calculations should be too time-consuming and in order to describe the complex in the solid state, these calculations were done on the geometry determined by X-ray crystallography. However, as the C-H, O-H and N-H distances were too short compared to what is usually observed in theoretical calculations (by X-ray crystallography they were assumed to be in a 0.95 -1.00 Å range, see above, while theoretical calculations give 1.05 to 1.09 Å), an optimization of the proton positions was done first. The HOMO and LUMO and the Mulliken charge distribution were then determined of these semi-optimized geometries.

In the two cases the theoretical infrared spectrum was obtained by summing the individual spectra of two acetate ions and the cationic copper complex. All calculations were done with the classical B3LYP method and the 6-31+G* basis set for all atoms.

Hirshfeld surface analyses.

Fingerprint plots were obtained with the CrystalExplorer program [30]. The contact types and their enrichment were analyzed with the program MoProViewer [31]. Moieties in the crystal packing not in contact with each other were selected in order to obtain an integral Hirshfeld surface around each molecule/ion. For compound (1), the Hirshfeld surface was computed for two copies of the asymmetric unit as the Cu atom lies on a special position and has a multiplicity of 2. For electrostatic energy calculations, the electron density of the molecules was modeled by transfer from the ELMAM2 multipolar atoms database [32]. In both compounds, the copper cation was set spherical with a charge of +2e while the asymmetric unit was set electrically neutral. The main geometrical features of the different chemical entities of [Cu(C5H7N3O)4](CH3COO)2 are reported in Tables 1 andS2. X-ray crystal structure analysis reveals that [Cu(ampymo)4](O2CMe) crystallizes in the monoclinic space group P42/n. The asymmetric unit consists of one Cu(II) cation, two ampymo ligands and one acetate anion (Fig. 1). In the atomic arrangement of this complex, the Cu(II) atom is coordinated in a square planar fashion by four nitrogen atoms N1, N1 In the atomic arrangement, the complex cations are arranged in periodic pillars of individual complexes that interact with their neighbors in the c-axis direction. The pillars are alternated with channels filled with the acetate anions (Fig. 2) that play a role of linking the neighboring complex cations through N2-H•••O3 and N5-H•••O4 hydrogen bonds (see Table 1) to form a 1-D corrugated chain running along the b-axis direction (Fig. 3). The donor acceptor distances N2 typical for ketonic oxygen [36,37]. In both cases, the tautomerism increases compound stability.

Results and Discussion

3.1.2. Structure description of [Cu(ampymo)2(H2O)3](O2CMe)2 (2)
The main geometrical features of the different chemical entities of complex (2) are given in Tables 2 andS3. The X-ray diffraction study of a single crystal of (2) shows that this compound crystallizes in the space group P21/n. The asymmetric unit consists of one Cu(II) cation, two ampymo ligands, three coordinated water molecules and two acetate anions (Fig. 4). The copper atom Cu(II) is penta-coordinated in a square pyramidal fashion by two pyrimidine nitrogen atoms (N1 and N4) as well as two water oxygen atoms (O3 and O4) in basal positions and one water oxygen atom O5 at the apex. The Cu-N bond lengths (Cu-N1 = 2.004(4) and Cu-N4 = 2.017(4) Å) are close to those reported for other complexes [36,37].

The apical Cu-O bond length (Cu-O5 = 2.463(4) Å) is larger than the basal Cu-O ones (Cu-O3 = 1.969(3), Cu-O4 = 1.966(3) Å). This fact may be ascribed to a Jahn-Teller distortion.

These bond lengths are comparable to corresponding values in similar coordination compounds [38,39] Similarly, N3-Cu-N1 angle amounts to 163.8(1)°, which is far from the ideal value 180°. These geometrical parameters indicate that the CuN2O3 entity has a distorted pyramidal geometry (Fig. 4). In the atomic arrangement, the cationic complexes [Cu(ampymo)2(H2O)3] 2+ are located parallel to the (a, b) planes at z = ½ n (Fig. 5), between which the acetate anions intercalate and connect them through hydrogen bonds to form a tridimensional network (Fig. 5 and Table 2). It is worth to note that 3d-metal complexes of the same ligand with different nuclearities and coordination modes were previously described in the literature [36,37,[40][41][42] (Table S4).

Hirshfeld surface analysis

Contacts of (1)

The Hirshfeld surface is representative of the region in crystalline space where molecules come into contact allowing the analysis of the chemical nature of intermolecular interactions.

Contacts of (2)

The chemical content of crystal (2) indicates a higher proportion of oxygen and polar hydrogen Hn/o atoms compared to compound (1), due to the hydration of the crystal by three water molecules (Table 3b). As a result, only 45% of the surface involves the C and Hc atom types.

The contacts between these hydrophobic atoms are slightly over-represented (E=1. The copper cation is more in contact with oxygen atoms, but the Cu•••N contact is still the most enriched one at ECuN = 4. 37. The Cu(II) Hirshfeld surface has nearly a cuboid shape, except that the face without ligand is convex (Fig. 7c).

Electrostatics of contacts

The scatterplot of the average electrostatic energy Eelec as a function of enrichment values is shown in Fig. 8

Quantum mechanical study of compound of (2)

The HOMO and LUMO orbitals were determined for the cationic complex and the two acetate groups interacting with it, in order to describe the hydrogen bonds between the CH3COO -groups of acetate and the N-H bonds of the organic ligands. Although it is difficult to determine discrete orbitals for a crystal (a better choice would be to perform periodic calculations which are highly time consuming) a good approximation of the gap between the occupied and unoccupied bands can be obtained by this way. The energies of the frontier orbitals of [Cu(ampymo)2(H2O)3](O2CMe)2 are listed in Table 4. Fig. 10 shows the HOMOs and LUMOs and the next orbitals for the title compound calculated at the B3LYP/6-31++G**.

Clearly the highest occupied orbitals are localized mainly on the acetate groups, while the lowest unoccupied orbitals are mainly on the two organic ligands around the copper. The gap between the LUMO and the HOMO is 4.13 eV. As for (1), this large energy gap characterizes a high chemical hardness and kinetic stability of the new coordination compound.

The molecular electrostatic potential of the title compound has been computed at the B3LYP/6-31++G** level and is shown in Fig. S3. As seen from this figure, the negative zones are mainly on the acetate groups, the positive zones being at the proton level of the N-H bonds.

IR spectroscopy

The IR absorption spectra of two compounds There is a good agreement between the experimental and calculated spectra even if the intensities are not similar but this is related to the line width, which is greater for some experimental lines due to the hydrogen bonds (see for example the bands at ca. 1580 and 1407 cm -1 in the two spectra). There is almost no difference between the experimental and calculated spectra after optimization of the protons positions. The correlation is good (R = 0.9974 for the spectrum of compound (1), R = 0.9989 for the spectrum of compound (2)), as shown in the correlation plot, Fig. S6, but some bands were omitted for which the calculated intensity is too low. Thus, the precision is well-sufficient to assign the experimental frequencies and to confirm the attributions proposed above.

Supplementary data

Crystallographic data for the structural analysis have been deposited at the Cambridge Crystallographic Data Centre, CCDC No 1877359 for (1) and CCDC No 1939573 for (2).

These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK: fax: (+44) 01223-336-033; e-mail: deposit@ccdc.cam.ac. Table 1 Hydrogen-bond geometry (Å, °) in complex (1).

Figure captions

D-H•

••A D-H H•••A D•••A D-H•••A N2-H2•••O3 0.
Symmetry codes: (i) -x+ 3 /2, -y+½, z; (ii) y+½, -x+1, z-½ .
Table 2 Hydrogen-bond geometry (Å, °) in complex (2).

D-H•

••A D-H H•••A D•••A D-H•••A N2-H2•••O8 0.
(i) -x, -y, -z+1; (ii) -x+1, -y+1, -z+1; (iii) x+½, -y+½, z+½; (iv) x-½ , -y+ ½, z-½ .
Table 3. Statistical analysis of intermolecular contacts on the Hirshfeld surface of compounds Table S2. Selected bond distances and angles (Å, º) in complex (1).

Symmetry code: (i) -x+ 3 /2,-y+ ½, z
Table S3. Selected bond distances and angles (Å, º) in complex (2). --------------------------------------------------------------------------- ---------------------------------------------------------------------------# _symmetry_cell_setting tetragonal _symmetry_space_group_name_H-M 'P 42/n' _symmetry_space_group_name_Hall '-P 4bc' _symmetry_Int_Tables_number 86 loop_ _symmetry_equiv_pos_as_xyz 'x, y, z' '-x+1/2, -y+1/2, z' '-y, x+1/2, z+1/2' 'y+1/2, -x, z+1/2' '-x, -y, -z' 'x-1/2, y-1/2, -z' 'y, -x-1/2, -z-1/2' '-y-1/2, x, -z-1/2' _cell_length_a 14.2120(10) _cell_length_b 14.2120(10) _cell_length_c 16.6729 --------------------------------------------------------------------------- ---------------------------------------------------------------------------# # ABSORPTION CORRECTION # #- --------------------------------------------------------------------------- --------------------------------------------------------------------------- --------------------------------------------------------------------------- #---------------------------------------------------------------------------- --------------------------------------------------------------------------- --------------------------------------------------------------------------- #----------------------------------------------------------------------------# # ATOMIC TYPES, COORDINATES AND THERMAL PARAMETERS # #- ---------------------------------------------------------------------------# loop_ _atom_type_symbol _atom_type_description _atom_type_scat_dispersion_real _atom_type_scat_dispersion_imag _atom_type_scat_source C C 0.0033 0.0016 'International Tables Vol C Tables 4. [START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF].6.8 and 6. 1.1.4' H H 0 0 'International Tables Vol C Tables 4. [START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF].6.8 and 6. 1.1.4' N N 0.0061 0.0033 'International Tables Vol C Tables 4. [START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF].6.8 and 6. 1.1.4

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1 .

 1 Preparation of [Cu(ampymo)4](O2CMe)2 (1)

3. 1 . X-ray diffraction study 3. 1 . 1 .

 111 Structure description of [Cu(ampymo)4](O2CMe) (1)

  . The sum of the basal bond angles around the Cu atom (N1-Cu-O3 = 92.42(14), O3-Cu-N4 =89.46(14), N4-Cu-O4 = 90.99(14), O4-Cu-N1 = 88.13(14)°) is close to 360°, showing that the basal atoms (N1, N4, O3 and O4) are coplanar. The N1-Cu-N5 angle [100.59 (17)°] for the CuN2O3 coordination polyhedron shows a significant deviation from the ideal value of 90°.

  These H-bonds are of the N-H•••O and O-H•••O type in which the imine nitrogen atoms and the oxygen atoms of the coordinated water molecules play a role as donors and the oxygen atoms of the acetate anions play a role as acceptors. The distances N•••O and O•••O are in the [2.682 (5) -2.935 (6) Å] and [2.678 -2.712 Å] ranges, respectively. Fig. 6 represents the projection along c-axis at z = 0 (the acetate anions are omitted for clarity) and shows that the water oxygen atom O5 at the apex and the oxygen atom O2 of the organic ligand of neighboring complex molecules are linked through four O-H•••O hydrogen bonds forming a cyclic hydrogen-bonded with graph-set motif 𝑅 (8). The donor•••acceptor distances O5•••O2 of these hydrogen bonds are 2.638 and 2.771 Å. The crystal packing is also stabilized by intramolecular hydrogen bonds N3-H•••O5 and N6-H•••O1. The bond lengths and the angles in the organic molecules of (2) are similar to those of the organic molecules in (1), showing that the organic molecules in the two compounds have the same characteristics.

  ). Globally hydrophobic contacts involving C and Hc represent 38% of the surface. The pyrimidine aromatic cycle does not display any parallel stacking; the C•••C contacts are, however, enriched as four pyrimidines tend to form tetrahedral cages (Fig. 7-a). C-H•••π weak H-bonds are slightly under-represented (E=0.92) and occur mainly between the methyl group of acetic acid and the pyrimidine heterocycle. The cross contacts between hydrophobic (C, Hc) atoms and charged atoms are all disfavored, as atoms from each subset prefer to interact within their own group. The N•••Cu coordination bonds represent less than 6% of the contact surface but are, by far, the most enriched at E=7.0. The central short spikes in the fingerprint plots originate from the N•••Cu interactions. Comparatively the O•••Cu contacts (d > 2.67 Å ) are at longer distances than the N•••Cu ones (Table S2); they are much less abundant and only very slightly enriched. The Hirshfeld surface around the Cu(II) cation has a square shape in the CuN4 plane and is bifacial and elongated in the orthogonal direction (Fig. 7-b). The strong O•••Hn hydrogen bonds constitute the second largest interaction surface and have the second largest enrichment (E=3.0), while the weak O•••Hc hydrogen bonds are the fourth interaction in proportion and are slightly under-represented.

  22), as they amount to 37% of the interactions. All the cycles in the crystal are close to be coplanar which results in a high degree of cycle stacking, the C•••C and C•••N contacts are particularly enriched while N•••N is well represented. The contact in (2) with largest surface is constituted by the quite over-represented strong hydrogen bonds O•••Ho/n.

1 )

 1 -a,b for the different contact types. The graphs confirm that the two crystal packings are maintained primarily by three types of strongly attractive interactions with high enrichment, namely the coordination interactions Cu-N and Cu-O, followed by the O•••Hn strong hydrogen bonds. In both compounds, self-interactions between charged species (Cu 2+ , N -, O -, Hn + ) which are repulsive are mostly avoided, except for N•••N, due to the stacking of the heterocycles. The very unfavorable Cu•••Ho/n contact in compound (2) is due to proximity effects with the Cu•••O coordination with water. The Hc•••Hc and C•••C contacts have low electrostatic energy but appear enriched due to the hydrophobic effect. Globally the two compounds show similar features in Fig. 8.3.3. Quantum mechanical studies 3.3.1. Quantum mechanical study of compound (Quantum chemical calculations were performed from the crystal data with the DFT method at the 6-31+G* basis set, by using the Gaussian 09 software package[START_REF] Frisch | Gaussian 09,Revision B.01 Gaussian[END_REF]. The energies of the frontier orbitals of the compound (1) are listed in Table4.Fig. 9 clearly shows that the highest occupied orbital (HOMO) is located on one of the two crystallographic independent organic ligands, specifically on the C4 carbon atom bearing a methyl group and on the adjacent C3 carbon, while the lowest unoccupied orbital (LUMO) is mainly located on the other organic ligand. The large HOMO-LUMO gap of 5.53 eV implies high kinetic stability and low chemical reactivity[START_REF] Seth | [END_REF]46].The Molecular Electrostatic Potential (MEP) surface is depicted on Fig.S2. Blue and red colors indicate the positive and negative potentials, respectively. As it can be seen, the electrostatic potential maps are color-coded and are subdivided into many regions where those various colors are used to identify different potentials. Intermediate potentials are assigned to colors according to the following color spectrum: red < orange < yellow < green < blue. and confirms the above results with the most negative part near copper and the surrounding with positive charges.

( 1 )

 1 and (2) are shown in Figs.S4-aand S5-a. The two spectra are similar and the most representative and characteristic vibrational modes of these compounds can be compared to those of similar compounds[47][48][49]. The bands in the high wavenumber region on the two spectra, between 3500 and 2800 cm -1 , correspond to the stretching vibrations of the NH and CH groups for (1) and to the stretching vibrations of the NH, CH and OH groups for (2). These groups are interconnected by a system of hydrogen bonds in the crystal[50]. Bands in the 1700 -1400 cm -1 region of the two spectra correspond to the stretching vibrations of the acetate (OCO) groups and of the C=C, C=N and C=O groups of the organic ligands and to the N-H, C-H and O-H bending vibrations[51]. The bands in the range 1300 -1000 cm -1 can be attributed to the valence vibrations of the C-C, C-N and acetate C-O groups. Bands in the 1000 -500 cm -1 region correspond to the out of plane bending modes of C-H, N-H, C-C and C-N groups and to deformation mode of acetate (OCO) group[52,[START_REF] Scheinmann | i -Cu1-N1 90.89[END_REF]. The calculated IR spectra are given in Figs. S4-b and S5-b.

  Two new Cu(II) complexes, [Cu(ampymo)4](O2CMe)2 and [Cu(ampymo)2(H2O)3](O2CMe)2, were synthesized as single crystals at room temperature and characterized by various physicochemical methods. According to the X-ray structural results, the different components are held together in the packing arrangement by means of several N-H•••O hydrogen bonds forming three-dimensional networks. The enrichment ratio, derived from the Hirshfeld contact surface analysis, allows determining which types of contacts are over-or under-represented in the two crystal packings. The coordination Cu-N and Cu-O interactions are the most attractive from an electrostatic energy point of view and are both quite enriched in the two compounds. Along with the strong and over-represented O•••Ho/n hydrogen bonds, these interactions can be identified as the driving force in the packing formation. The hydrophobic atoms (Hc and C) tend to interact within their subgroup, with a very small electrostatic energy. In both compounds, C•••C interactions are quite over-represented, due to non-parallel and parallel stacking in compound (1) and (2) respectively. DFT calculations allow the attribution of the experimental IR bands at low frequencies. Further studies of biological evaluation and magnetic properties of these Cu(II) complexes are in progress.

Fig. 1 .

 1 Fig. 1. ORTEP of the structure of [Cu(ampymo)4](O2CMe)2 with displacement ellipsoids drawn

Fig. 2 .

 2 Fig. 2. Projection of the structure of [Cu(ampymo)4](O2CMe)2 along the c-axis.

Fig. 3 .

 3 Fig. 3. View of the 1D chain {[Cu(ampymo)4](O2CMe)2n along c-axis.

Fig. 4

 4 Fig. 4 ORTEP of the structure of the [Cu(ampymo)2(H2O)3](O2CMe)2 compound with

Fig. 5

 5 Fig. 5 Projection of the structure of [Cu(ampymo)2(H2O)3](O2CMe)2 along the a-axis.

Fig. 6

 6 Fig. 6 Projection of the structure of [Cu(ampymo)2(H2O)3](O2CMe)2along the c-axis. Acetates

Fig. 7

 7 Fig. 7 (a) View of four pyridine groups forming a tetrahedral cage. (b) View of the Hirshfeld

Fig. 8

 8 Fig. 8 Scatterplot of average electrostatic energy <Eelec> as a function of enrichment for the

Fig. 9

 9 Fig. 9 Frontier orbitals in [Cu(ampymo)4](O2CMe)2.

Fig. 10

 10 Fig. 10 Frontier orbitals in [Cu(ampymo)2(H2O)3](O2CMe)2.

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

Fig. 7

 7 Fig. 7

Fig. 8

 8 Fig. 8

Fig. 9

 9 Fig. 9

Fig. 10

 10 Fig. 10

( 1 )

 1 and (2). The second row shows the chemical content on the Hirshfeld surface. The middle part shows the proportions Cxy of the different contact types and the lower part shows the Exy enrichment ratios of contact types. Reciprocal contact type X…Y and Y…X are merged. The main contacts and the most enriched ones are highlighted in bold. The hydrophobic (C, Hc) atoms and charged (Cu, N, O, Hn) atoms have been regrouped in the Table.

(

Fig . S2

 . Fig. S2 MEP surface of [Cu(ampymo)4](O2CMe)2.

Fig . S3

 . Fig. S3 MEP surface of [Cu(ampymo)2(H2O)3](O2CMe)2.

Fig. S4

 S4 Fig. S4 (a) Experimental Infrared absorption spectrum. (b) Calculated infrared absorption

Fig . S5

 . Fig. S5 (a) Experimental Infrared absorption spectrum. (b) Calculated infrared absorption

Fig . S6

 . Fig. S6 Comparison between experimental and calculated IR frequencies (a) of

#

  ----------------------------------------------------------------------------# #UNIT CELL INFORMATION # #-

  0208(3) Uani 1 1 d . . . C2 C 0.90732(11) 0.27147(11) -0.02585(8) 0.0209(3) Uani 1 1 d . . . C3 C 0.98339(11) 0.25191(11) 0.02854(8) 0.0226(3) Uani 1 1 d . . . H3 H 1.0318 0.297 0.0365 0.027 Uiso 1 1 calc R . . C4 C 0.98560(11) 0.16896(11) 0.06823(8) 0.0226(3) Uani 1 1 d . . . C5 C 1.05948(12) 0.14027(13) 0.12740(9) 0.0306(4) Uani 1 1 d . . . H5A H 1.1098 0.1874 0.1281 0.046 Uiso 1 1 calc R . . H5B H 1.0856 0.0791 0.1119 0.046 Uiso 1 1 calc R . . H5C H 1.0314 0.1356 0.1809 0.046 Uiso 1 1 calc R . . C6 C 0.60109(11) 0.26124(11) -0.25409(8) 0.0228(3) Uani 1 1 d . . . C7 C 0.68614(11) 0.39885(11) -0.22152(8) 0.0239(3) Uani 1 1 d . . . C8 C 0.63180(11) 0.44962(11) -0.28005(9) 0.0250(3) Uani 1 1 d . . . H8 H 0.6426 0.5149 -0.2884 0.03 Uiso 1 1 calc R . . C9 C 0.56570(11) 0.40443(11) -0.32291(9) 0.0248(3) Uani 1 1 d . . . C10 C 0.50528(12) 0.44903(13) -0.38593(10) 0.0320(4) Uani 1 1 d . . . H10A H 0.5229 0.4245 -0.4388 0.048 Uiso 1 1 calc R . . H10B H 0.5143 0.5174 -0.385 0.048 Uiso 1 1 calc R . . H10C H 0.4391 0.4344 -0.3753 0.048 Uiso 1 1 calc R . . C11 C 0.86064(11) -0.11546(11) 0.13750(8) 0.0238(3) Uani 1 1 d . . . C12 C 0.88287(12) -0.21682(11) 0.15623(9) 0.0264(3) Uani 1 1 d . . . H12A H 0.9487 -0.2299 0.1427 0.04 Uiso 1 1 calc R . . H12B H 0.8417 -0.2579 0.1246 0.04 Uiso 1 1 calc R . . H12C H 0.8726 -0.2286 0.2135 0.04 Uiso 1 1 calc R . . N1 N 0.83971(9) 0.20261(9) -0.03765(7) 0.0200(3) Uani 1 1 d . . . N2 N 0.91497(9) 0.10433(9) 0.05539(7) 0.0220(3) Uani 1 1 d D . . H2 H 0.9160(13) 0.0521(7) 0.0814(9) 0.026 Uiso 1 1 d D . . N3 N 0.77711(10) 0.05707(10) -0.00535(8) 0.0266(3) Uani 1 1 d D . . H3A H 0.7312(9) 0.0695(11) -0.0369(9) 0.032 Uiso 1 1 d D . . H3B H 0.7778(11) 0.0064(8) 0.0227(9) 0.032 Uiso 1 1 d D . . N4 N 0.66641(9) 0.30454(9) -0.20953(7) 0.0226(3) Uani 1 1 d . . . N5 N 0.55114(10) 0.30961(10) -0.30995(7) 0.0243(3) Uani 1 1 d D . . H5 H 0.5100(10) 0.2793(12) -0.3377(10) 0.029 Uiso 1 1 d D . . N6 N 0.58389(10) 0.17004(9) -0.24466(8) 0.0257(3) Uani 1 1 d D . . H6A H 0.6140(11) 0.1397(10) -0.2083(8) 0.031 Uiso 1 1 d D . .

  in the case of methyl groups. NH and NH2 groups were refined with fixed NH and H•••H distance restraints so that the NH2 groups were free to rotate and tilt with respect to the C-N bonds. Bond length restraints were set in accord with following table:

		methane	methyl	N-H	N-H2	H•••H (NH2)
	d(X-H) (Å)	0.95	0.98	0.86	0.86	1.49

Table 3a

 3a Table 3 shows the proportions Cxy of the different contact types. The contact enrichment ratio Exy is obtained by comparing the actual contacts Cxy with those computed as if all contacts had the same probability to form. An enrichment ratio larger than unity indicates that the contact type is favored and over-represented in the crystal [43].

	The hydrophobic atoms C and Hc constitute 56% of the Hirshfeld surface and Hc•••Hc
	contacts have the highest proportion at 17.7% (

Table 4 .

 4 a) Energy values of frontier orbitals in compounds (1) and[START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF]

	atom	Cu	N	O	Hn	Hc	C
	Surface %	3.5	8.6	16.9	15.2	35.5	20.4
	Cu	0.0
	N	5.9	1.1			contacts	%
	O	2.1	0.8	0.2
	Hn	1.0	0.8	15.8	1.2
	Hc	0.1	3.5	11.3	7.9	17.7
	C	1.5	2.2	3.7	2.8	12.5	8.0
	Cu	0.00
	N	7.00	1.84			enrichment
	O	1.18	0.31	0.08
	Hn	0.59	0.35	3.00	0.49
	Hc	0.02	0.64	0.93	0.73	1.42
	C	0.73	0.74	0.56	0.48	0.92	2.13
				(b)
	atom	Cu	N	O	Hn/o	Hc	C
	surface %	5.4	5.9	22.1	22.6	28.8	15.2
	Cu	0.0
	N	4.2	0.3			contacts %
	O	8.3	1.0	0.0
	Hn/o	2.9	1.2	21.4	1.1
	Hc	1.3	0.8	8.3	12.6	12.7
	C	0.8	2.7	4.2	4.1	7.0	5.1
	Cu	0
	N	4.37	1.06			enrichment
	O	2.18	0.45	0.01
	H	0.73	0.51	2.23	0.23
	Hc	0.27	0.28	0.69	1.03	1.65
	C	0.32	1.79	0.67	0.63	0.87	2.44

Table S4 .

 S4 Nuclearities and coordination modes of 3d-metal complexes with ampymo ligand.

	Cu1-N1 data_nasr_1_0m 2.023(1)	C3-C4	1.352(2)	C7-N4	1.384(2)
	Cu1-N4 C1-N3 C1-N1 C1-N2 C2-O1 C2-N1 C2-C3 _audit_creation_date 2.026(1) 1.323(2) 1.338(2) 1.362(2) 1.250(2) 1.386(2) 1.438(2) Compounds _audit_creation_method _audit_conform_dict_name C4-N2 C4-C5 C6-N6 C6-N4 C6-N5 C7-O2 _audit_conform_dict_version [Cd(NO3)2(ampymo)2] [40] _audit_conform_dict_location ftp://ftp.iucr.org/pub/cif_core.dic 1.377(2) 1.497(2) 1.328(2) 1.339(2) 1.358(2) 1.253(2) nuclearity 2016-04-05T12:00:50-00:00 C7-C8 1.439(2) C8-C9 1.344(2) C9-N5 1.380(2) C9-C10 1.498(2) C11-O3 'WinGX routine CIF_UPDATE' coordination mode Metal (1) cif_core.dic Metal (2) 2.4 1.260(2) C11-O4 1.274(2) C11-C12 1.507(2) mononuclear eight--coordinated _publ_requested_category FM
	[CdCl2(ampymo)2] [41] #		mononuclear hexacoordinated -
	[Cu(ampymo)4]SO4.4H2O [42]		mononuclear tetracoordinated -
	[Cu(ampymo)4]Cl2.5H2O [36]		mononuclear tetracoordinated -
	[Cu(ampymo)4][Cu(H2O)5](ClO4)4.6H2O [37]	binuclear	tetracoordinated pentacoordinated
	N1-Cu1	2.004 (3)	O4-Cu1-O3	176.43 (15)
	N4-Cu1 [Cu2(ampymo)4(H2O)3](ClO4)4.½H2O [37] 2.018 (3)	O4-Cu1-N1 binuclear tetracoordinated hexacoordinated 88.13 (14)
	O3-Cu1	1.969 (3)	O3-Cu1-N1	92.42 (14)
	O4-Cu1	1.966 (3)	O4-Cu1-N4	90.99 (14)
	O5-Cu1	2.463(4) Å	O3-Cu1-N4	89.46 (14)
	[Ni(ampymo)4][Ni(H2O)6](ClO4)4.5H2O [37]	N1-Cu1-N4 binuclear tetracoordinated pentacoordinated 163.80 (15)

  ' O O 0.0106 0.006 'International Tables Vol C Tables 4.[START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF].6.8 and 6.1.1.4' Cu Cu 0.3201 1.2651 'International Tables Vol C Tables 4.[START_REF]_atom_site_aniso_label _atom_site_aniso_U_11 _atom_site_aniso_U_22 _atom_site_aniso_U_33 _atom_site_aniso_U_23 _atom_site_aniso_U_13 _atom_site_aniso_U_12[END_REF].6.8 and 6.1.1.4'

	loop_
	_atom_site_label
	_atom_site_type_symbol
	_atom_site_fract_x
	_atom_site_fract_y
	_atom_site_fract_z
	_atom_site_U_iso_or_equiv
	_atom_site_adp_type
	_atom_site_occupancy
	_atom_site_symmetry_multiplicity
	_atom_site_calc_flag
	_atom_site_refinement_flags
	_atom_site_disorder_assembly
	_atom_site_disorder_group
	C1 C 0.84323(11) 0.12179(11) 0.00335(8) 0.

Acknowledgment

We would like to thank Professor Jin-Xiao Mi from Xiamen University (People's Republic of Chin) for the X-ray data collections of compound (2).

Supplementary Information

Crystal Structure, Hirshfeld surface analysis and physicochemical studies


Mélanie Cavalier

Azza Ben Sedrine

Lea Thevenet

Nadine Crouzin

Janique Guiramand

Marie-Céleste De

Jesus Ferreira

Catherine Cohen-Solal

Gérard Barbanel

Michel Vignes
email: michel.vignes@umontpellier.fr

Jésus Ferreira

Disturbance of Metabotropic Glutamate Receptor-Mediated Long-Term Depression (mGlu-LTD) of Excitatory Synaptic Transmission in the Rat Hippocampus After Prenatal Immune Challenge

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Uncontrolled infection during pregnancy can increase the risk for the occurrence of neurodevelopmental disease in children, including autism or attention deficit hyperactivity disorder [START_REF] Atladóttir | Maternal infection requiring hospitalization during pregnancy and autism spectrum disorders[END_REF][START_REF] Brown | Prenatal infection and schizophrenia: a review of epidemiologic and translational studies[END_REF][START_REF] Hagberg | Inflammation during fetal and neonatal life: implications for neurologic and neuropsychiatric disease in children and adults[END_REF]. To some extent, rodent models of prenatal immune challenge recapitulate the behavioral deficits observed in the course of these diseases. Exposing dams to bacterial LPS results in the occurrence of cognitive, social, and behavioral deficits in the offspring [START_REF] Boksa | Effects of prenatal infection on brain development and behavior: a review of findings from animal models[END_REF]. Such deficits are reminiscent of 'psychotic-like' conditions in humans. For instance, probing prepulse inhibition reflex, a hallmark of 'psychotic-like' estate, demonstrated that prenatal LPS could reduce the effectiveness of this reflex [START_REF] Boksa | Effects of prenatal infection on brain development and behavior: a review of findings from animal models[END_REF]. These cognitive/behavioral deficits are underlined by severe impairments, even loss, of synaptic plasticity as evidenced by the inability to trigger long-term potentiation (LTP) or long-term depression (LTD) of excitatory synaptic transmission in the hippocampi from these animals [START_REF] Lanté | Neurodevelopmental damage after prenatal infection: role of oxidative stress in the fetal brain Free[END_REF][START_REF] Lanté | Late N-acetylcysteine treatment prevents the deficits induced in the offspring of dams exposed to an immune stress during gestation[END_REF][START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF]. As we reported previously, these changes may originate from various molecular/cellular mechanisms including a disruption of the excitatory/inhibitory balance. Firstly, we have evidenced a reduction in GABAergic synaptic transmission. Interestingly, reinstating GABA with type 1 GABA transporter (GAT-1) blocker, i.e. tiagabine, restored LTD [START_REF] Batista Novais | Tiagabine improves hippocampal long-term depression in rat pups subjected to prenatal inflammation[END_REF]. Functional deficits of inhibitory transmission are matched with reduced GABAergic interneuron occurrence in CA3 hippocampal area [START_REF] Batista Novais | Tiagabine improves hippocampal long-term depression in rat pups subjected to prenatal inflammation[END_REF]. Secondly, synaptic NMDA receptor function is decreased after prenatal LPS challenge [START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF]. In the hippocampus, LTP and LTD are largely dependent on the activation of postsynaptic NMDA receptors [START_REF] Volianskis | Long-term potentiation and the role of N-methyl-D-aspartate receptors[END_REF]. The loss of these phenomena observed in LPS animals is time matched with the decrease of NMDA receptor expression in hippocampal neurons [START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF]. Nevertheless, the activity of other glutamate receptors may also be disturbed after prenatal stress leading to the occurrence of aberrant forms of synaptic plasticity. This seems to be the case of phospholipase C-coupled metabotropic glutamate receptors (mGlu1/5). Indeed, we observed that a form of LTD induced by paired-pulse afferent stimulation, which involves mGlu1/5 activation [10], was transiently converted into an LTP after prenatal stress. The pharmacological blockade of mGlu1 or mGlu5 receptors leads to the inhibition of this 'aberrant' LTP. By contrast, this 'aberrant' LTP was completely independent of NMDA receptor activation [START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF].

The direct activation of mGlu1/5 with selective agonist as dihydroxyphenylglycine (DHPG) leads to LTD (mGlu-LTD) in the rodent hippocampus [10][START_REF] Fitzjohn | DHPG-induced LTD in area CA1 of juvenile rat hippocampus; characterisation and sensitivity to novel mGlu receptor antagonists[END_REF][START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF][START_REF] Collingridge | Long-term depression in the CNS[END_REF]. This form of plasticity has received much attention as it is very sensitive to the occurrence of pathological states [START_REF] Collingridge | Long-term depression in the CNS[END_REF]. Indeed, animal models of multiple brain pathologies, including FMRX-linked mental retardation [START_REF] Bear | The mGluR theory of fragile X mental retardation[END_REF], drug addiction [START_REF] Knackstedt | The role of ventral and dorsal striatum mGluR5 in relapse to cocaine-seeking and extinction learning[END_REF][START_REF] Hoffmann | Long-lasting impairment of mGluR5-activated intracellular pathways in the striatum after withdrawal of cocaine self-administration[END_REF], autism [START_REF] D'antoni | Dysregulation of group-I metabotropic glutamate (mGlu) receptor mediated signalling in disorders associated with Intellectual Disability and Autism[END_REF] are displaying alterations of this form of plasticity. We have thus decided to test whether the mGlu-LTD in the CA1 area of the hippocampus was altered after immune prenatal stress and whether these putative alterations shared similarities with those unveiled for electrically-induced LTD.

Materials and methods

Drugs

(RS)-3,5-Dihydroxyphenylglycine (DHPG), D-2-amino-5-phosphonovalerate (D-AP5), 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), dizolcipine (MK-801), (S)-(+)-α-Amino-4-carboxy-2methylbenzeneacetic acid (LY 367385) and tiagabine hydrochloride were from Tocris (BioTechne). All other drugs were from Sigma-Aldrich (Merck).

Animal groups

All experiments were carried out in accordance with the European Community Council Directive of November 24, 1986 (86/609/ECC). This study was approved by the local branch of the 'Comit National de Reflexion Ethique sur l'Experimentation Animale' (CNREEA nu 36) under the reference CEEA-LR-12099. All efforts were made to minimize animal suffering and to reduce the number of rats used. Pregnant Sprague-Dawley rats (Centre d'Elevage Depré, St Doulchard, France) were used throughout this study. Animals were housed individually and randomly assigned to either of these experimental groups: (i) a group of control animals born to saline-injected dams (2 ml.kg -1 ip at the 19 th day of gestation GD19) (SAL rats) or (ii) a group of animals born to LPS-treated dams (500 mg.kg -1 ip at GD19) (LPS rats). LPS (from Escherichia coli, serotype O55:B5) was obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). After birth, the size of the litters was limited to ten pups. All animals were maintained on a 12-hr light/dark cycle with food and water ad libitum.

Electrophysiological recordings with micro-electrode array

Experiments were carried out on hippocampal slices (300 μm thickness) obtained from 11 to 180 day-old male offspring. After decapitation, brains were quickly dissected and placed in ice-cold buffer comprising 124 mM NaCl, 3.5 mM KCl, 25 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 1 mM CaCl 2 , 2 mM MgSO 4 , 10 mM D-glucose, and 10 mM HEPES (bubbled with O 2 /CO 2 , 95/5). Slices were then cut with a Vibratome (VT1000S; Leica, France) and maintained at RT for at least 1 h in the same buffer supplemented with 1 mM CaCl 2 . This supplemented buffer -also named extracellular medium -was used for further recordings. For electrophysiological recordings, slices were transferred to a MEA (MEA60; Multi Channel Systems, Reutlingen, Germany) continually superfused with the above described extracellular medium (flow rate 2 ml.min -1 ) and kept at 32°C. Unless otherwise stated, this medium included AP5 (50 µM) a NMDA receptors selective antagonist. Drugs were directly applied in this superfusion. MEA was positioned on the platform of an inverted microscope (DMLFS, Leica). MEAs comprised 60 extracellular electrodes [START_REF] Lanté | Late N-acetylcysteine treatment prevents the deficits induced in the offspring of dams exposed to an immune stress during gestation[END_REF]. The inter-electrode distance was 200 μm. Each individual electrode from the array could be used either as a recording or as a stimulatory electrode. A nylon mesh was positioned above the slice to obtain a satisfactory electrical contact between the surface of the slice and the electrode array. Stimulation was achieved with an external stimulator (STG-1004; Multi Channel Systems) by applying biphasic current pulses to one electrode of the array located in the Schaffer Collateral pathway of the hippocampus. Stimulation intensity (60 to 300 μA) and duration (70 to 200 μs) were adapted to avoid multiphasic responses due to an excessive stimulation [START_REF] Heuschkel | A three-dimensional multielectrode array for multi-site stimulation and recording in acute brain slices[END_REF]. Field excitatory postsynaptic potentials (fEPSPs) could then be recorded in the stratum radiatum of the CA1 subfield by all the remaining electrodes of the array at the same time. The fEPSP recorded by the electrodes adjacent to the stimulating one were used for further analysis (see [START_REF] Lanté | Low-frequency stimulation induces a new form of LTP, metabotropic glutamate (mGlu5) receptor-and PKAdependent, in the CA1 area of the rat hippocampus[END_REF]). Signals were recorded and analyzed (MC Rack; Multi Channel Systems). Baseline synaptic signals were evoked using a 0.066 Hz frequency. Slices displaying epileptic-like activity were discarded. The magnitude of the effects on synaptic transmission was determined by measuring slopes of fEPSPs, which were modified in a similar way to fEPSP amplitudes, as reported by others also using MEA recordings [START_REF] Kopanitsa | Recording long-term potentiation of synaptic transmission by three-dimensional multi-electrode arrays[END_REF]. Data are presented as means ± SEM on graphs plotting pooled data. Each individual ('n') corresponds to an experiment performed on a slice from an individual animal. Pooled data were obtained from animals originating from at least three independent litters in order to avoid possible litter-specific effects.

Statistical analyses

Statistical analyses were performed using Sigma Stat software. Single curves were analyzed using ANOVA on repeated measures, followed by Holm-Sidak method for multiple comparisons. Curve comparisons were performed using two-way ANOVA, followed by Holm-Sidak method for multiple comparisons. Means of plateau fEPSP amplitudes were compared using ANOVA followed by Fisher LSD method, or t-test when comparing two groups.

Results

In a first set of experiments (Figure 1), hippocampal slices from rats subjected to prenatal exposure either to saline (SAL-rats) or to LPS (LPS-rats) were treated with DHPG (100 µM). In the age range tested, i.e. 16-to 180-day-old rats, mGlu-LTD (LTD amplitude of 14 ± 6% of basal) was triggered by DHPG in SAL rats (n = 17). By contrast, an enduring potentiation of excitatory transmission (LTP amplitude of 52 ± 15% of basal) was observed consistently in LPS rats after DHPG application (n = 19). Therefore, it appears that mGlu-LTD is converted in an 'mGlu-LTP' after prenatal exposure to LPS.

All further experiments were carried out in the presence of AP5 (50 µM), a NMDA receptor antagonist, in order to eliminate NMDA receptor contribution to DHPG-induced actions and thus to evaluate mGlu1/5 receptor-mediated effects more specifically on synaptic transmission. In control animals (SAL, n = 20), mGlu-LTD was substantially lost under NMDA receptor blockade (Figure 2). In fact when comparing the whole curves obtained either in the presence or the absence of AP5 using two-way ANOVA, we found that a statistical significance could be observed 30 min post DHPG (p=0.05) which became stronger 40 min after DHPG (p=0.03). This could be attributable to an increasing recruitment with time of NMDA receptors for the expression of mGlu-LTD.

By contrast, mGlu-LTP in LPS animals (n = 29) was completely insensitive to such application of AP5 (Figure 2A andB). When separating data obtained in rats the age range 11-to 25-day-old from those obtained rats older than 25-day-old (up to 180-day-old), it appears that this DHPG-induced LTP was consistently observed throughout development (Figure 3).

The dependence of mGlu-LTD on mGlu1 or mGlu5 activation was further studied in 11-25 day-old SAL rats with selective antagonists of these receptors i.e. LY367385 (100µM, n = 7) [21] and MPEP (10µM, n = 5) [START_REF] Gasparini | 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist[END_REF], respectively. Both antagonists could block efficiently and individually the induction of mGlu-LTD (Figure 4A andB). We have next examined whether mGlu1 or mGlu5 receptor blockade could also prevent mGlu-LTP occurrence in LPS rats. LY367385 (100µM, n = 9) and MPEP (10µM, n = 6) were both able to inhibit mGlu-LTP occurrence (Figure 5A). The GAT-1 blocker tiagabine (20µM, n = 5) was further tested on the DHPG-induced synaptic plasticity in LPS animals. Indeed, we have previously demonstrated that prenatal LPS challenge induced a deficit in the inhibitory GABAmediated synaptic transmission [START_REF] Batista Novais | Tiagabine improves hippocampal long-term depression in rat pups subjected to prenatal inflammation[END_REF]. The application of tiagabine led to the inhibition of mGlu-LTP occurrence in rats subjected to prenatal stress (Figure 5B).

Finally, we have examined whether the type one cannabinoid receptor (CB1R)-associated LTD was also disrupted after prenatal stress. Indeed, CB1R and mGlu1/5 receptor activations are often associated in the regulation of synaptic plasticity, especially LTD in the hippocampus [START_REF] Izumi | NMDA receptors, mGluR5, and endocannabinoids are involved in a cascade leading to hippocampal long-term depression[END_REF]. The application of the selective CB1R agonist WIN55,212-2 (10µM) resulted in a long-lasting synaptic depression in both SAL and LPS rats. LTD amplitudes reached 44 ± 6% (n = 7) and 40 ± 8% (n = 5) in SAL and LPS rats, respectively. These magnitudes of LTD level were not significantly different from each other.

Discussion

We find here that DHPG-induces an enduring potentiation of the excitatory synaptic transmission in the CA1 area of the hippocampus of rats challenged in utero with LPS. In fact, this form of plasticity, which could be named 'mGlu-LTP', recapitulates the features of the synaptic plasticity induced electrically with paired-pulse low frequency stimuli (so called 'ppulse-LFS-LTP') in these animals [START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF]. Indeed, we observe that mGlu1/5 activation triggers LTP instead of LTD in LPS rats. This mGlu-LTP occurs independently of NMDA receptor activation, since it is not affected by AP5 application. In addition, similar to ppulse-LFS-LTP, it also requires mGlu1 and mGlu5 activation. Therefore, there is a clear dysfunction of the pathways activated by mGlu1/5 in LPS animals as shown by their direct activation by a selective agonist. Nevertheless, there is one noticeable difference in their timing of appearance between mGlu-LTP and the ppulse-LFS-LTP obtained by afferent stimulation: mGlu-LTP in LPS animals lasts over 25 day-old of age while ppulse-LTP was generally extinguished after this age [START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF]. This suggests that mGlu1/5 dysfunction is enduring but other mechanisms should take place with development to limit mGlu-LTP as revealed by the loss of ppulse-LTP induced by electrical stimulation after 25 day-old of age.

There are many molecular mechanisms which could account for the disruption of mGlu-LTD in LPS animals. Our data agree with reduction in GABA-mediated inhibitory transmission and/or in NMDA receptor expression. Firstly, GABA accumulation via GAT-1 blockade with tiagabine inhibits DHPGinduced fEPSP potentiation in LPS rats. This indicates that mGlu-LTP could be partially attributed to a reduced inhibitory transmission, which leads to uncontrolled excitation. We have reported that electrically-induced LTD could be rescued in LPS animals by tiagabine [START_REF] Batista Novais | Tiagabine improves hippocampal long-term depression in rat pups subjected to prenatal inflammation[END_REF]. Secondly, the reduction in NMDA receptors at synaptic sites could also be involved in this switch of plasticity. Indeed, blocking NMDA receptors results in the inhibition of mGlu-LTD in SAL animals, while mGlu-LTP is unaffected. Therefore, the presence of NMDA receptors could limit mGlu1/5 receptor activity, as shown previously by interaction studies between NMDA and mGlu5 receptors [START_REF] Perroy | Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors[END_REF]. The presence of NMDA and mGlu5 receptors at synapses is regulated by intracellular scaffold proteins including Shank and Homer. Moreover, mutations in Shank genes have been associated with forms of autism [START_REF] Guilmatre | The emerging role of SHANK genes in neuropsychiatric disorders[END_REF][START_REF] Iasevoli | The glutamatergic aspects of schizophrenia molecular pathophysiology: role of the postsynaptic density, and implications for treatment[END_REF]. Therefore, decreased expression of Shank could also account for the impairment of synaptic plasticity after prenatal stress. Alternatively, disruption of mGlu-LTD could also account for decreased functional interaction with cannabinoid CB1 receptors. Indeed, mGlu5 and CB1R agonists seem to activate common intracellular pathways leading to LTD in the CA1 area of the hippocampus [START_REF] Izumi | NMDA receptors, mGluR5, and endocannabinoids are involved in a cascade leading to hippocampal long-term depression[END_REF]. In our hands, CB1R-mediated LTD appears to be rather unaffected by prenatal exposure to LPS. This suggests that this prenatal stress leads to some rather specific alterations of the mGlu1/5 receptors-associated pathways.

The functional relevance of this switch in synaptic plasticity in LPS animals, i.e. from LTD to LTP, obtained both electrically [START_REF] Escobar | Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge[END_REF] or through the direct activation of mGlu1/5 remains to be elucidated at the behavioral level. In this line, mGlu1/5 activation is known to contribute to neurophysiological adaptations occurring during several forms of learning. For instance, mGlu5 activation contributes to the LTD evidenced in the hippocampus during spatial object recognition [START_REF] Goh | Endogenous hippocampal LTD that is enabled by spatial object recognition requires activation of NMDA receptors and the metabotropic glutamate receptor, mGlu5[END_REF]. Therefore, such a switch in plasticity could be deleterious for correct learning acquisition. In support of this hypothesis, we have reported that spatial learning in the Morris' water-maze was greatly impaired in male rats subjected to such prenatal immune stress [START_REF] Lanté | Neurodevelopmental damage after prenatal infection: role of oxidative stress in the fetal brain Free[END_REF].

The mGlu-LTD appears to be dysregulated in a wide array of animal models recapitulating mental retardation and autism [START_REF] D'antoni | Dysregulation of group-I metabotropic glutamate (mGlu) receptor mediated signalling in disorders associated with Intellectual Disability and Autism[END_REF] and drug addiction [START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF]. It thus provides a robust parameter to probe synaptic deficits. According to the model considered, this form of LTD can be either enhanced or inhibited. Interestingly molecules modulating mGlu5, especially positive allosteric modulators, are exhibiting beneficial effects in alleviating the symptoms observed in different models of psychosis as those obtained by NMDA receptor blockade with MK-801 [START_REF] Rook | Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents[END_REF] or by Shank-3 genetic deletion [START_REF] Vicidomini | Pharmacological enhancement of mGlu5 receptors rescues behavioral deficits in SHANK3 knock-out mice[END_REF]. Therefore, the direct modulation of mGlu1/5 receptors is becoming a promising route in the prospect of curing neurodevelopmental diseases. In addition, the dysfunction of mGlu1/5 receptors could also be attributed to some defects in receptor desensitizing mechanisms involving the GRK/Arrestin pathway. In this line, it has recently been shown that neuroinflammation induced by LPS exposure could sensitize mGlu receptor-associated responses through the downregulation of GRK2 protein [START_REF] Degos | G protein-coupled receptor kinase 2 and group I metabotropic glutamate receptors mediate inflammation-induced sensitization to excitotoxic neurodegeneration[END_REF]. Thus the dysfunction of mGlu1/5 receptors that we observe here after prenatal stress could be due not only to alterations in receptor activation but also to modified mechanisms of receptor trafficking regulation in neurons.

Legends for the figures

Figure 1: DHPG-evoked plasticity is altered in LPS-rats. DHPG (100µM) was applied for 10min on hippocampal slices from either control (SAL) or LPS rats aged 16 day-to 6 month-old. fEPSP were recorded as described in the material and method section. A: While a significant long-term depression of fEPSP was observed in SAL rats (n=17), a significant LTP was induced in LPS rats (n=19). Sample traces of fEPSP recorded either before (1 and 3) or 30min (2 and 4) after the application of DHPG, in SAL (1 and 2) or LPS (3 and 4) rats, respectively are shown (top of the graph). * p<0.05 versus basal fEPSP, using one way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons; § p<0.05 for comparison of LPS versus SAL, using two way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons. B: Developmental profile (from 16 day old to 1 year old) of DHPG-induced plasticity in SAL or LPS rats. Amplitudes of fEPSP were determined either 30 or 35 min following DHPG application for SAL or LPS rats, respectively.

Figure 2: D-AP5 prevents the DHPG-induced LTD in SAL rats but not the DHPG-induced LTP in LPS rats. A: D-AP5 (50µM) was applied throughout fEPSP recording of hippocampal slices from either control (SAL) or LPS rats aged 11 day-to 3 month-old. Per se D-AP5 did not modify fEPSP amplitude. DHPG was applied for 10min. # p<0.05 versus basal fEPSP, using one way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons. N.S = non-significant. § p<0.05 for comparison of LPS versus SAL, using two way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons. B: Developmental profile of DHPG-induced plasticity in SAL or LPS rats in the presence of D-AP5. C: Summary histogram: Amplitudes of LTD or LTP were determined on a-15min plateau either 30 or 35 min following the DHPG stimulation for SAL or LPS rats, respectively. Values were compared to control fEPSP baseline using t-test: **p<0.01; ***p<0.001.

Figure 3: Similar DHPG-induced LTP is observed in LPS rats aged either under or over 25 day old. A: D-AP5 (50µM) was applied throughout fEPSP recording of LPS rat hippocampal slices and DHPG was applied for 10min. Data were separated in two groups according to the age of the animal, i.e. rats aged between 11 and 25 day old (n=16), and rat aged over 25 day old (n=13). B: Amplitudes of LTP were determined on a-15min plateau, 35 min following the DHPG stimulation. Values were compared to control baseline using t-test: *p<0.05; **p<0.01; ***p<0.001. Comparisons were also performed between the groups using ANOVA followed by Fisher LSD method; any significant difference was observed.

100µM) was applied 10 min prior to DHPG which was added for a further 10min on SAL rat hippocampal slices. * p<0.05 versus basal fEPSP, using one way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons; N.S = non-significant. § p<0.05 for comparison of values obtained either in the absence or the presence of antagonist, using two way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons. B: Mean amplitudes of fEPSP were determined on a-15min plateau, 30 min following the DHPG stimulation. Values were compared to control baseline using t-test: *p<0.05. Comparison between values obtained either in the absence or the presence of the antagonist was performed using ANOVA followed by Fisher LSD method: #p<0.05. A: D-AP5 (50µM) was applied throughout fEPSP recording of hippocampal slices from LPS rats. MPEP (10µM) or LY367385 (100µM) was applied 10 min prior to DHPG which was added for a further 10min. B: Tiagabine inhibits the DHPG-induced LTP in LPS rats in the presence of D-AP5 or MK801. D-AP5 (50µM) or MK801 (10µM) was applied throughout fEPSP recording of hippocampal slices from LPS rats. Tiagabine (20µM) was applied 10 min prior to DHPG which was added for a further 10min. * p<0.05 versus basal fEPSP, using one way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons; NS = non-significant; § p<0.05 for comparison of recording either in the absence or in the presence of drug, using two way ANOVA followed by Holm-Sidak method for multiple comparisons.

Figure 4 :

 4 Figure4: MPEP and LY367385 inhibit the DHPG-induced LTD in SAL rats. A: Either MPEP (10µM) or LY367385 (100µM) was applied 10 min prior to DHPG which was added for a further 10min on SAL rat hippocampal slices. * p<0.05 versus basal fEPSP, using one way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons; N.S = non-significant. § p<0.05 for

Figure 5 :

 5 Figure 5: A: MPEP and LY367385 inhibit the DHPG-induced LTP in LPS rats in the presence of D-AP5.A: D-AP5 (50µM) was applied throughout fEPSP recording of hippocampal slices from LPS rats. MPEP (10µM) or LY367385 (100µM) was applied 10 min prior to DHPG which was added for a further 10min. B: Tiagabine inhibits the DHPG-induced LTP in LPS rats in the presence of D-AP5 or MK801. D-AP5 (50µM) or MK801 (10µM) was applied throughout fEPSP recording of hippocampal slices from LPS rats. Tiagabine (20µM) was applied 10 min prior to DHPG which was added for a further 10min. * p<0.05 versus basal fEPSP, using one way ANOVA for repeated measures followed by Holm-Sidak method for multiple comparisons; NS = non-significant; § p<0.05 for comparison of recording either in the absence or in the presence of drug, using two way ANOVA followed by Holm-Sidak method for multiple comparisons.

Figure 6 :

 6 Figure6: WIN55-212,2 induces LTD in both SAL and LPS-rats. WIN55-212,2 (10µM) was applied for 10min on hippocampal slices from either SAL (n=7) or LPS (n=5) rats. * p<0.05 versus basal fEPSP, using one way ANOVA followed by Holm-Sidak method for multiple comparisons.