Jean-Michel Camus

Dolores Morales

Jacques Andrieu

Philippe Richard

Rinaldo Poli

Pierre Braunstein

Frédéric Naud

Half-sandwich complexes of molybdenum-(III), -(IV) and -(V) with P-O and P-N bifunctional ligands Ph2PCH2X (X = 2-oxazolinyl, or C(O)NPh2)

The reaction of the ligands Ph2PCH2X (X = 2-oxazolinyl, I; or C(O)NPh2, II) with the half-sandwich molybdenum(III) precursors [Mo(η-C5R5)(µ-Cl)2]2 (R = H or Me) has been investigated. Ligand I reacts with both complexes to form the corresponding adducts [Mo(η-C5R5)Cl2(Ph2PCH2C3H4NO)] (R=H, 1; or Me, 2). The reaction between I and [MoCp*Cl4] (Cp*= η-C5Me5) affords [MoCp*Cl4(Ph2PCH2C3H4NOκ 1 P)] as a kinetic isomer, which then transforms quantitatively to [MoCp*Cl3(Ph2PCH2C3H4NO-κ 2 P,N)] + Cl - , 3. Ligand II reacts with [MoCp(µ-Cl)2]2 (Cp = η-C5H5) to afford the adduct [CpMoCl2{Ph2PCH2C(O)NPh2-κ 2 P,O}], 4, as an equilibrium mixture of two isomers. Longer reaction times in CH2Cl2 lead to the molybdenum(IV) by-product [MoCpCl3{Ph2PCH2C(O)NPh2-κ 2 P,O}], 5. While ligand II does not react with [MoCp*(µ-Cl)2]2, it does so in the presence of 1 equivalent of AgBF4 to afford [MoCp*Cl2{Ph2PCH2C(O)NPh2-κ 2 P,O}][BF4], 6. Further reaction with CH2Cl2 leads to [MoCp*Cl3{Ph2PCH2C(O)-NPh2-κ 2 P,O}][BF4], 7. The direct reaction between [MoCp*Cl4] and ligand II affords [MoCp*Cl4{Ph2PCH2C(O)-NPh2-κ 1 P}] 8 as a kinetic isomer which then slowly establishes a solventdependent equilibrium with the ionic isomer [MoCp*Cl3{Ph2PCH2C(O)NPh2-κ 2 P,O}] + Cl -. The solid state structure of compounds 5 and 7 has been determined by single crystal X-ray diffraction. The chemical, spectroscopic, and electrochemical data indicate that ligand II has a greater hemilabile character than ligand I.

Introduction

Molybdenum(III) half-sandwich complexes are known as mononuclear

[MoCpX2L2],

[MoCpXL3] + and [MoCpL4] 2+ and as dinuclear [Mo 2Cp2X4(µ-X)] -, [MoCp(µ-X)2]2, [Mo2Cp2L2-(µ-X)3] + and [{MoCpL2(µ-X)}2] 2+ and have shown compati-bility with a large array of ligands. Examples with X = halide, 1-5 hydrosulfido, alkane-and aryenethiolato, 6-9 amido, 10 dialkylphosphido, 11 phenyldiazo, 12 isocyanato, 13 azido, 14 alkyl, 15 and hydroborate units, 16 L=phosphine, 17-21 CO

22-26

isocyanides 27, 28 and MeCN, 27,29,30 L2 =diphosphines, 31-33 phosphine sulfides 34 and dienes, [35][36][37][38] and XL=phosphine-thiolato 34 and allyl 37-40 have been reported. Our own work in this area has been summarized in review articles. [41][42][43][44] Interest in these compounds ranges from modelling the activity of nitrogenase enzymes and hydrodesulfurization catalysts to the quest for new poly-merization catalysts. The possibility to link bifunctional ligands having a soft and a hard donor atom may open new opportunities in terms of chemical reactivity and catalysis by virtue of the hemilability concept and in terms of establishing new building blocks for the assembly of heterobimetallic architectures. 45, 46 We have extended the chemistry of this class of compounds to the bifunctional ligands 2-(diphenylphosphino-methyl)oxazoline I and diphenylphosphino-N,N-diphenyl-acetamide II, and report here our results, which also involve the generation of a few related species in the oxidation states IV and V. The complexes obtained are the first examples of this class where co-ordination by O-and N-based (except for the above mentioned MeCN complexes) L ligands is demonstrated.

Experimental

General

All reactions involving air-and moisture-sensitive organo-metallic compounds were carried out in a Jacomex glove-box or by the use of standard Schlenk techniques under an argon atmosphere. The solvents were dried by conventional methods (THF, toluene and pentane from sodium-benzophenone and CH2Cl2 from P4O10) and distilled under argon prior to use. 1 H NMR measurements were carried out on a Bruker AC200 spectrometer. The peak positions are reported with positive shifts in ppm downfield of TMS as calculated from the residual solvent peaks. The IR spectra were recorded on a Bruker IFS 66V spectrophotometer with NaCl optics. EPR measurements were carried out at the X-band microwave frequency on a Bruker ESP300 spectrometer. The spectrometer frequency was calibrated with diphenylpicrylhydrazyl (DPPH, g = 2.0037). Cyclic voltamograms were recorded with an EG&G 362 poten-tiostat connected to a Macintosh computer through MacLab hardware/software. The electrochemical cell was fitted with a Ag-AgCl reference electrode, a platinum disk working electrode and a platinum wire counterelectrode. [Bu4N]PF6 (ca. 0.1 M) was used as supporting electrolyte. All potentials are reported relative to the ferrocene standard, which was added to each solution and measured at the end of the experiments. The magnetic susceptibility measurements were carried out at room temperature with a Johnson Matthey magnetic balance which operates with a modified Gouy method, whereby the effect of the sample on the weight of the magnet, rather than the effect of the magnet on the weight of the sample, is used to retrieve the susceptibility information. The elemental analyses were carried out by the analytical service of the Laboratoire de Synthèse et d'Electrosynthèse Organométallique with a Fisons EA 1108 apparatus. [START_REF] Poli | Synthetic Methods of Organometallic and Inorganic Chemistry[END_REF] and the ligands 2-(diphenylphosphinomethyl)oxazoline 45 and diphenylphosphino-N,N-diphenylacetamide [START_REF] Andrieu | [END_REF] were prepared as described in the literature.

Compounds [MoCp*Cl4], 47 [MoCp*(µ-Cl)2]2, 48 [MoCp- (µ-Cl)2]2, 49,

Preparations

[MoCpCl2(Ph2PCH2C3H4NO)], 1. A solution of ligand I (0.160 g, 0.59 mmol) in 5 mL of CH2Cl2 was added to a suspension of [MoCp(µ-Cl)2]2 (0.137 g, 0.29 mmol) in 10 mL of CH2Cl2 at room temperature. The insoluble starting material disappeared after stirring for 30 min and the resulting brown solution was filtered through Celite. Addition of 20 mL of pentane gave complex 1 as a microcrystalline grey solid, which was filtered off, washed with pentane ( Carrying out the reaction between [MoCp(µ-Cl)2]2 (0.370 g, 0.80 mmol) and ligand I (0.370 g, 1.59 mmol) in THF as solvent (20 mL), led to the precipitation of compound 1 from the reaction mixture. Yield: 0.492 g, 61%. Dichloromethane solutions of this product exhibited an EPR spectrum identical to that of the product obtained directly from CH2Cl2.

[MoCp*Cl2(Ph2PCH2C3H4NO)], 2. To a solution of [MoCp*(µ-Cl)2]2 (0.319 g, 0.52 mmol) in 10 mL of CH2Cl2 was added a solution of I (0.284 g, 1.059 mmol) in 5 mL of CH2Cl2 at room temperature, followed by stirring for 3 h. The resulting green-brown solution was evaporated to dryness, the solid extracted with warm toluene, and the solution was filtered through Celite and cooled to room temperature to give complex 2 as a microcrystalline green solid. The product was isolated by filtration, washed with pentane ( An analogous preparative procedure was carried out from II (0.590 g, 1.49 mmol) and [MoCp(µ-Cl)2]2 (0.346 g, 0.75 mmol) in 25 mL of CH2Cl2, a difference being that the mixture was stirred overnight. After filtration through Celite, the filtrate was evaporated to dryness and the residue extracted with hot toluene (30 mL) and filtered. The precipitate which formed upon cooling was separated by decanting off the mother-liquor, dried and redissolved in CH2Cl2 (10 mL). Diffusion of a pentane layer (20 mL) afforded crystals of complex 5• CH2Cl2 (ca. 0.030 g) over 1 week, one of which was used for the X-ray analysis. Calc

[MoCp*Cl2{Ph2PCH2C(O)NPh2-κ 2 P,O}]BF4, 6.

A solution of II (0.327 g, 0.82 mmol) in 5 mL of CH2Cl2 was added to a solution of [MoCp*(µ-Cl)2]2 (0.250 g, 0.41 mmol) in 15 mL of CH2Cl2 at room temperature, followed by stirring for 3 h. NMR and EPR monitoring showed the absence of any interaction between the two reagents. The solution was then added to AgBF4 (0.161 g, 0.82 mmol) resulting in an immediate color change and the precipitation of a dark solid. After 30 min of stirring at room temperature the resulting mixture was filtered through Celite and the red-brown filtrate evaporated to dryness. Extraction of the residue with warm toluene (20 mL), followed by filtration and cooling to room temperature, gave complex 6 as red crystals (yield 0.17 g, 26%). Calc. for C36H37BCl2F4-MoNOP: C, 55.13; H, 4.75. Found: C, 55.19; H, 4.42%. IR (CH2Cl2): ν(CO) 1540; ν(BF4) 1061 cm -1 . 1 H NMR (CDCl3, 297 K): δ 16.0 (br s, w1/2 = 70, Ph), 12.3 (br s, w 1/2 =130, Ph), ca. 9.8 (sh, w1/2 = 100, Ph), 9.7 (br s, w1/2 = 35, Ph), 9.3 (br s, w1/2 = 30, Ph), 8.3-8.1 (br m, total w1/2 = 75, Ph), 7.4 (s, overlaps with solvent peak, Ph), 6.8 (br s, w1/2 = 30, Ph), 6.3 (br s, w1/2 = 110, Ph), 3.6 (br s, 15 H, w1/2 = 290, Cp*), -9.0 (br s, 1 H, w 1/2 = 300, CH2) and -65.8 (br s, 1 H, w1/2 = 400 Hz, CH2).

Recrystallization of complex 6 (0.070 g) by diffusion of a toluene layer (25 mL) into a CH2Cl2 solution (10 mL) over 1 week afforded crystals of [MoCp*Cl3-{Ph2PCH2C(O)NPh2-κ 2 P,O}]BF4, 7, which were used for the X-ray analysis. EPR (CH2Cl2): doublet with molybdenum satellites, g=1.979, aMo =44.0, a P =25.2 G.

[MoCp*Cl4{Ph2PCH2C(O)NPh2}], 8. A solution of II (0.045 g, 0.11 mmol) in 5 mL of THF was added at room temperature to a suspension of MoCp*Cl4 (0.043 g, 0.11 mmol) in 5 mL of THF. The solid immediately dissolved to yield an orange-brown solution. An EPR spectrum was recorded immediately, showing a main doublet with molybdenum satellites (g = 1.986, aMo = 43.0, aP = 26.7 G) and a smaller doublet at g=1.976, a P =26.0 Hz. The solution was stirred overnight and an EPR spectrum recorded again. The weaker doublet at g= 1.976 had increased (see Results section). The EPR properties of this solution did not further evolve upon more prolonged stirring. An IR spectrum of the equilibrium THF solution was also recorded (see Results section).

X-Ray analysis

Compound 5•CH2Cl2. A red crystal (0.20 × 0.16 × 0.12 mm) suitable for an X-ray analysis was obtained by layering n-pentane onto a saturated methylene chloride solution at room temperature. The crystal was mounted on an Enraf-Nonius KappaCCD diffractometer equipped with Mo-Kα radiation. Absorption corrections were applied to the data during inte-gration by the SCALEPACK 52 algorithm. The structure was solved via a Patterson search program [START_REF] Sheldrick | SHELXS and SHELXL[END_REF] and refined (space group P-1) with full-matrix least-squares methods [START_REF] Sheldrick | SHELXS and SHELXL[END_REF] based on |F 2 |. All non hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atoms of the complex were included in their calculated positions and refined with a riding model. Crystal data are collected in Table 1 and selected bond distances and angles are listed in Table 2.

Compound 7. A small brown crystal (0.22 × 0.10 × 0.05 mm) suitable for an X-ray analysis was obtained by layering toluene onto a saturated methylene chloride solution at room tem-perature. The crystal was mounted on an Enraf-Nonius KappaCCD using MoKα radiation. Although the final Fourier difference map exhibited strong electron density close to the molybdenum atom (1.98 and -1.02 e Å -3 ), any tentative to apply absorption corrections failed to fix the problem, thus no correction was applied. The structure was solved and refined (space group P21/n) as above.

CCDC reference number 186/2025. See http://www.rsc.org/suppdata/dt/b0/b002403l/ for crystal-lographic files in .cif format. Results and discussion (1)

The IR spectrum shows strong C=N stretching vibrations for the oxazoline ring at 1617 and 1616 cm -1 for complexes 1 and 2, respectively. These are 43-44 cm -1 red-shifted relative to the "free" ligand (1660 cm -1 ). Other N-co-ordinated oxazoline complexes also display red-shifted absorptions, for instance from 1661 to 1648 and 1649 cm -1 upon co-ordination of the bis(oxazolinyl)phosphine ligand PhP(CH2C3H4NO-2)2 to ruthenium(II) centres. [START_REF] Braunstein | [END_REF] Oxygen co-ordination (to our know-ledge yet unreported), on the other hand, would be expected to result in a smaller or even opposite shift of the C=N absorption. The IR evidence, therefore, agrees with N-co-ordination for the phosphinomethyloxazoline ligand. 21 For these reasons, we prefer to assign a cis relative arrangement to both compounds 1 and 2, although an unambiguous assignment can only be provided by a single crystal X-ray study. Unfortunately, suitable crystals for such a determination could not be obtained for either compound.

The cyclic voltammograms of compounds 1 and 2 show a reversible oxidation wave at 0.17 and -0.74 V vs. ferrocene, respectively. All compounds of the type [MoCpX2L2] show a one-electron oxidation process of which the potential and reversibility depend on the nature of X and L. For instance, a reversible process is observed in the -0.4 to -0.6 V range for [MoCpCl2(PR3)2] 41 and at -0.33 V for [MoCpCl2(dppe)], 32 while the same process is shifted to -1.1 V for [MoCpCl2(η 4 -C4H6)] 35 and to 0.12 V for [MoCpCl2-(Ph2PCH2CH2SMe)]. 34 Therefore, according to the cyclic voltammetric data, the relative ability of the Nco-ordinated oxazoline moiety in the chelate to stabilize the molybdenum(III) and -(IV) species involved in the oxidation process is similar to that of the S-coordinated -CH2SMe moiety. The negative shift observed on going from Cp to Cp* is consistent with the greater electron donating power of the latter. The magnitude of this shift (0.91 V), however, is quite large when compared to other Cp and Cp* bis(phosphine) complexes [0.32 V for [Mo(η-C5R5)Cl2(PMe3)2] and 0.25 V for [Mo(η-C5R5)Cl2(dppe)] on going from R = H to Me]. 26,56 A possible rationalization of this effect is to invoke a greater ability of the π-acidic phosphorus donors to buffer the electron density variations caused by the different cyclopenta-dienyl substitution. In an attempt to find a more convenient synthetic method for compound 2, ligand I was added to [MoCp*Cl4], yielding the corresponding adduct 3, eqn.

(2). Monitoring this reaction by EPR spectroscopy (Fig. 1) revealed the immediate formation of an intermediate, followed by the slow rearrangement (12 h) to the isolated product. Both compounds show similar hyperfine splittings for Mo and P. It seems reasonable to propose that the compound which forms immediately corresponds to an adduct with the P-co-ordinated monodentate ligand, A, with a structure related to those determined for [M(η-C5R5)Cl4(PR3)] (M = Mo or W), namely with the phosphine ligand located trans relative to the Cp* ligand. 57, 58 The final product, on the other hand, probably involves chelation of the ligand with displacement of a chloride ligand from the coordination sphere, as indicated in B. The structure of the cation would be identical to that determined for the cation of compound 7 containing ligand II, see below, whose EPR parameters are essentially identical to those of B and significantly different from those of the reaction intermediate A. Another related structure is that of [MoCp*Cl3{S2P(OEt)2}], 59 where the bidentate dithiophosphate ligand occupies the axial and one of the equatorial positions. The reduction of compound 3 (thermodynamic isomer B) with two equivalents of sodium failed to yield 2 and the characterization of the reduction product was therefore not pursued.

(b) Phosphine amide complexes

The addition reactions of the phosphine amide ligand II are more complex than those of ligand I. With [MoCp(µ-Cl)2]2 the reaction leads to the formation of the desired ligand adduct [MoCpCl2{Ph2PCH2C(O)NPh2-κ 2 P,O}], 4, which is however accompanied by the formation of a molybdenum(IV) by-product. The latter corresponds to the neutral trichloride derivative [MoCpCl3-{Ph2PCH2C(O)NPh2}], 5 (see below). The formation of this by-product is more pronounced when the reaction time in CH2Cl2 is longer, in agreement with a slow Cl atom abstraction from the solvent by the first reaction product 4. When the reaction was stopped soon after the complete comsumption of the [MoCp(µ-Cl)2]2 starting material (1 h) elemental analysis and cyclic voltammetry (see below) of the isolated product indicated only a minor contamination by 5. Compound 4 was also obtained by carrying out the reaction in THF, as confirmed by EPR spectroscopy and cyclic voltammetry, but the analytical purity of this sample was not superior to that of the reaction product from CH2Cl2.

(3) An additional complication is that the EPR spectrum of the molybdenum(III) product 4 appears as a distorted triplet (Fig. 2). However, this is interpreted as the overlap of two doublets that are assigned to two different isomers of the adduct [MoCpCl2{Ph2PCH2C(O)NPh2-κ 2 P,O}], 4 (see eqn. 3). Both materials obtained from CH2Cl2 or THF show identical EPR properties. Variable temperature EPR measurements did not improve the resolution. However, a satisfactory digital fitting of the spectrum confirms the co-ordination of just one P donor atom for both isomers and provides the spectral parameters as well as the relative proportion of the two species. The IR spectrum contains a single and strong ν(C=O) band at 1554 cm -1 , showing that both isomers have a similar environ-ment for the amide function. The "free" ligand 51 exhibits this absorption at 1658 cm -1 , while previously described co-ordination compounds with analogous phosphine amide ligands display absorptions around 1650 cm -1 when N-co-ordinated [e.g. for (Ph2P)2CHC(O)NPh2] 60 and around 1560 cm -1 when Oco-ordinated. [START_REF] Andrieu | [END_REF] Thus, the IR results document O-coordination for the ligand in both isomers of compound 4. Given that both species appear to contain the phosphine amide ligand in the same co-ordination mode (κ 2 P,O), we propose that these are stereoisomers differing by the relative configuration at the metal center. Since both cisoid and transoid arrangements of 4-atom chelating ligands have been reported for [Mo(η-C5R5)X2L2] complexes, for instance cisoid for [MoCpBr2(dppe)] 32 and transoid for the more sterically encumbered [MoCp*Cl (dppe)], 61 it seems reasonable to propose that these two alternative arrangements, shown as C and D in eqn. (3), are responsible for the isomerism of 4. The bulkier nature of ligand II would presumably force the structure to access the sterically less congested, but orbitally disfavored, transoid structure.

The cyclic voltammogram of compound 4 shows only one oxidation wave at E1/2 = 0.43 V. This observation indicates that the rate of interconversion of the two isomers of 4 is fast on the cyclic voltammetric timescale, while it is slow on the EPR time-scale. The same phenomenon has been observed for other MoCp III derivatives. 20 The E1/2 value is slightly higher than that of compound 1, suggesting that the amide function in II is a poorer donor than the oxazoline function in I.

As stated above, the molybdenum(IV) by-product 5 was also obtained, in addition to 4, when ligand II was treated with [MoCp(µ-Cl)2]2 in CH2Cl2 and crystallized selectively after long contact times in this solvent. Compound 5 has been characterized by 1 H NMR spectroscopy, cyclic voltammetry, elemental analysis (C, H, N), and by X-ray crystallography (see below). The cyclic voltammogram shows a reversible oxidation wave at E1/2 = 0.19 V, i.e. 0.24 V less positive than the oxidation of 4. This signifies that donation from one additional Cl - ligand more than compensates for the electron density loss associated to the one-electron oxidation. This situation is analogous to that of the corresponding [MoCpCln(dppe)] complexes (E1/2 = -0.33 V for n = 2 and -0.39 for the ax-eq isomer, i.e. isostructural with 5, with n = 3). 26 The 1 H NMR spectrum of 5 is consistent with a ground state diamagnetism, in agreement with its 18-electron configuration, but shows abnormally broad resonances for the Cp and methylene protons. The 31 P NMR resonance is also abnormally broad. A variable temperature study shows that these resonances sharpen and shift slightly upon cooling, consistent with a thermal equilibrium between the diamagnetic ground state and a higher energy paramagnetic species. This behaviour is not consistent with a fast equilibration between two diamagnetic species, because in that case the resonances would sharpen upon warming in Attempts to grow single crystals of compound 6 from CH2Cl2-toluene afforded instead crystals of the related oxidized trichloride species, [MoCp*Cl3{Ph2PCH2C(O)-NPh2-κ 2 -P,O}]BF4, 7, which is characterized by an EPR spectrum essentially identical with that of the final product of reaction (2), i.e. B. The cation of compound 7 has also been generated by direct interaction between [MoCp*Cl4] and ligand II, eqn. (5). EPR spectroscopy (Fig. 4) shows that the reaction follows a similar course to the corresponding reaction of [MoCp*Cl4] with ligand I, namely a molecular adduct G is formed as immediate, followed by a structural rearrangement to the ionic isomer H. The EPR parameters of the kinetic product G are essentially identical with those of A, while those of H are essentially identical with those of B and superimposable with those of 7. However, while the transformation of A to B [eqn. (2)] is quantitative, both the molecular and ionic isomers persist for compound 8, the neutral isomer remaining the predominant species at equilibrium. This is in agreement with a weaker chelating power of ligand II relative to ligand I. The presence of an isomeric equilibrium between monodentate and chelating forms is further supported by solution IR studies. A THF solution of complex 8 shows a major absorption at 1670 cm -1 for the uncoordinated amide function of G (cf. 1658 cm -1 for the "free" ligand) and a smaller absorp tion at 1540 cm -1 for the O-co-ordinated amide function of H (see Fig. 4).

(5)

(c) Crystal structures

The geometries of compound 5 and of the cation of 7 are shown in Figs. 5 and6, respectively, and the relevant bond distances and angles are compared in Table 2. As is immediately evident from the figures, both complexes adopt the same basic pseudo-octahedral geometry when considering the cyclopentadienyl ring as occupying a single co-ordination position. The three chloride ligands occupy three equatorial positions in both structures and the amide function is co-ordinated via the oxygen atom in both cases, confirming the indications obtained by IR spectroscopy. On the other hand, the orientation of the phosphine amide ligand is inverted in the two structures. For 5 the phosphorus atom occupies an equatorial position and the oxygen is in the axial position, while the reverse holds true for 7. The reason for this change may have a steric origin. For the more sterically encumbered 7 the bulkier PPh2 end of the chelating ligand is located farther from the bulky Cp* ligand. The less sterically congested 5, however, may have an electronic preference for the observed structure. An analysis of the previously reported pseudo-octahedral [MoCpCl3L2] (L2 = dppe or dmpe) complexes [START_REF] Stärker | [END_REF]65 shows that the axially co-ordinated P donor atom is less tightly bonded than the corresponding equatorial P atom, a situation that may be associated with the trans influence of the cyclopentadienyl ligand. Thus, the more strongly coordinated phosphorus donor in ligand II should have a greater tendency to occupy an equatorial position, leaving the labile oxygen end of the ligand to provide a supporting stabilization by co-ordinatively saturating the axial position.

(d) Comparison of the two ligands

Both ligands I and II contain, in addition to a Ph2P donor group, a nitrogen and an oxygen-based donor function. However, I chose binds selectively with the nitrogen function, II with the oxygen function. This behaviour can easily be understood on the basis of the resonance forms shown in Scheme 1, where an oxygen lone pair is seen to reinforce the donor power of the nitrogen function for I and the nitrogen lone pair reinforces the donor power of the oxygen function for II, as a result of a similar delocalization mechanism. Thus, in spite of a greater "oxophilicity" of molybdenum relative to the late transition metals, the phosphine oxazoline ligand I still binds preferencially with the nitrogen function and oxygen binding with this ligand remains undocumented.

Scheme 1

While ligand I adds selectively to [MoCp(µ-Cl)2]2 in CH2Cl2, II also provokes a chlorine abstraction process from the solvent. A similar chlorine abstraction was previously observed when treating [MoCp*Cl2-(PMe2Ph)2] with AlCl3 in CH2Cl2. 66 It was proposed that the abstraction of a chloride ion by AlCl3 produces the highly reactive 15-electron species [MoCp*Cl-(PMe2Ph)2] + , which is capable of abstracting a Cl atom in order to produce the more stable 16-electron species [MoCp*Cl2 (PMe2Ph)2] + . A similar mechanism may therefore take place for the formation of compound 5 (Scheme 2). This observation may therefore be taken as an indication of a greater hemilabile character of ligand II relative to I. This is also demonstrated by the equilibrium between the κ 1 P (major) and κ 2 P,O (minor) forms of compound 8 (eqn. 5), while for ligand I the kinetically accessed κ 1 P form of compound 3 transforms quantitatively to the κ 2 P,N form (eqn. 2). However, the hemilability of ligand II in compound 8 is obviously related to the presence of the strongly co-ordinating Cl - anion in the ionic form H. For the analogous salt 7 containing the less strongly co-ordinating BF4 -anion the EPR properties indicate that only the κ 2 P,O form is present in solution. The hemilabile properties of ligand II are also evidenced by a similar κ 1 -κ 2 equilibrium for com-pound 5 (eqn. 3).

It has been argued in a previous section that the presence of two geometric isomers for compound 4, while only one is observed for 1, could arise from steric factors. On the same basis, one can rationalize the absence of an interaction between [MoCp*(µ-Cl)2]2 and ligand II. The addition of neutral ligands to dinuclear cyclopentadienyldichloromolybdenum(III) complexes is known to be delicately balanced from the thermodynamic point of view by steric effects. 66 As shown in Scheme 3, the addition products have the same number of metal-ligand bonds as the reagent but four weaker Mo-Cl interactions are replaced by stronger Mo-L interactions. On the other hand, a metal-metal bond is lost. A quantitative reaction occurs for the Cp* precursor with L = PMe 3 or PMe2Ph, whereas no reaction occurs with the bulkier ligands PMePh2 and PPh3, 66 while the analogous precursor with the sterically less demanding Cp ring reacts with all the above ligands. 20 The stability of a Cp* product with Ph2PCH2CH2PPh2, whose steric bulk is similar to that of two PMePh2 ligands, illustrates how delicate this steric balance is. 61 Since ligand IIshould be less bulky than dppe, a weaker Mo-O=C(NPh2) interaction relative to the Mo-PPh2 interaction must also be invoked to rationalize the absence of an interaction between [MoCp*(µ-Cl)2]2 and ligand II.

Scheme 2

The stability of the the cationic molybdenum(IV) and -(V) systems 6 and 7 demonstrates the steric compatibility of ligand II with the [MoCp*Cl2] and even with the [MoCp*Cl3] moiety, which is made even more stringent by the reduced size of Mo IV and Mo V relative to Mo III . Consequently, the stability of the phosphine oxazoline Cp* complex 2 relative to the non-existent phosphine amide adduct may be attributed to the lower steric hindrance of the oxazoline ring, or to a greater strength for the Mo-oxazoline interaction, or to a combination of both factors.

Conclusion

The phosphine oxazoline and phosphine amide compounds I and II behave in similar but not identical ways as ligands toward half-sandwich complexes of Mo III , Mo IV and Mo V . The observed differences can be traced to differences in steric and electronic properties: the weaker donor ability and greater bulk of the Odonating amide function relative to the N-donating oxazoline function make the former a less tightly bonded bidentate ligand. The greater hemilability exhibited by the phosphine amide ligand II is the source of greater reactivity. Therefore, the molybdenum complexes with the phosphine amide ligand II have greater potential for use as building blocks for the construction of more complex architectures and for catalytic applications.

  CH 2 (d, J PH = 9 Hz) resonances were observed at δ 5.31 and 4.25, respectively. 31 P-{ 1 H} NMR (CDCl3, 297 K): δ 57.1 (w1/2 = 70 Hz). Cyclic voltammetry (CH2Cl2): reversible oxidation at E1/2 = 0.43 V.

  (a) Phosphinomethyloxazoline complexes Compounds 1 and 2 are obtained by addition of ligand I to the dinuclear complexes [Mo(η-C5R5)(µ-Cl)2]2 (R = H or Me) as shown in eqn. (1). The products have been characterized by C,H,N analysis, IR and EPR spectroscopic methods and cyclic voltammetry. All data are consistent with the mononuclear formulation shown with a chelating (P,N) ligand.

( 2 )

 2 The X-band EPR spectrum for each compound shows a dis-tinctive central doublet resonance (g = 1.972 for 1, 1.982 for 2) showing coupling to a single phosphorus atom (aP = 21.3 G for 1, 19.3 G for 2) flanked with satellites generated by the I = 5/2 molybdenum isotopes (25% abundance; aMo = 40.2 G for 1, 39.8 G for 2). The EPR spectra therefore establish phosphorus coordination for the ligand. The similarity of the coupling constants indicates that the two compounds adopt the same stereochemistry. It was previously reported for analogous Ph2PCH2CH2PPh2 (dppe) derivatives that a relative cis con-figuration in complexes [MoCpX2(dppe)] (as verified by X-ray crystallography for X = Br) 32 gave aP hyperfine constants in the 18-26 G range (26 G for the dichloride), while the structurally characterized trans dichloride derivatives [MoCpCl2(PR3)2] (PR3 = PMe3, PMePh2, PPh3 or PEt3) 19,20,55 have aP in the 9-16 G range 20 and complex [Mo{η-C5H3-(SiMe2CH2PPr i -1,3)2}Cl2], also showing a trans relative arrangement in the solid state, has aP = 15.2 G.

Fig. 1 .

 1 Fig. 1. EPR monitoring of the room temperature reaction between [MoCp*Cl4] and ligand I in CH 2Cl2. (a) After 15 minutes; (b) after 1.5 h; (c) after 12 h.

Fig. 2

 2 Fig. 2 Room temperature X-band EPR spectrum of compound 4 i n CH2Cl2 solution.

  the fast exchange limit (only one set of resonances), contrary to the observation. Previous studies have shown that 16electron derivatives of formula [MoCpCl2L2] + and [MoCp*Cl3L] (L = tertiary phosphine) adopt a spin triplet ground state. Therefore, we propose that the NMR properties result from a thermal equilibrium between the diamagnetic 18-electron structure observed in the solid state, E, and a paramagnetic 16electron structure resulting from deco-ordination of the amide function, F [eqn. (3)]. Complex [MoCp*(µ-Cl)2]2 does not show any reactivity with ligand II. This contrasts with both the reactivity of the Cp analogue with the same ligand and with that of the same MoCp* complex with ligand I (see above). However, when one equivalent of Ag + per Mo atom is added as the BF4 -salt to a CH2Cl2 solution containing [MoCp*(µ-Cl)2]2 and ligand II a redox process takes place and complex [MoCp*Cl2{Ph PCH C(O)NPh-κ 2 P,O}]BF4, 6, may be recovered from the solution, eqn. (4).

( 4 )

 4 Complex 6 has been characterized by elemental analysis and by IR and 1 H NMR spectroscopy. The IR spectrum shows evidence for O-co-ordination, with a ν(CO) band at 1540 cm -1 . The 1 H NMR spectrum (see Fig. 3) indicates paramagnetism. Related spin triplet 16-electron half-sandwich molybdenum(IV) complexes, e.g. [MoCpCl2(PMe3)2] + and [MoCp*Cl3(PMe3)], 19,62 exhibit similar shifts for the 1 H NMR resonances. On the basis of these precedents, we are able to assign the upfield shifts at δ -9.0 and -65.8 to the two inequivalent methylene protons and the strong resonance at δ 3.6 to the Cp* protons, while the 4 inequivalent phenyl groups yield 12 signals (11 of which are distinctly observed) that are all more or less shifted downfield by the electron spin density.

Fig. 3

 3 Fig. 3 Room temperature 1 H NMR spectrum of compound 6 in CDCl3 solution.

Fig. 4

 4 Fig. 4 Equilibrium spectral properties of compound 8 in THF solu-tion at room temperature: (a) EPR spectrum; (b) IR spectrum in com-parison with that of the "free" ligand.

Fig. 5

 5 Fig. 5 An ORTEP 63 view of a molecule of complex 5. Ellipsoids are shown at the 50% probability level.

Fig. 6

 6 Fig. 6 An ORTEP view of the cation of complex 7. Details as in Fig. 5. The shortening of the Mo-Cl distances on going from complex 5 to 7 follows the expected decrease of the metal ionic radius upon increasing the oxidation state. The corresponding lengthening of the Mo-P distance, on the other hand, is related to the structural change which places the phosphorus donor in the more tightly bonded equatorial position in 5 and in the less tightly bonded axial position in 7. Other related Mo-P distances are 2.542(1) and 2.553(1) Å (equatorial) for the molybdenum(IV) compound [MoCpCl (PMe2Ph)2], 62

and 2 .

 2 617(1) and 2.554(1) Å (axial) for the molybdenum(V) compounds [Mo(C5H4Me)Cl4(PH2Ar)] (Ar = 2,4,6-Pr i 3C6H2 or C6H5, respectively). The Mo-O distance changes in the opposite direction, as expected.

  3 × 5 mL) and dried in vacuo. = 0.17 V. Compound 1 is insoluble in all common hydrocarbon solvents and only slightly soluble in THF. After isolation as a solid from CH2Cl2 solution, it also exhibited sparing solubility in this solvent. It is quite air sensitive, becoming immediately brown upon exposure to air.

	Yield: 0.112 g, 38%. Calc. for C21H21Cl2MoNOP: C,
	50.32; H, 4.22; N, 2.79. Found: C, 50.44; H, 4.28;
	N, 2.63%. IR (Nujol mull): 1617 cm -1 . EPR (CH2Cl2):
	g = 1.972 (d with molybdenum satellites, aMo = 40.2, aP =
	21.3 G). Cyclic voltammetry (CH2Cl2): reversible
	oxidation at E1/2

MoCpCl2{Ph2PCH2C(O)NPh2-κ 2 P,O}], 4. To

		Et2O.
		[a
		solution of II (0.418 g, 1.06 mmol) in 5 mL of CH2Cl2
		was added a suspension of [MoCp(µ-Cl)2]2 (0.463 g,
		1.00 mmol). After stirring for 1 h the resulting red brown
		solution was filtered through Celite. The filtrate was
		evaporated to dryness and the residue extracted with
		toluene (20 mL) and filtered. Addition of 2 × 10 mL of
		pentane gave complex 4 as a brick-red powder, which
		was filtered off, washed with pentane (2 × 10 mL) and
		dried in vacuo. Yield: 0.225 g, 32%. Calc. for
		C31H27Cl2MoNOP: C, 59.35; H, 4.34; N, 2.23. Found: C,
		58.89; H, 4.34; N, 2.27%. IR (CH2Cl2): ν(CO) 1554 cm -
		1 . EPR (CH2Cl2): overlap of two doublets with
		molybdenum satellites; 1st species (55.1%), g = 1.977,
		aP = 11.7, aMo = 38.7 G; 2nd species (44.9%), g = 1.971,
		aP = 12.2 G, aMo = 37.8 G. Cyclic voltammetry (CH2Cl2):
		reversible oxidation at E 1/2 = 0.43 V. The
		voltammogram also showed a smaller reversible
		oxidation wave with E 1/2 = 0.19 V, due to the by-product
		5 (see below).
		3 × 5 mL)
	and dried in vacuo. Yield: 0.209 g, 35%. Calc. for
	C26H31Cl2MoNOP: C, 54.66; H, 5.47; N, 2.45. Found:
	C, 54.44; H, 5.33; N, 2.78%. IR (Nujol mull): 1616 cm -1 .
	EPR (CH2Cl2): g = 1.982 (d, with molybdenum satellites,
	aMo = 39.8, aP = 19.3 G). Cyclic voltammetry (CH2Cl2):
	reversible oxidation at E1/2 = -0.74 V (vs ferrocene).
	[MoCp*Cl4(Ph2PCH2C3H4NO)], 3. A solution of I
	(0.160 g, 0.59 mmol) in 5 mL of CH2Cl2 was added to
	a suspension of [MoCp*Cl4] (0.223 g, 0.59 mmol) in 20
	mL of CH2Cl2 at 0°C. The solution was warmed to room
	temperature and an aliquot immediately withdrawn for
	an EPR investigation:	doublet with molybdenum
	satellites, g = 1.986, aMo = 44.2, aP = 26.1 G. The solution
	was stirred overnight and then filtered through Celite.
	The filtrate was concentrated under reduced pressure to
	ca. 1 mL. Addition of 20 mL of pentane gave complex
	3 as a red microcrystalline solid, which was filtered off,
	washed with pentane (4 × 5 mL) and dried in vacuo.
	Yield: 0.220 g, 57%. Calc. for C26H31Cl4MoNOP: C,
	48.62; H, 4.86; N, 2.18. Found: C, 48.23; H, 4.80; N,
	2.09%. EPR: (CH2Cl2) g = 1.978 (d, with molybdenum
	satellites, aMo = 45.7, aP = 25.5 G); (THF or MeCN) g
	= 1.962 (broad d, aP =27.4 G). Cyclic voltammetry
	(CH2Cl2): reversible reduction at E1/2 = -1.096 V (vs.
	ferrocene). Compound 3 is insoluble in pentane and in

Table 1

 1 Crystal data and structure refinement parameters for compounds 5•CH2Cl2 and 7

		5•CH2Cl2	7
	Formula	C31H27Cl3MoNOP CH2Cl2 C36H37Cl3MoNOP BF4
	M	747.72	819.74
	T/K	110(2)	110(2)
	Crystal system	Triclinic	Monoclinic
	a/Å	11.1707(6)	12.354(1)
	b/Å	11.8110(6)	14.895(1)
	c/Å	12.2717(8)	19.937(1)
	α/°	88.024(3)
	β/°	80.438(2)	106.961(2)
	γ/°	79.718(3)
	V/Å 3	1570.9(2)	3509.1(4)
	Z	2	4
	µ/mm -1	0.922	0.70
	RC = Reflections	11333	24328
	collected
	IRC = Independent	6814 [R(int)=0.031]	8008 [R(int)=0.08]
	RC
	IRCGT = IRC and	5916	5910
	[I>2σ(I)]
	R1, wR2 for IRCGT	0.030, 0.065	0.064, 0.13
	For IRC	0.039, 0.069	0.096, 0.15

Table 2

 2 Selected bond distances (Å) and angles (°) for compounds 5•CH2Cl2 and 7

Acknowledgements

We are grateful to the Ministère de l'Education Nationale, de la Recherche et de la Technologie (MENRT) for support and a PhD grant (to J.-M. C. and F. N.), the Centre National de la Recherche Scientifique and the Conseil Régional de Bourgogne for support of this work.


Yukari Nakamura

Sophie Longueville

Akinori Nishi

Denis Herve

Jean- Antoine Girault

Yuki Nakamura

Jean-Antoine Girault
email: jean-antoine.girault@inserm.fr

Dopamine D1 receptor-expressing neurons activity is essential for locomotor and sensitizing effects of a single injection of cocaine

Keywords: sensitization, dopamine receptor, DREADD, 2-injection protocol of sensitization

Dopamine D1 receptors play an important role in the effects of cocaine. Here we investigated the role of neurons, which express these receptors (D1-neurons) in the acute locomotor effects of cocaine and the locomotor sensitization observed after a second injection of this drug, using the previously established two-injection protocol of sensitization. We inhibited D1-neurons using double transgenic mice conditionally expressing the inhibitory Gi-coupled designer receptor exclusively activated by designer drugs (Gi-DREADD) in D1-neurons. Chemogenetic inhibition of D1-neurons by a low dose of clozapine (0.1 mg/kg) decreased the cocaine-induced expression of Fos in striatal neurons. It diminished the basal locomotor activity and acute hyper-locomotion induced by cocaine (20 mg/kg). Clozapine 0.1 mg/kg had no effect by itself and did not alter cocaine effects in wild type mice. Inhibition of D1-neurons during the first cocaine administration prevented the sensitization of the locomotor response in response to a second cocaine administration ten days later. On day 11, inhibition of D1-neurons by clozapine stimulation of Gi-DREADD blocked cocaine-induced locomotion including in sensitized mice, whereas on day 12, in the absence of clozapine and D1-neurons inhibition, all mice displayed a sensitized response to cocaine. These results show that chemogenetic inhibition of D1-neurons decreases spontaneous and cocaine-induced locomotor activity. It prevents the induction and expression of sensitization in a two-injection protocol of sensitization but does not alter established sensitization. Our study further supports the central role of D1-neurons in mediating the acute locomotor effects of cocaine and its sensitization.

Introduction

Psychostimulants such as cocaine enhance locomotor activity and repeated cocaine exposure progressively increases the locomotor responses due to sensitization processes (review in [START_REF] Steketee | Drug wanting: behavioral sensitization and relapse to drug-seeking behavior[END_REF]. Locomotor sensitization to repeated drug administration is a complex adaptation that involves several neuronal populations and is correlated with aspects of addiction including the excessive pursuit and self-administration of psychostimulant drugs [START_REF] Vezina | Sensitization of midbrain dopamine neuron reactivityand the self-administration of psychomotor stimulant drugs[END_REF].

However, multiple drug exposures can promote the parallel development of tolerance that could interfere with behavioral sensitization [START_REF] Stewart | Tolerance and sensitization to the behavioral effects of drugs[END_REF]. Remarkably, a single cocaine administration induces a locomotor sensitization that increases during the first week without repetition (a property sometimes referred to as "incubation") and is long-lasting [START_REF] Valjent | Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum[END_REF][START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF]. This persistent ability of a single injection of cocaine or other drugs to increase locomotor response to a second injection several days later has been reported by several investigators [START_REF] Jackson | A single preexposure produces sensitization to the locomotor effects of cocaine in mice[END_REF][START_REF] Lin-Chu | Sensitization of rotational behavior produced by a single exposure to cocaine[END_REF][START_REF] O'neill | A single pretreatment with MK-801 or cocaine enhances their locomotor stimulant effects in rats[END_REF][START_REF] Robinson | Long-term facilitation of amphetamine-induced rotational behavior and striatal dopamine release produced by a single exposure to amphetamine: sex differences[END_REF][START_REF] Vanderschuren | A single exposure to amphetamine is sufficient to induce longterm behavioral, neuroendocrine, and neurochemical sensitization in rats[END_REF][START_REF] Vanderschuren | A single exposure to morphine induces long-lasting behavioural and neurochemical sensitization in rats[END_REF][START_REF] Weiss | Context-dependent cocaine sensitization: differential effect of haloperidol on development versus expression[END_REF] and evaluated in details as the twoinjection protocol of sensitization, TIPS [START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF]. TIPS avoids confusing effects of repetition of the manipulations which are investigated and is a simple protocol to explore the signaling mechanisms by which drugs of abuse exert long-lasting effects [START_REF] Corbillé | Role of Cannabinoid Type 1 Receptors in Locomotor Activity and Striatal Signaling in Response to Psychostimulants[END_REF][START_REF] Valjent | Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum[END_REF] as well as the role of synaptic long term potentiation in these persistent alterations [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF]. It has the advantage to allow simple manipulations during the unique initial injection and test their consequences on subsequent sensitization. Dopamine D1 receptor (Drd1) plays a major role in the rewarding properties of drugs of abuse including cocaine [START_REF] Self | Opposite modulation of cocaine-seeking behavior by D1-and D2-like dopamine receptor Agonists[END_REF]. Cocaine treatment stimulates the activity of D1-neurons in the striatum in vivo [START_REF] Luo | Acute cocaine induces fast activation of D1 receptor and progressive deactivation of D2 receptor striatal neurons: in vivo optical microprobe [Ca 2+ ]i imaging[END_REF]. SCH23390, a Drd1 antagonist, prevents locomotor sensitization [START_REF] Mccreary | Cocaine-induced behaviour: dopamine D1 receptor antagonism by SCH 23390 prevents expression of conditioned sensitisation following repeated administration of cocaine[END_REF]. In Drd1 knockout mice spontaneous locomotor activity is either unchanged [START_REF] Drago | D1 dopamine receptor-deficient mouse: cocaine-induced regulation of immediate-early gene and substance P expression in the striatum[END_REF][START_REF] Miner | Retained cocaine conditioned place preference in D1 receptor deficient mice[END_REF] or increased [START_REF] Xu | Dopamine D1 receptor mutant mice are deficient in striatal expression of dynorphin and in dopamine-mediated behavioral responses[END_REF][START_REF] Xu | Behavioral responses to cocaine and amphetamine administration in mice lacking the dopamine D1 receptor[END_REF] but in most reports the locomotor response to cocaine is lost [START_REF] Karlsson | Comparison of dopamine D1 and D5 receptor mice for cocaine locomotor sensitization[END_REF][START_REF] Xu | Behavioral responses to cocaine and amphetamine administration in mice lacking the dopamine D1 receptor[END_REF] as well as the ability of cocaine to induce Fos expression in striatal neurons [START_REF] Drago | D1 dopamine receptor-deficient mouse: cocaine-induced regulation of immediate-early gene and substance P expression in the striatum[END_REF][START_REF] Moratalla | Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor[END_REF]. Drd1 knockout mice also show reduced sensitized locomotor responses to cocaine [START_REF] Karlsson | Comparison of dopamine D1 and D5 receptor mice for cocaine locomotor sensitization[END_REF] and amphetamine [START_REF] Karper | Role of D1-like receptors in amphetamine-induced behavioral sensitization: a study using D1A receptor knockout mice[END_REF]. With the TIPS protocol, cocaine sensitization was absent in homozygous Drd1 knockout mice, and reduced in heterozygous mice [START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF]. However there is a limitation in the interpretation of knockout mice experiments, because the lack of Drd1 gene affects the expression of other genes [START_REF] Xu | Dopamine D1 receptor mutant mice are deficient in striatal expression of dynorphin and in dopamine-mediated behavioral responses[END_REF] and developmental alterations cannot be excluded. Targeted mutations in mice also showed the importance in sensitization of dopamine D2 (Drd2) (Solis et al., 2021) and glutamate NMDA (Grin1) receptors [START_REF] Beutler | Balanced NMDA receptor activity in dopamine D1 receptor (D1R)-and D2R-expressing medium spiny neurons is required for amphetamine sensitization[END_REF]. These experiments provide evidence about the role of receptors but do not explore the contribution of specific neuronal populations. While local administration of amphetamine into the VTA is sufficient to induce Drd1-dependent locomotor sensitization [START_REF] Vezina | D1 dopamine receptor activation is necessary for the induction of sensitization by amphetamine in the ventral tegmental area[END_REF], sensitization to systemic amphetamine was prevented in mice by ablation of dorsomedial striatum D2-neurons, but not D1-neurons [START_REF] Durieux | Differential regulation of motor control and response to dopaminergic drugs by D1R and D2R neurons in distinct dorsal striatum subregions[END_REF].

Metamphetamine sensitization was only delayed in mice by ablation of nucleus accumbens (NAc) shell D1-neurons [START_REF] Kai | Differential roles of dopamine D1 and D2 receptor-containing neurons of the nucleus accumbens shell in behavioral sensitization[END_REF]. Cocaine locomotor sensitization was markedly decreased in mice with neurotransmission blockade [START_REF] Hikida | Distinct roles of synaptic transmission in direct and indirect striatal pathways to reward and aversive behavior[END_REF] or optogenetic inhibition [START_REF] Chandra | Optogenetic inhibition of D1R containing nucleus accumbens neurons alters cocaine-mediated regulation of Tiam1[END_REF] of NAc D1-neurons. Potentiation of corticostriatal synapses onto NAc D1-neurons is a key component of locomotor sensitization in response to a single injection of cocaine [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF]. Chemogenetic inhibition of dorsal striatum nigral projection neurons also impaired amphetamine sensitization [START_REF] Ferguson | Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization[END_REF]). In addition, recent work reveals that striatal cholinergic interneurons also play a role in locomotor responses to cocaine, including sensitization (Hanada et al., 2018;[START_REF] Lewis | Dopaminergic control of striatal cholinergic interneurons underlies cocaine-induced psychostimulation[END_REF]. Thus, available evidence indicates that locomotor sensitization can be triggered by just a single exposure to psychostimulants and involves several neuronal populations in the striatum.

In the current study we addressed the global role of D1-neurons asking two simple questions: Does inhibition of D1-neurons alter i) the acute locomotor response to cocaine and ii) the induction or expression of sensitization in the TIPS protocol? To address these questions we used chemogenetics with designer receptor exclusively activated by designer drug (DREADDs, [START_REF] Roth | DREADDs for Neuroscientists[END_REF] and expressed hM4Di, an inhibitory DREADD, referred to below as Gi-DREADD, selectively in Drd1-expressing cells. Since recent work showed that DREADD stimulation with low doses of clozapine (CLZ) is faster and at least as efficient as clozapine-N-oxide (CNO), the commonly used ligand [START_REF] Gomez | Chemogenetics revealed: DREADD occupancy and activation via converted clozapine[END_REF], an observation confirmed in our laboratory [START_REF] Nakamura | fMRI detects bilateral brain network activation following unilateral chemogenetic activation of direct striatal projection neurons[END_REF], we used CLZ to activate hM4Di and inhibit D1-neurons.

Materials and Methods

Animals and treatments

We used heterozygous bacterial artificial chromosome (BAC) Drd1-Cre transgenic (Drd1-Cre) mice expressing Cre recombinase under the control of the dopamine D1 receptor gene (Drd1) promoter (GENSAT project, EY262 line), bred onto a C57BL/6J genetic background. Gi-DREADD (B6N.129-Gt(ROSA)26Sor tm1(CAG-CHRM4*,-mCitrine)Ute /J, a.k.a. R26-LSL-hM4Di-DREADDpta-mCitrine or R26-hM4Di/mCitrine) mice were purchased from the Jackson Laboratory (stock #026219). We crossed Drd1-Cre mice with Gi-DREADD mice. A subset of their offspring carried both Drd1-Cre and R26-hM4Di/mCitrine alleles, allowing effective removal of the stop cassette and expression of Gi-DREADD (mice referred to as D1-Gi-DREADD below, Fig. 1A). Males and females, 10-21-weekold at the beginning of the experiments, were used. Genotypes were determined by polymerase chain reaction (PCR) on mouse tail DNA samples. Cocaine-HCl (Sigma, France) was dissolved in 9 g L -1

NaCl solution (saline). CLZ (Sigma-Aldrich) was first dissolved in DMSO, and then diluted in saline (final DMSO concentration, 0.1 mL L -1 ). Cocaine (20 mg kg -1 ) and CLZ (0.1 mg kg -1 ) were administered by intraperitoneal injection in a volume of 10 mL kg -1 body weight. The dose of CLZ was chosen to be efficient for Gi-DREADD stimulation but without other identified pharmacological effects, according to previous reports [START_REF] Gomez | Chemogenetics revealed: DREADD occupancy and activation via converted clozapine[END_REF][START_REF] Nakamura | fMRI detects bilateral brain network activation following unilateral chemogenetic activation of direct striatal projection neurons[END_REF]. Mice were housed under a 12-hour light/12-hour dark cycle with free access to food and water. All animal procedures were approved by the Ministère de l'Education Nationale de l'Enseignement Supérieur de la Recherche (France, project APAFIS#8196-201704201224850 v1. All methods in this study were performed in accordance with the relevant guidelines and regulations.

Behavioral analysis

Locomotor activity was measured in a circular corridor with four infrared beams placed at every 90° angles (Imetronic, Pessac, France) in a low luminosity environment (locomotor activity box, LAbox). Counts were incremented by consecutive interruptions of two adjacent beams. Locomotor sensitization induced by a single cocaine injection was studied as in [START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF], as descibed in Fig. 1B-C. Mice were habituated to handling, test apparatus, and procedures for 3 consecutive days (Days -3 to -1). In this habituation period, mice received every day a first saline injection and were placed in the LA-box (T0 min). Thirty minutes later, they received a second saline injection (T30 min) and were put back in the LA-box for 1 h. On experimental days (Day 0, Days 10-12), mice were handled in the same manner, except that they were injected with the solutions indicated in Fig. 1B at T0 min and T30 min, respectively.

Tissue preparation and immunofluorescence

For the experiment in which the brains were used for immunohistofluorescence, D1-Gi-DREADD mice different from those used for sensitization experiments were treated as above on Day 0 but killed 90 min after cocaine injection (Fig. 2A) with a lethal dose of pentobarbital (500 mg kg -1 , i.p., Sanofi-Aventis, France) and perfused transcardially with 40 g L -1 paraformaldehyde in 0.1 M sodium phosphate buffer (Fig. 2A). Brains were post-fixed overnight at 4°C, cut into free-floating sections (30 μm) with a vibrating microtome (Leica) and kept at -20°C in a solution containing 300 mL L -1 ethylene glycol, 300 mL L -1 glycerol in 0.1 M sodium phosphate buffer. Immunolabeling procedures were as previously described [START_REF] Valjent | Involvement of the extracellular signal-regulated kinase cascade for cocaine-rewarding properties[END_REF]. Primary antibodies were rabbit Fos antibodies (1:1000, Santa Cruz, #sc-52) and mouse hemagglutinin (HA) antibodies (1:50, Sigma, #A-2095).

Secondary antibodies were anti-rabbit A488-labeled antibody (1:400, Invitrogen, #A-21206) and anti-mouse A555-labeled antibody (1:400; Invitrogen, #A-31570). Images were acquired using a Leica SP5-II confocal microscope at the Institut du Fer à Moulin Tissue and Cell Imaging Facility.

Statistical analysis

When normal distribution was not rejected by d'Agostino-Pearson and Shapiro-Wilk tests, data were analyzed with Student's t test or paired t-test or two-way ANOVA followed by post hoc comparisons as indicated in the figure legends. When normal distribution was rejected, analysis was done with non-parametric tests. All statistical analyses were performed using Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA). A threshold value for significance of p<0.05 was used throughout the study.

Results

D1-Gi-DREADD stimulation prevents cocaine-induced hyperlocomotion and Fos expression in the striatum

We first assessed the functionality of Gi-DREADD by testing the ability of CLZ to alter cocaineinduced locomotion and prevent cocaine-induced expression of Fos, a marker of activated neurons. D1-Gi-DREADD mice were treated with CLZ (0.1 mg/kg) or vehicle and 30 min later with cocaine (Fig. 2A). Cocaine markedly increased locomotion in the group of vehicle-pretreated mice whereas CLZ-pretreated mice displayed little locomotor activity (Fig. 2B). Brains were collected 90 min after cocaine injection and we quantified the percentage of cells expressing Gi-DREADD, revealed by HA immunoreactivity, a peptide tag of Gi-DREADD, which were also positive for Fos (Fig. 2C). We analyzed this percentage in the dorsolateral and dorsomedial striatum and in the NAc core and shell regions (Fig. 2D). In both vehicle-and cocaine-treated mice, most Fos-positive cells, but not all, coexpressed HA (and thus Gi-DREADD), indicating that Fos was mainly induced in D1-neurons as previously reported [START_REF] Bertran-Gonzalez | Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol[END_REF][START_REF] Guez-Barber | FACS identifies unique cocaine-induced gene regulation in selectively activated adult striatal neurons[END_REF][START_REF] Zhang | c-Fos facilitates the acquisition and extinction of cocaine-induced persistent changes[END_REF]. As compared with vehicle, CLZ pretreatment decreased Fos-positive/HA-positive ratio in the NAc shell, dorsomedial, and dorsolateral striatum (Fig. 2E). These results indicated that stimulating Gi-DREADD with CLZ inhibited the cocaine-induced response of striatal D1-neurons.

Inhibition of D1-neurons decreases basal and cocaine-induced locomotor activity

Next, using different groups of mice, we analyzed how inhibition of D1-neurons affected spontaneous locomotion and locomotor responses after the first and second cocaine injection (see Fig. 1B-C). On Day 0, during the 30-min period after CLZ pretreatment (groups 3-4) the basal locomotor activity of mice was decreased as compared with vehicle-pretreated mice (groups 1-2, Fig. 3A-B). In contrast CLZ pretreatment had no effect in wild type mice (WT, Fig. 3B, groups 5-8). During the 60 min after cocaine injection, locomotor activity was dramatically decreased in CLZ-pretreated D1-Gi-DREADD mice (group 4) as compared to vehicle-pretreated mice (group 2, Fig. 3A,C), whereas CLZ injection did not alter cocaine-induced hyper-locomotion in WT mice (Fig. 3C, group 8). Thus, inhibition of Gi-DREADD-expressing D1-neurons by CLZ decreased the spontaneous locomotor activity and cocaine-induced hyper-locomotion. Although sex-dependent differences in the response to cocaine have been reported [START_REF] Festa | Cocaine-induced sex differences in D1 receptor activation and binding levels after acute cocaine administration[END_REF][START_REF] Rivera | Sex-specific behavioral and neurogenic responses to cocaine in mice lacking and blocking dopamine D1 or dopamine D2 receptors[END_REF][START_REF] Kokane | Sex differences and the role of estradiol in mesolimbic reward circuits and vulnerability to cocaine and opiate addiction[END_REF], the sex of the mice, which was evenly distributed in all groups of mice used in these experiments, did not modify the responses (Figs. 1B,3D).

Inhibition of D1-neurons impairs the induction of cocaine locomotor sensitization

We then investigated the effects of chemogenetic inhibition of D1-neurons during the first injection of cocaine on locomotor sensitization. To test this effect we used the TIPS protocol which avoids confusing effects due to repeated drugs administration, and provides a robust assessment of sensitization [START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF]. On Day 10, we injected cocaine to all D1-Gi-DREADD mice (Fig. 3E). All groups displayed an increased locomotor activity after cocaine injection (Fig. 3E). In the groups that had not received CLZ on Day 0 (groups 1-2), locomotor response to cocaine on Day10 was significantly increased in the mice injected with cocaine for the second time (group 2) as compared to those that had previously received saline (group 1, Fig. 3E-F). This result confirmed the locomotor sensitization induced in the TIPS protocol. In contrast, in the mice pretreated with CLZ on Day 0 (groups 3-4), the responses to cocaine were not significantly different whether or not they had received cocaine a week before. Thus sensitization was blunted in group 4 mice that had received cocaine on Day 0 in the presence of CLZ (Fig. 3E-F). In WT mice (groups 5-8) the locomotor response to the second injection of cocaine was increased (groups 6, 8) as compared to mice previously injected with saline (groups 5, 7), whether they also had received on Day 0 CLZ (group 8) or vehicle (group 6, Fig. 3F).

Inhibition of D1-neurons blocks previously sensitized cocaine-induced locomotion

To assess the acute effects of D1-neuron inhibition on locomotor activity in mice previously exposed to cocaine, we injected on Day 11 the same mice as in Fig. 3 with CLZ, followed 30 min later by cocaine (Fig. 4A). In the four D1-Gi-DREADD groups (1-4), the locomotor responses (30-90 min) were lower than those observed the day before in the absence of CLZ (Day 10, compare Fig. 3 E-F and Fig. 4A-B) and there was no significant difference between groups on Day 11 (Fig. 4B). In contrast, in all groups of WT mice injected with CLZ (groups 5-8), cocaine induced a clear hyperlocomotion (Fig. 4B), ruling out a non-specific pharmacological effect of CLZ. Paired analysis showed that locomotor responses to cocaine on Day 11 were significantly decreased compared with those on Day10 in the four D1-Gi-DREADD groups (Fig. 4C), including group 2 which was clearly sensitized on Day 10 (Fig. 3D-E). These results showed that inhibition of D1-neurons strongly decreased the cocaine-induced hyper-locomotion, including in mice with already established cocainesensitized locomotion (group 2).

Inhibition of D1-neurons on Day 11 does not alter established cocaine sensitization on Day 12

Because cocaine TIPS is protein synthesis-and context-dependent [START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF] and context memory retrieval in the presence of a protein synthesis inhibitor can disrupt locomotor sensitization in rats [START_REF] Bernardi | Anisomycin disrupts a contextual memory following reactivation in a cocaine-induced locomotor activity paradigm[END_REF], we asked whether exposure of mice to the LA-box and cocaine in the presence of CLZ could have persistent effects on cocaine locomotor responses. We tested whether D1-neuron inhibition on Day 11 by CLZ in D1-Gi-DREADD mice affected the locomotor response to cocaine the next day (Day 12). On Day 12, all groups were injected with vehicle and 30 min later with cocaine. There was no significant difference in locomotor activity (30-90 min) between the 4 D1-Gi-DREADD groups (Fig. 4D,E). These results were similar to those observed in WT mice receiving the same treatment on Day 12 (groups 5-8, Fig. 4E). Paired analysis showed that the locomotor response to cocaine (30-90 min) on Day 12 was significantly increased in Groups 1, 3, and 4, as compared to Day 10 (Fig. 4F), indicating the existence of a sensitization step. No significant difference between Day 10 and 12 was observed in group 2 (Fig. 4F), suggesting that sensitization had reached a plateau in this group, in agreement with previous report showing that cocaine sensitization to the 2 nd cocaine injection is time-dependent and becomes maximum about 7 days after the first cocaine injection [START_REF] Valjent | Mechanisms of locomotor sensitization to drugs of abuse in a two-injection protocol[END_REF]. None of the groups displayed any decreased cocaineinduced locomotor activity on Day 12 as compared to Day 10, showing that inhibition of D1-neurons during cocaine exposure in paired context on Day 11 did not diminish previously established sensitization.

Discussion

In this study, we used double transgenic mice with conditional expression of Gi-DREADD in neurons that express Drd1 and showed that inhibition of these neurons decreased spontaneous locomotor activity and acute cocaine-induced hyper-locomotion. Inhibition of D1-neurons during the first cocaine administration blunted sensitization to a cocaine challenge ten days later. In addition, acute inhibition of D1-neurons prevented cocaine-induced locomotor activity in previously sensitized mice.

However, inhibition of D1-neurons concomitant with cocaine injection in the drug-associated environment did not diminish previously acquired sensitization.

For pharmacological activation of Gi-DREADD, we used low doses of CLZ instead of commonly used CNO. CNO induces cellular DREADD-mediated effects with different time-courses in the brain and peripheral organs [START_REF] Alexander | Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors[END_REF]. The effects of systemic administration of CNO on DREADD-expressing neurons appear to result mainly from the action of CLZ produced by its metabolic degradation, which crosses the blood brain barrier more efficiently and has a higher affinity for DREADDs [START_REF] Gomez | Chemogenetics revealed: DREADD occupancy and activation via converted clozapine[END_REF]. In a recent fMRI study of DREADD-induced activation of D1-neurons, we confirmed a more rapid and efficient effect of low doses of CLZ as compared to CNO [START_REF] Nakamura | fMRI detects bilateral brain network activation following unilateral chemogenetic activation of direct striatal projection neurons[END_REF]. Here low doses of CLZ (0.1 mg/kg, i.p.) had clear effects only in mutant mice expressing Gi-DREADD. Although CLZ has numerous endogenous targets at higher doses including serotonin, dopamine, and muscarinic acetylcholine receptors [START_REF] Bonaventura | High-potency ligands for DREADD imaging and activation in rodents and monkeys[END_REF][START_REF] Schotte | Occupancy of central neurotransmitter receptors by risperidone, clozapine and haloperidol, measured ex vivo by quantitative autoradiography[END_REF], in our experiments we did not observe any effect of the low dose of CLZ used in WT mice, ruling out off-target effects.

Cell-type-specific technologies allow better characterization of cell populations involved in the effects of psychostimulants [START_REF] Durieux | Targeting neuronal populations of the striatum[END_REF][START_REF] Yager | The ins and outs of the striatum: role in drug addiction[END_REF]. Optogenetic inhibition of striatal D1-but not D2-neurons inhibited the expression of cocaine-induced locomotor sensitization, (Lee et al., 2017). Repeated association of optogenetic inhibition of NAc D1-neurons with cocaine injection decreased sensitization [START_REF] Chandra | Optogenetic inhibition of D1R containing nucleus accumbens neurons alters cocaine-mediated regulation of Tiam1[END_REF]. Chemogenetic inhibition of dorsal striatum dynorphin-neurons (corresponding to D1) but not enkephalin-neurons (D2) impaired behavioral sensitization although it did not alter spontaneous activity or acute response to amphetamine [START_REF] Ferguson | Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization[END_REF]. In our study, we observed a strong effect of inhibiting D1-neurons on spontaneous locomotor activity and acute response to cocaine. These stronger effects may result from the wider inhibition of D1-neurons excitability throughout the brain, including in dorsal striatum, NAc and cerebral cortex, in contrast to the restricted inhibition in the dorsomedial striatum in the study of Ferguson et al. In addition, it is possible that the efficacy of CLZ to activate DREADD is higher and more rapid than that of CNO, as suggested by [START_REF] Gomez | Chemogenetics revealed: DREADD occupancy and activation via converted clozapine[END_REF].

Decreasing activity of the medial prefrontal cortex afferent to the NAc did not impair amphetamine sensitization [START_REF] Kerstetter | Corticostriatal afferents modulate responsiveness to psychostimulant drugs and drugassociated stimuli[END_REF]. In contrast, increasing activity of striatopallidal neurons (D2-neurons) with Gs-DREADD prevented amphetamine sensitization [START_REF] Farrell | A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons[END_REF]. This is in agreement with the results on mice lacking Drd2, a receptor exerting an inhibitory effect on D2-neurons, in which acute response to cocaine or to a Drd1 agonist (SKF81297) was blunted and locomotor sensitization to cocaine and amphetamine attenuated (Solis et al. 2021).

Combined with our current findings, these reports underline the importance of the balance between D1-and D2-neurons in regulating sensitization.

Although Drd1 is predominantly expressed in a subset of striatal projection neurons [START_REF] Gerfen | D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons[END_REF], it is also found at lower levels in some neurons of other regions such as prefrontal cortex [START_REF] Santana | Quantitative analysis of the expression of dopamine D1 and D2 receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex[END_REF]) and hippocampus [START_REF] Gangarossa | Characterization of dopamine D1 and D2 receptor-expressing neurons in the mouse hippocampus[END_REF] and some nonneuronal cells in and outside the brain [START_REF] Amenta | Light microscope autoradiography of peripheral dopamine receptor subtypes[END_REF][START_REF] Corkrum | Dopamine-evoked synaptic regulation in the nucleus accumbens requires astrocyte activity[END_REF]. These cells were potentially expressing Gi-DREADD and could have responded to CLZ stimulation, but not necessarily by an inhibition, as suggested for hippocampal astrocytes [START_REF] Nam | Activation of astrocytic μ-opioid receptor causes conditioned place preference[END_REF]. We therefore cannot formally exclude that the effects of CLZ were also involving cells outside the striatum.

Drug addiction is a complex disorder in which pathological memories play a critical role in the maintenance of addictive behaviors and relapse to drug seeking (reviews in Hyman et al., 2006;[START_REF] Robbins | Drug addiction and the memory systems of the brain[END_REF][START_REF] Tronson | Addiction: a drug-induced disorder of memory reconsolidation[END_REF]. Several rodent studies aimed to erase or weaken these drug-associated memories by inducing targeted disruption of memory reconsolidation processes. Memory reconsolidation is the process by which memories become labile under certain conditions of reactivation (retrieval) before being stabilized again and persistently stored in the brain (review in Lee et al., 2017). Drug-induced conditioned place preference was attenuated or erased by combining re-exposure to the drug-paired stimuli and/or the drug itself with inhibition of protein synthesis [START_REF] Valjent | Inhibition of ERK pathway or protein synthesis during reexposure to drugs of abuse erases previously learned place preference[END_REF][START_REF] Dunbar | Inhibition of protein synthesis but not β-adrenergic receptors blocks reconsolidation of a cocaine-associated cue memory[END_REF], ERK signaling [START_REF] Valjent | Inhibition of ERK pathway or protein synthesis during reexposure to drugs of abuse erases previously learned place preference[END_REF], D1 receptors [START_REF] Yan | Dopamine D1 and D3 receptors mediate reconsolidation of cocaine memories in mouse models of drug self-administration[END_REF][START_REF] Marion-Poll | Cocaine conditioned place preference: unexpected suppression of preference due to testing combined with strong conditioning[END_REF] or knockdown of the immediate early gene zif268/Egr1 [START_REF] Lee | Disrupting reconsolidation of drug memories reduces cocaine-seeking behavior[END_REF][START_REF] Lee | Cue-induced cocaine seeking and relapse are reduced by disruption of drug memory reconsolidation[END_REF][START_REF] Théberge | The basolateral amygdala and nucleus accumbens core mediate dissociable aspects of drug memory reconsolidation[END_REF]. Locomotor sensitization may undergo protein synthesis-dependent reconsolidation when the animals are briefly exposed to the drug-associated context without drug [START_REF] Bernardi | Anisomycin disrupts a contextual memory following reactivation in a cocaine-induced locomotor activity paradigm[END_REF]). Locomotor sensitization induced by a single injection of cocaine was shown to involve potentiation of excitatory synapses onto nucleus accumbens D1-neurons and was erased by optogenetic depotentiation of cortical inputs to these neurons [START_REF] Pascoli | Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour[END_REF]. Complete DREADD-induced inhibition of D1 neurons in the current study did not erase sensitization however, presumably because it did not modify synaptic input weights.

In conclusion, our results show that specific inhibition of D1-neurons strongly decreases spontaneous and cocaine-induced locomotor activity and alters locomotor sensitization induced by a single injection of cocaine. Our study suggests that selective manipulation of the excitability of these neurons is an interesting therapeutic approach in the context of addiction. 1-4) and wild type mice (WT, groups 5-8, independent experiments) were injected with vehicle (Veh, groups 1, 2, 5, 6) or CLZ (0.1 mg/kg, groups 3, 4, 7, 8) and placed in the locomotor activity box (circular corridor). Thirty min later, they were injected with saline (Sal, groups 1, 3, 5, 7) or cocaine (Coc,20 mg/kg,groups 2,4,6,8). (A) Timecourse of locomotor activity measured in 5-min bins in D1-Gi-DREADD mice. Repeated measures 2-way ANOVA, interaction, F(33, 407) = 3.25, p<10 -4 , time, F(11, 407) = 5.16, p<10 -4 , groups, F(3, 37) = 25.92, p<10 -4 . (B) Basal locomotor activity during the 30 min before cocaine injection, in D1-Gi-DREADD and WT mice after vehicle or CLZ pretreatment as indicated. CLZ decreased basal locomotion in transgenic mice (pooled groups 1 and 2 vs pooled groups 3 and 4, Mann-Whitney test, U = 21, p<10 -4 ) but not in WT mice (pooled groups 5 and 6 vs pooled groups 7 and 8, Mann-Whitney test, U = 654, NS). C) Total locomotor activity during the 60 min after cocaine injection. D1-Gi-DREADD mice (groups 1-4), Kruskal-Wallis test value = 25.2, p<10 -4 , Dunn's multiple comparisons test, 1 vs 2, p<0.05. Wild type mice (groups 5-8, independent experiment) Kruskal-Wallis test value = 50.8, p<10 -4 , Dunn's multiple comparisons test, 5 vs 6 and 7 vs 8, p<10 -4 . D) Same data as in C) but shown separately for males (M) and females (F). E-F) On day 10, all mice were injected with vehicle, put in the locomotor activity box and injected with cocaine (20 mg/kg) 30 min later. E) Time-course of locomotor activity measured in 5-min bins in D1-Gi-DREADD mice. Repeated measures 2-way ANOVA, interaction, F(33, 407) = 1.93, p = 0.002, time, F(11, 407) = 17.21, p<10 -4 , groups, F(3, 37) = 7.29, p = 6 10 -4 . F) Total locomotor activity during the 60 min after cocaine injection. D1-Gi-DREADD mice (groups 1-4), 2-way ANOVA, interaction, F(1, 37) = 5.93, p = 0.02, pretreatment on day 0, F(1, 37) = 2.06, NS, treatment on day 0, F(1, 37) = 13.62, p = 7 10 -4 , Sidak's multiple comparisons test, 1 vs 2, p<0.001, WT mice (groups 5-8, independent experiment), 2-way ANOVA, interaction, F(1, 75) = 0.24, NS, pretreatment on day 0, F(1, 75) = 1.83, NS, treatment on day 0, F(1, 75) = 16.92, p<10 -4 , Sidak's multiple comparisons test, 5 vs 6, p<0.05, 7 vs 8, p<0.01. In A and E each point correspond to a group mean ± SEM. In B, C, D, and F, each point correspond

Figure legends

Figure 1 .

 1 Figure 1. Experimental design of Gi-DREADD inhibition of D1 neurons in cocaine two-injection protocol of sensitization (TIPS). A) Gi-DREADD (hM4Di) expression in Drd1-expressing cells (D1-cells) of double transgenic Drd1-Cre x Gi-DREADD mice (referred to as D1-Gi-DREADD mice). Following Cre-mediated removal of an upstream floxed-STOP cassette, the HA-tagged Gi-DREADD-mCitrine mRNA is expressed in D1-cells. HA, hemagglutinin epitope tag, PTA, porcine teschovirus cleavage site. B) Groups of mutant and wild type mice used in the study. The number of mice and sex ratio is indicated for each group. C) Schematic schedule of behavioral experiments. Cocaine, 20 mg/kg, clozapine, CLZ, 0.1 mg/kg.

Figure 2 .

 2 Figure 2. CLZ decreases cocaine-induced locomotor activity and Fos expression in Gi-DREADD-expressing striatal neurons. A) Schematic experimental design. Vehicle or CLZ was injected 30 min before cocaine administration. The locomotor activity box is a circular corridor. Mice were killed 90 min after cocaine administration for immunofluorescence. B) Inhibition of D1-neurons by stimulating Gi-DREADD with CLZ prevents cocaine-induced hyper-locomotion. Locomotor activity was measured during 60 min after cocaine injection in mice pretreated with vehicle or CLZ as indicted. Data were collected in 5-min bins and shown for each mouse. Bars indicate means + SEM, n = 5 mice per group. Mann-Whitney's U = 0, p = 0.0079, ** p<0.01. C) Fosand HA-immunoreactivity 90 min after cocaine administration (20 mg kg -1 i.p.) were visualized by immunofluorescence and confocal microscopy. Representative images of double immunolabeling of Fos (green) and HA (red) in NAc shell. Merged image demonstrates colocalization (arrows) of HA-Gi-DREADD and Fos. Scale bar, 50 µm. D) The position of the striatal regions used for quantification (388 × 388 µm) is indicated by red squares on a mouse brain coronal section at 0.98 mm antero-posterior coordinate (after Paxinos and Franklin, 2001): dorsomedial (DM) and dorsolateral (DL) striatum, NAc core (C) and shell (S). E) The percentage of Fos-positive neurons among HA-positive neurons was analyzed in the 4 regions indicated in D. Each data point corresponds to the mean results of the right and left side for each region in one mouse (5 mice per group), with bars indicating means + SEM. Mann-Whitney's test, NAc core, U = 26, p<0.075, NAc shell, U = 18.5, p = 0.016, DM, U = 5, p = 2 10 -4 , DL, U = 18, p = 0.067. *p<0.05, *p<0.01, ***p<0.001.

Figure 3 .

 3 Figure 3. Gi-DREADD-induced inhibition of D1-neurons decreases acute cocaine-induced hyper-locomotion and cocaine sensitization in the TIPS protocol. A-D) On day 0, D1-Gi-DREADD mice (D1-DREADD, groups

  to a mouse, bars indicate means + SEM. NS, not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<10 -4 .

Figure 4 .

 4 Figure 4. Gi-DREADD-induced inhibition of D1-neurons blocks cocaine-induced locomotion in previouslysensitized mice without persistent effect on its further expression. A-C) On day 11, the same D1-Gi-DREADD and WT mice as in Fig.3were all injected with CLZ, placed in the locomotor activity box, and 30 min later, injected with cocaine (20 mg/kg). The time-course of locomotor activity was measured in 5-min bins. A) D1-Gi-DREADD mice, repeated measures 2-way ANOVA, interaction, F(33, 407) = 1.39, NS, time, F(11, 407) = 7.29, p<10 -4 , groups, F(3, 37) = 1.20, NS. B) Total locomotor activity during the 60 min after cocaine injection. D1-Gi-DREADD mice (groups 1-4), Kruskal-Wallis test value = 4.6, NS. WT mice (groups 5-8, independent experiment), Kruskal-Wallis test value = 6.25, NS. C) Pairwise comparison for each mouse of locomotor responses to cocaine on day 10 without CLZ pretreatment (data from Fig.3E) and on day 11 with CLZ (data from Fig.4B). Paired t-test, group 1, p = 0.001, group 3, p = 0.001, group 4, p = 0.038. Wilcoxon matched-pairs test, group 2, p = 0.001. D-F) On day 12, the same mice as in A were all injected with vehicle, placed in the locomotor activity box, and 30 min later, injected with cocaine (20 mg/kg). The time-course of locomotor activity was measured in 5-min bins. D) D1-Gi-DREADD mice, repeated measures 2-way ANOVA, interaction, F(33, 397) = 1.41, NS, time, F(11, 396) = 36.87, p<10 -4 , groups, F(3, 36) = 0.63, NS. E) Total locomotor activity during the 60 min after cocaine injection. D1-Gi-DREADD mice (groups 1-4), Kruskal-Wallis test value = 2.39, NS. Wild type mice (groups 5-8, independent experiment) Kruskal-Wallis test value = 0.76, NS. F) Pairwise comparison for each mouse of locomotor responses to cocaine on day 10 without CLZ pretreatment (data from Fig.3B) and on day 12 (data from Fig.4E). Paired t-test, group 1, p = 0.009, group 2, p = 0.235, group 3, p = 0.001. Wilcoxon matched-pairs test, group 4, p = 0.014. In A and D, each point correspond to a group mean ± SEM. In B, C, E, and F, each point correspond to a mouse. In B and E, bars indicate means + SEM. NS, not significant, * p<0.05, ** p<0.01, *** p<0.001.

Acknowledgments

Research in the laboratory of JAG was supported by Inserm and Sorbonne University and by grants from Fondation pour la Recherche Médicale (FRM DPA20140629798 and EQU201903007844) and ANR (Epitraces, ANR-16-CE16-0018). Equipment at the IFM was funded in part by Fondation pour la recherche sur le cerveau (FRC) and Rotary Espoir en Tête, and by DIM Cerveau et Pensée Région Ile-de-France. YN was recipient of an Uehara Memorial Foundation fellowship and a Fyssen Foundation fellowship. Cell imaging was done at the Institut du Fer à Moulin Tissue and Cell Imaging Facility and mice were bred and studied at the Institut du Fer à Moulin Rodent Breeding and Phenotyping Facility.

Data accessibility

Original data are accessible upon request

Competing Interests

The authors declare no competing interest.

Authors contributions

Yukari N carried out experiments, analyzed results and wrote the manuscript, SL and Yuki N carried out experiments and analyzed results, AN analyzed results, JAG supervised the work, DH, JAG, and Yuki N planned and conceived experiments, analyzed results and wrote the manuscript.

Abbreviations