Oleksandr Daniel A Díaz-Thomas
email: daniel.diaz-thomas@umontpellier.fr

Oleksandr Stepanenko

Michaël Bahriz

Stéphane Calvez

Thomas Batte

Cyril Paranthoen

Gilles Patriarche

Eric Tournié

Alexei N Baranov

Guilhem Almuneau

C Levallois

L Cerutti

3 µm interband-cascade resonant-cavity lightemitting diode with narrow spectral emission linewidth

Keywords: RCLED, midinfrared, interband-cascade

come

µm interband-cascade resonant-cavity light-emitting diode with narrow spectral emission linewidth

Introduction

The strong absorption of hydrocarbons and other pollutants in the window of atmospheric transparency between 3 and 4 µm makes this spectral region very attractive for environmental monitoring [START_REF] Tournié | Mid-infrared optoelectronics: materials, devices, and applications[END_REF]. Sb-based semiconductor light sources allow addressing such gas sensing applications in the mid-infrared (mid-IR). Over the last decade, significant progress has been achieved in mid-IR emitters employing Interband Cascade (IC) structures based on InAs/GaInSb type-II quantum wells, particularly efficient in the 3 -4 µm range [START_REF] Vurgaftman | [END_REF]. The luminescence of the IC active region enables both TE and TM light polarizations and therefore the ability to vertically extract the light from a planar cavity structures such as light emitting diodes (LEDs) [START_REF] Schubert E F, Gessmann | Kirk-Othmer Encyclopedia of Chemical Technology[END_REF], or vertical cavity surface emitting lasers (VCSELs) [START_REF] Coldren | Vertical-cavity Surface-emitting Lasers: Design, Fabrication, Characterization, and Applications[END_REF]. Resonant Cavity Light Emitting Diodes (RCLEDs) are attractive for the development of low-cost systems for gas analysis [START_REF] Böttger | [END_REF] Since their first demonstration in 1992 [6], only a few mid-IR RCLEDs, based on arsenide [7], phosphide [8] and antimonide compounds have been reported [9] [10], with only one IC-RCLED operating at RT [11]. Even if RCLEDs use less distributed Bragg reflectors (DBR) pairs than VCSELs, their electrical resistivity can induce a strong self-heating of the devices [12] and lead to a degradation of the performances. The use of an intracavity electrical contact has been proposed and successfully demonstrated in VCSELs [13][14][15] to circumvent this issue. In addition, in a RCLED, the current is commonly [11] injected through an annular anode contact pad leading to a non-negligible radial

a)

inhomogeneity. Even though several strategies used for Sb-based VCSELs such as a buried tunnel junction [13], or an underetched tunnel junction [14]

Experimental details

IC-RCLED design and epitaxy

Structural and optical characterizations

The crystalline quality of the grown structures shows that the satellite peaks for the RCLED are however observed to be larger than those of the conventional IC-LED. This could be attributed to the X-ray scattering within the metamorphic GaAs layer because of the high density of dislocations. arising at the GaSb/GaAs interface.

To confirm this hypothesis, the top GaAs was dry etched using inductively coupled plasma reactive ion etching (ICP-RIE) on a part of the sample.

Four etching steps of 250 nm each were performed and HRXRD was carried out between each step. Furthermore, the effect of these dislocations on the active zone of the device was studied using transmission electron microscopy (TEM). Figure 3 clearly demonstrates that the dislocations do not extend from the GaAs/GaSb interface down to the active region, contrarily to what it was observed in metamorphic GaAs/InP structures [23], and remain confined to the arsenide layers.

Devices fabrication

The epitaxial wafer was patterned with circular mesas with diameters ranging from 25 µm to 55 µm using standard optical photolithography and a combination of ICP-RIE and wet etching. The mesas were etched down to the InAsSb bottom contact layer. After this step, a thermal selective lateral oxidation process of the Al-rich AlGaAs layer was performed at temperature at conditions similar to those used for AlGaAs/GaAs structures reported in [21] [24]. The progress of the oxidation front was observed during the oxidation process with a real-time in situ monitoring optical system [25]. After oxidation, the aperture diameters spanned from 5 to 35 µm incremented by 2 µm.

This step was followed by surface passivation of the structures using a Si3N4 ICP-PECVD coating and then Ti/Au metallization of the InAsSb layer for the bottom contact. Afterwards, contact pads for top contacts and another Si3N4 passivation layer were deposited.

Finally, a two-pair ZnS/Ge dielectric Bragg mirror was deposited by RF sputtering and patterned by a lift-off process. The reflectivity seen from the cavity is calculated to be around 91%, a value slightly lower than the bottom DBR allowing a better extraction of the light. Figure 5 shows a schematic and a SEM cross section image of the complete RCLED structure at the end of the process flow. The oxide confinement aperture is clearly visible in Figure 5 and is of the same size as the top emission window defined by the contact ring, which ensures an efficient current confinement. In parallel, the IC-LED (without lateral confinement) was processed with mesa diameters of 100 µm and ring contacts width of 20 µm.

Electro-optical characterizations

Electro-optical measurements of the fabricated devices were performed under CW operation using a thermoelectrically-controlled stage.

  Due to a narrower emission linewidth [Sarcan F, Wang Y, Krauss T F, Erucar T, and Erol A 2020 Optics and Laser Technology 122 105888] such sources can offer better overlap with absorption features of the analyzed specie compared with the conventional LEDs and thus improved sensitivity [Al-Saymari F A, Craig A P, Lu Q, Marshall A R J, Carrington P J, and Krier A 2020 Optics Express 28 23338]. Usually, there is only one DBR in RCLEDS, placed below the active region. A simpler Bragg mirror with a few quarterwavelength dielectric layers deposited on the top of an RCLED structure is however useful to control the device properties. By adjusting its reflectivity the RCLED emission linewidth can be matched with the shape of the desired absorption band making their relative overlap to be little sensitive to measurement conditions and hence no sophisticated control is needed to compensate the eventual change. In the case of a VCSEL, the wavelength of the narrow laser emission line should be precisely fixed inside the absorption band to avoid signal fluctuations or scanned around it, which requires specific electronic circuits resulting in elevated equipment cost. The main motivation of this work was to develop light sources for specific spectroscopic applications which require an emission linewidth intermediate between that of conventional LEDs (few tens nm) and VCSELs (< 1 nm).

Figure 1 .

 1 Figure 1. a) Scheme of the IC-RCLED structure with the refractive index profile (black) and the EM intensity distribution (red). b) Energy band diagram of one stage of the active region simulated with Nextnano © , showing the cascade process.

Figure 1 .

 1 Figure 1.a) depicts a schematic diagram of the IC-RCLED structure and the intrinsic optical field

Figure 2 .

 2 Figure 2. a)- -2 HRXRD scans at the (0 0 4) reflection for the RCLED and reference LED structure. Inset: magnification on the satellite peaks -b) Evolution of the FWHM of the satellite peaks of the RCLED with etched GaAs.

  Figure 2.a shows for the RCLED structure, a

Figure 3 .

 3 Figure 3. Transmission electron microscopy crosssection views of the RCLED showing dislocations propagating above the GaSb/GaAs metamorphic interface

Figure 2 .

 2 b displays the evolution of the full width half maximum (FWHM) averaged over the 10 satellite peaks closest to the substrate peak. Upon removal of the GaAs layer, the FWHM of the satellite peaks from the IC-RCLED approaches the value of the reference IC-LED.

  the RCLED epitaxial structure are presented in Figure 4. The measurement was performed with a Brucker FTIR spectrometer under quasi-normal incidence. The stop-band is centered at 3.32 μm and the reflectivity dip related to the cavity resonance is at 3.26 μm. This wavelength detuning from 3.3 µm is attributed, based on reflectivity calculation, to a cavity thickness of about 3% smaller than the target value.

Figure 4 .

 4 Figure 4. Experimental and simulated top reflectivity spectrum of the RCLED epitaxial structure without the top dielectric DBR.

Figure

  Figure shows the CW P-I-V characteristics at 20°C for different oxide aperture diameters. The

Figure 5 .

 5 Figure 5. Schematic and Scanning Electron Microscopy (SEM)picture of a cross section of the IC-RCLED after processing

Figure 6 .

 6 Figure 6.c), shows the electroluminescence

  Figure shows the P-I-V and the electroluminescence (EL) spectra at different

Figure 6 .Figure 7

 67 Figure 6. (a) CW L -I -V characteristics, and (b) series resistance for RCLEDs as a function of the aperture diameter ranging from 5 µm to 35 µm. (c) Emission spectra of a 35 µm oxide aperture IC-RCLED and 100 µm mesa diameter IC-LED at 20°C in CW and simulated reflectivity of the whole IC-RCLED

3 .

 3 Conclusions summary, we have demonstrated a Mid-IR IC-RCLED combining the benefits of both antimonide and arsenide materials and of their processing technologies. The device emitting near 3.3 µm was grown on a GaSb substrate by MBE and operated in CW regime up to 80°C (setup-limited). An electro-optical lateral confinement was applied within the device structure by wet thermal oxidation of an AlGaAs layer metamorphically grown after the active region. It was demonstrated that this metamorphic layer did not affect the crystal quality of the active region of the device. Thanks to this optimal IC-RCLED configuration combining intracavity electrical injection and lateral confinement, the emission spectral width could be reduced to 20 nm, either a factor of 20 smaller compared to a conventional IC-LED. The narrow emission line and its weak shift with temperature make this device attractive for low-cost systems for gas analysis.

Acknowledgements

The authors would like to aknowledge the support from the Agence Nationale de la Recherche (ANR-16-CE24-0011); French Program on "Investment for the Future" (Equipex EXTRA, ANR-11-EQPX-0016) The authors are thankfull for the support from RENATECH (French Network of Major Technology Centers) and RENATECH+ and theirs LAAS-CNRS, Nanorennes and CTM technological facilities.


Ph.D. † Benjamin Galet

Ph.D. ♯ Manuela Ingallinesi

MSc Jonathan Pegon

Anh Do

Ph.D. † Philippe Ravassard

Ph.D Nicole § Faucon Biguet

M.D Rolando Meloni
email: rolando.meloni@upmc.fr

Gpr88 and psychiatric symptoms of Parkinson's disease

Keywords: GPR88, Parkinson's disease, non-motor symptoms, gene therapy, striatum, 6-OHDA

Background: In addition to motor disability, another characteristic feature of Parkinson disease is the early appearance of psychiatric symptoms, including apathy, depression, anxiety and cognitive deficits; treatments for these symptoms are limited by the development of adverse effects such as impulse-control disorders. In this context, we investigated the orphan G protein-coupled receptor 88 (GPR88) as a novel therapeutic target. Methods: We used lentiviral-mediated expression of specifically designed microRNA to knock down Gpr88 in a translational male rat model of early Parkinson disease obtained by dopamine loss in the dorsolateral striatum as a result of 6-hydroxydopamine lesions. We evaluated the impact of Gpr88 knockdown on the Parkinson disease model using behavioural, immunohistochemical and in situ hybridization studies. Results: Knockdown of Gpr88 in associative territories of the dorsal striatum efficiently reduced alterations in mood, motivation and cognition through modulation of the regulator of the G-protein signalling 4 and of the truncated splice variant of the FosB transcription factor. Knockdown of Gpr88 also reduced allostatic changes in striatal activity markers that may be related to patterns observed in patients and that provide support for an "overload" hypothesis for the etiology of the psychiatric symptoms of Parkinson disease. Limitations: Behavioural tests assessing specific cognitive and motivational parameters are needed to further characterize the effects of the lesion and of Gpr88 knockdown in early-stage and advanced Parkinson disease models, presenting more extensive dopamine loss. Additional studies focusing on the direct and indirect striatal output pathways are also required, because little is known about the signalling pathways regulated by GPR88 in different striatal cell types. Conclusion: GPR88 may constitute a highly relevant target for the treatment of the psychiatric symptoms of Parkinson disease.

Introduction

Parkinson disease is classically defined as a motor disorder related to the neurodegenerative loss of dopamine innervation of the striatum, but it also involves psychiatric symptoms that represent a major burden for patients' quality of life. 1 Many of these symptoms have been grouped as part of a "hypodopaminergic" syndrome, which includes apathy, depression, anxiety and cognitive impairment and affects up to 85%of patients with Parkinson disease. [START_REF] Sierra | Apathy and impulse control disorders: yin & yang of dopamine dependent behaviors[END_REF][START_REF] Schapira | Non-motor features of Parkinson disease[END_REF] Apathy in particular, defined as a decrease in motivational drive, has emerged as a core symptom of Parkinson disease and is often associated with cognitive impairment. [START_REF] Pagonabarraga | Apathy in Parkinson's disease: clinical features, neural substrates, diagnosis, and treatment[END_REF] A remarkable feature of these symptoms is that, because of the progressive nature of the neurodegeneration, they frequently emerge years before the onset of motor impairments, which appear when striatal dopamine loss exceeds 60% to70%. [START_REF] Darweesh | Trajectories of prediagnostic functioning in Parkinson's disease[END_REF]6 Thus, at the time of diagnosis, which is elicited by the appearance of motor symptoms, 37% of patients already have depression, 27% have apathy, 17% have anxiety and 20% to 40% have cognitive dysfunction, 6 redefining Parkinson disease as a quintessential neuropsychiatric disorder. [START_REF] Weintraub | Parkinson's disease: the quintessential neuropsychiatric disorder[END_REF] Dopaminergic medications can efficiently treat some of these aspects of Parkinson disease, [START_REF] Schapira | Non-motor features of Parkinson disease[END_REF][START_REF] Pagonabarraga | Apathy in Parkinson's disease: clinical features, neural substrates, diagnosis, and treatment[END_REF] but their effect is based on nonphysiologic pulsatile dopaminergic stimulation (similar to the effect of L-DOPA substitutive therapy for motor symptoms) and is thus hindered by the frequent development of "hyperdopaminergic" symptoms such as impulse-control disorders. [START_REF] Sierra | Apathy and impulse control disorders: yin & yang of dopamine dependent behaviors[END_REF] Furthermore, existing treatments for cognitive impairment have variable efficacy and limiting adverse effects. [START_REF] Svenningsson | Cognitive impairment in patients with Parkinson's disease: diagnosis, biomarkers, and treatment[END_REF] There is thus an unmet medical need for new treatments to manage these symptoms.

In this context, orphan G protein-coupled receptor 88 (GPR88) is emerging as a particularly suitable target. The GPR88 gene has been associated with bipolar disorder and schizophrenia, [START_REF] Zompo | Association study in three different populations between the GPR88 gene and major psychoses[END_REF] as well as with learning deficits and chorea. [START_REF] Alkufri | Deleterious mutation in GPR88 is associated with chorea, speech delay, and learning disabilities[END_REF] It is expressed mainly in the corticostriatal synapse of striatal medium spiny neurons, [START_REF] Massart | Striatal GPR88 expression is confined to the whole projection neuron population and is regulated by dopaminergic and glutamatergic afferents[END_REF] where it exerts an inhibitory activity through Gi/o coupling. [START_REF] Quintana | Lack of GPR88 enhances medium spiny neuron activity and alters motor-and cue-dependent behaviors[END_REF][START_REF] Ye | Orphan receptor GPR88 as an emerging neurotherapeutic target[END_REF] Indeed, Gpr88 knockout (KO) mice are hypersensitive to dopamine receptor type 2 agonists and display impaired affective, goal-directed, cognitive and sensorimotor behaviours. [START_REF] Quintana | Lack of GPR88 enhances medium spiny neuron activity and alters motor-and cue-dependent behaviors[END_REF][START_REF] Logue | The orphan GPCR, GPR88, modulates function of the striatal dopamine system: a possible therapeutic target for psychiatric disorders?[END_REF][START_REF] Meirsman | Mice lacking Gpr88 show motor deficit, improved spatial learning, and low anxiety reversed by delta opioid antagonist[END_REF] We have also reported that Gpr88 knockdown (Gpr88-KD) reduces behavioural alterations in a rat model of schizophrenia. [START_REF] Ingallinesi | Local inactivation of Gpr88 in the nucleus accumbens attenuates behavioral deficits elicited by the neonatal administration of phencyclidine in rats[END_REF] As well, we have more recently shown that Gpr88-KD decreases locomotor alterations in a rat model of Parkinson disease motor symptoms. [START_REF] Ingallinesi | Knock-down of GPR88 in the dorsal striatum alters the response of medium spiny neurons to the loss of dopamine input and L-3-4dyhydroxyphenylalanine[END_REF] Therefore, because GPR88 exerts inhibitory control over key neurotransmitter systems that are altered in Parkinson disease, we further hypothesized that inhibiting GPR88 in the striatum could have an effect on the psychiatric symptoms of Parkinson disease.

To evaluate GPR88 as a therapeutic target for the psychiatric symptoms of Parkinson disease, we first developed a translational model of early Parkinson disease in rats by reproducing the loss of dopamine that is observed in the first stages of the disease, specifically affecting sensorimotor territories of the striatum, [START_REF] Brooks | Imaging biomarkers in Parkinson's disease[END_REF] using stereotaxic injections of the 6hydroxydopamine (6-OHDA) toxin. We then implemented Gpr88-KD in the dorsolateral sensorimotor or dorsomedial associative areas of the striatum (the latter being associated with psychiatric symptoms in Parkinson disease [START_REF] Qamar | Presynaptic dopaminergic terminal imaging and non-motor symptoms assessment of Parkinson's disease: evidence for dopaminergic basis?[END_REF] ) and evaluated effects at the behavioural and molecular levels.

Methods

The study design is shown in Figure 1; details are provided in Appendix 1, available at jpn.ca/190171-a1.

Animals

Animal studies were authorized by an ethical committee (APAFIS reference #3669-2016011817516297) and conducted in the in-house specific pathogen-free animal facility (agreement reference B75- 13-19). Male Sprague-Dawley rats weighing 300 g at the beginning of experimentation were housed in standard conditions and handled throughout the study in compliance with the European Union 2010 animal welfare act, and the 2010/63 French directive. Procedures were reported following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.20

Stereotaxic injections of 6-OHDA

Intrastriatal stereotaxic injections were performed bilaterally with 4 μL of a solution of 6-OHDA (3 μg/μL, 12 μg total per hemisphere) in saline + 0.02% ascorbic acid (all chemicals from Sigma) or with a control solution (saline + 0.02% ascorbic acid only).

Lentivirus production

The lentiviral vectors coexpressing specifically designed microRNA (miR) and emerald green fluorescent protein were generated at the in-house iVector platform, using the BLOCKiT Pol II miR RNAi expression vector kit (Invitrogen) and injected 2 weeks after the 6-OHDA lesions were introduced.

Behavioural tests

The rats were tested for general motor and exploratory behaviour using an actimeter, for anhedonia using the sucrose preference test, for social interaction and selective attention using the social novelty discrimination task, for sensorimotor gating using the prepulse inhibition test, and for depression associated behaviour using the forced swim test.

Immunolabelling and in situ hybridization

After the end of the behavioural procedures (2 to 4 days later), the rats were anesthetized in an isoflurane chamber and decapitated. Their brains were rapidly removed and snap-frozen for 90 seconds in isopentane at -55°C (Carlo Erba Reagents). Coronal cryosections (12 μm) of the striatum were used to control for the presence and extent of emerald green fluorescent protein signal in the targeted areas. The slices were then post-fixed in 4% paraformaldehyde for 30 minutes at 4°C to perform immunolabelling and in situ hybridization experiments.

Statistical analysis

Data from the experiments were analyzed using Prism 6.0 (GraphPad Software Inc.) and implementing different tests as detailed in Appendix 1.

Results

6-OHDA stereotaxic injections induce dopaminergic denervation

The 6-OHDA injections in the dorsolateral striatum (DLS) reproduced the partial loss of dopaminergic projections to sensorimotor territories of the striatum (posterior putamen) observed in early Parkinson disease [START_REF] Redgrave | Goal-directed and habitual control in the basal ganglia: implications for Parkinson's disease[END_REF] by inducing a 48% mean reduction of tyrosine hydroxylase immunoreactivity over at least 1 mm on the anterior-posterior axis (t16 = 9.28, p < 0.001; multiple t tests followed by Holm-Sidak corrections; Fig. 2A andB). The neighbouring associative dorsomedial striatum (DMS) and limbic ventral striatum (VS) were not significantly affected by the lesion (DMS t16 = 1.82, p = 0.09; VS t15 = 0.43, p = 0.67; Fig. 2B).

Lentivirus-mediated knockdown efficiently reduced Gpr88 expression

The injections at multiple sites in the DLS or DMS of lentiviral vectors containing a control miR (miR-neg) or an miR directed against Gpr88 (miR-Gpr88) resulted in the transduction of the corresponding regions without spreading to the adjacent areas (Fig. 2C andD). Two-way analysis of variance (ANOVA) showed significant effects of Gpr88-KD (F2,52 = 28, p < 0.001), the striatal region (F1,52 = 10, p = 0.002) and a Gpr88-KD  striatal region interaction (F2,52 = 72, p < 0.001) on Gpr88 expression. The region-specific effect of Gpr88-KD was confirmed using Tukey post hoc multiple comparison tests (p < 0.001 in the DLS and DMS; Fig. 2E andF).

Partial dopaminergic depletion of the DLS reproduced psychiatric symptoms without inducing motor deficits

Motor behaviour was assessed at 2 and 4 weeks after 6-OHDA injections by measuring horizontal movements in the actimeter. This parameter was unaffected by dopamine loss at both time points (Fig. 3A; Appendix 1, Fig. S2A), as expected with a restricted lesion. [START_REF] Drui | Loss of dopaminergic nigrostriatal neurons accounts for the motivational and affective deficits in Parkinson's disease[END_REF] However, although stereotyped behaviour in the actimeter test was also preserved at both time points, 2-way ANOVA revealed a significant interaction of treatment  time on the number of rearings (F2,44 = 4.66, p = 0.015; Fig. 3A); these were significantly reduced by the lesion at the 15-minute time point (Sidak multiple comparison test, p = 0.013), indicating a decrease in novelty exploration behaviour, previously characterized in similar models of partial dopamine loss in the DLS. 24,25 Sensorimotor gating is altered in many basal ganglia disorders, including Parkinson disease, and has been linked to cognitive dysfunction in patients with Parkinson disease. [START_REF] Zoetmulder | Prepulse inhibition is associated with attention, processing speed, and 123I-FP-CIT SPECT in Parkinson's disease[END_REF] We assessed this parameter using the prepulse inhibition test. Although the partial lesion of the DLS increased the startle reaction in response to a loud auditory stimulus (110 dB; t10.21 = 2.396, p = 0.037, Welch t test; Fig. 3B, left panel), the level of sensorimotor gating (% prepulse inhibition) was preserved at all levels of prepulse intensity used (Fig. 3B, right panel), suggesting the presence of efficient compensatory mechanisms.

Affective parameters relating to anhedonia and depression (both frequently reported in

Parkinson disease) [START_REF] Schapira | Non-motor features of Parkinson disease[END_REF] were assessed using the sucrose preference and the forced swim tests, respectively. Sucrose preference and general consummatory behaviour, measured by daily food and water intake, were not affected by the 6-OHDA lesion (Fig. 3C; Appendix 1, Fig. S2B). However, the lesion did decrease the latency to immobility and increased immobility count in the forced swim test (latency t21.34 = 3.94, p < 0.001; immobility count t22.85 = 4.36, p < 0.001; Welch t tests; Fig. 3D), representative of a despair-like behaviour associated with depression.27 Finally, we used the social novelty discrimination task to evaluate deficits in motivation and selective attention (a core feature of Parkinson disease executive dysfunction). [START_REF] Svenningsson | Cognitive impairment in patients with Parkinson's disease: diagnosis, biomarkers, and treatment[END_REF] The loss of dopamine in the DLS decreased social interaction time during the first presentation phase (P1; t22.72 = 2.95, p = 0.007, Welch t test; Fig. 3E), an ecological indicator of apathy. [START_REF] Cathomas | The translational study of apathy-an ecological approach[END_REF] Further analyses then revealed a significant treatment  novelty status interaction in the discrimination phase of the test (P2; 2-way ANOVA: F1,48 = 8.995, p = 0.004); the lesioned animals failed to preferentially direct their attention to the novel juvenile, which resulted in a decreased discrimination ratio (t22.51 = 4.29, p < 0.001, Welch t test; Fig. 3E), an effect that was not due to a reduced level of interaction during the discrimination task (Appendix 1, Figure S2C).

Gpr88-KD in the DMS reversed behavioural deficits

Although 2-way ANOVA revealed no significant main effects of treatment or treatment Furthermore, whereas Gpr88-KD in either area had no effect on sucrose preference (Fig. 4C), inactivation in the DMS had an antidepressant-like effect in the forced swim test: it increased latency (effect of treatment F2,28 = 6.883, p = 0.004; Dunnett post hoc test miR-Gpr88-DMS v. miR-neg p = 0.003) and reduced immobility count (effect of treatment F2,28 = 3.736, p = 0.037; Dunnett post hoc test miR-Gpr88-DMS v. miR-neg p = 0.021; Fig. 4D). This reduction was specifically mediated by an increase in swimming behaviour (effect of treatment F2,28 = 3.524, p = 0.043; Dunnett post hoc test miRGpr88-DMS v. miR-neg p = 0.028; Appendix 1, Fig. S3A). We observed no effect on consummatory behaviour (Appendix 1, Fig. S3B).

In the DMS, Gpr88-KD also had a promotivational effect, as it increased the duration of social interaction (effect of treatment F2,28 = 3.938, p = 0.031; Dunnett post hoc test miR-Gpr88-DMS v. miR-neg p = 0.019; Fig. 4E). Finally, Gpr88-KD increased preferential interaction with the novel juvenile in the social novelty discrimination task (2-way ANOVA, treatment  novelty status interaction F2,56 = 3.162, p = 0.05) in both the DLS and DMS (p= 0.004 and p < 0.001, respectively; Sidak multiple comparison test; Fig. 4E). However, only the effect in the DMS was large enough to restore the discrimination ratio (effect of treatment F2,28 = 7.998, p = 0.002; Dunnett post hoc test miR-Gpr88-DMS v. miR-neg p = 0.001; Fig. 4E). However, this proattentional effect was not associated with an increase in total interaction time during the discrimination task (Appendix 1, Fig. S3C). It is worth noting that although significant effects of Gpr88-KD were observed mainly when targeting the DMS, inactivation in the DLS often also tended to reduce behavioural alterations.

Dopamine loss in the DLS induces molecular alterations in the adjacent DMS

The dopamine striatal loss in Parkinson disease or after a 6-OHDA lesion dysregulates the balanced activity of the 2 types of projecting medium spiny neurons -the direct stimulatory striatonigral (dMSN) and the indirect inhibitory striatopallidal (iMSN) pathways -inducing the symptoms of Parkinson disease. [START_REF] Gerfen | Modulation of striatal projection systems by dopamine[END_REF] Alterations in these pathways are associated with the downregulation of prodynorphin (Pdyn) in the dMSN and the upregulation of proenkephalin (Penk) in the iMSN, as well as a net increase in striatal glutamic acid decarboxylase 67 (Gad67) expression. [START_REF] Carta | Alterations in GAD67, dynorphin and enkephalin mRNA in striatal output neurons following priming in the 6-OHDA model of Parkinson's disease[END_REF] Therefore, we evaluated the expression levels of Pdyn and Penk, which reflect dMSN and iMSN activity, respectively, [START_REF] Sgroi | Opioidergic modulation of striatal circuits, implications in Parkinson's disease and levodopa induced dyskinesia[END_REF] and Gad67, which is considered a proxy for global levels of transmission of γ-aminobutyric acid in the striatum. [START_REF] Consolo | Increased striatal expression of glutamate decarboxylase 67 after priming of 6-hydroxydopamine-lesioned rats[END_REF] Strikingly, although the loss of dopamine was strictly limited to the DLS, we also observed significant alterations in the expression of these markers in the adjacent, intact DMS. Indeed, although the lesion decreased expression levels of Pdyn and increased those of Penk in the DLS as expected (Pdyn t21 = 5.551, p < 0.001; Penk t21 = 5.035, p < 0.001; multiple t tests followed by Holm-Sidak corrections, here and throughout the next sections; Fig. 5A), expression of both markers was upregulated in the unlesioned DMS (Pdyn t21 = 2.522, p = 0.020; Penk t21 = 2.709, p = 0.013; Fig. 5A), suggesting a local hyperactivity of dMSN and iMSN. Furthermore, Gad67 expression was also significantly upregulated throughout the dorsal striatum (DLS t21 = 4.095, p < 0.001; DMS t21 = 2.967, p = 0.007; Fig. 5A).

The regulator of G protein signalling 4 (Rgs4) is highly expressed in the brain and acts as a negative regulator of synaptic signalling. [START_REF] Gerber | Roles for regulator of G protein signaling proteins in synaptic signaling and plasticity[END_REF] In 6-OHDA models of Parkinson disease, inhibition of Rgs4 has antiparkinsonian effects: it reduces behavioural alterations by restoring long-term depression in the iMSN while reducing acetylcholine release from cholinergic interneurons. [START_REF] Lerner | RGS4 is required for dopaminergic control of striatal LTD and susceptibility to Parkinsonian motor deficits[END_REF][START_REF] Ding | RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion[END_REF] Moreover, the increase in the excitability of medium spiny neurons associated with Gpr88-KO is mediated by the downregulation of Rgs4. [START_REF] Quintana | Lack of GPR88 enhances medium spiny neuron activity and alters motor-and cue-dependent behaviors[END_REF] Thus, we used Rgs4 expression to further investigate the effects of the lesion and Gpr88-KD on intracellular signalling. The 6-OHDA lesion induced a trend toward a decrease in the expression of Rgs4 in the DLS (t22 = 1.965, p = 0.06) and significantly increased expression in the DMS (t22 = 2.794, p = 0.011; Fig. 5A). However, Gpr88 expression remained unaffected by the loss of dopamine in either striatal area (Appendix 1, Fig. S4).

We then used the truncated splice variant of FosB (ΔFosB), a transcription factor involved in neuropsychiatric disorders including Parkinson disease, [START_REF] Lindgren | Putaminal upregulation of FosB/DeltaFosBlike immunoreactivity in Parkinson's disease patients with dyskinesia[END_REF] as a final molecular readout, because its unique accumulation profile confers a long-lasting effect on the regulation of striatal gene networks. [START_REF] Nestler | DFosB: a transcriptional regulator of stress and antidepressant responses[END_REF] We found that ΔFosB expression was locally increased by the loss of dopamine in the DLS (t28 = 3.713, p = 0.002), but it was also significantly decreased in the unlesioned DMS (t28 = 3.338, p = 0.002; Fig. 5B).

Inactivation of Gpr88 in the DMS reverses alterations in Rgs4 and ΔFosB expression

Although Gpr88-KD had no significant effect on Pdyn expression, it significantly reduced the expression level of Penk in the DLS (t17 = 5.163, p < 0.001; Fig. 6A), suggesting a reduction of iMSN hyperactivity. Also, Gpr88-KD strongly reduced the expression of Gad67 and Rgs4 in the DLS and DMS (DLS t17 = 5.956, p < 0.001 for Gad67 and t20 = 6.404, p < 0.001 for Rgs4; DMS t19 = 4.319, p < 0.001 for Gad67 and t20 = 3.633, p = 0.002 for Rgs4; Fig. 6A).

Interestingly, Gpr88-KD had an opposing effect on ΔFosB expression in the DLS and DMS, in both cases reversing the 6-OHDA-induced alterations (t20 = 3.982, p < 0.001 and t22 = 3.452, p = 0.002, respectively; Fig. 6B).

Discussion

Although the acute dopamine loss in the 6-OHDA lesion model does not correspond to the progressive course of Parkinson disease, the partial bilateral striatal dopamine denervation most closely mimics the disease's early stage. [START_REF] Deumens | Modeling Parkinson's disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway[END_REF] Accordingly, we established that the loss of dopamine restricted to the DLS was sufficient -without modifying motor behaviour -to reproduce alterations relating to depression, apathy and attentional deficits, 3 of the most prevalent psychiatric symptoms at the time of Parkinson disease diagnosis, [START_REF] Pont-Sunyer | The onset of nonmotor symptoms in Parkinson's disease (the ONSET PD study)[END_REF] and that Gpr88-KD can reduce several of these deficits. Indeed, dopamine loss increased the immobility count in the forced swim test, an indicator of behavioural despair linked to depression. [START_REF] Slattery | Using the rat forced swim test to assess antidepressant-like activity in rodents[END_REF] It also induced a decrease in social interaction and rearing behaviour related to novelty exploration, [START_REF] Lever | Rearing on hind legs, environmental novelty, and the hippocampal formation[END_REF] 2 parameters that have been proposed as ecological and translational measures of apathy. [START_REF] Cathomas | The translational study of apathy-an ecological approach[END_REF] These results were also consistent with observations that the behavioural and social aspects of apathy are the most affected in Parkinson disease, and that they are specifically associated with depression. [START_REF] Ang | Differential impact of behavioral, social, and emotional apathy on Parkinson's disease[END_REF] Furthermore, apathy in Parkinson disease is frequently associated with cognitive impairment and dementia. [START_REF] Pagonabarraga | Apathy in Parkinson's disease: clinical features, neural substrates, diagnosis, and treatment[END_REF] In this regard, we also observed that the lesioned animals performed poorly in the social novelty discrimination task, suggesting an impairment of selective attention [START_REF] Terranova | SSR181507, a dopamine D(2) receptor antagonist and 5-HT(1A) receptor agonist, alleviates disturbances of novelty discrimination in a social context in rats, a putative model of selective attention deficit[END_REF] -a core component of the dysexecutive syndrome experienced by patients with Parkinson disease. [START_REF] Svenningsson | Cognitive impairment in patients with Parkinson's disease: diagnosis, biomarkers, and treatment[END_REF] However, we did not reproduce the deficits relating to anhedonia (sucrose preference) and sensorimotor gating (prepulse inhibition) that had been previously reported in other 6-OHDA models of Parkinson disease. [START_REF] Issy | Disturbance of sensorimotor filtering in the 6-OHDA rodent model of Parkinson's disease[END_REF][START_REF] Magnard | What can rodent models tell us about apathy and associated neuropsychiatric symptoms in Parkinson's disease?[END_REF] Anhedonia in Parkinson disease has been associated with alterations in the mesolimbic dopamine pathway, [START_REF] Husain | Neuroscience of apathy and anhedonia: a transdiagnostic approach[END_REF] and prepulse inhibition alterations require extensive dopamine loss, [START_REF] Issy | Disturbance of sensorimotor filtering in the 6-OHDA rodent model of Parkinson's disease[END_REF] suggesting that the lack of prepulse inhibition and sucrose preference alterations in our model were due to the restricted nature of the lesion.

Gpr88 and psychiatric symptoms of Parkinson's disease Importantly, although the inactivation of Gpr88 in the lesioned DLS tended to have an effect on some of the altered behaviours, only the knockdown in the intact associative DMS reversed all of the behavioural deficits induced by dopamine loss, suggesting an effect that could be related to functional cross-talk between these 2 regions. [START_REF] Gruber | Context, emotion, and the strategic pursuit of goals: interactions among multiple brain systems controlling motivated behaviour[END_REF] In the DMS, Gpr88-KD also increased sensorimotor gating in the prepulse inhibition test, indicating an increased efficiency of preattentive information processing, which could underlie the proattentional effect in the social novelty discrimination task. Interestingly, computational models of Parkinson disease psychosis have linked the emergence of hallucinations to deficits in such gating and attentional processes, [START_REF] Ffytche | Gpr88 and psychiatric symptoms of Parkinson's disease[END_REF] suggesting that Gpr88-KD may constitute a relevant target for evaluating this symptom in animal models.

The restricted loss of dopamine inputs to the sensorimotor territories of the striatum also altered the expression of molecular markers of neuronal activity, signalling and transcription beyond the lesioned area, significantly affecting associative territories. For instance, although the lesion locally induced a decrease in Pdyn and an increase in Penk and Gad67 expression levels (reproducing the imbalance in neuronal activity that has been characterized in Parkinson disease models [START_REF] Sgroi | Opioidergic modulation of striatal circuits, implications in Parkinson's disease and levodopa induced dyskinesia[END_REF][START_REF] Consolo | Increased striatal expression of glutamate decarboxylase 67 after priming of 6-hydroxydopamine-lesioned rats[END_REF] ), their expression was significantly elevated in the DMS, suggesting a compensatory overactivity of neurons in response to dopamine depletion in the DLS.

A previous study had reported that 6-OHDA lesions induced a decrease in Gpr88 mRNA and protein expression after 1 and 4 weeks,11 but we did not observe such an effect at 6 weeks, suggesting a time-dependent recovery of Gpr88 expression following dopamine loss. The Gpr88-KD in the lesioned DLS was associated with a significant decrease in the expression of Penk and Gad67, suggesting that Gpr88-KD preferentially affects iMSN, which is consistent with the receptor's relative enrichment in this neuronal type [START_REF] Massart | Striatal GPR88 expression is confined to the whole projection neuron population and is regulated by dopaminergic and glutamatergic afferents[END_REF] and the hypersensitivity to dopamine receptor type 2 agonists that has been consistently reported in Gpr88-KO mice. [START_REF] Quintana | Lack of GPR88 enhances medium spiny neuron activity and alters motor-and cue-dependent behaviors[END_REF][START_REF] Logue | The orphan GPCR, GPR88, modulates function of the striatal dopamine system: a possible therapeutic target for psychiatric disorders?[END_REF] Because GPR88 is also known to modulate enkephalin delta opioid receptor signalling, [START_REF] Meirsman | Mice lacking Gpr88 show motor deficit, improved spatial learning, and low anxiety reversed by delta opioid antagonist[END_REF] the reduction in Penk expression observed after Gpr88-KD may have resulted partly from a potentiation of this receptor's effects. In the DMS, Gpr88-KD reduced Gad67 overexpression without significantly affecting Penk or Pdyn, suggesting a potential decrease in γaminobutyric acid interneuron activity, [START_REF] Consolo | Increased striatal expression of glutamate decarboxylase 67 after priming of 6-hydroxydopamine-lesioned rats[END_REF] in which GPR88 expression has been also reported. [START_REF] Massart | Striatal GPR88 expression is confined to the whole projection neuron population and is regulated by dopaminergic and glutamatergic afferents[END_REF] A GTPase accelerating protein, RGS4 negatively regulates the signalling of Gi/o-and Gqassociated receptors such as dopamine receptor types 2 and 3, 47 metabotropic glutamate receptor type 5 [START_REF] Schwendt | RGS4 overexpression in the rat dorsal striatum modulates mGluR5-and amphetaminemediated behavior and signaling[END_REF] and muscarinic acetylcholine receptor M4. [START_REF] Ding | RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion[END_REF] Previous studies have reported decreases in striatal RGS4 mRNA in people with Parkinson disease [START_REF] Zhang | Transcriptional analysis of multiple brain regions in Parkinson's disease supports the involvement of specific protein processing, energy metabolism, and signaling pathways, and suggests novel disease mechanisms[END_REF] and in 6-OHDAlesioned rodents. [START_REF] Geurts | Altered expression of regulators of G-protein signaling (RGS) mRNAs in the striatum of rats undergoing dopamine depletion[END_REF] Although Rgs4 expression was slightly decreased in the DLS (p = 0.06), it was also strikingly elevated in the DMS. However, this overexpression was completely reversed by Gpr88-KD, an effect that was in line with findings from knockout mice. [START_REF] Quintana | Lack of GPR88 enhances medium spiny neuron activity and alters motor-and cue-dependent behaviors[END_REF][START_REF] Meirsman | Mice lacking Gpr88 show motor deficit, improved spatial learning, and low anxiety reversed by delta opioid antagonist[END_REF] Interestingly, in models of Parkinson disease, inhibition or loss of Rgs4 has been shown to have potent antiparkinsonian effects, [START_REF] Ko | RGS4 is involved in the generation of abnormal involuntary movements in the unilateral 6-OHDA-lesioned rat model of Parkinson's disease[END_REF] notably by restoring longterm depression in iMSN. [START_REF] Lerner | RGS4 is required for dopaminergic control of striatal LTD and susceptibility to Parkinsonian motor deficits[END_REF] However, translation to the clinic of these findings has been impeded by the high risk of adverse effects due to the ubiquitous presence of RGS4. Therefore, targeting GPR88 may constitute a unique strategy for bypassing these limitations, by inhibiting RGS4 with high spatial relevance, because GPR88 is exclusively expressed in the central nervous system and is mostly enriched in the striatum, where its expression levels match the typical pattern of dopamine loss. [START_REF] Van Waes | GPR88: A putative signaling molecule predominantly expressed in the striatum: cellular localization and developmental regulation[END_REF] The 6-OHDA lesion and Gpr88-KD also had contrasting effects on the expression of transcription factor ΔFosB. Although ΔFosB accumulation in Parkinson disease has been linked to the effects of L-DOPA treatment, [START_REF] Lindgren | Putaminal upregulation of FosB/DeltaFosBlike immunoreactivity in Parkinson's disease patients with dyskinesia[END_REF] we also observed an increase in the DLS of the lesioned rats. This unexpected effect could have been due to stimulation of hypersensitive D1 receptors in the DLS, resulting from a "volume transmission" effect from the unlesioned striatum. [START_REF] Taber | Volume transmission in the brain: beyond the synapse[END_REF] In the DMS, however, we found a significant decrease in ΔFosB expression, which was reversed by Gpr88-KD. Interestingly, the antidepressant effect of Gpr88-KD observed in the forced swim test may have been due to its modulation of ΔFosB, because fluoxetine is also known to increase the expression of this transcription factor in the DMS. [START_REF] Vialou | Differential induction of FosB isoforms throughout the brain by fluoxetine and chronic stress[END_REF] The finding that the knockdown of Gpr88 in the associative striatum reduced behavioural deficits and normalized the expression of Rgs4 and ΔFosB must be integrated with our other recent work showing that Gpr88-KD in the dorsal striatum has an antiparkinsonian-like effect in a model of the motor symptoms of Parkinson disease by regulating the expression of Pdyn, Penk and ΔFosB. [START_REF] Ingallinesi | Knock-down of GPR88 in the dorsal striatum alters the response of medium spiny neurons to the loss of dopamine input and L-3-4dyhydroxyphenylalanine[END_REF] Thus, although the models we used are not easily comparable, taken together our results indicate that GPR88 may represent an unique target for treating both the psychiatric and the motor symptoms of Parkinson disease and avoiding some of the shortcomings of current therapies.

Moreover, this study's findings may also provide insights into the etiology of the psychiatric symptoms of Parkinson disease. Indeed, although psychiatric symptoms have been linked to alterations in multiple neurotransmitter systems, [START_REF] Qamar | Presynaptic dopaminergic terminal imaging and non-motor symptoms assessment of Parkinson's disease: evidence for dopaminergic basis?[END_REF] clustering studies of de novo patients suggest that the loss of striatal dopamine in particular may play an important role in their emergence. [START_REF] Moccia | Dopamine transporter availability in motor subtypes of de novo drug-naive Parkinson's disease[END_REF] This idea is further supported by studies in rodent models, which have consistently shown that inducing extensive dopamine loss throughout the striatum could induce representative behavioural deficits. [START_REF] Magnard | What can rodent models tell us about apathy and associated neuropsychiatric symptoms in Parkinson's disease?[END_REF] Nevertheless, these models have not necessarily accounted for the frequent premotor appearance of these symptoms, when dopamine loss is limited mainly to the sensorimotor areas of the striatum. [START_REF] Brooks | Imaging biomarkers in Parkinson's disease[END_REF] In this regard, our results show that such a restricted loss of dopamine is in itself sufficient to reproduce these behavioural alterations in an animal model. However, these psychiatric aspects of Parkinson disease have typically been associated with dopamine dysfunction in associative networks (DMS), [START_REF] Qamar | Presynaptic dopaminergic terminal imaging and non-motor symptoms assessment of Parkinson's disease: evidence for dopaminergic basis?[END_REF] which are relatively preserved in the early stages of the disease, as well as in our model. Nevertheless, the overexpression of neuronal activity markers that we observed in the DMS suggests possible interactions between striatal areas. Interestingly, some functional MRI studies in people with Parkinson disease have also found that although functional connectivity decreased in the dopaminedeprived sensorimotor networks of the basal ganglia, it was increased in associative networks. [START_REF] Bell | Dopaminergic basis for impairments in functional connectivity across subdivisions of the striatum in Parkinson's disease[END_REF][START_REF] Helmich | Spatial remapping of corticostriatal connectivity in Parkinson's disease[END_REF] These increases are thought to reflect early Parkinson disease allostatic mechanisms, by which associative networks expand or strengthen their connectivity to compensate for the loss of dopamine in sensorimotor territories. Although the underlying mechanisms are currently unknown, recently characterized striatonigrostriatal cellular pathways may provide a system for such lateral transfer of information. [START_REF] Lerner | Intact-brain analyses reveal distinct information carried by snc dopamine subcircuits[END_REF] However, because of the resulting overlap of networks within limited striatal computational resources, it has been suggested that this compensatory mechanism could lead to a "neural bottleneck" or "overload" of associative networks, and an impairment in related behaviours. [START_REF] Redgrave | Goal-directed and habitual control in the basal ganglia: implications for Parkinson's disease[END_REF][START_REF] Helmich | Spatial remapping of corticostriatal connectivity in Parkinson's disease[END_REF] This hypothesis could explain the emergence of psychiatric deficits relating to cognition and motivation during the earliest stages of Parkinson disease and in the striatal 6-OHDA focalized-lesion model of Parkinson disease, before the loss of dopamine affects associative networks.

Although such imbalances in DMS/DLS dynamics are emerging as key players in the pathophysiology of basal ganglia disorders such as addiction [START_REF] Belin | Addiction: failure of control over maladaptive incentive habits[END_REF] and obsessive-compulsive disorder, [START_REF] Burguiere | Striatal circuits, habits, and implications for obsessive-compulsive disorder[END_REF] this study is, to our knowledge, the first to enquire into this concept in a rodent model of Parkinson disease.

Limitations

Several factors need to be investigated further. For instance, behavioural tests assessing specific cognitive and motivational parameters are needed to better characterize the effects of the lesion and of Gpr88-KD. Because the model reproduced aspects of early-stage Parkinson disease, it will also be necessary to assess the effects of the Gpr88-KD in models of advanced Parkinson disease, presenting more extensive dopamine loss and alterations in other neurotransmitter systems. Furthermore, although molecular experiments have provided some clues about the mechanisms involved, additional in-depth studies are required, because little is known about the signalling pathways regulated by GPR88 in different striatal cell types (dMSN/iMSN/interneurons).

Conclusion

Although the restricted expression of GPR88 makes it an ideal therapeutic target, no antagonist has so far been developed for this orphan receptor. It is thus crucial to develop lead identification efforts. Nevertheless, gene-therapy approaches have shown successful applications in Parkinson disease. [START_REF] Palfi | Long-term follow-up of a phase I/II study of ProSavin, a lentiviral vector gene therapy for Parkinson's disease[END_REF] Based on our findings, GPR88 gene therapy could constitute a highly relevant non-dopaminergic strategy for treating the psychiatric and the motor symptoms of Parkinson disease through striatal-level modulation of allostatic basal ganglia function.



  time interaction on the motor parameters measured by the actimeter, Gpr88-KD in the DMS reversed the deficit in rearing behaviour at the 15-minute time point (p = 0.020, Dunnett post hoc multiple comparison test; Fig.4A). It also increased sensorimotor gating without affecting startle amplitude (2-way ANOVA, effect of treatment on % prepulse inhibition, F2,78 = 9,79, p < 0.001; Dunnett post hoc test miR-Gpr88-DMS v. miR-neg, 70 dB p = 0.010 and 75 dB p = 0.025; Fig.4B).

FiguresFig. 1 :

 1 Figures

Fig. 2 :

 2 Fig. 2: Stereotaxic injections induce a restricted loss of nigrostriatal dopaminergic projections and an efficient knockdown of Gpr88 expression in discrete striatal areas. (A) Representative images of 6-OHDA-lesioned striata. Coronal sections stained for TH by immunofluorescence (+1.2 to +0.2 mm anterior to the bregma), superimposed on the Paxinos and Watson rat brain atlas for anatomic reference.22 (B) Quantification of the extent of TH signal loss in the DLS, DMS and VS (nsham + miR-neg = 7-8; n6-OHDA + miR-neg = 10). (C) Schematic

Fig. 3 :

 3 Fig. 3: Dopamine loss in the dorsolateral striatum reproduces psychiatric symptoms of Parkinson disease. (A) Horizontal, stereotyped and rearing behaviours measured during 3 consecutive 5-minute segments with the actimeter test 4 weeks after the 6-OHDA lesion (nsham + miR-neg = 14; n6-OHDA + miR-neg = 10). (B) Effects of the 6-OHDA lesion on the startle amplitude in response to a loud stimulus (110 dB) and on percent of prepulse inhibition at all tested prepulse intensities (70, 75 and 80 dB; nsham + miR-neg = 14; n6-OHDA + miR-neg = 7-10). (C) The 6-OHDA lesions did not alter the percent preference for the sucrose solution (nsham + miR-neg = 6; n6-OHDA + miR-neg = 7). (D) The 6-OHDA lesions did induce depressive-like behaviour in the forced swim test, as suggested by

Fig. 4 :Fig. 5 :

 45 Fig. 4: Gpr88 knockdown in the dorsomedial striatum reverses behavioural deficits. Effects of Gpr88 knockdown on the following: (A) horizontal, stereotyped and rearing behaviours in the actimeter test; (B) startle amplitude and percent of prepulse inhibition; (C) sucrose preference; (D) latency and immobility count in the forced swim test; (E) social interaction and novelty preference; (n6-OHDA + miR-neg = 10; n6-OHDA + miR-Gpr88-DLS = 9-11; n6-OHDA + miR-Gpr88-DMS = 8-10, except in the sucrose preference test, where n = 7-10 throughout the groups). Data are presented as mean ± standard error of the mean. For reference, a dashed horizontal line indicates the values from the control group (Sham + miRneg) that were presented in Figure 3. When we compared the 3 groups for 6-OHDA, we

Fig. 6 :

 6 Fig. 6: Gpr88 knockdown modulates markers of neuronal activity, signalling and transcription. (A) Expression of Pdyn, Penk, Gad67 and Rgs4. (B) Expression of ∆FosB in the DLS and DMS after Gpr88 knockdown (n6-OHDA + miR-neg = 12-14, n6-OHDA + miR-Gpr88 = 8-10. Values were normalized to those of the control group (Sham + miR-neg; shown in Fig. 5) and expressed as percent change. Data are presented as mean ± standard error of the mean, and were compared using multiple t tests with Holm-Sidak corrections. **p < 0.01, ***p < 0.001, ****p < 0.0001. ∆FosB = truncated splice variant of FosB; 6-OHDA = 6-hydroxydopamine; DLS = dorsolateral striatum; DMS = dorsolateral striatum; miR = micro RNA; miR-neg = micro RNA negative; miR-Gpr88 = micro RNA against Gpr88.

Acknowledgments:

The lentiviral vectors were produced by the inhouse iVector facility. In vivo experiments were carried out in the PHENOPARC core facility, and histological procedures were performed at the HISTOMICS platform, both also situated in the ICM. The authors thank Annick Prigent for her time and advice related to the histological processing of the samples.

Affiliation

: Biotechnology and Biotherapy team, ICM Brain and Spine Institute, Sorbonne University/INSERM U 1127/CNRS UMR 7225, CHU Pitie-Salpetriere, Paris, France (Galet, Ingallinesi, Pegon, Do Thi, Ravassard, Faucon Biguet, Meloni).

Funding: The research leading to these results has received funding from the program Investissements d'avenir ANR-10-IAIHU-06. B. Galet was supported by a scholarship from the French Ministry of Research, awarded by the Brain-Cognition-Behavior Doctoral School of the Sorbonne University. The PHENOPARC Core is supported by 2 Investissements d'avenir grants (ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS) and the Fondation pour la Recherche Medicale. The funding sources had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.


Hélène D Moreau
email: helene.moreau@curie.fr

Ana-Maria Lennon-Duménil

Paolo Pierobon
email: paolo.pierobon@curie.fr

"If you please… Draw me a cell" Insights from immune cells

"If you please… draw me a cell". Insights from immune cells

Introduction

"If you please… Draw me a sheep". Lost in the desert, you would have little difficulty drawing a sheep if the Little Prince asked you to. However, you might find it more difficult if he were to ask: "If you please… Draw me a cell." You might hesitate, depending on your background, as cells from multicellular organisms have diverse shapes and properties. If you had studied cancer-derived epithelial or fibroblast cell lines, you might draw a flat fried-egg-shaped adherent object.

Alternatively, you might draw an in situ epithelial cell: rectangular, static and polarised.

Immunologists would be more likely to draw a polarised, potentially motile cell, probably without focal adhesion, possibly a phagocytic or macropinocytic cell. It is even harder to draw "a" cell in the in vivo context, as cells from multicellular organisms reside in tissues with different physical and chemical properties that can influence cell behaviour. These observations led us to wonder what immunologists and cell biologists can learn from each other.

In multicellular organisms, most cells reach their final positions during embryonic development, and then fulfil their function at that site, or at least within the same tissue. An ability to migrate and to change their microenvironment is often considered a pathological hallmark of cancer cells, but immune cells also have these properties. What makes immune cells special is that each individual single immune cell can migrate between tissues, fulfilling specific functions in different microenvironments. To be able to do this, immune cells must be able: (i) to explore multiple environments (i.e. migrating in search of a danger signal originating from a pathogen or another cell), (ii) to gather and/or transmit information, generally by secreting cytokines or through direct cell-cell contact via an 'immune synapse' and (iii) to exert effector functions, by eliminating the pathogen such as through phagocytosis (macrophages, neutrophils), killing dangerous cells (cytotoxic T cells, natural killer (NK) cells), or secreting antibodies (B cells).

In this Essay, we will highlight the unusual features of immune cells relative to typical "textbook" adherent cells, focusing particularly on their cytoskeleton dynamics and cell mechanics. We also propose that, thanks to their unique properties, immune cells were selected during evolution to operate at spatial and temporal scales different from those of typical adherent cells.

Immune cells continually change morphology and polarity

Most cells acquire their overall shape early during development and do not undergo major shape changes, at least over short time scales (minutes). For example, neurons may grow and their synaptic connections may change during their lifetime, but they retain the same overall morphology.

The maintenance of cell shape is dependent on interactions between cells, which are regulated by junction molecules, such as cadherins, or on signals generated by interactions with the extracellular matrix that contains adhesion molecules such as integrins [START_REF] Lecuit | Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis[END_REF]. The most unusual feature of immune cells is, thus, their continually changing morphology, enabling them to adapt to the physical and biochemical features of the environments with which they come into contact. This ability is dependent on a highly dynamic actin cytoskeleton, the presence of which makes these cells an ideal model for cell biology studies.

Immune cells can change shape very rapidly. For instance, lymphocytes become elongated during migration with their shape tightly coupled to their speed [START_REF] Hons | Chemokines and integrins independently tune actin flow and substrate friction during intranodal migration of T cells[END_REF], but they round up again within seconds upon immobilisation [START_REF] Dustin | T-cell activation through immunological synapses and kinapses[END_REF]. It remains unclear what triggers the rounding of immune cells when they stop migrating, but calcium signalling has been implicated in T cells [START_REF] Donnadieu | Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium[END_REF][START_REF] Dustin | Antigen receptor engagement delivers a stop signal to migrating T lymphocytes[END_REF][START_REF] Negulescu | Polarity of T cell shape, motility, and sensitivity to antigen[END_REF]. Cell rounding may also occur very rapidly during the division of adherent cells. In this case, cell rounding is accompanied by a strong increase in cortical tension, allowing the cell to withstand the compressive stress of the surrounding tissue [START_REF] Kondo | Mitotic cell rounding accelerates epithelial invagination[END_REF][START_REF] Théry | Get round and stiff for mitosis[END_REF]. We can therefore hypothesise that the changes in cell shape of immune cells depend on the environmental stresses they face at a given time. What is the machinery that helps immune cells rapidly responding to external cues by reshaping? First, immune cells are the smallest and softest of all mammalian cells (together with metastatic cells!) [START_REF] Bufi | Human primary immune cells exhibit distinct mechanical properties that are modified by inflammation[END_REF]. Their relatively low rigidity may facilitate rapid and efficient changes in shape and mechanical (microrheological) properties. Interestingly, neutrophils have been recently reported to stiffen and shrink upon activation and then expand and soften again within minutes [START_REF] Bashant | Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming[END_REF]. Modification of rheological properties have also been observed upon antigen recognition in both B and T cells [START_REF] Zak | Single-cell immuno-mechanics: rapid viscoelastic changes are a hall-mark of early leukocyte activation[END_REF]. Second, under the microscope, most immune cells can be seen to be covered with microvilli, which constitute a major source of membrane material for rapid shape changes [START_REF] Cai | Visualizing dynamic microvillar search and stabilization during ligand detection by T cells[END_REF][START_REF] Jung | Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies[END_REF][START_REF] Majstoravich | Lymphocyte microvilli are dynamic, actin-dependent structures that do not require Wiskott-Aldrich syndrome protein (WASp) for their morphology[END_REF][START_REF] Shao | Static and dynamic lengths of neutrophil microvilli[END_REF]. The formation and stabilisation of microvilli depend on the physical properties of the membrane mediating the local reorganisation of actin-associated proteins and nucleators. Microvilli are present in several different types of immune cells, but have different dynamics in different cells.

Morphological changes are not restricted to the shape of the cell, but also concern its internal components. Developing tissues typically rapidly establish a polarity pattern [START_REF] Cowan | Acto-myosin reorganization and PAR polarity in C. elegans[END_REF], and most polarised cells, such as epithelial cells and neurons, do not change polarity during their lifetime. Indeed, cell polarity is often dependent on the micro-environment, and, as this environment tends to be stable for most cells, polarity is maintained over the lifespan of the cell [START_REF] Gundersen | Nuclear positioning[END_REF]. By contrast, the polarity of immune cells is continually challenged: while migrating in response to biochemical or mechanical cues and when polarising towards a cell to establish a synapse, or re-orienting to break the contact and follow another signal.

Below, we explore the role of changes in shape and polarity on the various functions accomplished by immune cells.

Immune cells migrate with minimal forces in complex environments

Another remarkable feature of immune cells is their ability to migrate. Classical textbook cells (e.g. HeLa cells) migrate through adhesion of their front end to the substrate, followed by pulling on the adhesion molecular "clutch" to move forward. However, they need to release the clutch at some point to keep moving forward [START_REF] Hu | Differential Transmission of Actin Motion Within Focal Adhesions[END_REF]. Strikingly, immune cells migrate mostly without integrin-mediated adhesion [START_REF] Friedl | CD4 + T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize[END_REF][START_REF] Lämmermann | Rapid leukocyte migration by integrin-independent flowing and squeezing[END_REF]. Their migration process is similar in this respect to that described for Dictyostelium [START_REF] Friedl | Plasticity of cell migration: A multiscale tuning model[END_REF], and this mode of migration is therefore described as amoeboid. This has been observed in confined environments in vitro and in vivo, and it enables immune cells to navigate through tissues for the purposes of immune surveillance.

Amoeboid migration depends on a particular organisation of the building blocks of the cytoskeleton, particularly those controlling actin nucleation and actomyosin contractility [START_REF] Chabaud | Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells[END_REF][START_REF] Jacobelli | Myosin-IIA and ICAM-1 Regulate the Interchange between Two Distinct Modes of T Cell Migration[END_REF][START_REF] Jacobelli | Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions[END_REF]. This type of migration is particularly efficient in confined environments, such as the interstitial space in tissues, in which immune cells move. The immune cell cytoskeleton is reorganised to generate forces that squeeze the cell, and particularly its nucleus, through constrictions [START_REF] Lämmermann | Rapid leukocyte migration by integrin-independent flowing and squeezing[END_REF][START_REF] Raab | ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death[END_REF][START_REF] Thiam | Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments[END_REF][START_REF] Wolf | Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force[END_REF]. Dendritic cells migrating in constricted conditions generally have three pools of actin: one at the front, one around the nucleus, with both branched filaments that are nucleated by Arp2/3 (Actin-Related Proteins ARP2 and ARP3) complex, and one at the back (actin bundles, nucleated by formins). The first drives macropinocytosis (i.e. environment sampling), the second squeezes the nucleus through constrictions (e.g. collagen pores or cell-cell junctions), and the third propels the cell forwards (Fig. 1). Myosin II at the back of the cell is thus involved in squeezing and propelling the cell, whereas the myosin II at the front of the cell promotes macropinosome maturation and the transfer of macropinosome content to lysosomes [START_REF] Bretou | Lysosome signaling controls the migration of dendritic cells[END_REF][START_REF] Chabaud | Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells[END_REF].

Adherent migration requires the generation of strong forces, which may slow the cells down. By contrast, non-adhesive modes of migration make use of frictional forces against the substrate. The ranges of stress generated by these two types of migration are very different (~100 Pa for adhesionmediated migration versus less than 1 Pa for cortical flow-driven migration [START_REF] Bergert | Force transmission during adhesion-independent migration[END_REF]).

The generation of smaller forces does not result in slower migration, because the cells do not need to detach from the substrate to move forward [START_REF] Lämmermann | Mechanical modes of 'amoeboid' cell migration[END_REF]. They have indeed less effective friction to overcome. They can even simply use the topography of the substrate to propel themselves, in the absence of any molecular link between the cytoskeleton and the substrate [START_REF] Reversat | Adhesion-free cell migration by topography-based force transduction[END_REF]. During interstitial migration, the generation of small forces acting against the environment may enable immune cells to detect weak environmental cues and to integrate local chemical and mechanical signals. For instance, neutrophils are able to follow the minimal hydraulic resistance path (barotaxis), thereby overriding chemotactic signals [START_REF] Prentice-Mott | Biased migration of confined neutrophil-like cells in asymmetric hydraulic environments[END_REF].

Conversely, immature dendritic cells overcome barotaxis by macropinocytosis, enabling space exploration; this feature is lost upon maturation, allowing mechanical (and not only chemical) guidance to lymph nodes. Barotaxis appears to not depend on any specific molecular sensor but rather to be the result of intrinsic properties of the actomyosin cytoskeleton [START_REF] Moreau | Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration[END_REF], suggesting that any cell is subjected to barotaxis, except cells that are endowed with an efficient fluid transport mechanics, such as immature dendritic cells and macropinocytic metastatic cells.

During extravasation, immune cells must counteract flow; they therefore need to generate more force, through integrin-mediated adhesion, which they achieve by changing their motility mode [START_REF] Shulman | Lymphocyte Crawling and Transendothelial Migration Require Chemokine Triggering of High-Affinity LFA-1 Integrin[END_REF][START_REF] Sixt | Cell adhesion and migration properties of β2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules: Relevance for leukocyte extravasation[END_REF].

Immune cells actively patrol their environment and test their local surroundings and neighbours

Cells in tissues depend on external factors (nutrients, growth factors, etc.) to survive. They sense these factors at the plasma membrane, or actively take them up. Immune cells use a similar strategy to sense their environment. Cytokines are sensed directly by membrane receptors, providing local information. However, immune cells often collect information over a whole tissue, or even across the whole body. They are therefore highly dependent on cell migration, as described above, to gather broadly distributed information, as well as on the local environment and cell scanning.

During migration, immune cells often undergo repolarisation, for instance to change direction, avoid obstacles or follow chemokine gradients. These repolarisation events occur over very short time scales [START_REF] Schumann | Immobilized Chemokine Fields and Soluble Chemokine Gradients Shape Migration Patterns of Dendritic Cells[END_REF]. Gradients of chemical signals (chemotaxis) provide a good trail to targets that is widely exploited in bacteria [START_REF] Berg | Physics of chemoreception[END_REF]. Immune cells make use of a similar mechanism, haptotaxis, i.e. following gradients of immobilised molecules, in which their orientation accuracy is precisely dependent on the signal:noise ratio of the chemokines [START_REF] Schwarz | Dendritic Cells Interpret Haptotactic Chemokine Gradients in a Manner Governed by Signal-to-Noise Ratio and Dependent on GRK6[END_REF][START_REF] Weber | Interstitial dendritic cell guidance by haptotactic chemokine gradients[END_REF]. Cell polarisation occurs through a biasing of actin polymerisation following the engagement of the chemotactic receptor (a system that is radically different from the run-and-tumble in bacterial chemotaxis). Actin-driven protrusions, such as lamellipodia, in particular, are thus highly efficient detectors of haptotactic gradients, but they are dispensable, as discussed above, for locomotion [START_REF] Leithner | Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes[END_REF]. Physical obstacles can also drive cell orientation, due to the load dependence of actin polymerization. Greater compression of the actin network (by an obstacle) results in a stiffening of this network, generating a force that act against the compression [START_REF] Mueller | Load Adaptation of Lamellipodial Actin Networks[END_REF]. Recently, the nucleus has been suggested to guide the cell towards the largest pore size, minimizing mechanical resistance to migration [START_REF] Renkawitz | Nuclear positioning facilitates amoeboid migration along the path of least resistance[END_REF].

Environmental patrolling is optimised not only by control over the direction of movement and its guidance by physico-chemical cues, but also by the coordination of migration and microenvironment testing. This rather general mechanism renders immune cells incredibly efficient at sensing their microenvironment, collecting information and transmitting it to other cells and at fulfilling their effector functions. In dendritic cells, for example, the uptake of large amounts of fluid through macropinocytosis for environmental scanning is antagonistic to migration. This antagonism results from the dependence of amoeboid migration and macropinocytosis on the same cytoskeleton building blocks, essentially the key player of actin contractility, myosin II, which is diverted from the back to the front of the cell to contract macropinosomes (Fig. 1) [START_REF] Lavi | Deterministic patterns in cell motility[END_REF]. As a result, dendritic cells display intermittent searching activity, with an alternation of locomotion and sensing phases [START_REF] Faure-André | Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain[END_REF]. This strategy has been shown to be efficient for the sampling of sparse targets [START_REF] Bénichou | Intermittent search strategies[END_REF]. The rate of switching between locomotion and sensing is optimised for the detection of a signal within hours over distances of several hundreds of microns, consistent with in vivo observations [START_REF] Chabaud | Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells[END_REF].

In general, cells in continuous motion also move very fast, their polarisation being reinforced by the retrograde actin flow responsible for locomotion itself. Consistently, the contractile machinery (myosin II) is also more polarized at the back of the cell in the fastest and most persistent cells [START_REF] Chabaud | Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells[END_REF]. More recently, spontaneous actin waves have been shown to generate the polarity cues and hence determine the diffusive versus persistent dendritic cell migration [START_REF] Stankevicins | Deterministic actin waves as generators of cell polarization cues[END_REF]. Immune cells spontaneously change polarity as a consequence of cortex instability or their differentiation and/or activation state. The alternation between low-and highpersistence states can be explained by the competition between the nucleation machineries for actin monomers (Fig. 1). This may help immune cells to build dynamic actin pools at different cellular locations, facilitating rapid repolarization. Intermittent migration requires the coordination of migration and function and leads an optimal environmental patrolling [START_REF] Chabaud | Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells[END_REF][START_REF] Lavi | Deterministic patterns in cell motility[END_REF]. Accordingly, mature dendritic cells, whose the main function is efficient migration to the lymph nodes to initiate immune responses, rather than tissue patrolling, lose the ability to engage myosin at their front. The actomyosin machinery therefore remains at the back of these cells, resulting in a downregulation of macropinocytosis, but promoting the continual rapid locomotion of these cells [START_REF] Vargas | Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells[END_REF]. Interestingly, T lymphocytes have also been reported to display intermittent migration, and appear to require the atypical motor protein myosin IG [START_REF] Gérard | Detection of rare antigen-presenting cells through T cell-intrinsic meandering motility, mediated by Myo1g[END_REF].

The local microenvironment is also tested by the constant scanning of neighbouring cells, as exemplified by T cells continually searching for their antigens. At a microscopic scale, these processes are dependent on dynamic membrane structures, such as protrusions, villi or ruffles (Fig. 1). These structures are mostly constitutive but can also be reinforced by specific signalling [START_REF] Pollard | Actin, a Central Player in Cell Shape and Movement[END_REF]. For example, lymphocyte microvilli are connected to specific signalling platforms and, at least for T cells, have been shown to scan the interaction area with another cell within seconds and then make a decision within one minute [START_REF] Brodovitch | T lymphocytes sense antigens within seconds and make a decision within one minute[END_REF]. The efficiency of this scanning process was recently reported to be dependent on the structure and dynamics of microvilli [START_REF] Cai | Visualizing dynamic microvillar search and stabilization during ligand detection by T cells[END_REF], actin-filled protrusions that move actively at the surface of T cells, thereby maximising surface coverage and minimising mutual overlapping. Microvilli have a residence time of three to six seconds in the absence of ligand. Once specific contact between T-cell receptor (TCR) and its ligand has been established, the individual microvilli are stabilized, potentially owing to TCR-ligand catch-bond behaviour [START_REF] Pullen | Mechanical feedback enables catch bonds to selectively stabilize scanning microvilli at T-cell surfaces[END_REF], allowing amplification of the signal and antigen discrimination. Remarkably, microvilli have been also

shown to release TCR-enriched vesicles and transmit information to antigen presenting cells [START_REF] Kim | T cell microvilli constitute immunological synaptosomes that carry messages to antigen-presenting cells[END_REF].

Immune cells gather and transmit information through synapses

When an immune cell finds a cognate partner, it can establish contact with this other cell through the so-called immunological synapse. This structure owes its name to the neurological structure, as both types of synapse mediate the direct transfer of information from one cell to another, and they have also been shown to have a similar organisation. In the immunological synapse, the lymphocyte and the antigen-presenting cell (or lymphocyte and target cell) (i) adhere to each other via a lamellipodium-like structure, (ii) are reorganised into a centrosymmetric configuration, with antigens located at the centre and adhesion molecules at the periphery, and (iii) subsequently detach from each other to resume their migration. Immune synapses are associated with various immunecell activation events, including antigen internalisation (in B cells), cell proliferation and the secretion of cytokine-containing vesicles (CD4 + T cells) or other molecules required for effector function, for cytolysis in the case of CD8 + T cells and NK cells, for example.

Immune synapses form and break down again within minutes. How can such drastic shape changes and molecular reorganisation occur so fast? Rapid shape changes make use of specific signalling cascades that amplify the primary signal, leading to a downstream reorganisation of the cytoskeleton building blocks within minutes. In the case of the immune synapse, antigen recognition by specific receptors (TCRs or BCRs) triggers a cascade involving kinases, such as PAK (p21-activated kinases) and PI3K (phosphatidylinositol 3-kinases), and small GTPases (e.g.

Cell division control protein 42 homolog CDC42, Ras-related C3 botulinum toxin substrate 2 RAC2, Vav) that ultimately remodels the actin cytoskeleton. In particular, a domination of WASP (Wiskott-Aldrich Syndrome protein), activity over PKCθ (Protein Kinase C Theta) has been reported to be crucial in T cells, to ensure the symmetry and stability of the immune synapse through a fine control of actin polymerization at the synapse [START_REF] Sims | Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse[END_REF]. This stabilization relies on an intracellular in-plane tension that is sustained at the synapse by WASP-dependent actin nucleation at TCR microclusters and interaction with Myosin-II. WASP degradation leads to a release of cytoskeletal tension and synapse breaking [START_REF] Kumari | Cytoskeletal tension actively sustains the migratory T-cell synaptic contact[END_REF]. WASP might also regulate receptor mobility in activated B cells (Rey-Suarez et al.). Signaling at the immune synapse further stimulates the local actomyosin reorganisation into a centripetal flow driven by contractile arcs that are stretched by radial actin bundles [START_REF] Yi | Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells[END_REF]. Like the pools of actin in migrating dendritic cells, the two classes of filaments are regulated by different nucleators (Arp2/3 versus formins), making it possible to group clusters of receptors together at the synapse [START_REF] Murugesan | Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse[END_REF]. Super-resolution techniques have shown the presence of a third central ramified network in between the immunological synapse and the nucleus, which might be involved in intracellular transport of TCRs [START_REF] Fritzsche | Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation[END_REF]. Whether this actin pool is similar to the actin cloud that tethers the MTOC to the nucleus of lymphocytes remain to be established [START_REF] Obino | Actin nucleation at the centrosome controls lymphocyte polarity[END_REF].

The rapidity of these signalling events and the subsequent responsiveness of immune cells may also be facilitated by their small size (large area:volume ratio) and the patterning of their membrane, which has been little studied. Indeed, these features might allow signals to be transduced rapidly from the membrane to the cytosol, as membrane compartmentalisation has been reported to accelerate chemical reactions and to improve control over these reactions. Also, decreasing dimensionality speeds up reactions. These general membrane properties combined with the underlying cytoskeleton dynamics favour aggregation and stabilise receptor clustering [START_REF] Gowrishankar | Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules[END_REF][START_REF] Su | Phase separation of signaling molecules promotes T cell receptor signal transduction[END_REF]. These particular signalling properties render immune cells highly responsive to extracellular signals and, therefore, very versatile.

The specific encounter between lymphocytes and antigen-presenting cells modifies their "environment", resulting in changes to both cell shape and cell polarity. During immune synapse formation, the entire network of microtubules (hence the MTOC) is re-oriented towards the site of cell-cell contact (Fig. 1). This polarity axis then reverts to its original state when the cells break contact and resume their migration [START_REF] Sims | Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse[END_REF]. During proliferation, in particular, lymphocytes eventually undergo asymmetric cell division along the axis putatively established during cell-cell contact [START_REF] Barnett | Asymmetric B cell division in the germinal center reaction[END_REF][START_REF] Chang | Asymmetric T lymphocyte division in the initiation of adaptive immune responses[END_REF][START_REF] Thaunat | Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity[END_REF]. This regulates the differentiation of T lymphocytes into memory cells, and lymphocyte polarity thus appears to determine their fate. Ancestral polarity mechanisms (typically the Cdc42-aPKC-PAR3/6 axes) that are common to most polarised cells are also at work in immune cells (see numerous reviews [START_REF] Krummel | Maintenance and modulation of T cell polarity[END_REF][START_REF] Russell | How polarity shapes the destiny of T cells[END_REF][START_REF] Yuseff | How B cells capture , process and present antigens : a critical role for cell polarity Antigen encounter and the role of presenting Cells[END_REF]). However, immune cells and the cells that form tissues differ in the speed in which polarity changes occur [START_REF] Reversat | Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse[END_REF][START_REF] Yuseff | Polarized Secretion of Lysosomes at the B Cell Synapse Couples Antigen Extraction to Processing and Presentation[END_REF]. The rapid changes in polarity observed in immune cells may be due to a particular feature of the cytoskeleton, i.e. the high capacity of their centrosome to nucleate actin [START_REF] Farina | The centrosome is an actin-organizing centre[END_REF][START_REF] Obino | Actin nucleation at the centrosome controls lymphocyte polarity[END_REF]. In B lymphocytes, F-actin is nucleated by Arp2/3 at the centrosome and this nucleation decreases upon lymphocyte engagement, probably due to Arp2/3 recruitment to the immune synapse. The downregulation of actin nucleation at the centrosome facilitates its physical detachment from the nucleus, allowing its rapid polarisation to the synapse (Fig. 1). Actin recruitment to the centrosome has recently been shown to depend on the small protein HSBP1, which promotes the local assembly of the actin nucleator WASH at the centrosome [START_REF] Visweshwaran | The trimeric coiled coil HSBP1 protein promotes WASH complex assembly at centrosomes[END_REF].

Immune cells have played an instrumental role in improving our understanding of the role of mechanical aspects in the fine sensing of the cell environment. Immune cells may use mechanical mechanisms to steer their migration (see above), and in interactions with other cells. Antigen recognition triggers macroscopic forces at the T-cell synapse [START_REF] Hui | Dynamic microtubules regulate cellular contractility during T-cell activation[END_REF][START_REF] Husson | Force generation upon T cell receptor engagement[END_REF]. More locally, at the B-cell synapse for example, the rate of loading on the receptor (its pulling speed) mediates affinity-based discrimination between antigens [START_REF] Natkanski | B cells use mechanical energy to discriminate antigen affinities[END_REF][START_REF] Spillane | B cell antigen extraction is regulated by physical properties of antigen-presenting cells[END_REF]. In T cells the force experienced by the TCR at the synapse is independent of antigen affinity due to adaptation of the actin flow [START_REF] Javanmardi | Cytoskeletal Control of Antigen-Dependent T Cell Activation[END_REF]. In cytotoxicity synapses, forces are generated that increase cell adhesion and the efficiency of perforation [START_REF] Basu | Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing[END_REF]. Interestingly, the TCR responds as catch bonds [START_REF] Liu | Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling[END_REF], which increase affinity for the ligand when pulled apart from it. It remains unclear what effect this has on T-cell biology and whether similar mechanisms may be at work in other lymphoid cells. More generally, mechanosensing plays a key role in cell-to-cell communication within the immune system. The properties of the actin cortex are modified by the inflammatory state of the immune cell [START_REF] Bufi | Human primary immune cells exhibit distinct mechanical properties that are modified by inflammation[END_REF], and signalling is closely related to the rigidity of the substrate with which the cell interacts [START_REF] Judokusumo | Mechanosensing in T lymphocyte activation[END_REF][START_REF] O'connor | Substrate Rigidity Regulates Human T Cell Activation and Proliferation[END_REF][START_REF] Saitakis | Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivityDifferent TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity[END_REF][START_REF] Shaheen | Substrate stiffness governs the initiation of B cell activation by the concerted signaling of PKCβ and focal adhesion kinase[END_REF].

Similarly, B cells have been recently shown to protrude actin rich structures where antigens are extracted when contacting soft substrates, i.e. whose stiffness matches the one of APCs (Kumari et al., 2019). Formation of such protrusions involve both Arp2/3 and formins [START_REF] Bolger-Munro | Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and b cell activation[END_REF][START_REF] Kwak | Intrinsic properties of human germinal center B cells set antigen affinity thresholds[END_REF][START_REF] Roper | B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments[END_REF] and ressemble those previously described in CD4+ T cells [START_REF] Sage | Antigen Recognition Is Facilitated by Invadosome-like Protrusions Formed by Memory/Effector T Cells[END_REF]. The function of the immunological synapse (both B-APC and T-APC) therefore relies on coordinated actin structures that display distinct organizations and subcellular localizations and are regulated by the physical properties of APCs. These different actin pools might be unique to immune cells or, alternatively, be related to the actin structures extensively characterized in adherent cells such as focal adhesions, podosomes and invadosomes, and could therefore be used to learn more about their regulation.

Conclusion: immune cells as a new playground for cell biologists

As discussed here, the highly dynamic properties of the cytoskeleton-membrane interface of immune cells allow these cells to modify their shape, polarity and locomotion in response to environmental cues and to fulfil their function. They also make these cells a powerful study model, and studies of these cells have recently challenged a number of classical cell biology concepts developed from studies of adherent cell lines. Immune cells have a number of features in common with cancer cells. Do the particular features of immune cells also observed in cancer cells facilitate the spread of these cells to healthy tissues? Some of the analogies between the two cell types appear particularly pertinent: first, cancer cells become highly migratory, like immune cells, and second, cancer cells also often become macropinocytic, enabling them to take up huge amounts of extracellular nutrients to satisfy their high metabolic needs [START_REF] Commisso | Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells[END_REF]. Do these similarities result from similarities in cytoskeletal and biophysical properties between immune cells and cancer cells? To what extent are these two sets of cells similar and how do they differ?

The most crucial characteristic of immune cells is probably the efficiency with which they perform particular functions. Most of the mechanisms described above have also been observed in other systems, but they have been explicitly linked to optimal behaviour in immune cells: optimal rates of switching between locomotion and sensing, optimal numbers and dynamics in environmental scanning by microvilli, optimal loading rate and the force for discriminating antigens. This tight control over their cellular functions makes immune cells good models for studies of fundamental cell biology and cell mechanics. Unfortunately for the Little Prince, it isn't really possible to draw a stereotypical cell. Cell biology is far too complex to be captured in a simple sketch. Maybe, as for the sheep in le Petit Prince, it is best to draw a box and leave biologists to continue exploring what is hidden inside!

Figure 1 legend

Draw me an immune cell. Artistic view of the different actin pools and associated functions in immune cells. The actin pool at the back (organised into cables, formin-mediated) is responsible for amoeboid cell migration, the pool around the nucleus (branched, Arp2/3 mediated) is responsible for translocation through constrictions, and the pool at the front (branched) is responsible for macropinocytosis, phagocytosis, environment sensing and the formation of the immune synapse.

Actin has been found around the microtubule-organising center (MTOC), playing a role in the polarisation of this structure. Competition for actin monomers acts as a switch between different functions.

Acknowledgements

We thank the members of the Lennon team for critical reading and suggestions. This work was supported by grants from the DCBIOL Labex (ANR-10-IDEX-0001-02-PSL and ANR-11-LABX-0043), the ANR (PhyMax), the Fondation pour la Recherche Médicale and the Institut National du Cancer to A.-M.L.-D.