domain: chim spi

text:
Syed Kaabir

Catherine Sella

Vadim Dilhas

Barbara Jacková

Marina Mariconti

Clara Gomez-Cruz

Laurent Thouin

Mathieu Morel

Damien Baigl

Ayako Yamada
email: ayako.yamada@ens.psl.eu

Sedimentation-based confinement of individual Giant Unilamellar Vesicles in microchamber arrays with a dynamically exchangeable outer medium

Giant unilamellar vesicles (GUVs) are an ideal model to study cellular membrane functions in vitro, yet difficult to manipulate due to their fragile nature, especially when subjected to dynamic change of their external microenvironment. Here, we introduce an original microfluidic concept for constrain-free confinement of individual GUVs in microchambers with a dynamically exchangeable outer medium. With this method, GUVs self-confine in an array of laterally separated microchambers by sedimentation, avoiding any mechanical constrain and membrane deformation while allowing time-resolved microscopy observation. A microfluidic channel above the chambers allows a diffusion-based exchange of the GUV outer medium that can be completed in a few seconds for fast-diffusing molecules to about one minute for large proteins in a viscous medium. We numerically establish the geometric and flow parameters optimizing medium exchange while preventing GUV from lifting out. We experimentally demonstrate that different aqueous solutions separated by air plugs can be flowed into the channel by taking advantage of a polydimethylsiloxane-based hydrophilic channel wall. We also exploit the possibility to manipulate microliter sample volumes and dynamically control the external environment of GUV for in situ observation of membrane binding protein cell-free expression. We find in particular that the membrane-targeting sequence of Bacillus subtilis MinD binds to GUVs and induces extensive membrane tubulation. This technically simple method offers a robust way to confine GUVs and dynamically control their outer medium, thus constituting an ideal platform to study the spatio-temporal response of reconstituted membranes and/or synthetic cell studies subjected to dynamic microenvironments.

Introduction

GUV, a cell-sized closed phospholipid bilayer membrane, has been extensively exploited as both synthetic cells and in vitro model of cell and cell organelle membranes. [1][2][3][4] GUVs are manipulable individually by micropipette under a precise control of membrane tension, which enables, for example, characterizing physicochemical properties of the membrane via submicron-diameter membrane tubes pulled out from a GUV for several tens of microns. [5][6][7] Their deformability, in turn, makes them difficult to handle in a high through-put manner, in particular, with changes of their environment. In this context, microfluidics technology has been employed to capture a large number of GUVs using microstructures. [8][9][10][11] However, those systems either are highly complex in fabrication and in manipulation, or do not allow GUVs to undergo deformations while being trapped or being free from mechanical constraint. As we reported previously, microwells etched on the ceiling of a microfluidic channel allows us to trap and release GUVs by elastic energy gradient. However, the system is sensitive to the membrane tension of GUVs and thus not suitable for assays accompanied by GUV deflation. 9 Herein, we introduce a microfluidic concept, in which a large number of individual GUVs selfconfine by sedimentation in microwells covered with channels allowing exchanges of their outer medium. The thin bottom layer of the microwells allows dynamic microscopy observations of GUVs at a high magnification, while a water reservoir placed closely above the channels prevents evaporation through the channel wall during hours of biochemical reactions, such as gene expression at 37 °C. 12 Microwells have been widely used, for instance, to capture single or multiple cells, GUVs as mentioned above, or to reconstitute cellular architecture in vitro. 9,[13][14][15][16] Various materials, such as polydimethylsiloxane (PDMS), hydrogels, or UV-curable resins have been used for microwells. In this study, we establish a novel protocol of a simpler fabrication of microwells made of a photoresist, notably without the issue of autofluorescence both under visible and UV light excitation.

To enable sequential exchanges of the outer medium of GUVs in a small volume, while keeping the device immobile on a microscopy stage, air plugs can be used to separate different solutions in single tubing connected to a pump. 17,18 However, due to the hydrophobic nature of conventional microfluidic channel surfaces, e.g., PDMS, introduction of air in a channel is usually undesired since it leaves air bubbles pinned at the channel surface. A smart solution has been proposed by Gokaltun, et al., in which the PDMS surface was turned hydrophilic simply by adding a block copolymer of dimethylsiloxane and ethylene oxide to PDMS. 19 By combining those methodologies, we demonstrate that the outer medium of GUVs can be exchanged multiple times in our device under microscopy observation. Moreover, a volume of the outer medium as small as 10 µL being required, the device is advantageous for biochemical assays targeting model membranes with precious reagents. During the medium exchange, however, GUVs tend to escape from shallow microwells due to a lift force induced by the medium flow above the microwells. To find an optimal range of well depth and flow velocity, numerical simulations have been performed following the strategy used in the study on spheroid trapping by Rousset, et al. 20 Lastly, the applicability of our device to in situ biochemical reaction in the presence of GUVs is investigated. As a proof of concept, a cell-free gene expression has been performed, which is challenging because of the high osmolality of the medium and hours of incubation process at 37 °C. A membrane-targeting sequence of Bacillus subtilis MinD, bsMTS, is expressed using the cell-free system in the presence of GUVs and is allowed to interact with their membrane.

We demonstrate that YFP-conjugated bsMTS is successfully expressed in the device in the presence of GUVs and binds to the bilayer membrane containing a negatively charged lipid. Moreover, spontaneous membrane tubulation from the GUVs occurs upon the expression of YFP-bsMTS.

Results and discussion

Microfluidic device characterization

As illustrated in Figure 1a, a thin photoresist layer containing microwells was covered by a PDMS layer containing a microfluidic circuit consisting here in two parallel straight channels.

To avoid evaporation through PDMS, a water reservoir was added at the vicinity of the channels, covering most of the channel areas except their extremities. 12 A photograph of the assembled microfluidic device is shown in Figure 1b. The microwells had a diameter of 40 µm and were distributed at 40 µm intervals, covering an area of 15 mm × 25 mm as illustrated in Figure S1a in Supporting Information. The two channels were designed to have a width and a length of 3 mm and 2 cm, respectively, smoothly connected to their inlets and outlets as depicted in Figure S1b in Supporting Information. Each channel covered ca. 1.1•10 4 microwells. The final depths of the microwells were smaller than the thickness of the original photoresist dry films used for a master mold fabrication due to compression during dry film lamination and PDMS mold replication processes: the wells fabricated with 50 µm-and 100 µm-thick films had a depth of 43.0 ± 0.2 µm and 91.5 ± 0.1 µm, respectively. The channels fabricated from 50 µm-and 100 µm-thick films had a final height of 46.3 ± 0.4 µm and 100.7 ± 3.2 µm, respectively, corresponding to volume channels of 3.1 µL and 6.8 µL, respectively. As described in Supporting Information, microwells were fabricated in OrmoStamp photoresist by soft lithography directly on a thin cover glass slide. Although a thin layer of the photoresist was remaining at the bottom of the microwells, its thickness, 6.5 ± 5.2 µm, was small enough to allow high magnification microscopy observations of GUVs inside the microwells. It is worth noting that the microwell layer can be fabricated rapidly in less than 20 min once a PDMS mold is obtained, and that OrmoStamp was selected as the material for the microwells among other UV sensitive resins because of its low autofluorescence both under visible and UV light excitation.

GUV trapping

GUVs containing a sucrose solution were left to sediment in an outside medium with a 1:1 (v/v) mixture of the sucrose and a glucose solution at a same osmolality. Due to the density difference, GUVs sedimented at the bottom of the channel and some of them further fell into the microwells, leading to spontaneous GUV confinement in laterally isolated microchambers with an open ceiling for medium exchange. The GUVs that remained outside the wells were carried away upon fluid exchange as depicted in Figure 1c. Figure 1d shows a representative image of GUVs composed of L-α-phosphatidylcholine from chicken egg (EPC) and a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Texas Red DHPE), trapped in the microwells after the channel was rinsed with a solution without GUV. It shows that, after their sedimentation in the wells, the GUVs remained confined under the medium replacement in the upper microfluidic channels.

To estimate the necessary time for GUVs to sediment in the microwells, an evaluation following an approximation made by Haberman et al. was employed. 20,[START_REF] Haberman | Motion of rigid and fluid spheres in stationary and moving liquids inside cylindrical tubes[END_REF] The terminal velocity U of a sphere with a radius r moving in a still medium in an infinite cylinder with a radius R can be described as

𝑈 = 2 9 ⁄ • 𝑟 2 𝑔(𝜌 𝑜 -𝜌 𝑖 )(1 + 𝜎)/𝜇𝐾(1 + 2𝜎 3) ⁄
where g is gravitational constant, ρo (1.030•10 3 kg m -3 ) and ρi (1.037•10 3 kg m -3 ) are the densities of the outer and inner medium of the sphere, respectively, σ is the viscosity ratio of the outer to the inner medium, µ is dynamic viscosity of the outer medium, and K is a wall correction factor, which is a function of σ and 𝛿 = 𝑟/𝑅. [START_REF] Haberman | Motion of rigid and fluid spheres in stationary and moving liquids inside cylindrical tubes[END_REF] Viscosities of 1:1 (v/v) mixture of sucrose and glucose solutions with an osmolality of 300 mOsm used for most of the experiments and 1.72 Osm used for the cell-free gene expression experiments were measured as 1.18 and 5.19 mPa s, respectively. Although lipid bilayer membranes in disordered phase are fluidic with certain viscosities, [22][23][24][25][26] when a GUV sediments vertically, there is no membrane flow due to the axial symmetricity that prevents circulation of lipids in the membrane. 27 We thus assumed σ = 0, which gives 21

𝑈 = 2 9 ⁄ • 𝑟 2 𝑔(𝜌 𝑜 -𝜌 𝑖 )/µ𝐾 and 𝐾 = (1 -0.75857𝛿 5 )/(1 -2.1050𝛿 + 2.0865𝛿 3 -1.7068𝛿 5 + 0.72603𝛿 6 )
The graph in Figure 2a shows the value of U plotted against GUV diameter, 2r, while the microwell diameter, 2R, is fixed to 40 µm and µ = 1.2 (magenta). For a comparison, the result when a GUV sediments in a still infinite medium, i.e., K = 1, is shown in green.

In Figure 2b, the time necessary for a GUV to sediment for 40 µm in a 40 µm-diameter infinitely long cylinder, T40 µm, is plotted against GUV diameter in magenta, together with the case K = 1 in green. For GUVs larger than 2 µm, T40 µm is smaller than 600 s, which is indicated with a blue line in Figure 2b. After introduction in a channel, GUVs were thus allowed to sediment for 10 min in a still medium, and this process was repeated twice. The size distribution of GUVs right after the electro-formation, with an average diameter of 4.4 ± 6.1 µm (n = 2984), is shown in magenta in Figure 3a. Interestingly, the peak of the size distribution of GUVs after trapping is shifted toward greater values as shown in yellow in Figure 3a, with an average diameter of 8.5 ± 5.6 µm (n = 1471). Due to a longer time necessary for small GUVs to sediment into microwells, GUVs with a diameter of a few µm can be excluded, in agreement with the result shown in Figure 2b.

The number of GUVs trapped in single microwells was also investigated. As shown in Figure 3b, a majority of the wells contained either 0 or 1 GUV (n = 3256), following a

Poisson distribution with λ = 0.54 as a mean number of GUV per well, as indicated with black dots. Such a trapping efficiency allows us to easily get a significant number of microwells containing single GUVs with a suitable size for microscopy observation, e.g., 15-30 µm in diameter.

Molecular diffusion in the microwells upon medium exchange

To estimate the necessary time to replace the medium in the microwells, 10 µM fluorescein in phosphate buffered saline (PBS) was firstly introduced in a 46.3 µm-high channel, then PBS without fluorescein was subsequently flowed by a micro-peristaltic pump under microscopy observation. Figure 4a shows a series of fluorescence microscopy images taken at the edge (top)

and center (bottom) of the channel at different time points. The flow velocity at the center of the channel induced by the peristaltic pump was measured to be 6.49 ± 1.02 mm s -1 by tracking fluorescently labelled GUVs flowed in the channel in a separate experiment. Note that this flow velocity corresponds to ca. 0.90 µL s -1 , which is also comparable to manual injection of medium to the device by micropipette. The fluorescence intensities in microwells at different positions were measured and plotted in Figure 4b for the microwells with two different depths. The distance between the microwell center and the channel edge is indicated with the color code on the right. The fluorescence decreases rapidly in particular at the channel center (black curves)

as expected in a laminar flow due to a maximal linear flow velocity at the center of the channel.

The results demonstrate that a few tens of second is enough to replace small molecules such as fluorescein in the microwells. The fluctuation in the curves is due to the fluctuation of flow velocity created by the peristaltic pump. Despite this issue, the peristaltic micro-pump is advantageous compared to a syringe pump or a pressure pump, since it can be directly placed on the microscopy stage closely connected to the device, and its flow rate as well as flow direction can be easily controlled, allowing us to control fluids swiftly without looking away from the device.

Sequential medium exchange with air plugs

Next, the possibility to sequentially introduce different mediums to the device with GUVs inside the microwells was investigated. To separate different solutions, ca. 10 µL air plugs were inserted between 10 µL of the solutions in a tubing connected to the peristaltic pump, as depicted in Figure 5a. 17 with white arrowheads. Importantly, when pure PDMS was initially used for the device, some microwells were covered by air that remained in the channel due to the hydrophobic nature of PDMS and OrmoStamp. To avoid the air pinning, 0.25% (w/w) dimethylsiloxane-(60-70% ethylene oxide) block copolymer (PDMS-EtO) was added to PDMS to turn the channel surface hydrophilic. 19 Therefore, with PDMS-EtO, the air plugs were smoothly moved throughout a 46.3 µm-high channel, without leaving air inside the channel as demonstrated in Figure 5c. This property was also significantly advantageous when the device was used manually without air plugs. The PDMS-EtO modification served as an effective preventive measure against the introduction of air bubbles. Furthermore, undesired air bubble accidentally introduced in the device could be easily removed by flowing a solution with micropipette, in direct contrast to traditional PDMS featuring hydrophobic channel walls, where trapped air bubbles could potentially inflict damage on the sample and/or alter the fluid flow.

Numerical simulations of molecular diffusion and the lift force on GUVs in the microwells

Next, we evaluated molecular diffusion upon medium exchange as well as the lift force that made GUVs escape from the microwells in silico using COMSOL Multiphysics 6.0 software. As depicted in Figure S4 times, validating the model used for the simulations and confirming that fast-diffusing entities can be quickly exchanged in a few tens of seconds in the system. Another set of parameters was tested to simulate for a larger molecule in a viscous medium, as is the case for the cell-free gene expression experiment. For instance, the largest protein involved in the cell-free gene expression system, T7 polymerase, is 99 kDa, and the viscosity of the sugar solution to reach the same osmolarity as the cell-free system was 5.2 mPa s. As shown in the right panel in Figure 6a, a longer time is necessary for a large molecule to be carried away from the microwell with a large depth. Nonetheless, the required time falls in a range of a minute, which is negligible compared to characteristic times involved in gene expression. The micro-scale vertical confinement and the free diffusion allow a fast exchange of the outer medium of the confined GUVs, ranging from a few seconds for fast-diffusing molecules to a minute for large proteins. Next, we established the conditions for which GUVs remained confined while subjected to a flow in the upper microfluidic channels. This was done by computing the lift force Flift exerted on the GUVs by the flow and calculating the critical flow velocities, at which the lift force Flift on a GUV equals the gravitational force 𝐹 𝑔 = 4𝜋𝑟 3 𝑔(𝜌 𝑖 -𝜌 𝑜 ) 3 ⁄ . 20 Figure 6b shows the resulting phase diagram, in which each line depicts the critical flow velocity as a function of the GUV diameter and for different well depths. Below each line, the colored regions represent the parameter ranges suitable for GUV trapping, i.e., 𝐹 𝑙𝑖𝑓𝑡 ≤ 𝐹 𝑔 . In a range of typical flow velocity made by the peristaltic micro-pump or by manual pipetting, i.e., up to several mm s -1 , microwells with a depth of 70 µm or larger can trap GUVs at a high efficiency. This confirms the experimental results with 91.5 µm-deep wells shown in Figure 5b.

Cell-free gene expression of membrane binding protein in the presence of GUVs

Finally, the system was applied for in situ cell-free expression of a membrane binding protein in the presence of GUVs in the microwells. The cell-free gene expression system is composed of purified components necessary for gene transcription and expression machineries of Escherichia coli. 28,29 Here, fluid manipulations were done manually by micropipette, which took ca. 15 -20 s for each fluid injection. Since the cell-free gene expression system is sensitive to dilution, a combination of a larger channel height (100.7 ± 3.2 µm) and smaller well depth (43.0 ± 0.2 µm) was exploited for a better fluid exchange inside microwells, even though the GUV trapping efficiency is lower with shallow wells as shown in Figure 6a right and Figure 6b. GUVs were composed of a 4:1 (mol/mol) mixture of 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) and a negatively charged lipid, 1,2-dioleoyl-sn-glycero-3-phospho-

(1'-rac-glycerol) (DOPG), with a small fraction of Texas Red DHPE. After GUV sedimentation, the solution in one channel was replaced with a cell-free gene expression medium containing a plasmid DNA, nucleotides, amino acids, and proteins necessary for transcription and translation machineries, as well as chaperons and disulfide bond enhancers as illustrated in Figure 7a. The same medium without plasmid DNA was introduced in the other channel. The plasmid DNA was coding for a membrane-targeting sequence (MTS) of B. subtilis MinD conjugated with PhiYFP (YFP-bsMTS) as its amino acid sequence depicted in Figure 7b. After introduction of the cell-free expression medium, the entire chip was incubated at 37 °C in a humidified chamber for 3.5 h prior to confocal microscopy observation. The fluorescence of the GUV membrane and YFP-bsMTS were observed with excitation wavelengths at 543 nm and 488 nm, and emission wavelengths at 599-797 nm and 518-571 nm, respectively. Surprisingly, a significant number of membrane tubes were formed from single GUVs as shown in Figure 7c, whereas in the control channel without plasmid DNA, GUVs kept their spherical shape after 3.5 h incubation at 37 °C as shown in Figure 7d. We also confirmed that in the absence of the negatively charged DOPG, YFP-bsMTS did not bind to the GUV membrane (data not shown). Spontaneous membrane tubulation from GUVs have been observed in different contexts, caused by a local charge gradient, [30][31][32] anchoring of amphiphilic molecules, 33 transbilayer charged lipid asymmetry, 34 or by asymmetric binding of proteins. 35,36 MinD, together with MinC, is a key player in the positioning of division protein at midcell in B. subtilis. 37 It has been shown that negatively charged lipid is enriched in a spiral shape along the longitudinal axis of the bacteria and MinD colocalizes with the lipid spiral via its bsMTS that is in a shape of ahelix. 38 However, bsMTS or MinD has never been shown to create or favor a positive membrane curvature. Interestingly, Stachowiak et al. demonstrated that even proteins unrelated to membrane curvature can drive membrane tubulation by protein-protein crowding. 36 Our case, yet to be investigated further, may fit in this criterion.

Conclusion

We have developed a microfluidic device, with a reasonably quick fabrication protocol and of easy handling, to capture GUVs by spontaneous sedimentation into microwells at the bottom of microfluidic channels, covered with a water reservoir to avoid evaporation through the channel wall. The device allows dynamic observation of GUVs at a high magnification under sequential medium changes. The device is compatible with fluorescence microscopy observation with an excitation at UV or visible wavelength. Optimal conditions of well depths and flow velocity were searched in silico, supporting our experimental results. In situ cell-free gene expression of a membrane binding protein in the presence of GUVs was successfully performed in the device, which led to membrane tubulation from the GUVs. With optimal conditions, the device is applicable to a wide range of biochemical and biophysical assays using GUVs as a model membrane, as well as targeting other microscale objects such as colloid particles, single cells, and purified cell organelles.

  , 18 Figure 5b shows the series of microscopy images taken at different steps, where different solutions were sequentially introduced into a 46.3 µm-high channel as follows. After GUV trapping, PBS (solution 1) was introduced in the channel as shown in the top left panel, where the fluorescence of GUVs composed of EPC and Texas Red DHPE is colored in red. Under microscopy observation, the solution was pushed throughout the channel by micropump, and replaced with air, leaving the solution 1 inside the microwells as shown in the upper, second left panel in Figure 5b. Subsequently, 10 µM fluorescein in PBS (solution 2), PBS (solution 3), and 10 µM fluorescein in PBS (solution 4) were introduced with air plugs in between. The fluorescence of fluorescein is shown in blue in Figure 5b. When the 43.0 µmdeep wells were used, a part of GUVs, in particular with a greater size, were lost during this process as shown in Figure S3 in Supporting Information. This issue was overcome by increasing the well depth. As shown in Figure 5b, with 91.5 µm-deep microwells, the majority of GUVs remained trapped after the sequential medium exchanges as indicated

  in Supporting Information, the 3D geometry of the model included 9 microwells with a depth h and a diameter of 40 µm were positioned at a 40 µm interval at the bottom of a 300 µm-wide, 46.3 µm-high, and 400 µm-long channel geometry. A solid sphere as a model of a GUV with a diameter d was positioned in the central microwell, at 1 µm distance from the microwell wall of the upstream side and of the bottom. The left panel in Figure6ashows the evolution of average fluorescein concentration in a microwell with time, as a result of its diffusion from the well under a constant flow of medium without fluorescein introduced at the channel inlet at a velocity of 6.0 mm s-1. The well depth was varied as indicated with a color code, while the viscosity µ of the medium was set to be 1.0 mPa s. The curves for a well depth of 43.0 µm (black) and 91.5 (red) correspond to the experimental conditions shown in the left and right panel of Figure 2b with black lines, respectively. Those curves and the experimental data are plotted on a same graph in Figure S5 for direct comparison. The experimental and simulation results give similar profiles and characteristic exchange

FiguresFigure 1

 1 Figures

Figure 2 .

 2 Figure 2. a) Terminal velocities U of a GUV sedimenting in a 40 µm-diameter cylinder (magenta) and in an infinite medium (green) are plotted against GUV diameter. The right panel shows an enlarged view of the magenta curve. b) Times necessary for a GUV to sediment for a distance of 40 µm in a 40 µm-diameter cylinder (magenta) and in an infinite medium (green) are plotted against GUV diameter. Ten minutes (blue line) is enough for a majority of GUVs to sediment for 40 µm in the cylinder.

Figure 3 .

 3 Figure 3. a) Size distribution of GUVs before (magenta) and after trapping (yellow). b) Number of microwells having trapped 0, 1, 2, 3 or 4 vesicles (n = 3256). The black dots represent expected counts of a Poisson distribution with a mean number of GUV per well, λ = 0.54.

Figure 4 .

 4 Figure 4. a) Fluorescence microscopy images of microwells at the edge of a channel (top) and at the center of the channel (bottom) at different time points under flow. The channel was initially filled with a fluorescein solution prior to the introduction at t = 0 of a non-fluorescent solution at a flow rate ca. 0.90 µL s -1 . b) Fluorescence intensities normalized by maximum values at t = 0 in the microwell areas plotted against time. The distance between the microwell center and the channel edge is indicated by the color code on the right.

Figure 5 .

 5 Figure 5. a) Scheme of the experimental setup. Non-fluorescent and fluorescent solutions, separated by air plugs, were introduced alternately into the microfluidic device containing GUVs by peristaltic micro-pump. b) Fluorescence microscopy images (GUV and fluorescein in red and blue, respectively) merged with phase-contrast microscopy images (grey) taken at different steps of the sequential medium exchanges using a microfluidic device with 91.5 µmdeep microwells. White arrowheads indicate the microwells containing GUVs before (top right) and after (bottom right) the medium exchanges, while a yellow arrowhead indicates a microwell where a GUV was lost during the operation. c) A series of images of an entire 46.3 µm-high channel, in which blue and red dyes separated by air plugs in a tubing were sequentially flowed by peristaltic micro-pump. Solutions were successfully exchanged without leaving air bubble in the channel.

Figure 6 .

 6 Figure 6. a) Molecular concentration change in a microwell with different depths under flow simulated by COMSOL. Results of fluorescein (diffusion coefficient D = 4.3•10 -10 m 2 s -1 ) in water (left) and 99 kDa protein (D = 6.0•10 -11 m 2 s -1 ) in a viscous medium (right), starting from an initial concentration of 10 µM and 100 nM, respectively. At t > 0, the same medium without fluorescein nor protein was introduced at the channel inlet at an average flow velocity fixed at 6.0 mm s -1 . The viscosity µ is indicated for each system. b) Phase diagram plotting critical flow velocity at which the lift force equals the gravitational force on a GUV simulated by COMSOL against the relative GUV diameter (DGUV) compared to the microwell diameter (Dwell = 40 µm)for different well depths. The viscosity of the medium was set to 1.0 mPa s.

Figure 7 .

 7 Figure 7. a) Scheme of the in situ cell-free gene expression in the presence of GUVs in the microfluidic device. b) Amino acid sequence of YFP-conjugated bsMTS coded on the plasmid DNA. c) Bright field images showing a microwell containing a GUV (gray), and confocal microscopy images of the GUV membrane (white), and YFP-bsMTS (yellow) after 3.5 h of gene expression at 37 °C. d) Results of the control experiment performed in the parallel channel on the same microfluidic device as c) without plasmid.

Acknowledgements


Myriam Abarkan

Giulia R Fois

Caroline Vouillac-Mendoza

Serge H Ahmed

PhD Karine Guillem
email: karine.guillem@u-bordeaux.fr

Altered neuronal activity in the ventromedial prefrontal cortex drives nicotine intake escalation

Keywords: nicotine escalation, addiction, prefrontal cortex, in vivo neuronal activity, optogenetic

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Nicotine is the main substance responsible for tobacco addiction which remains the most prevalent addiction in the world today and a leading cause of preventable morbidity and mortality worldwide [START_REF]WHO report on the global tobacco epidemic: raising taxes on tobacco[END_REF]. Among people who smoke tobacco cigarettes, many escalate their daily consumption [START_REF] Birge | What Proportion of People Who Try One Cigarette Become Daily Smokers? A Meta-Analysis of Representative Surveys[END_REF] and transition to a state of tobacco addiction [START_REF] Weinstein | Mood variability and cigarette smoking escalation among adolescents. Psychology of addictive behaviors[END_REF][START_REF] Villanti | Preventing Smoking Progression in Young Adults: the Concept of Prevescalation[END_REF][START_REF] Conklin | The return to smoking: 1-year relapse trajectories among female smokers[END_REF][START_REF] Doubeni | Early course of nicotine dependence in adolescent smokers[END_REF]. In rodents, escalation of drug use is typically observed with prolonged daily access to several intravenous drugsincluding cocaine [START_REF] Ahmed | Transition from moderate to excessive drug intake: change in hedonic set point[END_REF][START_REF] Ahmed | Neurobiological evidence for hedonic allostasis associated with escalating cocaine use[END_REF][START_REF] Ahmed | Persistent increase in the motivation to take heroin in rats with a history of drug escalation[END_REF], heroin [START_REF] Ahmed | Persistent increase in the motivation to take heroin in rats with a history of drug escalation[END_REF][START_REF] Lenoir | Heroin-induced reinstatement is specific to compulsive heroin use and dissociable from heroin reward and sensitization[END_REF][START_REF] Lenoir | Supply of a nondrug substitute reduces escalated heroin consumption[END_REF][START_REF] Kenny | Conditioned withdrawal drives heroin consumption and decreases reward sensitivity[END_REF][START_REF] Mcnamara | Trait-like impulsivity does not predict escalation of heroin self-administration in the rat[END_REF][START_REF] Vendruscolo | Escalation patterns of varying periods of heroin access[END_REF] and methamphetamine [START_REF] Mandyam | Methamphetamine selfadministration and voluntary exercise have opposing effects on medial prefrontal cortex gliogenesis[END_REF][START_REF] Schwendt | Extended methamphetamine self-administration in rats results in a selective reduction of dopamine transporter levels in the prefrontal cortex and dorsal striatum not accompanied by marked monoaminergic depletion[END_REF][START_REF] Kitamura | Escalation of methamphetamine self-administration in rats: a dose-effect function[END_REF][START_REF] Wee | Effect of aripiprazole, a partial dopamine D2 receptor agonist, on increased rate of methamphetamine self-administration in rats with prolonged session duration[END_REF]. In contrast, unless subjected to different access conditions [START_REF] Cohen | Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence[END_REF][START_REF] George | CRF-CRF1 system activation mediates withdrawal-induced increases in nicotine selfadministration in nicotine-dependent rats[END_REF], rats do not show nicotine intake escalation following prolonged access to the drug [START_REF] Kenny | Nicotine self-administration acutely activates brain reward systems and induces a long-lasting increase in reward sensitivity[END_REF][START_REF] Paterson | Prolonged nicotine dependence associated with extended access to nicotine self-administration in rats[END_REF]. The neuroadaptations underlying escalation of nicotine intake therefore still remain poorly understood.

Clinical neuroscience research has shown that the ventromedial prefrontal cortex (vmPFC) is important for controlling and inhibiting drug seeking and motivation [START_REF] Goldstein | Drug addiction and its underlying neurobiological basis: neuroimaging evidence for the involvement of the frontal cortex[END_REF]. Specifically, neuroimaging studies have consistently found changes in the vmPFC in chronic cigarette smokers [START_REF] Sutherland | Chronic cigarette smoking is linked with structural alterations in brain regions showing acute nicotinic drug-induced functional modulations[END_REF][START_REF] Sutherland | Insula's functional connectivity with ventromedial prefrontal cortex mediates the impact of trait alexithymia on state tobacco craving[END_REF][START_REF] Franklin | The effects of chronic cigarette smoking on gray matter volume: influence of sex[END_REF][START_REF] Hanlon | Lower subcortical gray matter volume in both younger smokers and established smokers relative to non-smokers[END_REF][START_REF] Brody | Differences between smokers and nonsmokers in regional gray matter volumes and densities[END_REF], notably a decrease in gray matter volume and in basal neuronal activity.

These changes could explain why they experience difficulty in controlling their consumption of cigarettes when they want to quit or cut down. Moreover, preclinical research on animal models of drug addiction also supports an involvement of the vmPFC in drug-taking andseeking behaviors [START_REF] Bossert | Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin[END_REF][START_REF] Van Den Oever | Prefrontal cortex plasticity mechanisms in drug seeking and relapse[END_REF][START_REF] Warren | Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories[END_REF][START_REF] Moorman | Differential roles of medial prefrontal subregions in the regulation of drug seeking[END_REF][START_REF] Lubbers | Prefrontal gamma-aminobutyric acid type A receptor insertion controls cue-induced relapse to nicotine seeking[END_REF][START_REF] Peters | Extinction circuits for fear and addiction overlap in prefrontal cortex[END_REF]. For instance, vmPFC inhibition reduces methamphetamine and heroin seeking after extinction or cocaine seeking after abstinence [START_REF] Bossert | Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin[END_REF][START_REF] Moorman | Differential roles of medial prefrontal subregions in the regulation of drug seeking[END_REF][START_REF] Koya | Role of ventral medial prefrontal cortex in incubation of cocaine craving[END_REF][START_REF] Rocha | Role of the prefrontal cortex and nucleus accumbens in reinstating methamphetamine seeking[END_REF]. However, we do not know how the activity of vmPFC neurons is modified during prolonged access to nicotine self-administration and, if so, whether these alterations contribute to the development and maintenance of nicotine intake escalation.

Here we set out to address these questions by recording in vivo vmPFC neuronal activity in a newly developed rat model of nicotine intake escalation. First, we report that access to unusually high doses of nicotine (i.e., from 30 µg to 240 µg/kg/injection) can precipitate a rapid and robust escalation of intake that was associated with an increased motivation to take the drug. Such escalation of nicotine intake induces long-lasting changes in the basal activity of vmPFC neurons measured 24h after the last self-administration session and also in their activity during ongoing intravenous nicotine self-administration. Specifically, after escalation of nicotine intake, basal vmPFC neuronal activity increased above pre-escalation and control activity levels, while in contrast nicotine intake during ongoing nicotine selfadministration restored these neuronal changes. Finally, simulation of the restoring effects of nicotine using in vivo optogenetic inhibition of vmPFC neurons caused a selective deescalation of nicotine self-administration. Overall, these data suggest that part of the motivation for using nicotine during escalation might be to restore post-escalation changes in vmPFC basal neuronal activity.

Material and Methods

Subjects

A total of 88 adult male Wistar rats were used (225-250 g at the beginning of experiments, Charles River, Lyon, France). Two rats did not complete the experiment because of loss of catheter patency, thereby leaving 78 rats for final analysis (39 for the nicotine escalation experiment, 10 for the in vivo electrophysiology experiment, and 37 for the optogenetic experiment). Rats were housed in groups of

Nicotine self-administration and escalation procedures

Fourteen identical operant chambers (30 x 40 x 36 cm) were used for nicotine selfadministration testing and training (Imétronic, Pessac, France). Each chamber was equipped with two retractable metal levers on opposite panels of the chamber and a corresponding white cue light above each lever. Under deep anesthesia (mixture of ketamine 100 mg/kg and xylazine 15 mg/kg, i.p), rats were surgically prepared with an indwelling silastic catheter (Dow Corning Corporation, Michigan, USA) in the right jugular vein that exited the skin in the middle of the back about 2 cm below the scapulae. After surgery, rats were flushed daily with 0.2 ml of an ampicillin solution (0.1 g/ml) containing heparin (300 IU/ml) to maintain patency.

After 7 days of recovery, rats were first habituated during 2 3-h daily sessions to the experimental chambers with no lever or light cue presented. Then rats were trained to press a lever to self-administer a unit dose of nicotine (30 µg/kg/injection free base; 40 µl in 1s) under a final fixed-ratio 1 schedule of reinforcement during 3-h daily sessions. Selfadministration sessions were run 5-6 days/week. All self-administration sessions began with extension of the operant lever and ended with its retraction after 3 h. Intravenous delivery of nicotine began immediately after completion of the lever press and was accompanied by illumination of the light cue above the lever for 20 s. Responses during the light cue were recorded but had no programmed consequence.

Rats trained to self-administer nicotine for 15 days were then divided in two groups: one noescalation (No ES, n = 12) and one escalation groups (ES, n = 27). In the no-escalation group, the unit dose of nicotine remained at the initial dose of 30 µg/kg/injection for 30 additional sessions, while in the escalation group the unit dose of nicotine available progressively increased to 120 µg and then to 240 µg/kg/injection for 15 sessions at each dose. Unit doses of nicotine were presented in ascending order to reduce the risk of accidental overdose or overconsumption with subsequent aversive reactions to the drug [START_REF] Fowler | Intravenous nicotine self-administration and cue-induced reinstatement in mice: effects of nicotine dose, rate of drug infusion and prior instrumental training[END_REF].

This increase in the unit dose of nicotine available was done by increasing the injection volume (to 160 and 320 µl), a procedure known to speed up the escalation process [START_REF] Lenoir | Heroin-induced reinstatement is specific to compulsive heroin use and dissociable from heroin reward and sensitization[END_REF][START_REF] Mantsch | Effects of extended access to high versus low cocaine doses on self-administration, cocaine-induced reinstatement and brain mRNA levels in rats[END_REF].

Twenty-four hours after the last escalation day, rats were tested under a progressive ratio schedule of reinforcement during which the response requirement for nicotine reinforcement was increased according to the following sequence: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, etc. [START_REF] Richardson | Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy[END_REF].

The progressive ratio session ended after 1 h had elapsed without a drug delivery reward or after a maximum of 6 h. The last ratio completed during the session, called the breaking point, was used to measure the intensity of drug seeking. Rats were then exposed to 3 additional self-administration session days (either 30 µg or 240µg/kg/injection according to the group) and then to a single sensitization test session during which all rats were exposed to the unit dose of 30 µg/kg/injection. Nicotine-induced behavioral excitement was observed and evaluated after the first nicotine injection using a four point-ranked score (0: no effect; 1: mild head tremor and straub tail; 2: apparent tremors in extended regions, 3: severe tremors and convulsions-like responses) [START_REF] Kunisawa | Nicotine evokes kinetic tremor by activating the inferior olive via alpha7 nicotinic acetylcholine receptors[END_REF] during the 15 days of nicotine self-administration at the highest dose of 240 µg/kg/injection. Both the incidence and the behavioral score were quantified across days and compared using a Chi X 2 or an ANOVA respectively.

Sucrose self-administration

Eight identical operant chambers (30 x 40 x 36 cm) were used for sucrose selfadministration (Imétronic, Pessac, France). Each chamber was equipped with one retractable metal lever, a white cue light above the lever and a drinking cup nearby. Rats were trained to lever press for sucrose (i.e., 0.28 ml of 10% sucrose over 10 s) under a fixedratio (FR) 1 time-out 10s schedule of reinforcement during 14 daily sessions before optogenetic testing. Each session began with extension of the operant lever and ended after a maximum of 40 rewards or after 1 h, whatever came first. Responses during the light cue were recorded but had no programmed consequence.

In vivo electrophysiological recordings

To assess whether nicotine intake escalation altered vmPFC neuronal activity, we used in vivo electrophysiology to record single-unit activity in the vmPFC. An independent group of rats (no-escalation and escalation, n = 5 for each group) were surgically prepared with a catheter in the right jugular vein and an array of 16 teflon-coated stainless steel microwires (MicroProbes Inc, Gaithersburg, MD) in the vmPFC [AP: + 2.5 to + 4.5 mm, ML: 0.3 to 1.2 mm, and DV: -4.8 mm relative to skull level], as previously described [START_REF] Girardeau | Relapse to cocaine use persists following extinction of drug-primed craving[END_REF].

Voltage signals from each microwire array were band-pass-filtered, such that activity between 150 and 8,000 Hz was analyzed as spiking activity. Data were amplified, processed and digitally captured using commercial hardware and software (Plexon Inc, Dallas, TX).

Single units were discriminated with principal component analysis (Offline sorter, Plexon Inc, Dallas, TX). The quality of recorded units was ensured with an interspike interval criterion (>1 ms) and a signal:noise criterion (>3X noise band). Neurons were classified into putative interneurons and pyramidal neurons according to the waveform spike width and average firing rate. Waveforms with durations shorter than 300 µs were classified as putative interneurons, and waveforms with durations longer than 300 µs were classified as putative pyramidal neurons, as previously described [START_REF] Girardeau | Relapse to cocaine use persists following extinction of drug-primed craving[END_REF][START_REF] Guillem | Orbitofrontal and insular cortex: neural responses to cocaine-associated cues and cocaine self-administration[END_REF]. We recorded a total number of putative interneurons (n = 14) and pyramidal neurons (n = 240) across all recordings days. Due to the small number of interneurons, data analysis exclusively focused on putative pyramidal neurons. We analyzed a total 81 pyramidal neurons during Habituation, 79 neurons during Initial self-administration and 80 neurons during Final self-administration.

Electrophysiological data were analyzed using NeuroExplorer (Plexon Inc., Dallas, TX).

After 1 week of recovery, rats were trained through the same nicotine intake escalation as described above. Electrophysiological recordings were conducted several times for each individual rat on the following days: on the habituation day before escalation (Hab), on the last day of nicotine self-administration acquisition (30 µg/kg/injection for both no-escalation and escalation groups; Initial SA), and on the last day of nicotine self-administration escalation (30 µg/kg/injection and 240 µg/kg/injection for both no-escalation and escalation respectively; Final SA). Recording sessions were identical to self-administration sessions except that they also included an initial 30-minute predrug period -during which the chamber was dark and no session events occurred -to allow measurement of basal tonic neuronal activity before onset of nicotine intake [START_REF] Guillem | Orbitofrontal and insular cortex: neural responses to cocaine-associated cues and cocaine self-administration[END_REF]. Tonic vmPFC neurons firing rates were measured during the 30-min pre-drug period before onset of nicotine self-administration (i.e., Before SA) and during the 3-h nicotine self-administration session (i.e., During SA) and normalized using the transformation of log10(x + 1) to reduce the skew of the firing rate distribution. Tonic neuronal activity was determined for each individual rat and averaged across rats. Individual neurons were tested for a significant and stable session-change in firing (increase or decrease) during the 3-h nicotine self-administration session relative to the pre-drug period using a Mann-Whitney test [START_REF] Guillem | Orbitofrontal and insular cortex: neural responses to cocaine-associated cues and cocaine self-administration[END_REF][START_REF] Guillem | Acute effects of nicotine amplify accumbal neural responses during nicotine-taking behavior and nicotine-paired environmental cues[END_REF]. Neurons were also tested for phasic long-duration change in firing (increase or decrease) that occurred slowly after, and relative to, firing in the 2 min before the nicotine reinforced lever press [START_REF] Peters | Infralimbic prefrontal cortex is responsible for inhibiting cocaine seeking in extinguished rats[END_REF]. At the end of the study, histological procedures were used to identify the location of all wire tips used to record neurons. Coronal sections showing the location of microwires electrodes tips from which vmPFC neurons were recorded are shown in Figure 3a.

In vivo optogenetic procedures

Viral infection of vmPFC pyramidal neurons was conducted in an independent group of rats (n = 26) trained to self-administered nicotine. Then, under isoflurane anesthesia, light-gated opsins viruses encoding the archaerhodopsin-3.0 protein (no-escalation and escalation, n = 8 for each group; CaMKII-α-eArchT3.0-eYFP; 5.5 x 10 12 viral molecules/mL; UNC Vector Core) or a control virus (no-escalation and escalation, n = 5 for each group; CaMKII-eYFP 5.5 x 10 12 viral molecules/mL; UNC Vector Core) were infected bilaterally in the vmPFC (AP: +3 mm; ML: ± 1.4 mm; DV: -4.5 from skull, angle 10°) to selectively infect pyramidal projecting neurons. A total of 0.5µl/hemisphere was injected at a rate of 0.1 µl/min for 10 min using an Hamilton syringe mounted in an infusion pump after which the injector will be left in place for an additional 10 min to allow virus diffusion. Optic fibers (200/230 µm core diameter Plexon Inc., Dallas, TX) were implanted bilaterally slightly above the injection site (~0.3 mm) to ensure illumination of the transduced neurons and secured to the skull screws and dental cement. Seven days after surgery, rats were retrained to nicotine self-administration escalation for 20 additional days before being testing for optogenetic manipulations. Optoinhibition of vmPFC neurons after nicotine escalation (250 ms pulse width at 1Hz) [START_REF] Martin-Garcia | Frequency of cocaine self-administration influences drug seeking in the rat: optogenetic evidence for a role of the prelimbic cortex[END_REF] during the 10-min pre-drug period before the onset of nicotine self-administration was assessed twice in two separate 1h nicotine self-administration sessions (no light and light conditions, order counter-balanced between groups) at the lowest dose of 30 µg/kg/injection to ensure sufficient number of presses and injections. Each implanted optic fiber was connected to a LED module (550 nm) mounted on a dual LED commutator connected to an optogenetic controller (PlexBright, Plexon Inc., Dallas, TX). A separate group of rats (n = 8) was similarly injected with eArchT3.0 in the vmPFC and trained to self-administer sucrose 10% under FR1 for 14 days. Optogenetic inhibition of vmPFC neurons (250 ms pulse width at 1Hz during the 10-min baseline period) was first assessed in two separate FR1 sucrose sessions (no light and light conditions, order counter-balanced). Then the FR was increased to 3 to further increase response rate in comparison to the lower rate during initial FR1 training, and opto-inhibition again assessed in two separate FR3 sucrose sessions (no light and light conditions, order counter-balanced). Efficiency of optical stimulation in inhibiting neuronal vmPFC pyramidal neurons activity was controlled in an independent group of rats injected with eArchT3.0 (n= 3) using optrodes by simultaneously recording in vivo vmPFC neuronal activity before and after optogenetic stimulation (250 ms pulse width at 1Hz during 5 min). At the end of the experiments, rats were perfused transcardially with 4% paraformaldehyde (PFA). Brains were removed, serially cut on a cryostat (50 µm) and stored in PBS as floating sections for immunohistochemistry. Brains floating sections were first put in a blocking solution with Normal Donkey Serum 5% in PBS-Triton 0.3% (PBST) during 90 min and then incubated with rabbit anti-GFP antibody (1:2000, Invitrogen, A11122) and mouse anti-CAMKII antibody (1:500, Thermo Fisher, MA1-048) overnight at room temperature (RT).

Sections were next washed with PBST and revealed with donkey anti-rabbit antibody conjugated to Alexa 488 (1: 1000, Invitrogen, A21206) and donkey anti-mouse antibody conjugated to A568 (1: 1000, Invitrogen, A10037) for 90 min at RT. Sections were finally mounted in antifading Vectashield medium with 4,6-diamidino-2-phenylindole DAPI (Vector Laboratories), coverslipped and imaged on an epifluorescence microscope (Olympus).

Histological and immunological procedures were used to verify the opsin expression and identify the location of all optic fibers. Coronal sections showing the location of the tips of the optic fibers within the vmPFC are shown in Figure 4a.

Data Analysis

Data were subjected to one-way or two-way ANOVAs with repeated measures, followed by

Results

Robust escalation of nicotine intake in rats exposed to unusually high doses of nicotine self-administration

We first developed a new three stages model of nicotine intake escalation (see Material and Methods; Fig. 1). Briefly, one group of rats had access to nicotine self-administration at the unit dose of 30 µg/kg/injection (no-escalation group, No ES, n = 12), while in the other group the unit dose of nicotine available progressively increased from 30 µg to 120 µg and to 240 µg/kg/injection (escalation group, ES, n = 27). Though drug intake was similar in both groups at the initial dose of 30 µg/kg/injection, nicotine intake increased with the unit dose of nicotine (F(44,1628) = 8.44; p < 0.001; Fig. 1a). At the dose of 120 µg/kg/injection, all rats immediately raised their drug intake to reach within 3 days a stable level of nicotine intake that they maintained thereafter across daily sessions (F(44,1144) = 19.7; p < 0.001; 15 th vs 16 th session; p < 0.01 and 18 th vs 30 th session; NS; Fig. 1a). Next, when the dose was enhanced to the final highest dose of 240 µg/kg/injection, rats further increased their intake but did so gradually over time (31 st vs 45 th session; p < 0.001; Fig. 1a). In contrast, in the no-escalation group, nicotine intake increased at the beginning of the training but remained stable during the rest of the procedure (F(44,484) = 4.41; p < 0.001; 1 st vs 7 th session; p < 0.05 and 15 th vs 45 th session; NS) . At the end of the escalation procedure, the level of nicotine intake (i.e., average over the last 3 sessions at each dose) was 3.5 times higher in the escalation group than in the no-escalation group (F(2,74) = 19.7; p < 0.001; Fig. 1b). Finally, rats in the escalation group showed a higher motivation to obtain nicotine as revealed both by elevated break point in a progressive-ratio reinforcement schedule (F(2,74) = 19.7; p < 0.001; Fig. 1c), and increased number of lever presses (F(1,37) = 11.6; p < 0.001; Fig. 1d) and increased number of nicotine injections when decreasing the nicotine dose back to 30 µg/kg/injection (F(1,37) = 9.4; p < 0.01; Fig. 1e). Moreover, this increase in nicotine intake was particularly evident during the first 10 minutes (F(17,663) = 3.61; p < 0.001; Fig. 1f), a drug-loading behavior that reflect an acquired need state for a higher level of nicotine intoxication.

During the course of escalation of nicotine intake, rats in the escalation group exposed to the highest dose of nicotine (240 µg/kg/injection) began to exhibit convulsive-like behavior after the first nicotine injection of the session day, a reaction not seen in the no-escalation group with the lowest dose of nicotine. Nicotine is known to evoke motor excitement including straub tail, tremors and convulsive responses [START_REF] Miner | Strain comparison of nicotine-induced seizure sensitivity and nicotinic receptors[END_REF][START_REF] Swan | The effects of tobacco smoke and nicotine on cognition and the brain[END_REF][START_REF] Foll | Effects of nicotine in experimental animals and humans: an update on addictive properties[END_REF][START_REF] Iha | Nicotine Elicits Convulsive Seizures by Activating Amygdalar Neurons[END_REF]. We thus evaluated nicotine-induced behavioral excitement after the first daily nicotine injection using a four point-ranked score (see Material and Methods) [START_REF] Kunisawa | Nicotine evokes kinetic tremor by activating the inferior olive via alpha7 nicotinic acetylcholine receptors[END_REF]. The majority (roughly 75%) of the rats exhibited convulsivelike responses during the first days of self-administration at 240 µg/kg/injection (Fig. 2a), suggesting that nicotine has important negative-side effects. However, the incidence (Chi X 2 (n=20, dl =14) =76.66; p < 0.001; Fig. 2a) as well as the intensity (F(14,266) = 7.81; p < 0.001; Fig. 2b) of these convulsions decreased over repeated sessions presumably because of the development of a tolerance to nicotine-induced convulsive responses. These changes were, however, not correlated to escalated levels of nicotine intake (r = -0.29; NS). Importantly, despite these convulsions, rats not only continued to self-administer the high dose of nicotine, but were also even faster to initiate drug use at session onset in comparison to controls (1 st injection latency: F(44,1628) = 2.56; p < 0.001; Fig. 2c andF(2,78) = 4.79; p < 0.01; Fig. 2d). Thus, apparently, the convulsions induced by the high dose of nicotine were not aversive, an interpretation further supported by the observation within the escalation group that rats with the more severe convulsive-like responses were also the faster to initiate drug intake (r = -0.42; p < 0.05, data not shown).

Escalation of nicotine alters vmPFC basal neuronal activity and its activity during ongoing nicotine self-administration

We next assessed changes in vmPFC neuronal activity during escalation of nicotine intake using in vivo electrophysiological recordings in another group of rats (n = 10; Fig. 3a). Tonic vmPFC neuronal activity was measured i) after overnight abstinence during a 30-min predrug period before onset of nicotine self-administration (i.e., Before SA) and ii) during the 3-h nicotine self-administration session (i.e., During SA). In the no-escalation group vmPFC neuronal activity before SA decreased rapidly after the initial sessions of nicotine intake (F(2,102) = 3.47; p < 0.05; Hab vs Initial SA, p < 0.05; Fig. 3b) and remained at the same level until the end of the procedure (Initial vs Final SA, NS; Fig. 3b), indicating that repeated nicotine self-administration inhibited basal vmPFC neuronal activity. In contrast in the escalation group, though an initial decrease in basal activity at the lowest dose of nicotine, vmPFC basal activity after nicotine escalation increased above pre-escalation levels (F(2,132) = 4.48; p < 0.05; Hab vs Initial SA, p < 0.05; Initial SA vs Final SA, p < 0.01; Fig. 3b) and above no-escalation activity levels observed at the end of the procedure (F(1,76) = 4.91; p < 0.05; Fig. 3b), suggesting a compensatory increase of initially depressed vmPFC neuronal activity after nicotine escalation.

In addition to these basal neuronal adaptations, ongoing nicotine self-administration also directly affected vmPFC neuronal activity. It decreased overall firing rate activity during the entire 3-h nicotine self-administration session (i.e., During SA) compared to pre-dug baseline period (i.e., Before SA) in both groups (F(1,67) = 12.4; p < 0.001 and F(1,88) = 74.9; p < 0.001 for no-escalation and escalation groups, respectively; Fig. 3b,c), indicating that nicotine had inhibitory effects on vmPFC neuronal activity. However, while this inhibitory effect remained constant during repeated nicotine self-administration in the no-escalation group (F(1,67) = 1.13; NS; Fig. 3b), it intensified with repeated high doses of nicotine in the escalation group (F(1,88) = 29.07; p < 0.001; Initial SA Before vs During SA, NS; Final SA Before vs During SA, p < 0.01; Fig. 3b). Consistent with this, there was significantly more neurons showing a sustained decrease in firing rate during the self-administration session relative to the pre-drug baseline period in the escalation than in the no-escalation group (escalation 80% vs no-escalation 44%; Z = 3.37; p < 0.001; Fig. 3d), indicating stronger and more inhibitory neuronal responses in escalated than in non-escalated rats. Importantly, nicotine intake restored post-escalation changes in vmPFC neuronal activity as it brought the activity back to pre-escalation (Initial SA during SA vs Final SA during SA; NS; Fig. 3b) and no-escalation levels (F(1,76) = 13.50; p < 0.001; No ES Final SA vs ES Final SA; NS; Fig. 3b). Further quantification of the difference in firing activity before versus during SA (i.e., ∆ overall firing activity) revealed that escalated rats with higher level of nicotine intake than non-escalated rats (F(1,4) = 41.8; p < 0.01; Fig. 3e) exhibited a higher vmPFC ∆ overall firing activity (F(1,4) = 10.8; p < 0.05; Fig. 3f). This was further confirmed by looking at the longduration changes in firing time-locked to nicotine self-injections (Fig. 3g,h). For most of the neurons it consisted of a change in firing rate, typically a decrease, within 1 or 2 min after the reinforced lever press followed by a progressive reversal of that change (Fig. 3g).

Importantly, there were two times more neurons exhibiting long-duration decreases in firing after nicotine injection in the escalation than in the no-escalation group (47% and 78% for noescalation and escalation groups respectively, Z = 3.04; p < 0.01; Fig. 3h). Together, these results suggest that nicotine intake induced larger vmPFC hypoactivity after nicotine escalation, and that this inhibitory effect can restore post-escalation changes in vmPFC neuronal activity.

Restoring basal vmPFC neuronal activity with in vivo optogenetic inhibition of vmPFC pyramidal neurons decreases nicotine self-administration in escalated rats

Based on these observations, we hypothesized that rats will escalate their drug intake in order to inhibit their vmPFC activity and thus to restore their basal vmPFC neuronal activity.

If so, then mimicking the restoring effects of nicotine using in vivo optogenetic inhibition of vmPFC neurons during ongoing nicotine self-administration should decrease nicotine intake in escalated rats through a hedonic allostasis mechanism. To test this hypothesis, an adenoassociated virus (AAV) targeting the pyramidal neurons and encoding the archaerhodopsin (CaMKII-ArChT3.0-eYFP) was bilaterally injected into the vmPFC with bilateral implantation of chronic optic fibers in the vmPFC (see Material and Methods; Fig. 4a). Efficiency of optical stimulation in inhibiting vmPFC pyramidal neurons activity was first controlled in an independent group of rats injected with ArChT3.0 (n = 3) in which vmPFC pyramidal neurons activity was strongly reduced by light application (F(2,22) = 14.2; p < 0.001; Fig. 4b). We next tested whether decreasing vmPFC pyramidal neurons activity could alter nicotine selfadministration in two separates 1h nicotine self-administration sessions (no light and light conditions; see Material and Methods) at the lowest dose of 30 µg/kg/injection. As expected from above, at this dose, rats in the escalation group pressed more (training condition effect: 5b) in a separate group of rats trained to lever press under a FR1 schedule of reinforcement. In addition, after initial training under a FR1, the same rats were tested with a FR3 to further increase the response rate, but despite this, behavior remained unchanged during vmPFC optogenetic inhibition (F(1,7) = 0.04; NS; Fig. 5c).

Discussion

Access to unusually high doses of nicotine precipitated a rapid and robust escalation of nicotine intake in rats that progressed despite the emergence of convulsion-like behavior in the majority of rats. In addition, once established escalated levels of nicotine intake were associated with an increased motivation to take the drug and an increased daily drug-loading behavior at session onset, and were defended when the unit dose of nicotine was decreased. These phenomena were previously observed with other drugs after escalation ( [START_REF] Ahmed | Transition from moderate to excessive drug intake: change in hedonic set point[END_REF]; for a systematic review, see [START_REF] Ahmed | Toward an evolutionary basis for resilience to drug addiction[END_REF]) and are thought to represent a behavioral signature of an increased motivation for higher states of intoxication. Escalation of nicotine intake had been difficult to obtain previously [START_REF] Kenny | Nicotine self-administration acutely activates brain reward systems and induces a long-lasting increase in reward sensitivity[END_REF][START_REF] Paterson | Prolonged nicotine dependence associated with extended access to nicotine self-administration in rats[END_REF][START_REF] O'dell | Extended access to nicotine self-administration leads to dependence: Circadian measures, withdrawal measures, and extinction behavior in rats[END_REF][START_REF] Valentine | Self-administration in rats allowed unlimited access to nicotine[END_REF], except under specific access conditions, such as, for instance, intermittent access to extended periods of nicotine self-administration, separated by long periods of forced abstinence (i.e., 21-23-h sessions every 24-72 h) [START_REF] Cohen | Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence[END_REF][START_REF] George | CRF-CRF1 system activation mediates withdrawal-induced increases in nicotine selfadministration in nicotine-dependent rats[END_REF]. As shown here, previous difficulties in inducing robust escalation of nicotine intake are also probably due, at least partly, to the use of low unit doses of nicotine, though other factors are also involved.

Transition to nicotine addiction is thought to result from neuroadaptative changes that increase the need to continue using tobacco [START_REF] Tiffany | What can dependence theories tell us about assessing the emergence of tobacco dependence?[END_REF][START_REF] Koob | Plasticity of reward neurocircuitry and the 'dark side' of drug addiction[END_REF]. Here we report that escalation of nicotine intake induced long-lasting changes in vmPFC basal neuronal activity (i.e., activity recorded before onset of nicotine self-administration session). Specifically, after escalation of nicotine intake, basal vmPFC neuronal activity increased above pre-escalation and noescalation activity levels. Similar alterations in mPFC neurons firing rate have been previously observed with repeated psychostimulant treatments [START_REF] Homayoun | Bursting of prefrontal cortex neurons in awake rats is regulated by metabotropic glutamate 5 (mGlu5) receptors: rate-dependent influence and interaction with NMDA receptors[END_REF][START_REF] Moghaddam | Divergent plasticity of prefrontal cortex networks[END_REF] or after withdrawal from psychostimulants self-administration [START_REF] Parsegian | Methamphetamine self-administration produces attentional set-shifting deficits and alters prefrontal cortical neurophysiology in rats[END_REF][START_REF] Sun | Repeated cocaine self-administration alters processing of cocainerelated information in rat prefrontal cortex[END_REF]. Together, these results suggest that the post-escalation basal changes in neuronal activity could indicate the instauration of a new allostatic state in the vmPFC during drug intake escalation, a neurophysiological adaptation that might be common to escalation of intake across different drugs.

Ongoing nicotine self-administration also altered vmPFC neuronal activity. Despite an heterogeneous population response, there was a net decrease of vmPFC neuronal activity during the entire self-administration session compared to pre-dug baseline period, as well as during few minutes after the nicotine self-injection. Importantly, greater and more sustained inhibitory responses were observed in rats with escalated levels of nicotine intake, suggesting that this nicotine-induced vmPFC hypoactivity may contribute to the motivation to take the drug during nicotine intake escalation. As such, we hypothesized that rats will escalate their nicotine intake in order to inhibit their vmPFC neuronal activity in an attempt to compensate for basal changes in vmPFC neuronal activity, and that interventions simulating these restoring effects of nicotine on vmPFC neuronal activity should thus decrease escalated levels of nicotine use. Consistent with our hypothesis, the optogenetic inhibition of vmPFC pyramidal neurons before or at the onset of nicotine self-administration strongly decreased nicotine self-administration selectively in rats that had escalated their nicotine intake, thus suggesting that part of the motivation for using nicotine during escalation might be to restore post-escalation changes in vmPFC basal neuronal activity. Importantly, this effect was specific to nicotine intake escalation as it was not observed in the no-escalation group or in rats trained to respond at high rate for sucrose. Though conducting the converse experiment with optical excitation of vmPFC neurons may add to this conclusion, this would require being able to reproduce reliably with optogenetic stimulation the complex changes in vmPFC neuronal activity that were observed after nicotine intake escalationa delicate task that is beyond the scope of the present study (see also, below).

The finding that vmPFC optogenetic inhibition decreased nicotine intake is consistent with previous report showing that vmPFC inhibition reduced methamphetamine and heroin seeking after extinction or cocaine seeking after abstinence [START_REF] Bossert | Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin[END_REF][START_REF] Koya | Role of ventral medial prefrontal cortex in incubation of cocaine craving[END_REF][START_REF] Rocha | Role of the prefrontal cortex and nucleus accumbens in reinstating methamphetamine seeking[END_REF][START_REF] Moorman | Prefrontal neurons encode context-based response execution and inhibition in reward seeking and extinction[END_REF]]. However, opposite behavioral outcomes have also been reported [START_REF] Peters | Infralimbic prefrontal cortex is responsible for inhibiting cocaine seeking in extinguished rats[END_REF][START_REF] Lalumiere | Neural circuit competition in cocaineseeking: roles of the infralimbic cortex and nucleus accumbens shell[END_REF], suggesting a more complex role for the vmPFC in drug-taking and seeking behaviors. Interestingly, growing evidence indicates that separate, but intermingled, neural ensembles within vmPFC selectively encode different and even opposite behaviors [START_REF] Warren | Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories[END_REF][START_REF] Moorman | Differential roles of medial prefrontal subregions in the regulation of drug seeking[END_REF][START_REF] Koya | Role of ventral medial prefrontal cortex in incubation of cocaine craving[END_REF]. Though we did not determine which vmPFC neuronal ensembles are involved, the heterogeneous vmPFC neuronal responses observed here during nicotine self-administration is consistent with this conceptualization.

Finally, it is important to stress that our interpretation is not inconsistent with other possible interpretations of nicotine intake escalation. For instance, abstinence from nicotine is also known to produce robust affective withdrawal symptoms, including increased anxiety-like behavior [START_REF] George | CRF-CRF1 system activation mediates withdrawal-induced increases in nicotine selfadministration in nicotine-dependent rats[END_REF][START_REF] Hughes | Symptoms of tobacco withdrawal. A replication and extension[END_REF][START_REF] Cohen | Extended access to nicotine leads to a CRF1 receptor dependent increase in anxiety-like behavior and hyperalgesia in rats[END_REF], symptoms that have been associated with increased probability for escalating smoking behavior [START_REF] Doubeni | Early course of nicotine dependence in adolescent smokers[END_REF][START_REF] Dierker | Early emerging nicotine-dependence symptoms: a signal of propensity for chronic smoking behavior in adolescents[END_REF]. Moreover, vmPFC has been implicated in anxiety regulatory processes [START_REF] Phelps | Contributions of the amygdala to emotion processing: from animal models to human behavior[END_REF][START_REF] Quirk | Prefrontal involvement in the regulation of emotion: convergence of rat and human studies[END_REF] and vmPFC lesion or inactivation reduced anxiety-like behaviors [START_REF] Wall | Infralimbic D1 receptor agonist effects on spontaneous novelty exploration and anxiety-like defensive responding in CD-1 mice[END_REF]. Thus, it is also possible that rats during overnight abstinence exhibited an increased level of anxiety [START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF][START_REF] Balerio | Delta9tetrahydrocannabinol decreases somatic and motivational manifestations of nicotine withdrawal in mice[END_REF] that they seek to relieve through increased nicotine use resumption. In addition, post-escalation changes in basal vmPFC neuronal activity can result from altered glutamatergic [START_REF] Kalivas | The neural basis of addiction: a pathology of motivation and choice[END_REF][START_REF] Knackstedt | The role of cystine-glutamate exchange in nicotine dependence in rats and humans[END_REF] and/or cholinergic signaling [START_REF] Koukouli | Nicotine reverses hypofrontality in animal models of addiction and schizophrenia[END_REF]. Indeed, withdrawal from nicotine self-administration down-regulates metabotropic glutamate receptors (mGluRs) function in the mPFC [START_REF] Kenny | NMDA receptors regulate nicotine-enhanced brain reward function and intravenous nicotine selfadministration: role of the ventral tegmental area and central nucleus of the amygdala[END_REF][START_REF] Liechti | Metabotropic glutamate 2/3 receptors in the ventral tegmental area and the nucleus accumbens shell are involved in behaviors relating to nicotine dependence[END_REF], and NMDA or mGluR5s antagonists increase mPFC neurons firing [START_REF] Homayoun | Bursting of prefrontal cortex neurons in awake rats is regulated by metabotropic glutamate 5 (mGlu5) receptors: rate-dependent influence and interaction with NMDA receptors[END_REF][START_REF] Homayoun | Activation of metabotropic glutamate 2/3 receptors reverses the effects of NMDA receptor hypofunction on prefrontal cortex unit activity in awake rats[END_REF][START_REF] Jackson | NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex[END_REF]. Moreover, chronic nicotine administration also leads to a rapid desensitization of nicotinic acetylcholine receptors (nAChRs) in the mPFC that are known to regulate the neuronal activity of mPFC pyramidal neurons [START_REF] Poorthuis | Layerspecific modulation of the prefrontal cortex by nicotinic acetylcholine receptors[END_REF][START_REF] Poorthuis | Layer-specific interference with cholinergic signaling in the prefrontal cortex by smoking concentrations of nicotine[END_REF][START_REF] Buisson | Chronic exposure to nicotine upregulates the human (alpha)4((beta)2 nicotinic acetylcholine receptor function[END_REF].

To sum up, our findings add to growing evidence that drug intake escalation induces changes in vmPFC neuronal function and suggest that interventions aimed at correcting these changes may prove useful in the treatment of addiction.

Legends

  2 and were maintained in a light-(reverse lightdark cycle), humidity-(60 ± 20%) and temperature-controlled vivarium (21 ± 2°C), with water and food available ad libitum. All behavioral testing occurred during the dark phase of the light-dark cycle. Home cages were enriched with a nylon gnawing bone and a cardboard tunnel (Plexx BV, The Netherlands). All experiments were carried out in accordance with institutional and international standards of care and use of laboratory animals [UK Animals (Scientific Procedures) Act, 1986; and associated guidelines; the European Communities Council Directive (2010/63/UE, 22 September 2010) and the French Directives concerning the use of laboratory animals (décret 2013-118, 1 February 2013)]. The animal facility has been approved by the Committee of the Veterinary Services Gironde, agreement number B33-063-922.

  Tukey post hoc tests where relevant. For the optogenetic study, data were subjected to three-way ANOVAs with training condition (escalation or no-escalation) and virus type as between factors and light condition (light or no-light) as repeated measures, followed by Tukey post hoc tests where relevant. Percentage of neurons were compared using the two-proportion z-test. Statistical analyses were run using Statistica, version 7.1 (Statsoft Inc., Maisons-Alfort, France).

F( 1 ,

 1 22) = 23.28; p < 0.001; Fig 4c) and self-administered more nicotine than those in the noescalation group (F(1,22) = 15.31; p < 0.010; Fig. 4d). Importantly, vmPFC optogenetic inhibition (250 ms pulse width at 1Hz) during the 10-min pre-drug period before the onset of nicotine self-administration strongly decreased the total number of lever presses (training condition X virus type X light condition interaction: F(1,22) = 4.66; p < 0.05; ArChT-ES-No light vs ArChT-ES-Light, p < 0.01; ArChT-No ES-No light vs ArChT-No ES-Light, NS; Fig. 4c) and the total number of nicotine injections selectively in the escalation group injected with the archaerhodopsin but not in the no-escalation group (training condition X virus type X light condition interaction: F(1,22) = 4.17; p < 0.05; ArChT-ES-No light vs ArChT-ES-Light, p < 0.05; ArChT-No ES-No light vs ArChT-No ES-Light, NS; Fig. 4d). This effect was particularly pronounced during the first 10 minutes of nicotine self-administration corresponding to the drug-loading behavioral period for both the number of lever presses (F(5,55) = 3.10; p < 0.01; ArChT-ES-No light 10min vs ArChT-ES-Light 10min, p < 0.01; Fig. 4e) and the number of nicotine injections (F(5,55) = 3.65; p < 0.05; ArChT-ES-No light 10min vs ArChT-ES-Light 10min, p < 0.01; Fig. 4f). Similar results were obtained when the vmPFC optogenetic inhibition was performed during the first 10-min of nicotine self-administration (F(5,55) = 2.42; p < 0.05; ES-No light 10min vs ES-Light 10min, p < 0.01 and F(5,55) = 2.42; p < 0.05; ES-No light 10min vs ES-Light 10min, p < 0.01; data not shown). Importantly, this was specific to nicotine escalation as vmPFC optogenetic inhibition had no effect on responding for sucrose (F(1,7) = 2.59; NS; Fig.

Figure 1 .

 1 Figure 1. Nicotine intake escaladed with increasing doses of nicotine. (a) Schematic

Figure 2 .

 2 Figure 2. Nicotine escalation induced convulsive-like behavior. (a) Incidence and (b)

Figure 3 .

 3 Figure 3. Changes in vmPFC neuronal activity during nicotine intake escalation. (a)

Figure 4 .

 4 Figure 4. In vivo optogenetic inhibition of vmPFC decreased nicotine self-

Figure 1 Figure 5

 15 Figure 1

Funding and Disclosure

This work was supported by the French Research Council (CNRS), the Université de Bordeaux, and the French National Agency (ANR-15-CE37-0008-01; K.G.). The authors have nothing to disclose.

Author contributions