I Gifuni

L Lavenant

J Pruvost

A Masse

Recovery of microalgal protein by three-steps membrane filtration: Advancements and feasibility

Keywords:

Microalgae are recognized as source of proteins for nutraceuticals, food additives and bioactive peptides. Also, the exploitation of the protein fraction can contribute to reach the profitability of the microalgal biorefinery. Among the available technologies, membrane filtration is one of the most sustainable and cost-effective technique for the protein recovery and purification. This study proposes a three-step filtration for the recovery, the purification and the concentration of proteins extracted from microalgal biomass. The selected microalgal strain was Chlorella sorokiniana. The proposed membrane process includes: 1) Prefiltration (0.22 μm -ceramic membrane) to remove cell fragments; 2) Diafiltration (0.22 μm -ceramic membrane) to recover the proteins retained in the first step; 3) Concentration (3 kDa -ceramic membrane) to concentrate the proteins and wash out salts and small sugars. The overall filtration process allowed the recovery of 12% of the initial proteins. The resulting proteins are uncoloured which suggests interesting industrial application. The reproducibility of the process was demonstrated by three different replicates. Finally, different strategies, for improving the economic viability of the refinery chain, have been proposed and discussed in the article.

Introduction

Microalgae are widely studied as sources of renewable feedstock for food and energy production [START_REF] Vanthoor-Koopmans | Biorefinery of microalgae for food and fuel[END_REF][START_REF] Enzing | Microalgae-based Products for the Food and Feed Sector: An Outlook for Europe[END_REF]. The biorefinery approach for the valorisation of several microalgal components, remains the most accredited solution to reduce the costs and drive investments. The feasibility to refine the biomass has to be proved from a technical as well as economic point of view. As already seen, the biorefinery should allow the recovery of most of the microalgal components without causing harm to the other fraction [START_REF] Gifuni | Current bottlenecks and challenges of the microalgal biorefinery[END_REF]. Then, mild and low-cost techniques are advisable.

Membrane filtration is one of the techniques widely applied in several industrial fields, from the desalinization and purification of the water to the recovery of high value and fragile compounds [START_REF] Massé | Seawater ultrafiltration: role of particles on organic rejections and permeate fluxes[END_REF][START_REF] Balti | Concentration and purification of Porphyridium cruentum exopolysaccharides by membrane filtration at various cross-flow velocities[END_REF][START_REF] Patel | Separation and fractionation of exopolysaccharides from Porphyridium cruentum[END_REF]. No chemical addition is needed and the operating conditions (temperature, shear stress) are relatively soft. The membrane filtration is already used at large scale and it constitutes a mature technology. Membrane filtration was investigated, for example, for TAG (Triacylglycerol) separation [START_REF] Giorno | Purification of triacylglycerols for biodiesel production from Nannochloropsis microalgae by membrane technology[END_REF][START_REF] Gong | Carotenoids from microalgae: a review of recent developments[END_REF], phycoerythrin and polysaccharides purification, toxins and proteins recovery [START_REF] Ba | Haematococcus pluvialis soluble proteins: extraction, characterization, concentration/fractionation and emulsifying properties[END_REF][START_REF] Safi | Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration[END_REF][START_REF] Zaouk | Filterability of exopolysaccharides solutions from the red microalga Porphyridium cruentum by tangential filtration on a polymeric membrane[END_REF]. The membrane filtration can be considered as one of the best techniques preserving the functional and nutritional properties of the proteins [START_REF] Ursu | Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris[END_REF] and it was also proved to remove or reduce the strong chlorophyll colour and taste [START_REF] Schwenzfeier | Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp[END_REF]. The proteins recovery by membrane filtration generally occurs after the microalgal cells disruption. The latter is also a crucial step which influences the recovery yield and the degradation of the proteins. Safi et al. [START_REF] Safi | Aqueous extraction of proteins from microalgae: effect of different cell disruption methods[END_REF] demonstrates that the best compromise between the quantity of proteins released and the quality of the proteins is ensured by mechanical disruption method, such as bead milling.

The separation of the microalgal components from the whole bead milling lysate was tested by Safi et al. [START_REF] Safi | A twostage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption[END_REF]. They proposed a strategy of two-step membrane filtration for the separation of microalgal components. They used 100 kDa and 10 kDa polyethersulfone membranes for the separation of a model suspension reproducing cells lysate of T. suecica. Some proteins were lost after filtrations and chlorophyll was poorly separated from proteins. Moreover, fouling seemed to be important for all the pressure tested (0.69-2.07 bar). The approach seemed interesting and merit to be pursued in order to reduce the loss of proteins as well as the fouling. Other authors worked on proteins separation from microalgal lysate after centrifugation focusing also on the characterization of the recovered proteins [START_REF] Ursu | Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris[END_REF][START_REF] Schwenzfeier | Effect of charged polysaccharides on the techno-functional properties of fractions obtained from algae soluble protein isolate[END_REF]. These studies demonstrated that the separation process and their operating conditions not only influence the recovery yield of proteins, but also the proteins quality (especially the emulsifying property). In particular, the emulsifying activity was attributed to soluble fraction of the microalgal protein.

Safi et al. [START_REF] Safi | Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration[END_REF] proposed another strategy composed of disruption and centrifugation steps followed by membrane filtration and diafiltration for the recovery of soluble proteins from N. gaditana. Three different membranes cut off, 300, 500 and 1000 kDa were tested to identify the optimal cut off for recovering proteins in the permeate. The best performances for proteins recovery and fouling mitigation were observed for 300 kDa membrane. The fouling of the membrane is often a key issue to be resolved in view of a large-scale production. The fouling can act on the reduction of the flow rates as well as the selectivity/retention of the process and it is largely influenced by the upstream disruption process.

Our previous work [START_REF] Zinkoné | Bead milling disruption kinetics of microalgae: process modeling, optimization and application to biomolecules recovery from Chlorella sorokiniana[END_REF] optimized the bead milling disruption for the recovery of dense and insoluble particles (i.e. starch) in the pellet -after a mild centrifugation-and proteins in the supernatant. This strategy aimed to valorise the entire biomass. The present study focuses on the recovery of the proteins from the supernatant by membrane filtration. The membrane filtration process was designed in three steps: prefiltration (concentration mode) and diafiltration on 0.22 μm membrane then concentration on 3 kDa membrane in order to retain cells debris, increase the transmission of proteins through the membrane, and concentrate the proteins, respectively. The repartition of sugars and chlorophyll during the filtration steps was also analysed.

Material and methods

The experimental work covered the entire production chain form the cultivation of the microalgal strain to the protein purification, the biochemical analysis of the fractions obtained and the analysis of the process performances.

The work comprises: 1) the cultivation of the microalga in a photobioreactor (PBR) and harvesting in continuous mode, 2) the disruption of the microalgal biomass by bead milling, 3) the centrifugation of the disrupted biomass for the separation of big and dense particles in the pellet; 4) Analysis of the supernatant features for the design of the filtration step; 5) three-step membrane filtration for the recovery, purification and concentration of proteins in the supernatant; 6) biochemical characterization of the filtration samples and filtration products.

Microalgal culture

The green microalga Chlorella sorokiniana was cultivated in continuous mode in a 5 L flat panel PBR using modified BBM medium [START_REF] Kandilian | Control of incident irradiance on a batch operated flat-plate photobioreactor[END_REF]. About 2 L of biomass, with a concentration of 1 g L -1 were harvested every 24 h and directly processed in bead milling for cell disruption, without previous concentration.

Cell disruption for proteins extraction

The inlet of the bead milling was the untreated culture at biomass concentration of 1 g L -1 . Disruption was performed in Dyno-mill multi lab from Willy A Bachofen AG (Muttenz, Switzerland). The temperature was maintained at 20 °C, the flow rate was set at 200 mL min -1 and the filling ratio of the grinding media at 80%.The bead milling operated in continuous mode as described by [START_REF] Kandilian | Control of incident irradiance on a batch operated flat-plate photobioreactor[END_REF]. The bead milling conditions have been selected in the previous study [START_REF] Zinkoné | Bead milling disruption kinetics of microalgae: process modeling, optimization and application to biomolecules recovery from Chlorella sorokiniana[END_REF]. In particular, for the microalgal strain used in this study, glass beads of 0.6 mm were selected, and the rotational speed of the milling motor was fixed at 14 m s -1 . Based of the disruption kinetic reported in the previous study, a residence time of 4 min was used to reach 90% of cell disruption.

Centrifugation for preliminary clarification

The disrupted biomass suspension was centrifuged for 5 min at 2500g by Sorvall™ LYNX 6000 superspeed centrifuge, (Thermo Scientific, US), in order to recover most of the starch and insoluble particles in the pellet. The supernatant was collected for 3 days before to reach enough volume to perform filtration experiment (7 L). To preserve the proteins during the storage, the supernatant was heated at 60 °C for 1 min for protease inactivation, then 0.02% m/v of sodium azide was added for inhibition of bacterial growth. Three batch of supernatant were produced in order to carry out the membrane filtration on three biological replicates and demonstrate the reproducibility of the process.

Preliminary analysis of the supernatant: DLS and SDS-PAGE

The supernatant produced by disruption and centrifugation was analysed for particles and protein size to define rationally the choice of the membrane cut off. The dynamic light scattering (DLS) was used to measure the size distribution of the particles suspended in the supernatant. The supernatant sample was measured without treatment and after filtration with 0.8, 0.45 and 0.22 μm cellulose filter. The preliminary filtration, with cellulose filter, allow to reduce the interferences and to have a clearer signal by the instrument. The proteins present in the supernatant were analysed by SDS-PAGE in order to determine the molecular mass of the soluble proteins under denaturing conditions. The SDS_PAGE protocol followed [START_REF] Carfagna | Changes in cysteine and O-acetyl-lserine levels in the microalga Chlorella sorokiniana in response to the S-nutritional status[END_REF]. 20 μL of protein solution were loaded on 10% SDS gel. Protein standard (26614 Thermo Scientific) was used as marker of molecular weight. The gel was stained overnight using Comassi Brilliant blue R250. The de-staining was carried out with distilled water. DLS and SDS-PAGE were also performed on the final products of the filtration: permeate and retentate of the three steps.

Membrane filtration process 2.5.1. Description of the equipment

Membrane filtration was performed on a pilot-scale equipment: Microlab40 pilot plant by VMA Industries (France), already described in [START_REF] Denis | Concentration and Pre-purification With Ultrafiltration of a R-phycoerythrin Solution Extracted From Macro-algae Grateloupia Turuturu: Process Definition and Up-scaling[END_REF]. The filtration plant was equipped by tubular ceramic membranes Inside CeRAM, Cell 60, 7 channels from TAMI Industries (France) characterized by a surface area of 0.032 m 2 . Two different membrane were used: MWCO of 0.22 μm for microfiltration and MWCO of 3 kDa for ultrafiltration. The water permeability of the selected membrane was 140 ± 12 and 16 ± 3 L h -1 m -2 bar -1 for the 0.22 μm and 3 kDa, respectively.

Description of the process

The membrane process for the protein recovery was composed of three steps operated as showed in the Fig. 1: 1) Prefiltration with 0.22 μm membrane. It was aimed to remove the cell fragments and retain them in the retentate while the proteins passed through the membrane. The final volume reduction factor was equal to about 10 (initial volume 7 L). 2) Diafiltration of the retentate of the step 1 with 0.22 μm membrane.

In this step, 6 dia-volumes of demineralized water were added for recovering residual proteins from the retentate. Prefiltration and diafiltration steps on the 0.22 μm membrane were consecutives meaning no washing step was carried out between prefiltration and diafiltration. 3) Concentration of the protein rich permeates produced in the step 1 and 2, with a 3 kDa membrane. Here, the permeates of the previous two steps were mixed and concentrated 10 folds (initial volume of 8 L).

Description of the operating conditions

The filtration was carried out at 20 °C by means of a cooling system connected to the pilot plant. The transmembrane pressure (TMP) was 2-5 bar, which corresponds to the minimal operating pressure for the Microlab40 in microfiltration and ultrafiltration mode. The recirculation flow rate was fixed at 250 L h -1 , that means a crossflow velocity inside the channel lumen equal to 2.5 m s -1 . The duration of each filtration step varied between 2 and 5 h as function of the feed volume. Every 20 min the permeate flux was measured and a permeate sample was collected for the biochemical analysis.

After each trial, the membranes were washed with 5 L of demineralized water at room temperature, 2 L of water at 50 °C and 2 L of NaOH (15 g L -1 , pH = 12) and flushed with demineralized water at 20 °C until the pH of the permeate was equal to the demineralized water pH. Then, the water permeability of the membrane was measured. A new filtration experiment was conducted only if at least 80% of the initial permeability was recovered after cleaning.

Calculations

Permeate flux (J) during the filtration was calculated according to the following equation:

= × J V t A permeate filtration (1)
The volume of permeate (V permeate ) was reported in L; ∆t is the time necessary to collect the permeate volume (h); A filtration is the filtration area (m 2 ).

The permeate flux will be reported as function of the volume reduction factor (VRF). VRF is defined as:

= VRF V V retent te 0 a (2)
where V 0 is the initial volume of the supernatant to be treated (L) and V retentate is the volume of the retentate in the feed tank at the instant of the sampling.

During a concentration step, the mean retention factor (TR) of the proteins was calculated by the following equation:

= C C VRF r TR 0 (3)
Here, C r is the protein concentration in the retentate (g L -1 ), C 0 is the protein concentration in the initial feed solution (g L -1 ), and VRF is the volume reduction factor. It is worth noting that TR can change during the filtration. We reported the TR corresponding to the mean retention rate during the operations.

For the diafiltration step, the relative flux and the number of diavolume are considered. The relative flux (J/J 0 ) is defined as the ratio between permeate flux of the diafiltration and the permeate flux at the end of the prefiltration step.

The number of dia-volume (DV) is the relative volume of demineralized water added (V water ) during the process with respect to the initial volume in the feed tank (V r ):

= DV V V water r ( 4
)
The protein yield or protein recovery rate (Y) was calculated independently for the three filtration steps and for the entire filtration process:

= Y C V C V prefiltration p p 1 1 0 0 = Y C V C V diafiltration p p r r 2 2 1 1 = + + Y C V C V concentration r r 3 3 1 2 1 2 = Y C V C V tot r r 3 3 0 0 (5)
Here, C and V represent protein concentration and the volume respectively (g L -1 ; L). The subscript i; f; p; r refers to the initial and final solution, the permeate and the retentate. The superscript 1; 2; 3 refers respectively to the prefiltration, diafiltration and concentration steps; 1 + 2 for the solution resulting from the mixing of the permeates from the prefiltration and diafiltration steps. The protein yield of the full process is reported as Y tot .

Quantification of proteins, sugars, and chlorophyll

Protein concentration was quantified using BCA kit assay by Thermo Scientific. The quantification of the proteins was performed on the sample before the filtration, for the samples collected during the filtrations and in the final permeate and retentate recovered for the three filtration steps. The sensitivity of the method allows detecting proteins from 0.05 to 1 g L -1 . Samples that were more concentrated were opportunely diluted. The analysis was performed in duplicates for each sample collected.

Sugars and chlorophyll were assayed in the initial supernatant and in the final permeate and retentate of each filtration step to have an indication of the repartition of these molecules during the filtration. Sugars were quantified by Dubois's method. Pigments were assayed according to the protocol reported by Safi et al. [START_REF] Safi | A twostage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption[END_REF]. Chlorophyll and carotenoids concentration were determined using the equations already reported in [START_REF] Pruvost | Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application[END_REF]. The analysis were performed in duplicates for each sample collected.

Statistical analysis

The membrane filtration tests were conducted on three biological replicates. The three different biological replicates were presented on the same plot with the aim to show the reduced variability and the robustness of the process. The biochemical analysis were conducted in duplicates and the standard deviation between the analytical duplicated was calculated using Statgraphics Sigma Express.

Results and discussion

The analysis of the results is focused on the filtration process since the cell disruption, proteins extraction and centrifugation have been deeply discussed in our previous paper [START_REF] Zinkoné | Bead milling disruption kinetics of microalgae: process modeling, optimization and application to biomolecules recovery from Chlorella sorokiniana[END_REF]. The previous work showed that, for a limited energy input in the bead milling followed by mild centrifugation, important amount of metabolites are recovered in the supernatant in the form of micro-particles. In such microparticles even non soluble proteins and pigments are put in suspension in the liquid phase. This phenomenon allows the recovery of 80% of the total proteins in the supernatant and 75% of the initial pigments. Then membrane filtration was applied to purify the proteins recovered in the supernatant.

The choice of the membrane cut off for prefiltration and concentration step (Fig. 1) has been made on the basis of a preliminary DLS and SDS analysis of particles and proteins present in the supernatant.

The mean particle diameter, detected by DLS, was 0.6 μm. The proteins detected by the SDS-PAGE have a size between 55 and 15 kDa, as highlighted by the red square in Fig. 2. A light band around 25 kDa is only detectable in the no diluted sample. Similar results were already found by [START_REF] Ursu | Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris[END_REF][START_REF] Schwenzfeier | Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp[END_REF]. The highlighted bands correspond to subunit of Rubisco (55 and 15 kDa) and other proteins involved in the photosynthesis.

Then, the MWCO selected for the prefiltration step was 0.22 μm: 3 times less than the particles detected by DLS. Such MWCO ensures the retention of the particles and helps the purification of the soluble proteins.

The MWCO selected for the ultrafiltration step was 3 kDa: more than 3 times lower than the smallest detected proteins (15 kDa). This MWCO has been chosen to retain most of the proteins.

Prefiltration

In the Fig. 3, the results of the prefiltration step are reported. In particular, the Fig. 3.A shows the permeate flux as function of the VRF. The permeate flux of the test 2 was not registered. The permeate flux decreases slowly with the VRF and it attains an almost constant value of 70 L m -2 h -1 from VRF of 6.The values of the permeate fluxes here reported are considered in line with the current microfiltration process involving proteins, like milk filtration [START_REF] Al-Akoum | Casein micelles separation from skimmed milk using a VSEP dynamic filtration module[END_REF]. In the Fig. 3.B the concentration of proteins in the retentate is reported. The relationship between protein concentration and the VRF follows a power law, reported in the Eq. ( 3). The exponent represents the mean retention rate of the proteins (TR). It was estimated to be equal to 0.78. The concentration of the proteins recovered in the permeate slightly increases during the prefiltration step. The retention rate varied from the beginning to the end of the filtration from 0.71 to 0.88 respectively (Fig. 3.C). The final recovery yield of this step was 23 ± 5%. The retention of a part of proteins could find explanation in different hypothesis. I) A fouling (cake, adsorption, pore blocking), formed by the components of the supernatant, is established and it limits the transmission of proteins through the membrane. II) The proteins released in the supernatant after the disruption are still linked to small cell debris that cannot pass through the membrane. III) The protocol for the storage (heating at 60 °C for 1 min) or the storage itself (10 days at 4 °C) causes aggregations protein-protein or protein-debris and the final size of the aggregate cannot pass through the 0.22 μm membrane. IV) proteins can be adsorbed on membrane, pipes, valves, etc. of the pilot plant.

Diafiltration

The Fig. 3.D shows the permeate flux during the dialfiltration step. Around seven dia-volumes (DV) were added. The goal of the diafiltration is to continue the transmission of proteins through the 0.22 μm membrane. Indeed, it seems not acceptable to lose too much protein during the concentration and purification chain. The permeate flux was almost constant during the diafliltration, while the protein concentration within the permeate decreases from 0.05 g L -1 at the beginning of the test, to 0.01 g L -1 after 5 DV (results not shown). The quantity of proteins recovered for each diavolume of water added was also calculated (data not showed). After 3 DV, no relevant increase of the Fig. 2. The volume distribution of the particles in the supernatant (filtration feed) on the left. Gel electrophoresis of the proteins in the supernatant, under denaturing conditions, on the right. Red squares on the gel highlight the bands corresponding to the detected proteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) protein's recovery was found. The proteins recovery rate of the diafiltration was equal to 14 ± 3%. These results are comparable with that obtained by Safi et al. [START_REF] Safi | Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration[END_REF] using high-pressure disrupter (16, 10, 4% with 300, 500 and 100 kDa membrane).

Concentration

The Fig. 4 reports the monitored parameters of the concentration step. The Fig. 4.A shows the trend of the permeate flux (J) along the VRF. The flux decreases of about 15% at the VRF of 2, then it remains constant until the end of the process. Low deviation is noted among the three different tests performed. The Fig. 4.B shows the increase of the protein concentration in the retentate (C r ) as the VRF increases. The mean retention factor (TR) is 0.91 then, most of the proteins are retained. Anyway, the proteins yield in the concentrate was 54 ± 3%, while 31% of the proteins are recovered in the permeate. A proteins loss of 15 ± 2% was calculated, probably due to the dead volume of the filtration plant and/or adsorption on the membrane surface. According to the protein size reported in the Fig. 2, almost complete retention of the proteins was expected in the concentration step. The loss of proteins in the permeate fraction could be associated to degradation into small peptides during the storage and the membrane process.

Process performance

The characterization of the permeate and retentate of each filtration step has been carried out in order to assess the process efficiency. In the Table 1, the mass balance and the composition of each fraction are reported. Thus, the amount of sugars that could not be separated from the proteins is an important information, as well as the content of chlorophyll that gives to the protein isolate a green colour. We can notice that the chlorophyll is removed with the prefiltration/ diafiltration steps and uncoloured permeate solution is obtained after the first two steps. This is a promising result, since the green colour is not desirable for all the food-applications. Similar separation was obtained by Safi et al. [START_REF] Safi | A twostage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption[END_REF] by ultrafiltration (100 kDa). In this study the retention of the chlorophyll was obtained by a microfiltration step (0.1 μm) probably because of c the storage protocol. In fact, the heating could cause aggregation of the chlorophyll complex to other molecules and their retention by the membrane. Otherwise, the complex chlorophyll-proteins can be adsorbed on the membrane. However, not only the pigments are retained but also most of the proteins (as already reported in [START_REF] Garcia | Selective and energy efficient extraction of functional proteins from microalgae for food applications[END_REF][START_REF] Garcia | Techno-functional properties of crude extracts from the green microalga Tetraselmis suecica[END_REF]). The retained proteins are reasonably structural proteins, mainly unsoluble. The sugars retained by the 0.22 μm membrane are probably represented by polysaccharides: little starch granules that are not able to be separated in the previous centrifugation, damaged starch granules that lose their granular structure during the disruption step, other polysaccharides produced by the algae or simple sugars (mono-, di-saccharides). The sugars that are recovered in the retentate of the concentration step (on 3 kDa) are probably part of glycoproteins or oligosaccharides, while the sugars of permeate are mainly simple soluble sugars. The final yield of protein recovery after the entire filtration process was 12 ± 3%, which is in line with other studies focused on the recovery of soluble proteins from microalgae [START_REF] Safi | Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration[END_REF][START_REF] Garcia | Selective and energy efficient extraction of functional proteins from microalgae for food applications[END_REF][START_REF] Garcia | Techno-functional properties of crude extracts from the green microalga Tetraselmis suecica[END_REF]. Even if the low yield seems to be strongly influenced by the storage protocol, these results are preliminary and fundamental for the effective integration of the membrane filtration in the microalgal biorefinery. It gives many starting points for further tests and the introduction of a storage protocol is not far from the reality. Indeed, many times the microalgal production platform is not localized near the processing platform. Then logistic problem forces the use of a storage strategy. The results of this study suggest that many factors should be considered when delicate molecules, such as proteins, want to be recovered. Furthermore, in deep analysis of molecules size, moleculesmolecules interaction and target molecules activity during the biomass processing should be performed for the achievement of the biorefinery concept.

Additional studies are required for the optimisation of the membrane material, molecular weight cut off and operating condition of the filtration plant (pressure, velocity). Nevertheless, based on the permeate flux values during each step as well as retention performances, the use of three step membrane filtration remains relevant.

It should be noted that, in this study, special attention was payed to the process repeatability. To the author knowledge, experimental triplicates in this field are not common and the variability raises doubt among scientist and manufacturer about the reproducibility of the microalgal fractionation.

Here, the same filtration experiment was carried out three times using different batches of supernatant (feed) coming from different cell disruption and centrifugation tests. The reproducibility of the flow rate and the proteins recovery yield demonstrates the robustness of membrane filtration technologies and supports its application in industrialization of the microalgal biorefinery. Ceramic membranes showed the ability of recovering their permeability after contact (and/ or adsorption) of organic materials such as microalgal cell debris.

Analysis of the operative costs of the three-steps membrane filtration

The results of this research study refer to a lab-scale equipment characterized by a working volume of 10 L and membrane filtrating area of 0.032 m 2 . Improved performances could be obtained, in larger scale equipment, notably in terms of energy consumption.

A preliminary analysis of the operating costs of the proposed process was carried out in this section and strategies of cost reductions were proposed and simulated.

The preliminary analysis of the costs considered a single batch of 10 L, using 0.032 m 2 and producing 0.5 g of proteins in 6 h (present study). The increase of the production was calculated as fold-increase of the 3 connected parameters: volume-membrane surface-protein production. Then, the increase of the plant volume to 1 m3 implies the use of 3.2 m2 of membranes and the production of 500 g of proteins each 6 h.

The main operating costs (OPEX) involved in the operation of a filtration plant are: labour costs, electricity, water, consumables (reagents for membrane cleaning) and analytics costs (biochemical characterization of the obtained fractions).

Assuming the data obtained from the three-step filtration experiments, the operating time of each step was calculated. Simple excel model was built for obtaining the operating cost of a single batch. The ratio between the operating cost of the batch and the quantity of proteins produced per batch, gives the operating cost of the process per gram of protein produced.

The labour cost was calculated as product of the average grosshourly wage for a technician in Europe (21 €/hour) and the overall process time per batch (including cleaning and preparation of the equipment) [START_REF] Olivieri | A Techno-economic Analysis of Biodiesel Production From Microalgae[END_REF].

The electricity cost was calculated according to the power requirement of each operation unit and the working time for a single batch. An average price of 0.14 € per kWh of electricity was considered.

The water cost was also calculated as the amount of water required for the preparation and the cleaning of the filtration plant, based on the laboratory experiments. The price of 4 €/m 3 of water was considered for the calculations (http://www.publicpolicyarchive.ie).

The consumables were considered as all the reagents for the equipment cleaning and all the disposable materials for sampling and for the protection of the operator.

Analytical costs are the costs associated to the biochemical analysis of each fraction produced. Broadly, the routine quality control and process assessment analysis of a production plant.

The distribution of the different operating cost, for the three-step membrane filtration, is reported in the Fig. 5.A. Clearly, the main operating cost is the labour cost. Common strategy for labour cost

Table 1

Mass balance and composition of the different fraction obtained during the 3 steps membrane filtration. The values are the results of an average of three different experiments (biological replicates, n = 3); the standard deviation between the biological replicates was ≤10%. Note that proteins lost occurs because of degradation processes and/or adsorption on the membrane surface (total proteins loss 32.8%). reduction is automation of the production line, but in this case one single operator is considered and even automatized production requires human control. The most appropriate strategy, in this case, is the increase of the production volume, using the same labour power. The increase of the production volume involves the increase of the production plant capacity: bigger equipment, higher filtration area, higher energy, water, and consumable consumption. However, energy and consumables costs do not increase linearly with the scale-up.

A simulation of the production performances at increasing production volume is presented in the Fig. 5.B. The power requirement for the scale-up of filtration equipment was calculated based on the advices of the filtration plant suppliers. The Fig. 5.B shows that the operating cost per gram of protein produced, suddenly decreases with the increase of the production volume and it attains a reasonably lower price from 1000 L (1 m 3 ) filtration plant. Further reduction of the operating costs could be attained by the elimination of proteins loss during the process (7.6%, as mentioned in the previous paragraph), or by increasing the proteins solubility and then the yield of the overall process.

Accordingly, three scenarios were simulated and compared:

1) Production volume of 100 m 3 and protein recovery yield of 12% (as per lab experiments) 2) Production volume of 100 m 3 and elimination of the protein loss during the filtration process (32.8%, see Table 1) 3) Production volume of 100 m 3 and doubling of the protein recovery yield.

The scenario 2 (reduction of protein loss) could be achieved, at industrial level by using some technical facilities: 3 filtration units working simultaneously on the 3 steps to limit the storage and filtration time; backwash to limit absorption of the proteins in the cake; controlled temperature in the plant for reducing proteins degradation. Indeed, proteins are especially sensible molecules which can easily lose their structure and functionality once extracted from the cells.

The scenario 3 could be achieved by improving the proteins solubilization during the extraction step and optimizing the filtration parameters of the step 1.

The results of the proteins production per batch and the operating cost per gram of protein produced, are presented in the Fig. 5.C. The operating cost could be reduced from 5.3 to 2.6 €/g if the proteins yield could be doubled. The calculated cost per gram of proteins does not claim a comparison with the current protein sources available in the market (whey, soya, pea). Here, the authors simply provide a global analysis of the operating costs for a three-steps membrane filtration and strategies for costs reduction. Moreover, beyond the mass of protein it is important to consider the functionality and activities of proteins. These kinds of proteins could exhibit particular properties, different from the soya, whey, pea proteins.

Conclusions

A membrane filtration chain was suggested for the recovery and the preliminary concentration of the soluble microalgal proteins. The integration of the filtration strategy with the optimized disruption step and mild centrifugation, really contribute to the fulfilment of the microalgal biorefinery. The hydraulic performances of the membrane filtration chain are promising; the fouling seems not such important and the reproducibility of the process was also demonstrated by three different replicates. The final recovery yield of proteins was 12% and the recovered proteins were uncoloured, which is particularly appealing for food supplements application. Improvement of the recovery yield could be attained introducing additional steps, between cell disruption and membrane filtration, in order to increase the solubility of the proteins in the aqueous phase. This field is still in its infancy, but it has great potential. However, reasonable process design could be obtained with an in deep characterization of the structure, the size, and the interaction of the different molecules in the suspension obtained after the disruption. Fig. 5. A) Distribution of the operating cost (OPEX) for the 3 steps membrane filtration process B). Operating cost (€/g of proteins produced) as function of the plant volume (L). C) Proteins production (kg) and relative operating cost (€/g of produced proteins) for 3 different scenarios simulated: 1) production volume of 100 m 3 ; 2) production volume of 100 m 3 and elimination of the protein loss during the process (see Table 1); 3) production volume of 100 m 3 and doubling of proteins yield.

Fig. 1 .

 1 Fig. 1. Schematic representation of the three-step filtration process.

Fig. 3 .Fig. 4 .

 34 Fig.3. Prefiltration results about A) permeate flux (J, L h -1 m -2 ); B) protein concentration in the permeate (Cp, g L -1 ); C) protein concentration in the retentate (Cr, g L -1 ) as function of the volume reduction factor (VRF); D) Diafiltration step, J/J 0 is reported as function of the dia-volumes added (DV). NOTE: In this graphs, Cp and Cr is referred to the protein concentration in the permeate and retentate of the single sample at defined VRF. Blue dotted curves report the mean trend of J, Cp, Cr and J/J0 according to the mathematical model fitting the 3 biological replicates (n = 3: Test 1,2,3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Acknowledgements

This work was performed thanks to the contribution of the Italian "Ministero dell'Istruzione, dell'Università e della Ricerca", which financed the scholarship, and the internal funding of the GEPEA "Laboratoire de Génie des Procédés-Environnement-Agro-alimentaire" which covered the material costs.

Statement of informed consent

No conflicts, informed consent, or human or animal rights are applicable to this study.

CRediT authorship contribution statement

-I. Gifuni: Writing Original Draft -Experimenter -L. Lavenant: Experimenter -J. Pruvost: Supervisor -A. Massé: Experimenter -Supervisor

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Loïc Magrou

Pascal Barone

Nikola T Markov

Gwylan Scheeren

Herbert P Killackey

Pascale Giroud

Michel Berland

Kenneth Knoblauch

Colette Dehay
email: colette.dehay@inserm.fr

Henry Kennedy
email: henry.kennedy@inserm.fr

Unique Features of Subcortical Circuits in a Macaque Model of Congenital Blindness

Keywords: connectivity, deafferentation, hybrid cortex, primate, pulvinar

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

Introduction

There is a major functional reorganization of the cortex during development in the congenitally blind human. Braille reading has been shown to activate the primary visual cortex, area V1, in early blind individuals [START_REF] Sadato | Activation of the primary visual cortex by Braille reading in blind subjects[END_REF][START_REF] Cohen | Functional relevance of cross-modal plasticity in blind humans[END_REF][START_REF] Buchel | Different activation patterns in the visual cortex of late and congenitally blind subjects[END_REF]). In addition, the visual cortex of the congenitally blind has been observed to support higher cognitive functions including language and numerical processing [START_REF] Cohen | Functional relevance of cross-modal plasticity in blind humans[END_REF][START_REF] Roder | Speech processing activates visual cortex in congenitally blind humans[END_REF][START_REF] Amedi | Transcranial magnetic stimulation of the occipital pole interferes with verbal processing in blind subjects[END_REF][START_REF] Bedny | Language processing in the occipital cortex of congenitally blind adults[END_REF][START_REF] Burton | Recognition memory for braille or spoken words: an fMRI study in early blind[END_REF][START_REF] Watkins | Language networks in anophthalmia: maintained hierarchy of processing in 'visual' cortex[END_REF][START_REF] Kanjlia | Absence of visual experience modifies the neural basis of numerical thinking[END_REF][START_REF] Crollen | Recruitment of the occipital cortex by arithmetic processing follows computational bias in the congenitally blind[END_REF]. It has been hypothesized that such functional reorganizations reflect a global metamodal cortical function, where computations are defined by a particular local network, and where the reorganized brain function reflects a conserved cortical structure accommodating the changes in the inputs from the sensory periphery. Hence according to this theory, developmental deafferentation could induce a higher cognitive function in an early area of the visual cortex by the accommodation of possibly innately determined, long-distance inputs [START_REF] Pascual-Leone | The metamodal organization of the brain[END_REF][START_REF] Sur | Development and plasticity of cortical areas and networks[END_REF][START_REF] Merabet | Neural reorganization following sensory loss: the opportunity of change[END_REF][START_REF] Bedny | Evidence from blindness for a cognitively pluripotent cortex[END_REF][START_REF] Amalric | On the role of visual experience in mathematical development: evidence from blind mathematicians[END_REF].

In contrast to the metamodal theory, there is ample evidence from invasive animal experimentation that deafferentation during development leads to widespread changes in the adult brain. Recent work in mouse using genetic deletion has allowed major advances in understanding how modification of input from the sensory periphery shapes the identity of cortical neurons [START_REF] Chou | Geniculocortical input drives genetic distinctions between primary and higher-order visual areas[END_REF]Pouchelon et al. 2014) as well as that of thalamic nuclei [START_REF] Frangeul | A cross-modal genetic framework for the development and plasticity of sensory pathways[END_REF]. Although the plastic changes that underpin cross-modal compensation are not fully understood, it is generally agreed that they engage activity-dependent processes during brain development [START_REF] Bavelier | Cross-modal plasticity: where and how?[END_REF][START_REF] Ruthazer | Insights into activity-dependent map formation from the retinotectal system: a middle-ofthe-brain perspective[END_REF]. Genetic deletion studies in rodent provide a conceptual framework for interpreting the developmental plasticity and cross-modal plasticity [START_REF] Frangeul | A cross-modal genetic framework for the development and plasticity of sensory pathways[END_REF]. Further, studies in human provide strong evidence of a reorganization of the large-scale network in the cortex of the congenitally blind. Whole-brain networks of cortical thickness in sighted and early blind individuals suggest that the congenital blind exhibit an extensive reorganization of anatomical networks defined in this manner [START_REF] Hasson | Congenital blindness is associated with large-scale reorganization of anatomical networks[END_REF]). More recently this issue has been addressed in a macaque model of congenital blindness where anophthalmia is surgically induced in utero by bilateral enucleation [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]. This model of congenital blindness builds on earlier investigations, which explored the impact of sensory input from the retina on the specification of the primary visual cortex and where the absence of the retina during development led to part of the primary visual cortex acquiring profound changes in its cytoarchitecture to form a so-called hybrid cortex combining histological and connectivity features of striate and extrastriate cortex [START_REF] Rakic | Specification of cerebral cortical areas[END_REF][START_REF] Dehay | Maturation and connectivity of the visual cortex in monkey is altered by prenatal removal of retinal input[END_REF]Dehay et al. 1996a;Dehay et al. 1996b). Investigation of the cortical network in this model of congenital blindness showed a 6-fold expansion of the spatial extent of local connectivity in the hybrid cortex (HC) and a surprisingly high location of the HC in a computational model of the cortical hierarchy [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]). In the anophthalmic, the set of areas projecting to the HC, areas V2 and V4 does not differ from that of normal adult controls, but there is a highly significant increase in the relative cumulative weight of the ventral stream areas input to the early visual areas. These findings show that although occupying the territory that would have become primary visual cortex, the HC exhibits features of a higher-order area, thus reflecting a combination of intrinsic and extrinsic factors on the cortical specification [START_REF] Rakic | Specification of cerebral cortical areas[END_REF][START_REF] Dehay | Maturation and connectivity of the visual cortex in monkey is altered by prenatal removal of retinal input[END_REF]. Understanding the interaction of these contributing factors will shed light on the neurobiology of blindness.

Are these changes in the cortical network solely responsible for the functional reorganization observed in congenital blindness? Intrigued by the changes in dimensions of the thalamic nuclei in the anophthalmic monkey [START_REF] Rakic | A novel cytoarchitectonic area induced experimentally within the primate visual cortex[END_REF]Dehay et al. 1996a), in the present study we have examined the subcortical projections to the cortex of anophthalmic monkey. This revealed a massive reduction of the projections from the lateral geniculate nucleus to the HC. This reduction of afferents from the major thalamic relay of the visual system was counterbalanced by a significant increase in projections from the pulvinar complex to the HC. The marked changes from the pulvinar to the cortex contrasted with the observations that projections from other nuclei including the intralaminar thalamic nuclei, the amygdala, and the claustrum were similar across experimental and control cases.

Materials and Methods

We examined the connectivity of the cortex in two 25-day old and one 10-month-old macaque, respectively, that had undergone retinal ablation at E58) and E73 (Table 1). In these three experimental anophthalmic animals, we made six tracer injections (three in the HC, one in area V2, and two in area V4), a fast blue (FB) and a diamidino yellow (DY) injection in each and we compared the results to 10 injections made in 8 adult controls.

Anesthesia and Surgery

The present study is based on observations following bilateral enucleation performed in three monkey fetuses and contrasted to eight normal controls. The enucleated fetuses were carried to term and after birth injected with retrograde tracers (DY and FB) at different postnatal ages (Table 1). Pregnant cynomolgus monkeys (Macaca fascicularis) received atropine (1.25 mg, i.m.), dexamethasone (4 mg, i.m.), isoxsuprine (2.5 mg, i.m.), and chlorpromazine (2 mg/kg, i.m.) surgical premedication. They were prepared for surgery under ketamine hydrochloride (20 mg/kg, i.m) anesthesia. Following intubation, anesthesia was continued with 1-2% halothane in an N20/02 mixture (70/30). The heart rate was monitored, and the expired CO2 maintained between 4.5% and 6%. Body temperature was maintained using a thermostatically controlled heating blanket. Between embryonic day 58 (E58) and E73 and using sterile procedures a midline abdominal incision was made, and uterotomy was performed. The fetal head was exposed, bilateral eye removal performed, and the fetus replaced in the uterus after closing the incisions. The mother was returned to her cage and given an analgesic (visceralgine, 1.25 mg, i.m.) twice daily for 2 days. All fetuses were allowed normal development until term (E165).

Injections of Retrograde Tracers

Identical medication, anesthesia, and monitoring procedures were used as described above. Tracer injections were placed in the HC, area V2 and area V4. Injections were made by means of Hamilton syringes in a stereotypic fashion. Following injections, artificial dura mater was applied, the bone f laps were closed, cemented and the scalp stitched back into position.

All injections in the anophthalmic brain were confined to the cortical gray matter (Supplementary Figure S1 andS2). Sideby-side injections in target areas of retrograde tracers revealed the topology of connectivity in source areas. Such side-by-side injections were made in the HC in the lower part of the medial operculum in case BB181 (Fig. 1), corresponding in normal cortex to area V1subserving parafoveal visual field [START_REF] Gattass | Visual topography of V1 in the Cebus monkey[END_REF]. In case BB122 a single injection was made in V2 near the lip of the lunate sulcus (Fig. 2) where foveal visual field is represented in the normal cortex [START_REF] Gattass | Visual topography of V2 in the macaque[END_REF]. Finally, a pair of very large injections was made on the dorsal part of the prelunate gyrus (Fig. 3), spanning the central and peripheral representation of area V4 in the normal brain [START_REF] Li | Attention and eye movement related activation of neurons in the dorsal pr elunate gyrus (area DP)[END_REF].

The full extent of labeled neurons was charted in subcortical strucutures.

Animal Euthanasia

After 10 to 12 days of recovery that allows optimal retrograde labeling of neurons projecting to the pick-up zone, animals were anesthetised with ketamine (20 mg/kg, i.m.) followed by a lethal dose of Nembutal (60 mg/kg, i.p.) and perfused through the heart with a 1.25% paraformaldehyde and 1.5% glutaraldehyde solution. After fixation, perfusion was continued with a 10-30% sucrose solution to provide cryoprotection of the brain.

Data Acquisition

Depending on the enucleation case, parasagittal (BB181) or horizontal (BB122 and BB169) sections (40-μm thick) were cut on a freezing microtome and at least 1 in 3 sections were stained for Nissl substance. Controls were cut in horizontal and coronal planes. Sections were observed in UV light with oil-immersion objectives using a Leitz fluorescence microscope equipped with a D-filter set (355-425 nm). High precision maps were made using Mercator software running on Exploranova technology, coupled to the microscope stage. Controlled high-frequency sampling gives stable neuron counts despite the curvature of the cortex and heterogeneity of neuron distribution in the projection zones of individual areas [START_REF] Vezoli | Quantitative analysis of connectivity in the visual cortex: extracting function from structure[END_REF]Markov et al. 2014b) Characteristics of neurons labeled with FB or DY are described by Keizer and colleagues [START_REF] Keizer | Diamidino yellow dihydrochloride (DY 2HCl): a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell[END_REF]. Area limits and layer 4 were marked on the charts of labeled neurons. These neurons were then attributed to areas using our atlas based on landmarks and histology and counted according to that parcellation (Markov et al. 2014a).

Statistical Analysis

All statistical analyses were performed in the R statistical environment (R Development Core Team 2016) with additional tools from the MASS, aods3, and betareg packages [START_REF] Venables | Modern applied statistics with S[END_REF][START_REF] Cribari-Neto | Beta regression in R[END_REF][START_REF] Lesnoff | aod: Analysis of Overdispersed Data[END_REF]. Each injection gave rise to retrogradely labeled neurons, which were plotted and compared against those of normal animals, injected at anatomically equivalent locations. As previously defined [START_REF] Markov | Weight consistency specifies regularities of macaque cortical networks[END_REF], the fraction of labeled neurons (FLN) is the proportion of cells located in a given source area with respect to the total number of labeled neurons in the cortex. The connectivity profile is defined by the FLN values for each of the structures labeled from the injected target area.

FLN

The distribution of FLN values has been successfully modeled previously by a negative binomial distribution [START_REF] Markov | Weight consistency specifies regularities of macaque cortical networks[END_REF]Markov et al. 2014a). This can be performed using a generalized linear model (GLM) with a fixed dispersion parameter [START_REF] Mccullagh | Generalized linear models[END_REF]. In brief, the logarithm of raw neuron counts obtained from each area enter the model as a response variable and the logarithm of the total number of labeled neurons across all areas is used as a fixed offset.

In this way, the model coefficients estimate FLN values. The dispersion parameter of the negative binomial serves to capture the overdispersion frequently observed in data based on counts [START_REF] Scannell | On variability in the density of corticocortical and thalamocortical connections[END_REF][START_REF] Markov | Weight consistency specifies regularities of macaque cortical networks[END_REF]. We initially estimated the dispersion parameter for individual areas obtaining values between 2.2 and 7.2 and for subsequent testing used an average value of 4. We then fit this model to compare connection strengths (i.e., FLN values) between normal (i.e., nonenucleated) and enucleated animals. As explanatory variables, we used a 2level factor, Group (Normal/Anophtalmic) and a 2-level factor, Area for the labeled areas projecting on the target injection. The linear predictor in the GLM includes the main effects of both factors and their interaction. Confidence intervals of 95% were computed to assess the significance of the difference based on the model-fitted estimates.

Results

In the present study, we report on the subcortical labeling observed following all the six tracer injections performed in three anopthalmics. The marked features following in utero bilateral enucleation that include changes in cortical area dimensions and cytology and the gross reduction of the lateral geniculate nucleus critically depend on the age of enucleation [START_REF] Dehay | The effects of bilateral enucleation in the primate fetus on the parcellation of visual cortex[END_REF]Dehay et al. 1996a;Dehay et al. 1996b). All three experimental cases showed the extensive changes in cortical cytoarchitecture, gross morphology, and connectivity that were reported elsewhere [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]. Injections of tracer in anophthalmics and in 12 normal controls were aimed at regions housing areas V1, V2, and V4. The increase in the number of controls was to increase statistical power in estimations of the quantitative effects of subcortical reorganization in the absence of the retina.

The nonvisual thalamus was examined at high magnification in all three anophthalmics for back-labeled neurons. Overall the subcortical structures containing labeled neurons were identical in the experimental and control groups.

Injection Sites

We describe and quantify the injection sites in controls and experimental cases in order to validate comparing the extent of labeling in subcortical structures. The injections of tracers were made in the region of the cortex that is located on the operculum and that in the normal animal corresponds to area V1 but following early in utero removal of the retina assumes hybrid features combining traits observed normally in areas V1 and V2 (Fig. 1). Injections in presumptive area V2 were made adjacent and posterior to the lunate sulcus (Fig. 2) and in area V4 on the prelunate gyrus (Fig. 3).

Quantitative Changes in Subcortical Distribution of Labeling

In Figures 4,5, and 6 we compare subcortical labeling in controls and anophthalmics following injections in HC in Figure 4, area V2 in Figure 5 and area V4 in Figure 6. In the illustration of the results, controls have been chosen such that they have the same plane of section as the experimental animals so as to facilitate comparisons of labeling across cases.

One anophthalmic animal received side-by-side injections in the HC and was sectioned in the parasagittal plane. These injections led to extensive labeling in pulvinar (Fig. 4), where the DY-and FB-labeled neurons were considerably intermingled, in contrast to the labeling in the normal control where in section 148 in Figure 4B, the two sets of labeled neurons were tightly grouped but segregated. The extent of the labeled neurons in the anophthalmic was very widespread occupying all of the subdivisions of the pulvinar and hence stretching from the most lateral section containing the pulvinar (section 115 Fig. 4A) to the most medial section (section 143). This strongly contrasted with the control (Fig. 4B), where the lateral-most regions of the pulvinar from sections 134 to 144 were entirely devoid of labeling and where labeling was restricted to sections 146-148. Comparison of the distribution of labeling in the pulvinar of the experimental and control cases shows that injection of HC leads to considerably more widespread distribution of labeled neurons in lateral, medial, and inferior subdivisions compared to that found following injection in area V1 in controls. It would seem that the additional labeling in the experimental case concerned more extensively the lateral subdivision than the medial subdivision.

Following injection in the HC, outside of the pulvinar, labeling in the greatly reduced lateral geniculate nucleus was extremely limited in the anophthalmic and comparatively extensive in the normal. In the experimental case, labeled lateral geniculate neurons were concentrated on sections 119, with occasionally labeled neuron retching as far as section 127. As in the pulvinar, the two populations of labeled neurons were once again largely intermingled. These observations in the lateral geniculate nucleus of the anophthalmic contrasted with the observation of a much stronger labeling in the lateral geniculate nucleus of the control, with consistent labeling stretching from The anophthalmic brain that received an injection of Fast Blue in area V2 was cut in the horizontal plane and showed strong labeling in the pulvinar largely located in the lateral subdivision of the pulvinar and stretching from sections 75 to section 97 (Fig. 5A). In this animal, there was also a DY injection in the HC that largely coincided with the labeling from the area V2 injection, but was more restricted and largely located in sections 85-89. In this animal, very few labeled neurons were observed in the lateral geniculate nucleus in sections 93 to 103. Compared to the anophthalmic, labeling in the pulvinar in the control brain shown in Fig. 5B was much more compact and large numbers of labeled neurons were found in the lateral-interior subdivisions in the characteristic v shape that has been reported elsewhere [START_REF] Ungerleider | Visuotopic organization of projections from striate cortex to inferior and lateral pulvinar in rhesus monkey[END_REF][START_REF] Kennedy | A double-labeling investigation of the afferent connectivity to cortical areas V1 and V2 of the macaque monkey[END_REF]. Comparison of the distribution of labeling in the pulvinar of the experimental and control cases shows that injection of area V2 in the experimental case leads to a more widespread distribution of labeled neurons in primarily the lateral subdivisions compared to that that found following injection in area V2 in controls.

The anophthalmic brain that received injections of tracer in area V4 had relatively widespread labeling in the pulvinar (Fig. 6A). However, the area V4 control shown in Figure 6B also showed relatively widespread pulvinar labeling with cells from both injections showing intermingling and it was difficult to define a distinct pattern in the anophthalmic with respect to the control. Comparison of the distribution of labeling in the pulvinar of the experimental and control cases shows that injection of area V4 in the experimental case leads to a more widespread distribution of labeled neurons in primarily the lateral subdivisions compared to that found following injection in area V4 in controls.

A Selective Increase in Pulvinar Originating Projections

The distributions of labeled neurons reported above suggest that there might be significant changes in the weight of subcortical projections of subcortical inputs to the cortex following the removal of the retina. To address this issue, we calculated the FLN for the individual projections (Fig. 7). Side-by-side injections in the HC (BB181) were close enough (less than 1 mm apart) to be considered a single cumulated injection for the following quantitative analysis (individual values are available in Table 2). This showed a significant increase in the FLN of the pulvinar projection to HC compared to the pulvinar projection to area V1 in the normal control. There were no significant differences in the FLN values of the pulvinar projections to either V2 or V4. The projection from the lateral geniculate nucleus was also influenced by the removal of the retina; in the enucleate, compared to the normal control, there was a decrease in the projection of the lateral geniculate projection to area V1 and area V2. Are these changes in the visual thalamus echoed by other changes in subcortical structures projecting to the early visual areas? To address this, we quantified the FLN in claustrum, amygdala, and the intralaminar thalamic nuclei and found no significant differences between control and experimental cases (Fig. 7).

To assess the effect of enucleation on labeling in the pulvinar, illustrated in Figures 4 and8, we estimated the quantitative spread of labeling in that structure. In each section with pulvinar labeling, the smallest polygon encompassing all marked cells was drawn and its area was computed. The volume of labeling for each case was then interpolated from the series of mea-sured surfaces. To account for the effect of injection size on the labeling spread, the volume of the uptake zone for each case was also measured and used as a covariate in statistical tests. A linear model (LM) was used to test for differences in pulvinar of the volume of labeling spread between normal controls and anophthalmic animals. The cube root of labeling spread volume was taken as the response variable, following a Box-Cox test to assess the best transformation for normality of the data [START_REF] Box | An analysis of transformations (with discussion)[END_REF][START_REF] Venables | Modern applied statistics with S[END_REF]. For V1/HC, the difference in spread between normal and anophthalmic groups was significant (P < 0.001, t = 6.4, degrees of freedom (df) = 9), but not for V2 (P = 0.12, t = 2.62, df = 2) and V4 (P = 0.9, t = -0.09, df = 3). These results indicate an effect of anophthalmia on the spread of labeling in the pulvinar for V1/HC but not for V2 and V4, despite the suggestion in Figure 5 of a differential spread for V2. The enucleated V2 injection, however, is based on a single case and so any test of its difference from the normal is likely to be of low statistical power.

Our results show that in the absence of the retina there is a significant increase in the strength of the projection of the pulvinar to the HC, which is accompanied by a significant increase in the spatial extent of labeling in the pulvinar. The injections in experimental and controls were carried out in a stereotypical fashion. Variations of the uptake zone are not predicted to impact on the FLN values, which are normalized with respect to the total number of labeled neurons. However, a variation of the size of the injection and the resulting pickup zone could impact on the spread of labeled neurons that we observe in the pulvinar. To answer this potential concern, we included the size of uptake zones in our analysis of the spread of labeling in the pulvinar as a covariate. Results shown in Figure 8A and respective tests (see P values above) take into account the effect of injection size. Moreover, an analysis of variance could not reject the simple model that size of the uptake zone had no influence on the results. Finally, we compared quantification of the uptake zones, which showed that uptake zones of all injections did not show significant differences.

Discussion

Subcortical labeling in the anophthalmic brains was quantitatively but not qualitatively different from subcortical labeling in the controls. Although the absence of the retina did not lead to the appearance of novel thalamocortical pathways, it did profoundly modify the strength of the projection from the pulvinar complex, which showed significantly increased numbers of labeled neurons with a spatially expanded distribution. This increase in the pulvinar projection to the HC was accompanied by a reduction in the projection of the geniculocortical pathway. The expansion of the pulvinar projection to the HC appeared to be the unique expansion of ascending pathways as quantification of the projections of the claustrum, amygdala, and interlaminar nucleus revealed no differences between experimental and control groups.

Primate and Nonprimate Models of Congenital Blindness

There have been a number of investigations in congenitally blind rodents that show many similarities with the present findings.

Tracers injected to the visual cortex of congenitally anophthalmic mice found point-to-point connectivity in the dorsal lateral geniculate nucleus, and while retrogradely labeled neurons were not reported in nonvisual thalamus there appeared to be an increase in the incidence of labeled neurons in the lateral posterior thalamic nuclei [START_REF] Godement | Thalamic afferents to the visual cortex in congenitally anophthalmic mice[END_REF][START_REF] Kaiserman-Abramof | The thalamic projection to cortical area 17 in a congenitally anophtalmic mouse strain[END_REF]. Given that the lateral posterior thalamic nucleus is thought to be evolutionarily related to the pulvinar these results in mutant eyeless mice agree with the present findings. Similar findings have been observed in blind mole rats, animals which have adapted to a nonvisual lifestyle [START_REF] Cooper | Visual system of a naturally microphthalmic mammal: the blind mole rat, Spalax-Ehrenbergi[END_REF].

The present findings contrast with those obtained in a recent study on the effects of bilateral enucleation in Mondelphis domestica, a South American marsupial [START_REF] Kahn | Massive cross-modal cortical plasticity and the emergence of a new cortical area in devel-opmentally blind mammals[END_REF][START_REF] Karlen | Early blindness results in abnormal corticocortical and thalamocortical connections[END_REF]). Enucleation at postnatal day 4 led followed by tracers injected in area 17 led to retrograde labeling of neurons in subcortical nuclei associated with somatosensory (ventral posterior nuclei), auditory (medial geniculate nuclei), motor (ventrolateral nucleus) systems and limbic/hippocampal systems (anterior dorsal and anterior ventral nuclei [START_REF] Karlen | Effects of bilateral enucleation on the size of visual and nonvisual areas of the brain[END_REF]. These findings are very different from what is observed in the anophthalmic monkey where subcortical afferents were restricted to the visual thalamus. A recent study in mouse where geniculocortical pathways were genetically deleted in utero likewise failed to find projections to the visual cortex from nonvisual thalamus [START_REF] Chou | Geniculocortical input drives genetic distinctions between primary and higher-order visual areas[END_REF] The widespread thalamic projections from nonvisual thalamus to the visual cortex in the anophthalmic marsupial brain [START_REF] Karlen | Effects of bilateral enucleation on the size of visual and nonvisual areas of the brain[END_REF], not found in the present study, could be due to the differences in developmental timing with respect to retinal ablation. There are two arguments against this hypothesis. Firstly, while the enucleation at 4th postnatal day in the opossum is at an earlier developmental stage than the enucleation in the macaque at embryonic (E) day 70 [START_REF] Workman | Modeling transformations of neurodevelopmental sequences across mammalian species[END_REF], in both species thalamic innervation of the visual cortex is incomplete and generation of supragranular layers has not yet commenced [START_REF] Rakic | Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition[END_REF][START_REF] Robinson | The visual pathways of eutherian mammals and marsupials develop according to a common timetable[END_REF][START_REF] Finlay | Linked regularities in the development and evolution of mammalian brains[END_REF][START_REF] Molnar | Development of thalamocortical projections in the South American gray short-tailed opossum (Monodelphis domestica)[END_REF]. Secondly, in both species the ages when the retinae were removed lead to profound and very similar modifications of the cortex, with the generation of a novel cortical region referred to as area X in the marsupial study and HC in the present and earlier macaque studies [START_REF] Rakic | Specification of cerebral cortical areas[END_REF][START_REF] Dehay | Maturation and connectivity of the visual cortex in monkey is altered by prenatal removal of retinal input[END_REF][START_REF] Kahn | Massive cross-modal cortical plasticity and the emergence of a new cortical area in devel-opmentally blind mammals[END_REF].

The discrepancy between widespread thalamic projections in the anophthalmic opossum and restricted thalamic projections in the anophthalmic macaque (present study) is difficult to explain. It echoes similar differences found in the cortex where nonvisual sensory areas were found to project to the visual cortex in the congenitally blind opossum [START_REF] Karlen | Early blindness results in abnormal corticocortical and thalamocortical connections[END_REF] but not in the congenitally blind macaque [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]). Elsewhere we have argued that at the level of the cortex these differences could be due to the stabilization of developmental transient projections that have been observed in the opossum [START_REF] Karlen | Early blindness results in abnormal corticocortical and thalamocortical connections[END_REF], and which do not exist in the developing macaque [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]. However, such transient widespread projections from different sensory thalamic nuclei have not been reported in any species and therefore cannot be easily evoked to explain the apparent opossum-macaque differences.

Developmental Specification of the Visual Pathways

Developmental specification of visual pathways depends on an interplay between genetic and extrinsic mechanisms. Previously, we reported that subsequent to early removal of the retina there were profound modifications of the dimensions, cytology, and connectivity of visual cortical areas cortical areas [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]. The areal specification is based on morphogens and secreted signaling molecules and extrinsic inputs relayed to the cortex by thalamocortical axons [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] De La Rossa | In vivo reprogramming of circuit connectivity in postmitotic neocortical neurons[END_REF][START_REF] Geschwind | Cortical evolution: judge the brain by its cover[END_REF][START_REF] Borello | The logistics of afferent cortical specification in mice and men[END_REF]. The role of thalamic axons in arealisation is a multistep hierarchical process involving events at progenitor and neuronal levels [START_REF] Dehay | Cell-cycle control and cortical development[END_REF][START_REF] Chou | Geniculocortical input drives genetic distinctions between primary and higher-order visual areas[END_REF]Pouchelon and Jabaudon 2014;[START_REF] Moreno-Juan | Prenatal thalamic waves regulate cortical area size prior to sensory processing[END_REF].

The present study finds a considerable modification of the subcortical input to the cortex in an experimental model of blindness resulting from an absence of the retina during in utero development. We examined the input to the cortex in addition to that from the pulvinar from a number of subcortical structures including the amygdala, the claustrum and interlaminar nuclei of the thalamus. We only found a modification of the input to the cortex from the pulvinar. The restriction of change to the projection of the pulvinar could be related to recent findings concerning the timetable of pulvinar innervation of the cortex during development [START_REF] Bourne | Hierarchical development of the primate visual cortex, as revealed by neurofilament immunoreactivity: early maturation of the middle temporal area (MT)[END_REF][START_REF] Warner | Preservation of vision by the pulvinar following early-life primary visual cortex lesions[END_REF][START_REF] Bourne | Plasticity of visual pathways and function in the developing brain: is the pulvinar a crucial player?[END_REF].

In rodent, genetic depletion of the thalamic input to the primary visual area failed to influence the specificity of thalamic projections to the cortex [START_REF] Chou | Geniculocortical input drives genetic distinctions between primary and higher-order visual areas[END_REF]). These findings support observations in eyeless mice [START_REF] Godement | Thalamic afferents to the visual cortex in congenitally anophthalmic mice[END_REF] suggesting that matching of thalamic inputs to different sensory areas is relatively genetically constrained. There is now considerable evidence that ascending pathways to the cortex in rodents specifies cortical areas. During development genetic deletion of geniculocortical axons suppressed the differentiation of gene expression that distinguishes between area V1 and high-order areas [START_REF] Chou | Geniculocortical input drives genetic distinctions between primary and higher-order visual areas[END_REF]. More specifically in rodent, thalamocortical axons have been shown to instruct the identity of postsynaptic identity of layer-4 neurons in a modalityspecific fashion (Pouchelon et al. 2014;Pouchelon and Jabaudon 2014). These ascending influences have also been shown to impact on descending corticothalamic pathways. During normal development, hierarchical wiring ensures that the cortical layers 5 and 6 differentially innervate first-order thalamic nuclei such as the lateral geniculate and higher-order nuclei such as the pulvinar. Loss of the sensory periphery in rodent leads to crosshierarchical corticothalamic rewiring [START_REF] Grant | The regulation of corticofugal fiber targeting by retinal inputs[END_REF]. In an elegant series of experiments, the team of Denis Jabaudon showed that such cross-hierarchical rewiring could provide a cross-modal genetic framework underlying the development and plasticity of sensory pathways [START_REF] Frangeul | A cross-modal genetic framework for the development and plasticity of sensory pathways[END_REF]). This work suggests that higher-order thalamic nuclei identity might be a ground state feature and that first-order nuclei identity reflects a periphery input. Because communication across the cortex is ensured by direct cortico-cortical pathways, as well as by corticothalamic-cortical loops, where cortical area interaction is ensured by loops via the higher-order thalamic nuclei [START_REF] Guillery | Thalamic relay functions and their role in corticocortical communication: generalizations from the visual system[END_REF][START_REF] Sherman | Thalamus plays a central role in ongoing cortical functioning[END_REF], cross-hierarchical rewiring could reflect an adaptive feature underlying evolutionary processes and cross-modal plasticity. With respect to the present findings, the question that now would need to be addressed is whether the modifications of the pulvinar projection in our experimental animals represent a consequence of a top-down influence.

If the modification of the pulvinar projections to the cortex that we report here are the consequences of top-down processes, why have they not been reported in the numerous lesion experiments that have been carried out in rodents? Elsewhere [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF], we speculate that differences in primate and rodent on the role of thalamic afferents to the cortex could reflect the early and prolonged thalamic innervation of the germinal zones in the primate compared to the rodent [START_REF] Dehay | Cell-cycle control and cortical development[END_REF][START_REF] Dehay | The outer subventricular zone and primate-specific cortical Complexification[END_REF].

Relevance of Developmental Effects of Retinal Ablation in Macaque on Congenital Blindness in Human

Following injection in the HC, the extension of labeled neurons into all subdivisions of the pulvinar in the present results suggests that the projection of the pulvinar to HC includes regions of the pulvinar that normally project beyond areas V1 and V2. The fact that the spread appears more extensive into lateral compared to medial subdivisions suggests that the pulvinar expansion in the absence of the retina concerns more territory that normally to extrastriate cortical regions than that to frontal regions. Following injection in area V2, the extension of labeling into the lateral subdivision of the pulvinar suggests that territory projecting to the extrastriate cortex was strongly implicated in the expansion of the pulvinar projection to area V2. Nevertheless, the area V2 in the experimental animal showed labeled neurons in the medial pulvinar suggesting that some neurons in the expanded projection concerned territories that would be involved in projections to frontal regions in the normal brain. Following injections in area V4, while there was a suggestion of a possibly greater spread of labeling in the anophthalmic, the location seemed to be grossly comparable in control and experimental brains.

In the anophthalmic macaque the topography of corticocortical connectivity and global organization of the ventral and dorsal streams are largely conserved [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF], which is also thought to be the case in human congenitally blind [START_REF] Pietrini | Beyond sensory images: objectbased representation in the human ventral pathway[END_REF][START_REF] Ptito | Recruitment of the middle temporal area by tactile motion in congenital blindness[END_REF][START_REF] Matteau | Beyond visual, aural and haptic movement perception: hMT+ is activated by electrotactile motion stimulation of the tongue in sighted and in congenitally blind individuals[END_REF]Striem-Amit et al. 2012a;Striem-Amit et al. 2012b;[START_REF] Striem-Amit | Functional connectivity of visual cortex in the blind follows retinotopic organization principles[END_REF][START_REF] Van Den Hurk | Development of visual category selectivity in ventral visual cortex does not require visual experience[END_REF]. These findings support an early developmental specification of the functional streams [START_REF] Deen | Organization of high-level visual cortex in human infants[END_REF][START_REF] Livingstone | Development of the macaque face-patch system[END_REF]. Further, in the anophtalmic brain, we observe en expansion of the ventral pathway that could reflect cross-modal plasticity [START_REF] Ptito | Recruitment of the middle temporal area by tactile motion in congenital blindness[END_REF]). However, in addition to the expansion of the ventral stream, there are important differences in the organization of the cortex in the congenitally blind macaque model, notably the extensive intrinsic connectivity of HC coupled to its high location in the cortical hierarchy. The relatively high position in the cortical hierarchy and the conservation of an extensive local connectivity in the HC could ensure the long-time constants, which would be required for the observed higher cognitive functions of the deafferentated cortex of the blind [START_REF] Honey | Slow cortical dynamics and the accumulation of information over long timescales[END_REF][START_REF] Chaudhuri | A large-scale circuit mechanism for hierarchical dynamical processing in the primate cortex[END_REF][START_REF] Tomasello | Visual cortex recruitment during language processing in blind individuals is explained by Hebbian learning[END_REF].

The present results show that in addition to the extensive reorganization of the cortex there are important changes in the projections to HC, namely an important increase in the weight of the projection of the pulvinar and a spatial expansion of cortical projecting neurons in this structure. Although the increase in weight is coherent with findings in eyeless mice, the greatly expanded spatial distribution is more unexpected [START_REF] Godement | Thalamic afferents to the visual cortex in congenitally anophthalmic mice[END_REF]. These findings could have a profound impact on cortical processing theories given a current understanding of the role of information processing by the pulvino-cortical interactions [START_REF] Arcaro | Organizing principles of pulvino-cortical functional coupling in humans[END_REF].

The understanding of the dynamic role of the pulvinar in orchestrating communication between cortical areas has come to the fore in the last 30 years [START_REF] Sherman | My prolonged collaboration with Ray Guillery[END_REF]. There is considerable anatomical evidence that areas that share direct cortical connections are also interconnected via their anatomical connections through the pulvinar [START_REF] Shipp | The functional logic of cortico-pulvinar connections[END_REF]. These cortical pulvinar loops are thought to play a critical role in active visual processing via gating information outflow [START_REF] Purushothaman | Gating and control of primary visual cortex by pulvinar[END_REF]) and synchronizing interconnected areas, thereby influencing the allocation of attention [START_REF] Saalmann | The pulvinar regulates information transmission between cortical areas based on attention demands[END_REF]. The role of the pulvinar in regulating communication between areas is intrinsically tied to the rich interconnectivity with the cortex. The present findings reveal an important expansion of the neurons projecting to HC compared to the projection to the normal area V1 (Figs 4 and8). Because cortical pulvinar loops directly depend on the overlapping of the afferent and efferent connections in the pulvinar, the much-enlarged distribution of pulvinar neurons projecting to the HC is expected to allow communication through a more widespread cortical network in the anophthalmic compared to the controls. Recent work in the Kastner lab suggests that dorsal pulvinar is functionally linked to frontal, parietal and cingulate areas and ventral pulvinar to occipital and temporal regions [START_REF] Arcaro | Organizing principles of pulvino-cortical functional coupling in humans[END_REF]. Figure 4 shows that whereas in the control there are no labeled neurons in dorsal pulvinar, in the anophthalmic there are numerous labeled neurons in this region. Arcaro et al showed that the cortical coupling in the dorsal pulvinar is relatively insensitive to low-level stimuli and is rather involved in higher-order information integration over extended time windows. These findings suggest that the HC in the congenitally blind could have an extensive interaction with frontoparietal networks with a broad range of time scales. This would introduce the possibility for information processing which would be eminently suited to its extensive intrinsic connectivity suggesting it could allow neuronal responses over seconds, which we have speculated would be required for the higher cognitive functions of the deafferented cortex of the blind [START_REF] Honey | Slow cortical dynamics and the accumulation of information over long timescales[END_REF][START_REF] Chaudhuri | A large-scale circuit mechanism for hierarchical dynamical processing in the primate cortex[END_REF][START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF].

Conclusion

The extensive functional reorganization found in the congenitally blind human cortex has led to the expectation that there will be widespread structural changes with the novel, ectopic pathways from different sensory modalities. The macaque model of congenital blindness reported here and elsewhere [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF] shows that aside from projections from the subiculum and entorhinal cortex there are no ectopic cortical or subcortical connections to the deafferented cortex. However, given that the interareal network is very dense [START_REF] Markov | Cortical high-density counterstream architectures[END_REF], we have taken the precaution of measuring the strength of connections and this shows major changes both in the cortex and in the subcortical projections. The major increase observed in the pulvinar input to the deafferented cortex, along with the modifications that we find in the cortical hierarchy and intrinsic connectivity of HC, could have important functional consequences in the blind brain. The role of corticalsubcortical cortical loops involving higher-order thalamic nuclei in communication across the cortex is now well established. The increased size and spatial spread of the pulvinar projection to hybrid cortex will facilitate communication between far-distant cortical areas, perhaps subserving specific and unique sensorymotor associations in the blind macaque. These long-distance interactions may concern the dorsal and ventral stream, the latter enjoying an increased connectivity with the HC [START_REF] Magrou | How areal specification shapes the local and interareal circuits in a macaque model of congenital blindness[END_REF]).

Figure 1 .

 1 Figure 1. Anatomical drawings showing the injection sites in area V1 in the normal brain and in HC in the anophthalmic brain. ( A) Tracer injection sites in HC in anophthalmic brain (case BB181LH). Upper-row left cartoons showing the location of sections containing injection sites; right low-power view of sections B and E. Bottom-row numbered sections spanning the injection sites. (B) Tracer injection sites in area V1 in a control (case M071RH). Convention as in (A). Yellow, DY pick-up zone; blue, FB pick-up zone.

Figure 2 .

 2 Figure 2. Anatomical drawings showing the injection sites in area V2. (A) Tracer injection sites in area V2 in the anophthalmic brain (case BB122LH). (B) Tracer injection sites in area V2 in a control (case M081RH). Conventions as in Fig. 1.

Figure 3 .

 3 Figure 3. Anatomical drawings showing the injection sites in area V4. (A) Tracer injection sites in area V4 in the anophthalmic brain (case BB169LH). (B) Tracer injection sites in area V4 in the normal control (case BB119LH). Conventions as in Figure 1.

Figure 4 .

 4 Figure 4. Anatomical drawings showing distributions of labeled cells in subcortical structures following injections in HC/V1. (A) anophthalmic case BB181LH, side-byside injections in the HC. (B) Corresponding normal control M071RH with a comparable injection site in V1. Blue dots, FB; red dots, DY; green dots, doubly labeled cells.For abbreviations of area names see Glossary.

Figure 5 .

 5 Figure 5. Anatomical drawings showing distributions of labeled cells in subcortical structures following an injection in V2. ( A) anophthalmic case BB122LH, single injection in V2. (B) Corresponding normal control M081RH with comparable injection site in V2. See Figure 1 for color coding, for abbreviations of area names see Glossary.

Figure 6 .

 6 Figure 6. Anatomical drawings showing distributions of labeled cells in subcortical structures following an injection in V2. (A) anophthalmic case BB169LH, side-by-side injections in V4. (B) Corresponding normal control BB119LH with comparable side-by-side injection sites in V4. See Figure 1 for color coding, for abbreviations of area names see Glossary.

Figure 7 .

 7 Figure 7. Effect of enucleation on subcortical FLN. Log scale dot plot of FLN. Enucleates, black circles; normal controls, black dots; upper-panel, injections in normal striate cortex (V1) and HC (1anophtalmic, six normals); middle-panel, injections in area V2 (1anophtalmic, three normals); bottom panel, injections in area V4 (two enucleates, three normal). For abbreviations of area names see Glossary.

Figure 8 .

 8 Figure 8. Effect of enucleation on the spread of labeling in the pulvinar. (A) Dot plot of the volume of labeling spread in the Pulvinar. (B) Dot plot of the volume of the matching injection sites. Enucleates, black circles; normal controls, black dots. Injections in V1/HC: three enucleate, nine controls; in area V2: 1anophtalmic, four normals; in area V4: two anophthalmics, four normals. " * * * " indicates significance with a P-value of 0.000126 (see Methods). The case of anophthalmic animal BB122LH is indicated as an outlier for visual reference. For abbreviations of area names, see Glossary.

Table 1

 1

	Experimental cases
	days
	days
	days
	days

Table 2

 2 Neuron counts for relevant subcortical structures

	HC

© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Funding

LABEX CORTEX (ANR-11-LABX-0042 to H.K.), Université de Lyon (ANR-11-IDEX-0007 to H.K.), CORE-NETS (ANR-11-BSV4-501 to H.K.), ARCHI-CORE (ANR-14-CE13-0033 to H.K.), CORNET (ANR-15-CE32-0016 to H.K.), PRIMACOR (ANR-14-CE13-0036; all operated by the French National Research Agency [ANR]), and FRM Equipe (DEQ20160334943; operated by the Foundation for Medical Research).

Author's Contribution CD, HK proposed the project; PG, MB, HK surgical intervention; PB, PG, CD histological processing; LM, PB, NTM, GS, HPK, PG, CD data acquisition; LM, PB, NMT, GS, KK, HK data analysis; KK computational modeling; LM, CD, HK wrote the paper. All authors contributed to editing the final document.