Abdoul Karim Boubacar

Mohamed Gafsi

Nicole Sibelet

Toudou Adam

Laurent Gazull

Pierre Montagne

Amah Akodewou

Régis Peltier
email: régispeltier@cirad.fr

Abdoul Karim Boubacar

Economic importance of fuelwod in family resources is not a sufficient trigger factor for farmers to restore their parklands in south-western Niger

Keywords: Fuelwood, Biodiversity, Parklands, Sahel, Faidherbia albida, Farmer Managed Natural Regeneration

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

Introduction

Over the past forty years, the Sahel has suffered several droughts, notably in 1973-1974and 1983-1984. In Niger, this phenomenon, coupled with an increase in the population from 5,102,990 inhabitants in 19775,102,990 inhabitants in to 17,129,076 in 20125,102,990 inhabitants in (RGPH 2012) ) has led to a serious deterioration in forest cover (Data for the 5th census in 2022 are not yet available). It was estimated that the surface area of forests in Niger will have shrunk from 1.94 Mha in 1990 to 1.08 Mha in 2020, a 44% decrease in 30 years (FAO 2020). Many rural communities are facing shortages of fuelwood and service wood, as well as declining soil fertility [START_REF] Pieri | Fertilité des terres de savanes[END_REF]. This depletion has forced farmers to modify their farming practices by drastically reducing fallow periods, overexploiting wood resources and accelerating the clearing of land for agriculture to meet the increasing demand generated by the growing population.

According greater attention to Sahelian Agroforestry Systems (AFS), and in particular parklands (Pelissier 1980), to sustainably meet the needs of rural communities has thus become a pressing challenge [START_REF] Peltier | Quelles évolutions pour la consommation d'énergie domestique et la gestion des ressources naturelles dans les bassins d'approvisionnement des grandes villes du Sud ?[END_REF]. Several development projects and actions by nongovernmental organizations (NGOs) have been undertaken to restore parkland using assisted natural regeneration (ANR) practices [START_REF] Montagne | Protection de la régénération naturelle de Faidherbia albida. Evaluation a posteriori du projet Gao Dosso au Niger[END_REF][START_REF] Larwanou | Pratiques sylvicoles et culturales dans les parcs agroforestiers suivant un gradient pluviométrique nord-sud dans la région de Maradi au Niger[END_REF][START_REF] Pasiecznik | Restoring African Drylands[END_REF].

Parklands in dry Africa, especially those with Faidherbia albida, are well-known AFS [START_REF] Peltier | Les Parcs à Faidherbia[END_REF]. These AFS are part of a pastoral system, as livestock graze under the parklands in the dry season (October to May, when the Faidherbia trees bear their leaves and fruits), drop crop residues, graze on branches pruned by pastoralists and on the pods of this legume and make their droppings. Although animal droppings are simply left on the ground, the fact that animals congregate in the shade of trees during the hot hours leads to trampling by the herd, which allows a significant portion of the droppings to be incorporated into the soil, where their nitrogen is released. These AFS are also part of rainfed cropping systems. Rainfed crops (sorghum, millet, cotton, etc.) are grown during the rainy season (June to September, when the Faidherbia trees lose their leaves), reducing their shade. The crops benefit from fertilization by livestock dung and nitrogen enrichment by the fall of leaves and twigs. Since planting trees in cropland and pasture land is very difficult (young trees need to be protected, sometimes irrigated etc.) this is why farmers favour natural regeneration by ANR, without protection or irrigation, simply "hold the axe" at the time of clearing so as not to cut down the young tree or its shoots, and then prune it in the following years, to favour its growth in height. This technique was rebaptized under the name, Farmer Managed Natural Regeneration (FMNR), in particular by [START_REF] Haglund | Dry land tree management for improved household livelihoods: Farmer managed natural regeneration in Niger[END_REF]. These efforts have made it possible to preserve some wood resources, and even to ensure the regreening of certain overexploited areas [START_REF] Baggnian | Impact des modes de gestion de la Régénération Naturelle Assistée des ligneux (RNA) sur la résilience des écosystèmes dans le Centre-Sud du Niger[END_REF].

Until recently, assessments of global forest resources were mostly limited to forests sensu stricto (uncultivated and with dense canopy cover), without considering trees and bushes on agricultural land. In a 2014 study, the FAO roughly projected that "land with tree cover" (including cultivated) in Niger would represent 8 Mha in 2015, compared to 4.15 Mha in 1990, meaning an increase of 48% of the surface area (no information on tree density was noted). In 2013, Herrmann and Tappan show the vegetation impoverishment despite greening in Senegal. In 2020, Brandt et al. have mapped the crown of trees more than 3m 2 in size, in dry West-Africa, including Sahel, by satellite imagery.

Few studies indicate the contribution of agroforestry trees to the rural economy, particularly in Niger. However, master plans for the supply of domestic fuel in the towns of Maradi, Zinder and Niamey (SDACDMZ 2015; SDACDN 2017) have shown that parklands account for a growing proportion of the production of fuelwood marketed in Niger [START_REF] Montagne | Evolutions de l'offre d'énergie domestique à Niamey, à Ouagadougou et à Bamako[END_REF].

The production of fuelwood, for sale or self-consumption, is among the strategies developed by rural producers.

The trees and wooded areas that villagers can access in their territory create a standing value that can take different forms: land reserves for exploitation and cultivation, timber and poles that can be sold according to a flexible calendar, an energy resource that can be sold continuously, a forage resource that can be rented out or exchanged for services [START_REF] Griffon | En quoi l'agroforesterie peut-elle contribuer à la révolution doublement verte ? Bois et Forêts des[END_REF][START_REF] Petheram | The wealth of the dry forests: can sound forest management contribute to the millennium development goals in Sub-Saharan Africa? CIFOR Livelihood Brief[END_REF].

The present research, carried out in 3 villages in 2015-2019, aims to: i) assess the importance of fuelwood (particularly that harvested in parklands) in farmers' incomes; ii) measure the state of tree resources in parklands; and iii) determine farmers' strategies and practices regarding the trees in their fields. These data are then compared and discussed to verify the following hypothesis: "The importance of fuelwood in farmers' incomes has led to a dynamic of restoration and management of tree resources in their parklands". The results should make it possible to guide the actions of a future fuelwood management project in the Niamey supply basin and to provide elements to support a modification of the law regulating the management of agroforestry trees in Niger.

Only one research activity on AFS was carried out in the area under consideration during the period 2015-2019 and, at the request of the Ministry of Environment, it focused on the description of woody stands and their production of wood energy [START_REF] Boubacar | Ressources arborées des territoires cultivés et leur importance dans les stratégies familiales de subsistance des exploitants agricoles dans le bassin d'approvisionnement en bois-énergie de Niamey, Niger. Thèse de l'université de Toulouse 2 Jean Jaurès[END_REF]. It was planned that other trainees or PhD students would work on soil fertility, cultivation practices and the production of non-wood forest products (fruits, nuts, etc.), but this was not possible due to the rapidly deteriorating security situation in the area. The many other products and services that these parklands provide to local communities, the ecological processes that lead to the mortality of adult Faidherbia, or other social processes that could lead to reduced interest on the part of these communities in restoring their parklands, will have to be studied when conditions and means allow it..

Methods

Study area

Three villages were selected in 3 different communes in the fuelwood supply basin of the city of Niamey. In each village, socioeconomic surveys were conducted to understand how farms operated. An inventory of the parklands was carried out to determine the structure and floristic composition of the tree stands and to situate the phenomenon of natural regeneration of different species in the cultivated area. The villages chosen for the study were Safa (commune of Koygolo), Kobi (Tondikandia) and Tientiergou (Say) (Fig. 1). These choices were motivated by the fact that Safa was a site of the Gao-Dosso project which worked to reconstitute parklands using ANR [START_REF] Montagne | Protection de la régénération naturelle de Faidherbia albida. Evaluation a posteriori du projet Gao Dosso au Niger[END_REF] in the 1980s. In Kobi, no project intervention has been documented. In Tientiergou, a village association was created in 1991 for managing natural forests and marketing fuelwood [START_REF] Rives | Fifteen Years of Forest Community Management in Niger: from a Technician's Dream to Social Reality[END_REF]).

In the 3 study areas, crop and livestock farming are the main socio-economic activities. There is an increasing rainfall gradient from Kobi to Tientiergou, but the impact of rainfall on tree vegetation is complicated by the variety of soils in each village (Tab. 1).

Socioeconomic study of the share of fuelwood in farmers' incomes Ninety comprehensive semi-structured interviews [START_REF] Sibelet | Qualitative survey methods applied to natural resource management[END_REF] were conducted with farm heads and other farm members available. The sampling aimed to explore the diversity of farms in terms of size and principal production activity. Three focus group discussions were conducted, one in each village.

The main variables studied are: activities practiced (crop and livestock productions, non-farming activities), means of production (agricultural surface area, available paid and family labors, agricultural equipment), technical-economic results (quantities produced and sold, selling prices, income from non-farming activities), household size and the objectives set for establishing and managing their parklands, the origin and quantities of wood collected and the quantity of wood sold [START_REF] Gafsi | Mesure des performances économiques. Exploitations agricoles familiales en Afrique de l'Ouest et du Centre : enjeux, caractéristiques et éléments de gestion[END_REF]. The total household income of each farm and the contribution to this income of each crop and livestock production and non-farming activities were assessed. "Household income" includes the value of the family consumption of resources generated on the farm or collected from surrounding areas. This made it possible to obtain the percentage of the overall income coming from fuelwood. Non-farming income in the region includes farm work wages, salaried activity outside agriculture, trade, and income from the sale of fuelwood. "Fuelwood income" is equal to the quantity of wood collected (sold and/or self-consumed) valued at the local market price of the wood. This quantity was determined on the basis of the weekly consumption (in bundles) of each farm studied. In addition, the locations where fuelwood is collected were recorded.

Inventory of trees in three parklands

Landsat 2016 digital satellite images covering each of the communes studied were used to estimate the total surface area of the parklands. Given the resources available, the zones to be inventoried were delineated over an area of about 5,000 ha per commune. Such a zone covered, for example, about one-sixth of the AFS area in the commune of Koygolo (Fig. 2).

The inventory was carried out by a systematic one-degree tree inventory with a sampling rate of 1.5%, which represents the ratio between the surface area of the sample and that of the selected zone (CIRAD 2015). The survey design was developed using a mapping tool (ArcView GIS software) and shows plots arranged on a square grid with 772 m between lines and 772 m between plots. Their pre-calculated geographical coordinates were integrated into a GPS.

The circular sampling units cover one hectare (a circle with a radius of approximately 56.42 m). In each commune, 75 plots were inventoried, for a total of 225 plots. On each plot, dendrometric data (species, trunk diameter at chest height, tree crown diameter and trunk height) were collected for all individuals with a height greater than or equal to 3 m.

To assess natural regeneration (NR), circular plots with a radius of 7 m (154 m 2 ) and the same center as the sampling units also were set up. All woody species less than 3 cm in diameter were systematically surveyed and recorded at the level of the NR study plot. The data collected were managed and processed using an Excel spreadsheet to calculate the parameters of interest.

Results

Origin and quantity of fuelwood harvested

The two sources of fuelwood mentioned by the greatest number of interviewees in the three villages are the shrub and tree formations on the plateaus (forests) and parklands (Fig. 3).

In Tientiergou, fuelwood is mainly harvested in a forest stand located on the plateau that is managed by the "Rural Wood Market" (the village association). In Safa and Kobi, parklands are the main source of fuelwood for rural people through the pruning of agroforestry trees and the harvesting of dead trees (Tab. 2). The proportion of species in the fuelwood was not studied.

By considering the average weight of a wood bundle (20 kg) and the number of inhabitants in the villages, the average self-consumption of wood per person per day was calculated to be 0.35 kg in Safa, 0.48 kg in Kobi and 1.39 kg in Tientiergou. The respondents explain this great difference in consumption by the availability of the resource (abundant in Tientiergou, very rare in Safa). Other causes could not be found.

Diversity of total household income and contribution of fuelwood

The composition of total household income reflects the strategy developed by farmers to limit the risks inherent in agriculture by diversifying food and cash crops and seeking off-farm income (Tab. 3).

In Kobi and Safa, almost half of the farmers' income comes from agricultural production based on market gardening. This is due to the favorable conditions for agricultural production: a shallow water table and the fertile alluvial soils in the Dallol river valley, and commercial outlets (weekly markets in the surrounding area). In Kobi, potatoes are produced, and in Safa, fresh vegetables (salad, cabbage, tomatoes...) and a legume, the cowpea (Vigna unguiculata). These products are mainly sold to generate cash resources, part of which is used to buy millet (Pennisetum glaucum), the staple cereal consumed by the local population and one which is rarely produced in sufficient quantities to meet household needs.

In Tientiergou, non-farming activities contribute most to total family income. This is due to the proximity of Niamey (salaried activities, trade ...) and the lack of potential for large-scale market gardening. Agricultural income is dominated by livestock husbandry linked to the customs and knowledge of the majority of farmers belonging to the Fulani ethnic group. Tientiergou is the only site where fuelwood accounts for an important share of income due to the existence of a forest stand on the plateau managed by the Rural Wood Market. In the other two villages, where wood mainly comes from parklands and is mostly self-consumed, fuelwood for sale accounts for a much smaller share of household income.

Farmers' strategies and practices regarding the trees in their fields Due to strong government directives regarding tree protection, especially agroforestry, and regular reforestation campaigns, farmers are wary when talking about tree management and censor their own words. The present study was difficult to carry out because the principal investigator, the first author of this article, is an officer of the Ministry of Environment, the agency responsible for both tree promotion efforts and punishing non-compliance with regulations. During the first formal surveys, the majority of farmers adhered to the official line, saying that they never cut or prune an agroforestry tree and they systematically conserve regeneration. In Safa, they recalled the ANR efforts they had made when the Gao-Dosso project was being implemented; in Safi, they said that the NGO SOS-Sahel also had encouraged them to practice ANR. However, this discourse was contradicted by the facts of the inventories, which indicated a high rate of pruning and a low rate of ANR. The actual situation was therefore gradually revealed through some slips of sentences, sometimes during the semi-structured individual interviews once trust had been established between the interviewer and interviewee.

According to several farmers, the Ministry of Environment (commonly referred to under its former name, "Water and Forests"), is impeding the rational management of trees. This is attested to by a farmer from Safa who states: "What's the point of leaving and protecting trees that you can't even use as you please?" Another says: "The agents apply the law too rigorously and it needs to be reviewed". One participant adds: "We are aware of the importance of these trees in our fields. We don't want the trees to die or disappear, which is why we have left and maintained them. But once the trees are grown, it is forbidden to do even a maintenance pruning, and if you do, you are fined". Another point of discontent mentioned concerns the authorization granted by forestry officers to timber operators to cut down dead trunks of Faidherbia albida trees in farmers' fields; according to farmers, these operators pay a formal or informal fee to government agents without any monetary compensation to those who cultivate or own the land). In another area altogether, several farmers also reported increasing mortality of mature Faidherbia albida without being able to identify the cause.

With regard to pruning, numerous disputes between Fulani breeders and field owners were reported in Kobi. The former sought to let their livestock forage on trees, particularly F. albida, and the latter sought to save their trees.

Evaluation of tree resources by inventory

Floristic composition of adult trees and regeneration

Twenty-four (24) woody species were present in the adult stand of the parklands in the three study sites and twenty-one (21) species were inventoried with regard to regeneration. Some tree species (Celtis integrifolia, Detarium microcarpum, Diospyros mespiliformis, Prosopis africana) represented in large and medium diameters, but whose regeneration is not ensured in any village, are endangered; this is all the more problematic for local communities as the species in question are multipurpose species.

Tree density in agroforestry areas

Densities are similar in the parklands inventoried in Kobi and Tientiergou (5.9 and 5.4 individuals/ha). In contrast, density is higher in Safa (8.4 ind. /ha), particularly for F. albida (4.7 ind. /ha) (Tab. 4).

Stand population structure: distribution of trees according to size

The analysis of the diameter structure of trees and shrubs in the wooded parklands shows an asymmetric "bellshaped" distribution centered on class 2 (20-29 cm) in all of the study areas (Fig. 4).

Small (classes 1: 10 cm ≤ diameter‹ 20 cm) and large individuals (class 4 and above: diameter ≥ 40 cm) are little represented in the stands.

Nevertheless, the inventories revealed a significant regeneration of trees less than 3 cm in diameter. In Tientiergou and Safa, this natural regeneration has a density of about 2,000 trees/ha, compared to 500/ha in Kobi.

The density of regeneration of Faidherbia albida is sufficient to be able to carry out ANR in Safa and Kobi (approximately 40 trees/ha), whereas this would be more difficult in Tientiergou (approximately 8 trees/ha).

Discussion

Contribution of fuelwood to farmers' household income

The income from fuelwood, although modest compared to other sources, is part of a diversification strategy that is essential for the maintenance and future of farms. Agricultural activities, including animal husbandry, represent on average 66% of total household income, compared to 34% for non-farming activities. These data are consistent with those found in the literature. Indeed, in sub-Saharan Africa, non-agricultural sources of income generally represent approximately 30 to 50% of household revenue [START_REF] Reardon | Using evidence of household income diversification to inform study of the rural nonfarm labour market in Africa[END_REF]. No doubt the proximity of the three villages studied to the capital and the existence of a decent road network play a role in the importance of income from non-agricultural activities. However, this study does not allow us to measure the importance of these factors.

The income from fuelwood is more important in Tientiergou (10% of average household income) than in Kobi and Safa (2-3% of household income). This shows that when wood is available (e.g., proximity to a forest), it makes a significant contribution to rural people's livelihoods. However, the simple calculation of the share of fuelwood in farmers' income does not adequately reflect the social importance of fuelwood or characterize its scarcity. In situations of scarcity, such as in Kobi and Safa, wood accounts for a very low share in household incomes because other non-monetizable products (straw, leaves, twigs, manure, etc.) are used to replace the missing wood. When this is not possible, households reduce the number of meals they cook (often only one cooked meal per day in Safa, choice of dishes requiring little cooking, e.g. briefly boiled millet flour) and no longer boil drinking water, which causes social, health and ecological problems (SDACDN 2017). Self-consumed firewood is most commonly woman's work, and depending on the availability of fuelwood resources can take up a large proportion of the daily time available. In addition, having to walk long distances to gather firewood exposes women to sexual assault. Both of these factors make a regular supply of fuelwood on, or close to, the farm more valuable than its financial value alone would suggest.

The results of this study reveal a paradoxical situation: restoring parkland through ANR is an action that would benefit all of the actors on the territory in the long term; however, in the short term, individual interests prevail in an economy of survival. In future studies, the issue of governance will need to be explored further, most certainly in the light of Elinor Ostrom's work on common pool resource management. Local governance systems with a collective discipline to manage agroforestry parklands have existed in the past and have been successful. It will be necessary to study what has degraded them, and what can be done to restore them. By working on a larger scale at the country level, it will be possible to better assess whether it is the projects, government and NGO actions and subsidies that explain the higher density of F. Albida origin in some villages, as was found in only two villages (Safa and Kobi) in the present study.

Floristic composition and density

F. albida is the most represented species in the agroforestry parks in Kobi and Safa, where it accounts for about 50% of the trees, and almost half of these trees have a diameter between 20 and 29 cm. In Safa, where the density of F. albida is close to 5 trees/ha, it may be assumed that these trees were conserved during the Gao-Dosso ANR support project, which was implemented between 1981 and 1985 [START_REF] Montagne | Protection de la régénération naturelle de Faidherbia albida. Evaluation a posteriori du projet Gao Dosso au Niger[END_REF]. The project encouraged farmers through a system of subsidies to protect the natural regeneration of F. albida where this was possible, and helped them to plant seedlings where existing tree stands and soil conditions were not conducive to natural regeneration. In Kobi, the efforts of the NGO SOS-Sahel also seem to have had some success (2.5 Faidherbia/ha). Such efforts were not made in Tientiergou (0.7 Faidherbia/ha).

Moreover, the toposequence around Safa and Kobi includes a dry valley (Dallol Bosso) characterized by a deep, clay-loam soil, well supplied with groundwater or by temporary flooding. This explains the density of natural regeneration of F. albida (about 40 indiv/ha in Safa and Kobi), much higher than in Tientiergou (8 indiv/ha) where the soil is shallow above a lateritic breastplate or comes from the dementing of a lateritic breastplate. The current composition of tree-covered parklands can therefore be explained, on the one hand, by the ecological conditions favoring natural regeneration, and, on the other, by the willingness of farmers to assist this, often supported in the past by extension projects.

Twenty-four species are present in the parklands on the three study areas, whereas at the level of young regeneration, only 21 species were inventoried. Even though these data should be verified over larger areas, they show that certain species which exist in large and medium diameters, but whose regeneration is not protected, are tending to disappear. This suggests that a new effort to restore parklands through ANR could ensure the survival of several, but not all, species. For the species for which there is no longer any regeneration, the survival of these species could only be achieved through planting. when there is no voluntary destruction of the good (the tree), simply by not compensating for the wear and tear of time (in other words, by not replacing old trees which die).

To revive the restoration of Faidherbia parklands, the first step required would be to resume awarding subsidies to those practicing ANR or planting (which would be a way to demonstrate public interest in these actions). At the same time, or in a second step, the right to manage and harvest the trees that people have protected or acquired should be ceded back to these individuals. However, local communities should maintain the right to arbitrate possible conflicts, for example between livestock herders and farmers, as was mentioned, for other common goods, by Ostrom (1990). We can also take into account the conclusions of [START_REF] Stith | Lamb Special Collection: Climate Variability and Its Impacts, Ways of Knowing: Traditional Knowledge as Key Insight for Addressing Environmental Change[END_REF] who consider that "it is essential to take into account the relationships between development projects and local communities, kinship and social networks, leadership across various spheres, and the resilience of indigenous environmental knowledge in future analyses that explore the complicated suite of factors that have influenced the Sahel Regreening" [START_REF] Pasiecznik | Restoring African Drylands[END_REF].

It should be noted that the first results of the present study were discussed during the National Colloquium on "Management of forest resources in peri-urban territories and climate change", which was held from 31/10 to 2/11/2017 in Niamey [START_REF] Boubacar | Ressources arborées et viabilité des exploitations agricoles familiales dans le bassin en bois-énergie de Niamey (Niger)[END_REF]. This most certainly contributed to alerting the government to the ongoing degradation of agroforestry parks. Several members of this symposium contributed to the drafting of Decree No. 2020-602/PRN/ME/SU/DD (République du Niger 2020). This decree states in particular the following points: i) Article 4: Every field owner has the right to identify and maintain, plant and exploit the trees of his choice in his field, and ii) Article 7: The owner has the exclusive right to exploit the green wood of the agroforestry parks in his field. However, at the time of the study, the concrete effects of the application of this decree could not be studied. Unfortunately, in March 2022, the text of decree n°2020-602, regulating the practice of Assisted Natural Regeneration in Niger, has not been published either on the internet or on the websites of government institutions. This decree has not yet been disseminated to those who are supposed to enforce the law (the agents of the forestry brigades); its signature came at a time when a committee was set up to revise law 2004-040 on the forest regime in Niger. The aim of this revision was precisely to take better account of agroforestry parklands. So this decree alone cannot change the situation in the field without it being integrated into the law itself because it is the basis for the work of the field agents.

Conclusion

Economic importance of fuelwod in family resources is not a sufficient trigger factor for farmers to restore their parklands in south-western Niger, but could support village's interest for sustainable management of natural forests.

Income diversification is crucial for most rural households in the Niamey region. Fuelwood plays a critical role in this diversification when the resource remains abundant and is well managed, as is the case in Tientiergou (10% of total income). On the other hand, in the villages of Kobi and Safa, where fuelwood comes essentially from furtively pruning the few trees scattered across the parklands, fuelwood accounts for a low share of income (less than 4%) and cannot cover all self-consumption needs. This generates shortages and forces the population to use various plant residues that then can no longer serve to fertilize fields with organic matter.

The parklands studied have a low biodiversity (7 to 17 species per village), low density (5 to 8 individuals/ha), scarce and often dying mature trees and few young trees (diameter less than 19 cm). This indicates that ANR, which projects had promoted from 1980 to 2000, is being practiced by farmers less and less, and proves that these parklands are no longer being restored. The mortality of old trees is in effect no longer being compensated by ANR.

The findings of this work clearly show that one cannot continue to accept past, very optimistic reports that the ANR is functioning perfectly in Niger and that the majority of parklands are in the process of regeneration.

On the contrary, it is clear that quite a few parklands are becoming severely degraded. To explain their loss of motivation for ANR, farmers' groups pointed to conflicts between farmers, who once protected trees, and livestock herders, who now come onto their fields to prune trees. Farmers feel that the pruning they do themselves on their own trees is moderate and does not harm the trees' survival. Another explanation put forward by several farmers concerns the formal and informal fines that environmental officials impose on farmers who prune the trees in their own fields, even if they themselves or their relatives were the ones who conserved them. They find this prohibition all the more unjust since the same officers allow outside loggers to come and harvest timber from dead trees in their fields. The fact that wood has a significant economic value is not enough to trigger action to protect young trees, because problems of formal or informal taxes, abusive authorisations, and conflicts between farmers and herders, which make it impossible for the farmer to make a linear link between increasing the number of trees in his fields and being able to harvest more wood in 20 years' time, must also be addressed.

In the absence of a large-scale study, the work carried out on three villages does not, at this stage, make it possible to offer definitive solutions for the entire country. However, it should make it possible to alert public authorities and aid agencies of the need to launch research and development actions to reverse this trend, which is threatening the survival of rural communities and some of the fuelwood supply chains serving the country's capital. The research should focus on the social processes behind the population's diminished interest in restoring parklands and on the causes of mortality of mature trees (climate change, overexploitation of water tables, diseases, etc.). Legislative and development actions should focus, among other things, on regulating negotiations between farmers and livestock herders, clarifying the law on agroforestry tree management and its application, and on incentive mechanisms for farmers practicing ANR. Table 1: Characteristics of the study areas1 1 In the Dallol or the river valley, the roots of mature trees have access to groundwater; low rainfall consequently does not limit tree growth. In contrast, tree growth on the plateaus is dependent on rainfall. The vegetation found in the valleys and on plateaus, and the productivity of the wood resources, therefore differ.

Fig. 1 :Fig. 2 : 472 Fig. 3 :Fig. 4 :

 1247234 Fig. 1: Location of the study villages and the parklands inventoried

The authors would like to thank the farmers from Niger who dedicated their time to participate in this collective research and the entire team and partners of the FONABES Project, including the administrative staff of the Agence Française de Développement and of the Ministére de l'Europe et des Affaires Etrangères in Niamey and Paris and the Ministry of the Environment, Urban Health and Sustainable Development in Niger who supported this study.

Stand population structure: distribution by tree diameter Concerning F. albida, regeneration is weak but significant (40 individuals/ha in Safa and Kobi), while class 1 (10 to 19 cm) is very poorly represented. This this would suggest that farmers allow very few trees from natural regeneration to grow to a diameter of 10 cm or more. On the basis of the available results concerning the average diameter growth of F. albida (1.5 cm/year, according to [START_REF] Depommier | Croissance de Faidherbia albida dans les parcs du Burkina Faso[END_REF]), one might think that few regenerations have therefore been preserved over the past decade. But it is also possible that some farmers would like to regenerate more Faidherbia but are unable to do so because of livestock damage or conflicts with herders.

Whatever the cause, it can be seen a regression in the use of ANR compared to the very positive results that were observed during the 2000s (Reij and Botoni 2009). In the villages studied, it should be noted that farmers are not able to keep as many young trees as in the past. Concerning the increasing mortality of old Faidherbia, some farmers mentioned the lowering of the water table and attacks by wood boring insects, these hypotheses could not be verified. A team from the University of Maradi is working on the Faidherbia dieback [START_REF] Tougiani | Faidherbia albida (Delile) Tree Dieback Effects on Crop Production in the Parkland Agroforests of Southwestern Niger[END_REF]).

The authors have, through inventories in the Dogondoutchi area and surveys of farmers, shown the mortality rate of F. albida in agroforestry parklands and that this has an impact on agricultural production. However, the scientific causes of this mortality phenomenon remain unclear.

Farmers' loss of interest in ANR

Although the restoration of wooded parklands is considered collectively useful, theories concerning the management of common property [START_REF] Ostrom | Governing the Commons: The Evolution of Institutions for Collective Action[END_REF][START_REF] Agrawal | Common property institutions and sustainable governance of resources[END_REF]) cannot be applied to explain individual farmers' loss of interest in the restoration of these parklands. The decision to keep a young tree found among all of the plants regenerating on a field, when clearing it prior to sowing, is a private decision made by the farmer.

Nor can economic theories on the management of private property like a "good parent" be applied. This is because a significant part of the future benefits of a tree that is conserved, namely wood and aerial fodder, are not at the farmer's free disposal, and may even be considered to be common goods given that the farmer must pay a fee to the State to use them, and is fined if s/he does not comply with this legal obligation.

The appropriation by the State of the use of a good whose creation is based on a private initiative leads to the disengagement of these private individuals and the progressive disappearance of the good. This holds true even


Loïc Angrand

Samir Takillah

Isabelle Malissin

Asma Berriche

Chloe Cervera

Rosalie Bel

Quentin Gerard

Julie Knoertzer

Rachid Baati

Joseph P Kononchik

Samir Takillah

Bruno Megarbane

Karine Thibault
email: karine.thibault@def.gouv.fr

Gregory Dal Bo

Persistent brainwave disruption and cognitive impairment induced by acute sarin surrogate sub-lethal dose exposure

Keywords: nerve agent, sarin surrogate, 4-nitrophenyl isopropyl methylphosphonate, sublethal exposure, brain waves, neuroinflammation AUC: area under the curve CA1: cornu ammonis 1of hippocampus electrocorticographic EEG: electroencephalographic GFAP: glial fibrillary acidic protein HF: hippocampal formation HPS: Hemalun-Phloxin-Safran staining IBA1: ionized calcium binding adaptor molecule

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

Introduction:

The 2010s have been marked by the resurgence of organophosphorus (OP) chemical warfare agent (CWA) use in various scenarios. Sarin was reported in numerous attacks between 2013 and 2017, causing deaths and casualties in both civilian and military populations [START_REF] Amend | Diagnostics and treatment of nerve agent poisoning-current status and future developments[END_REF][START_REF] John | Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network[END_REF]. Two others nerve agents (NA), VX and Novichok were used in targeted attacks, leading to the death of Kim Jong Nam in 2017 [START_REF] Amend | Diagnostics and treatment of nerve agent poisoning-current status and future developments[END_REF] and the direct poisoning of Sergei and Yulia Skripal in 2018, and Alexei Navalny in 2020 [START_REF] Amend | Diagnostics and treatment of nerve agent poisoning-current status and future developments[END_REF][START_REF] Chai | Novichok agents: a historical, current, and toxicological perspective[END_REF][START_REF] Greathouse | Acetylcholinesterase Inhibitors Toxicity[END_REF]. Despite the progress to eliminate chemical weapon stockpiles, the ongoing tangible threat of OP derivative compounds has persisted. In addition, the important use of OP containing pesticides still accounts for more than 1 million poisonings worldwide [START_REF] Abou-Donia | Sarin (GB, O-isopropyl methylphosphonofluoridate) neurotoxicity: critical review[END_REF][START_REF] Eddleston | Novel Clinical Toxicology and Pharmacology of Organophosphorus Insecticide Self-Poisoning[END_REF].

OP compounds are irreversible cholinesterase (ChE) inhibitors that lead to a toxic accumulation of acetylcholine in synapses and neuromuscular junctions. After exposure to some OPs, a large range of symptoms occur in victims including fasciculation, hypersecretion, and muscle twitching, which can culminate in respiratory distress, epileptic seizure, and death [START_REF] Abou-Donia | Sarin (GB, O-isopropyl methylphosphonofluoridate) neurotoxicity: critical review[END_REF]. When exposed to high doses of OP, death results from respiratory failure due to muscles paralysis and bronchoconstriction, though it can be avoided if emergency therapy (atropine sulfate and an oxime, a strong nucleophile able to reactivate OP-inhibited acetylcholinesterase (AChE)) is quickly administered. However, the OP effects in the central nervous system (CNS) may not be stopped quickly enough by this intervention if it is delayed, and their neurotoxicity may lead to epileptic seizures and subsequent brain damage [START_REF] Dail | Novel Brain-Penetrating Oxime Acetylcholinesterase Reactivators Attenuate Organophosphate-Induced Neuropathology in the Rat Hippocampus[END_REF][START_REF] De Araujo Furtado | Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats[END_REF]. While the neurotoxic effects of lethal or supralethal NA doses have been extensively studied [START_REF] Grauer | Single whole-body exposure to sarin vapor in rats: long-term neuronal and behavioral deficits[END_REF][START_REF] Schultz | Caramiphen edisylate as adjunct to standard therapy attenuates soman-induced seizures and cognitive deficits in rats[END_REF][START_REF] Testylier | Cerebral edema induced in mice by a convulsive dose of soman. Evaluation through diffusion-weighted magnetic resonance imaging and histology[END_REF], the cerebral impact of acute sub-lethal doses of NA have been poorly investigated. Also, due to the heterogeneity of intoxication protocols used, controversy persists on the neurophysiological changes and cellular alterations of those doses. Though single sub-lethal doses of soman failed to induce marked histopathological damage in exposed animals [START_REF] Baille | Acute exposure to a low or mild dose of soman: biochemical, behavioral and histopathological effects[END_REF], anxious behavior and cognitive deficits could be transiently observed following NA exposure [START_REF] Baille | Acute exposure to a low or mild dose of soman: biochemical, behavioral and histopathological effects[END_REF][START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF][START_REF] Genovese | Behavioral evaluation of rats following low-level inhalation exposure to sarin[END_REF][START_REF] Kassa | The influence of sarin on various physiological functions in rats following single or repeated low-level inhalation exposure[END_REF][START_REF] Pearce | The effects of acutely administered low dose sarin on cognitive behaviour and the electroencephalogram in the common marmoset[END_REF], which contrasts with the report that no memory impairment was observed below a threshold of 15% neuronal loss [START_REF] Filliat | Memory impairment after soman intoxication in rat: correlation with central neuropathology. Improvement with anticholinergic and antiglutamatergic therapeutics[END_REF]. It is also interesting that electroencephalographic (EEG) or electrocorticographic (ECoG) recordings seem to be only transiently disturbed if 50% of brain cholinesterases are inhibited [START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF][START_REF] Pearce | The effects of acutely administered low dose sarin on cognitive behaviour and the electroencephalogram in the common marmoset[END_REF]. Neural oscillatory patterns were altered in the first hours following soman exposure in mice, with an increase in delta power coupled to a decrease in theta [START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF]. At later stages, these changes in power are compensated within the first 24h after exposure, and only sleep stages are still altered up to 1 week [START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF]. Nevertheless, sub-lethal sarin doses induced longterm EEG/ECoG abnormalities in marmoset [START_REF] Van Helden | Low levels of sarin affect the EEG in marmoset monkeys: a pilot study[END_REF]) consistent with the reports on Tokyo's victims [START_REF] Yanagisawa | 4-6 Hz background activity and amplitude of 100/200 µV; Grade 3: Wakefulness stage at 3 Hz background activity and amplitude of 150/200 µV; Grade 4: Wakefulness stage with monomorphic and monotonous delta 2.5 Hz background activity and amplitude of 200 µV. The table highlights the number of evaluated animals, the severity and the grade reached depending of the dose of NIMP. (D) Examples of nonfiltered ECoG recording during the baseline (left) and 6 days post-0.5 LD50 NIMP exposure showing a "spike and wave"-like discharge. (E-G) Diagrams of the changes in brainwave distribution at different time periods following NIMP exposure over 1-month[END_REF]. Furthermore, sub-chronic VX exposure at 0.3LD50 was determined to be the threshold to elicit fluctuations in theta and alpha waves 4 weeks after the initial exposure in rats [START_REF] Bloch-Shilderman | Determining a threshold sub-acute dose leading to minimal physiological alterations following prolonged exposure to the nerve agent VX in rats[END_REF].

Therefore, more extensive characterization of the acute and long-term changes in EEG/ECoG recordings of sub-lethal doses of NA is needed.

One of the major limitations studying NA neurotoxicity is based on the restrictions for obtaining CWA. Considering the re-emergence of NA threat, the use of surrogates offers an alternative to extend investigating methods to unauthorized laboratories. A promising sarin surrogate was obtained by the synthesis of 4-nitrophenyl isopropyl methylphosphonate (NIMP) [START_REF] Meek | Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates[END_REF][START_REF] Ohta | New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes[END_REF]. This non-volatile compound is safer to use, efficiently inhibits cholinesterases, and is more potent and more stable in solution than diisopropyl fluorophosphates (DFP) [START_REF] Chambers | Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX[END_REF][START_REF] Chambers | Novel brain-penetrating oximes for reactivation of cholinesterase inhibited by sarin and VX surrogates[END_REF][START_REF] Meek | Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates[END_REF]. In vivo challenges with sub-lethal and/or lethal doses of NIMP elicit signs of OP intoxication, seizure-like behavior, and significant neuronal damage in rats [START_REF] Chambers | Novel brain-penetrating oximes for reactivation of cholinesterase inhibited by sarin and VX surrogates[END_REF][START_REF] Dail | Novel Brain-Penetrating Oxime Acetylcholinesterase Reactivators Attenuate Organophosphate-Induced Neuropathology in the Rat Hippocampus[END_REF]. Furthermore, NIMP exposure notably results in cellular injury and astrocytic activation in the piriform cortex and hippocampus, which are some of the most affected cerebral regions by NA [START_REF] Pringle | Neuroprotection From Organophosphate-Induced Damage by Novel Phenoxyalkyl Pyridinium Oximes in Rat Brain[END_REF]. Nonetheless, a deeper characterization of NIMP exposure will be needed to confirm its potency to mimic CWA.

The aim of this study was to test NIMP exposure in mice, report symptoms following a single exposure of different sub-lethal OP doses, and characterize a typical EEG/ECoG 'fingerprints' at key-points after intoxication. We thus evaluated the behavioral effects of four different doses (0.3, 0.5, 0.7 and 0.9 LD50) of NIMP during the first hour following mice exposure. Cerebral cholinesterase inhibition, neuroinflammation and signalling changes were evaluated at different time points up to 30 days post-exposure. In parallel, ECoG were recorded for 24 hours for each dose to differentiate the acute signs of intoxication and the brainwave changes at early stages. Afterwards, telemetric ECoG recording was performed to follow the perturbations over 1 month. At 1 month after NIMP exposure, potential cognitive impairments were evaluated.

Material and methods

Animals

All experimental procedures were approved by the French "Service de Santé des Armées" animal ethics committee according to the applicable French legislations (Directive 2010/63/UE, décret n°2013-118). Adult male Swiss mice (Janvier Labs, France) aged from 6 to 8 weeks were housed, four per cage, with a 12h/12h light/dark cycle temperature-controlled room (22 ± 2°C), food, and water available ad libitum. After 7 days of acclimation period, the animals were brought to their dedicated experiment. All animals were 9-week old at the OP challenge.

NIMP exposure

4-nitrophenyl isopropyl methylphosphonate (NIMP, Fig sup . 1) used as a sarin surrogate was synthesized by Rachid Baati (Université Strasbourg, ICPEES UMR CNRS 7515, France) as previously described [START_REF] Meek | Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates[END_REF], with almost identical purity, determined by 1 H NMR (mass %: 92%). The day of the experiment, mice received a single subcutaneous injection (10ml/kg) of NIMP (LD50 = 0.63mg/kg), freshly diluted in 0.9% NaCl at one of 4 sub-lethal doses (0.3, 0.5, 0.7, or 0.9 LD50). Control mice (CTL) received a similar vehicle injection without NIMP. For the preceding hour and the days following NIMP exposure the weight of the mice was monitored and behavioral assessment was conducted to evaluate their recovery (Fig. sup. 2).

Behavioral observation

Intoxication severity scale:

Several typical signs of cholinergic toxicity such as hypersecretion, tremor, loss of posture, etc., have been extensively described [START_REF] Bloch-Shilderman | Determining a threshold sub-acute dose leading to minimal physiological alterations following prolonged exposure to the nerve agent VX in rats[END_REF][START_REF] Senanayake | A scale to assess severity in organophosphorus intoxication: POP scale[END_REF][START_REF] Shih | The effects of choline on soman-induced analgesia and toxicity[END_REF]; with a modified Racine's scale used to evaluate epileptic behavior [START_REF] De Araujo Furtado | Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats[END_REF][START_REF] Enderlin | Characterization of organophosphate-induced brain injuries in a convulsive mouse model of diisopropylfluorophosphate exposure[END_REF][START_REF] Phelan | Pilocarpine-induced status epilepticus in mice: A comparison of spectral analysis of electroencephalogram and behavioral grading using the Racine scale[END_REF]. Considering that the lowest doses used in our study would elicit more subtle signs, all mice were observed and videotaped during the first hour postintoxication. All behavioral changes observed in NIMP exposed mice, compared to control animals, were noted with their onset, and considered as observable signs of intoxication. Finally, 13 signs of intoxication were selected, grading animals from normal (=0) to death (=12). For this study, 336 mice were evaluated to include 83 control animals.

Novel object recognition (NOR) test:

Mice were placed in an empty open-field (OF) box for a 10min habituation period. Before and 1 month after NIMP exposure, mice of 3 groups (CTL n=15; 0.5 LD50 n=15; 0.9 LD50 n=17) were placed in the same OF box with 2 identical objects, allowed to explore for 5 min, then returned to their home cage. After 1 hour, the mice were placed in the OF containing 1 familiar object and a novel object and allowed to explore for 5 min. The objects were placed at the same locations as the first session. All NOR sessions were videotaped and the exploration time was analyzed using EthoVision XT software (Noldus, NL).

Surgery

Two different ECoG procedures were used in this study that followed the same surgery protocol. Seven-week old mice were anesthetized in an induction box using isoflurane (5% in oxygen at L/m). They were injected with buprenorphine (Bupredine®, 0.1 mg/kg, i.p.), and prepared for surgery. Their head was shaved and lidocaine (Lurocaine®, 5mg/kg, s.c.) was applied on their scalp before being placed in a stereotaxic apparatus and maintained with inhalation of a mixture of isoflurane (2%) and oxygen (100%).

Wired ECoG recordings were used for 24-hour recordings (70 mice divided in five groups). After skin incision, 3 holes were drilled with the recording holes positioned over the left and right parietal cortices (AP -2mm; L ±1.75mm) and the third one over the cerebellum (AP -6.5mm) for reference. In each hole a stainless steel screw was inserted and bipolar cortical electrodes with ground (MS333/2-BIU/SPC, Plastics One, U.S.A.) were connected to the screw. Composite resin was wrapped around skull screws to secure the device.

For Telemetric ECoG (22 mice divided in 2 groups), only 2 holes were drilled over the parietal cortices (same coordinates). The screws were connected to flexible leads of a biopotential transmitter (ETA-F10, DSI, USA) subcutaneously implanted in the mouse's flank according to the manufacturer's protocol. The screws were wrapped with adhesive resin cement and covered with sterilized silicone paste. Then, the scalp was sewed.

All the mice were permitted to recover until regaining pre-surgery body weight and returned to their home cage for a 2-week complete recovery period before being recorded and exposed to NIMP.

ECoG recording and power spectra analysis

For wired ECoG recordings, 4 animals per experimental day were connected via a cable to a data acquisition system for a 24h recording period. Cerebral electrical activity was recorded using the customized Brain Quick EEG system (MicroMed, Treviso, Italy).

Baseline activity was recorded for 1 hour before NIMP exposure.

For telemetric recording, 8 animals were recorded simultaneously using DataQuest acquisition software (DSI, USA). In addition to ECoG, subcutaneous temperature, animal activity, and transmitter power were also recorded. Baseline cerebral activity was recorded for 4 hours the day before the intoxication challenge and 1 hour before intoxication. Mice were recorded continuously for the first 24 hours and then for 2 hours session per day the rest of the first week. Following the first week, two 2-hour recording sessions were conducted per week. Data were analyzed using Matlab (Matworks®, USA) built-in functions and chronux toolbox [START_REF] Bokil | Chronux: a platform for analyzing neural signals[END_REF]. ECoGs were (i) acquired at 512Hz and offline band-pass filtered at 0.1-128Hz with zero-phase shift filter function (zero-phase digital filtering filtfilt function), and (ii) detrended using a local linear regression (locdetrend function: window-size 1s, overlap 0.5s) to remove slow drifts, and (iii) notch-filtered (iirnotch function) with notch located at 50Hz to remove possible power line noise. Power spectrum of ECoG data was calculated using the multi-taper timefrequency spectrum method (mtspecgramc function) with time-bandwidth product of 30 and 59 Slepian sequences of orthogonal data tapers (windows-size 30s, 10s overlap).

The ECoG power spectrum was divided in the following bands: delta (0.5-4 Hz), low theta (4-8 Hz), high theta (8-12 Hz), low beta (12-16 Hz) and high beta (16-25 Hz) using Neuroscore software (DSI, St-Paul, MN, USA). Mean power was calculated for each band for 30s epochs. For each animal, data from telemetric recording are normalized to the pre-intoxication baseline recording values. Spontaneous electrical reactivity was defined by the modifications in the background activity (acceleration or slowing) in relation to the spontaneous fluctuations in arousal level. Encephalopathy was stratified using Markand's classification [START_REF] Markand | Electroencephalography in diffuse encephalopathies[END_REF].

Cholinesterase activity and multiplex biomarker assays

At different time points (6 and 24 hours, 3 and 7 days, and 1-month) after NIMP exposure, animals were deeply anaesthetized with pentobarbital, a sample of blood was collected, and the mice were transcardially perfused with cold 15 ml of 0.9% NaCl.

Brains were quickly removed and dropped in cold saline before being sliced in 2mm thick coronal sections. Hippocampus were micro-dissected, collected in Eppendorf tubes and frozen on dry ice. The hippocampal samples were homogenized in 50 mM phosphate buffer (pH 7.4)/0.5% Tween using a bead mill homogenizer (OMNI International) with 1.4 mm ceramic beads and centrifuged at 10,000 X g (4 °C) for 10 min.

The resulting supernatants were stored at -80°C. Total protein concentrations were determined using a DC Protein Assay (Bio-Rad).

Cholinesterase inhibition assay: AChE activity was determined using the Ellman method by adding 5 µl of hippocampus sample to 0.22 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB, Sigma Aldrich) plus 0.1 mM ethopropazine hydrochloride (Sigma Aldrich) to inhibit butyrylcholinesterase. After a 15min baseline measurement, 1 mM acetylthiocholine (Sigma Aldrich) was added and the reaction between thiocholine and DTNB was monitored for 30 min at 412 nm and 25 °C by a microplate reader. All samples were assayed in duplicate. The ChE activities of the hippocampal samples were normalized with total protein concentration for each sample. The final results were expressed as percentages of average control activity.

Milliplex multiplex assays: Cerebral concentrations of IL-1β, IL-6, MIP-1α, TNF-α, and VEGF were obtained by using the Milliplex MCYTMAG-70K-PX32. NF-κB hippocampal levels were analyzed using Milliplex 48-681MAG (EMD Millipore, USA) and the Bio-Plex MAGPIX multiplex reader (Bio-Rad, USA).

Histology

At different time points (24 hours, 7 days, and 1-month) post NIMP exposure, mice were deeply anaesthetized with pentobarbital, and transcardially perfused with cold 10 ml of 0.9% NaCl, followed by 30 ml of 4% paraformaldehyde (PFA) in phosphate buffer.

Brains were quickly removed and immerged in cold 4% PFA for overnight post-fixation.

The brains were cryoprotected 24-48h in cold 20% sucrose and then frozen in -40°C isopentane. All brains were sliced in 14 µm coronal sections using a cryostat and the slices were sequentially mounted on Superfrost+ slides (VWR, USA). For 1 slide per animal, common Hemalun-Phloxin-Safran (HPS) staining was conducted to evaluate neuronal damage and neuroanatomic changes. HPS labeled slices were imaged with a digital microscope system (K1300, Hirox, CN). For each animal, 3 slices containing septal hippocampus were analyzed. Immunofluorescent labelling with mouse anti-GFAP (1/1000, MAB360, Millipore) and rabbit anti-IBA1 (1/1000, Wako, USA) were detected by anti-mouse 488 and anti-rabbit 555 respectively (both at 1/500, Invitrogen, USA) to analyze astrocytic and microglial activity. The fluorescently labelled CA1 part of the septal hippocampus was imaged using an automated Leica DM6000 B research microscope (Leica Microsystems, DE), analyzing 3 slices per animal. All acquisitions were performed with the same acquisition setup. Stereotaxic consistency between animals was provided by using the reference mouse brain atlas [START_REF] Gf | The Mouse Brain In Stereotaxic Coordinates[END_REF].

Analysis was performed using the ImageJ software. To quantify Iba1 or GFAP staining, we performed an area fraction analysis. Briefly, a threshold is set, and pixels in the image whose value lie above the threshold are converted to white, while pixels with values below the threshold are converted to black. The threshold was determined to obtain clear area representing IBA1 or GFAP labelling in control animals. The same threshold was applied on each image of all animal groups. This analysis measured the area of labelling in regions of interest.

Statistics

Data were expressed as means ± standard error of the mean (SEM) and analyzed using

Results

Intoxication severity scale:

To discriminate between the most common intoxication signs induced by sublethal doses of NIMP, we conducted preliminary experiments over the first hour following NIMP exposure. We identified 22 signs that elicited significant behavioral changes compared to the CTL mice. To avoid experimental biases, 6 "treatment-blinded" experimenters noted the occurrence and the onset of signs. Data compilation for the 4 studied sub-lethal doses of NIMP allowed the selection of the 12 most prevalent behavioral changes, and established a severity scale based on the appearance percentage and time of each sign (Fig. 1B-E). Animals that did not show any behavioral perturbation were scored 0. Selected signs were classed and mice were attributed the highest number reached after NIMP exposure (Fig. 1B). The lowest dose (0.3 LD50) caused fasciculation restricted to the injection site in less than half of the animals, and the mice recovered quickly. At this dose no other intoxication sign was observed and the mice were scored 1 based on our severity scale. Almost all mice exposed to 0.5 LD50 of NIMP displayed fasciculation, stereotypies (chewing) and first signs of respiration difficulty (yawning). One fifth of 0.5 LD50-exposed mice showed transient muscle weakness and excessive thoracic kyphosis with a few animals at this dose also presenting light head bobbing and rare muscular spasms. With the following dose (0.7 LD50), the majority of animals quickly presented the first 6 signs as well as involuntary twitching that persisted over the 1-hour observation period. When exposed to this dose, a few animals also showed peripheral epileptiform behaviors that were differentiated into a short-time period (head or upper body part convulsion <5s, with rest periods) and episodic total body myoclonus (>10s, with calm periods) lasting for few minutes (< 30 min). Note that we used "convulsion" to describe the intense uncontrolled tremors when electrical seizure cannot be ensured. One mouse exposed to 0.7 LD50 of NIMP showed generalized convulsions that lasted more than 30 min. Finally, the highest tested dose (0.9 LD50) provoked all the previously cited symptoms in a short time (< 20 min) and was also characterized by the incidence of long-lasting convulsions in 78% of animals. In half of 0.9 LD50 exposed mice, bulging eyes were observed, but only in animals with long lasting convulsions that preceded generalized convulsions. Only 5 animals exposed to the highest dose died, and all of them presented the 11 first symptoms.

NIMP exposed animals used for ECoG recording were also evaluated on the same scale, and for the same intoxication doses no significant difference was observed in the score severity between animals with and without surgery (Fig. 2B,3B).

Cholinesterase activity:

Several brain areas including the hippocampus, thalamus, amygdala, and piriform cortex are known to be particularly sensitive to NA exposure. We chose to focus on HF due to its involvement in learning and memory processes as well as brain rhythm generation [START_REF] Amilhon | Theta Rhythm in Hippocampus and Cognition[END_REF][START_REF] França | Beta2 oscillations (23-30 Hz) in the mouse hippocampus during novel object recognition[END_REF][START_REF] Shi | Repeated low-dose exposures to sarin disrupted the homeostasis of phospholipid and sphingolipid metabolism in guinea pig hippocampus[END_REF]. Measurement of cerebral ChE activity following NIMP exposure was conducted at different time points after exposure on intact animals (without surgery). The lowest dose (0.3 LD50) did not show significant difference in ChE activity compared to CTL mice (Fig. 2C). Otherwise, the other sub-lethal doses (0.5; 0.7; 0.9 LD50) of NIMP induced robust hippocampal AChE inhibition at 6 and 24 hours after exposure in a dose-dependent manner (ranging from 75 to 90% respectively). These results seemed to be in agreement with the mild signs observed in 0.3 LD50 mice, as opposed to the other doses, which elicited clear intoxication symptoms in NIMP exposed animals. Similar inhibition patterns were observed in blood samples regarding the different doses of NIMP evaluated (Fig. sup.

3). Measurement of hippocampal AChE activity in the long-term showed a persistent, significant inhibition on animals exposed to 0.5 or 0.9 LD50 of NIMP at up to 1 week, while recovery was still incomplete at 1-month post exposure (Fig. 3C).

Cortical activity modifications:

Short-term ECoG evaluation was conducted over 24 hours. Baseline cortical activity was recorded for 1-hour prior to NIMP challenge to ensure that EEGs were consistent across the 5 different groups studied (Fig. 2D). After NIMP exposure, or vehicle for CTL, cortical activity was recorded for 23 hours. Of the 77 animals recorded, only 57 presented suitable and complete recordings and were kept for brainwave analysis. Mice exposed to 0.3 LD50 did not present any change in cortical frequencies ranges in their power spectrum when compared to the CTL and their own baseline (Fig. 2E,F). In contrast, power spectrum for the complete period (24 h) of ECoG recordings showed a significant decrease of relative power in animals exposed to 0.5 or 0.9 LD50 of NIMP (Fig. 2E). In addition, spectrogram representation for the 3 doses showed a switch of theta rhythm dominance for delta rhythm, which lasted in a dose-dependent manner (Fig. 2F). The fall in all brainwave power spectrum was observed at different time periods in animals exposed to 0.5, 0.7 and 0.9 LD50 of NIMP, with significant decrease in high theta (8-12 Hz), low beta (12-16 Hz), and high beta (16-24 Hz) without recovery at 24h (Fig. 2G). In addition, brainwave distribution analysis showed an increase of delta rhythm (0.5-4 Hz) during the first 6 hours following 0.7 and 0.9 LD50 exposure. This correlated to a decrease in high theta rhythm (Fig. 2H). Interestingly, no electrical seizure was observed in any NIMP exposed animals during the 24-hour recording, even if they presented long-lasting convulsions.

Cortical activity modifications were then evaluated for up to 1-month on mice exposed to 0.5 or 0.9 LD50. In this experiment, animals were their own control, so baseline activity was recorded during 2 periods, 24 hours and 1-hour before NIMP intoxication. A closed, blinded investigation of spontaneous cerebral activity was conducted by a neurologist. Four mice of each group were analyzed, and 2 out of the four mice treated with NIMP at 0.5 LD50 exhibited encephalopathy that peaked at 11 and 12 min after injection. Encephalopathy was moderate in two mice (maximum grades of 1 and 2), lasting 100 min and 24 h, respectively. The other two mice presented no changes in their ECoG cerebral activity. All four mice treated with NIMP at 0.9 LD50 exhibited more severe encephalopathy (maximum grade of 3 in three mice and 4 in one mouse), that occurred 6 (in 2 mice), 7, and 8 min after the injection (Fig. 3C). Again, no electrical seizures were observed at any selected time point over the month following exposure. However, sporadic "spike and wave"-like bursts were observed in 5 mice exposed to 0.5 LD50 and 8 mice exposed to 0.9 LD50, which persisted over 30 days (Fig. 3D). Similar to wired animals, delta brainwave (0.5-4 Hz) distribution was significantly increased during the first 24 hours in the 0.9 LD50 exposed animals, but returned to normal 48 hours after exposure (Fig. 3E). In contrast, low theta (4-8 Hz) and high theta (8-12 Hz) brainwaves were persistently altered over the month in animals exposed to 0.9 LD50 (Fig. 3F,G). Once again, 0.5 LD50 exposure elicited mitigated brainwave perturbations, however a significant increase in low theta rhythm was still observable 30 days post-intoxication (Fig. 3F). No persistent modifications were observed in beta rhythms in any animal.

Histological modifications:

Morphological evaluation was conducted at 2 different time points after NIMP exposure. Seven days post-intoxication, a significant swelling was observed in hippocampal formation (HF) for both NIMP doses tested (Fig. 4B). The ratio of HF over total hemisphere areas was increased by 21% (±0.03) and 20% (±0.04) on 0.5 and 0.9 LD50 exposed animals, respectively, when compared to the CTL (Fig. 4C). However, this morphological difference was no longer evident 30 days post-NIMP exposure, as HF over the total hemisphere area ratio was equivalent to the CTL. In addition, microscopic analysis did not reveal any histopathologic difference between NIMP-exposed mice and the CTL.

Extensive examination of astrocytic reactivity in the hippocampus showed a slight, but non-significant, increase (44% ±25 and 42% ±9) of GFAP labelling in 0.5 and 0.9 LD50 NIMP exposed mice, respectively (Fig. 4D-G). This trend was not apparent 30 days post-NIMP exposure. Furthermore, 7 days post-intoxication, NF-κB expression was significantly increased in 0.5 LD50-exposed animals (+ 104% NIMP ±40), and slightly increased in 0.9 LD50-exposed animals (+ 45% NIMP ±21) (Fig. 4H). Again, these differences in NF-κB expression were not observed 30 days post-intoxication.

Induced neuroinflammation:

Cerebral cytokine levels were evaluated using Luminex technology [START_REF] Moncunill | Quantification of multiple cytokines and chemokines using cytometric bead arrays[END_REF]. As suspected, sub-lethal doses of NIMP did not induce robust inflammation in mice hippocampus. Surprisingly, an early and transient increase in TNF-α and VEGF expressions was observed only in 0.5 LD50 mice 24h after NIMP exposure (Fig. 5B).

Nevertheless, even if no significant cytokine level increase was present 7 days postintoxication, the macrophage inflammatory protein 1-α (MIP-1-α or CCL3) expression was slightly increased in both groups (+48% ±37 for 0.5 LD50; +116% ±43 for 0. 9LD50 mice; Fig. 6B). In addition, microglia reactivation was significantly increased in 0.9 LD50 mice hippocampus at 24 hours, and persisted for up to 30 days post-intoxication (Fig. 6C-E). Microglia were moderately activated by the lower dose of NIMP, as a slight and transient increase was observed at 24 hours and 7 days post-intoxication in the 0.5 LD50 group (Fig. 6C).

Short-term memory impairment:

Considering that neuroinflammatory responses could alter cognitive functions, memory, and hippocampal synaptic activity [START_REF] Barrientos | Neuroinflammation in the normal aging hippocampus[END_REF][START_REF] Jin | Silibinin rescues learning and memory deficits by attenuating microglia activation and preventing neuroinflammatory reactions in SAMP8 mice[END_REF][START_REF] Marciniak | The Chemokine MIP-1α/CCL3 impairs mouse hippocampal synaptic transmission, plasticity and memory[END_REF], the mice of each group were subjected to recognition memory tests. Two sessions of object recognition tests were conducted before and 1 month after NIMP challenge (Fig. 6F). This test relies on a rodent's natural propensity to explore novelty and showed that all mice increased exploration (72.4% ± 3.9 for CTL; 75.4% ± 3.5 for 0.5 LD50; 72.4% ± 3.9 for 0.9 LD50) of the novel object during the baseline session before intoxication (Fig. 6G). One month after NIMP exposure, the exploration performance for the novel object significantly decreased in NIMP intoxicated animals (58.2% ± 2.3 for 0.5 LD50; 54.7% ± 2.3 for 0.9 LD50), while CTL mice kept their interest for the novel object (69.1% ± 3.6 for CTL) (Fig. 6G). No velocity modification was observed in any groups.

Discussion

Despite extensive efforts to ban the toxins, sarin and other CWA are still a major concern as evident by their use in the Syrian conflict and in targeted assassination attempts. In addition, follow-ups on victims exposed to sarin (Sugiyama et al., 2020) and to sarin combined with sulphur mustard (Talabani et al., 2018) have reported the prevalence of long-term neurological complications; suggesting the need to extend experimental research in order to identify biomarkers for novel targets and develop more effective antidotes to treat OP NA exposures. Considering that CWA restrictions limit the opportunity to investigate their effects, one of our goals was to confirm the reliability of a sarin surrogate (NIMP) in mice, and to develop a suitable animal model for studying long-term sub-lethal OP exposure effects. So far, research using NIMP exposure has been limited to rats. Developing a murine model will offer a good alternative and expand the breath of projects that use transgenic mice [START_REF] Marrero-Rosado | Ketamine as adjunct to midazolam treatment following somaninduced status epilepticus reduces seizure severity, epileptogenesis, and brain pathology in plasma carboxylesterase knockout mice[END_REF].

Initial in vivo research demonstrated that rats subjected to high sub-lethal or lethal doses of NIMP reproduced some typical features of NA intoxication, such as facial stereotypes, head and body tremors, fasciculations, myoclonic convulsions, seizure-like behaviors, and respiratory distress [START_REF] Chambers | Testing of novel brain-penetrating oxime reactivators of acetylcholinesterase inhibited by nerve agent surrogates[END_REF][START_REF] Chambers | Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX[END_REF][START_REF] Meek | Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates[END_REF]. Furthermore, NIMP challenges at high sub-lethal or lethal doses were able to respectively induce an astrocytic reactivation and a neurodegeneration in rats' piriform cortex and hippocampus; 2 cerebral regions that are particularly sensitive to OP intoxication [START_REF] Dail | Novel Brain-Penetrating Oxime Acetylcholinesterase Reactivators Attenuate Organophosphate-Induced Neuropathology in the Rat Hippocampus[END_REF][START_REF] Pringle | Neuroprotection From Organophosphate-Induced Damage by Novel Phenoxyalkyl Pyridinium Oximes in Rat Brain[END_REF]. The authors concluded that NIMP fits the requirements of a sarin surrogate in both in vitro and in vivo studies [START_REF] Chambers | Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX[END_REF].

As an initial step, we established a scoring scale to characterize the intoxication severity by challenging mice with 4 sub-lethal doses of NIMP. We determined that 0.5 LD50 is the threshold dose for NIMP to elicit significant behavioral changes, including facial stereotypes and yawning in the majority of exposed animals, and muscular weakness and thoracic kyphosis in 25% of them. This scale allows objective discrimination of the observed toxic symptomatology. Increasing the dose of NIMP results in more severe signs of intoxication (e.g. tremors, convulsions, eye bulb outgrowth). These symptoms were present in a time-dependent manner and in an increasing number of mice as the dose increased. Nevertheless, the threshold determined with our scale contrasted with some studies using sub-lethal doses of sarin in which no toxic signs were observed below 0.4 and 0.5 LD50 [START_REF] Hulet | The dose-response effects of repeated subacute sarin exposure on guinea pigs[END_REF][START_REF] Oswal | Low-dose sarin exposure produces long term changes in brain neurochemistry of mice[END_REF][START_REF] Scremin | Delayed neurologic and behavioral effects of subtoxic doses of cholinesterase inhibitors[END_REF]. Taking into account species differences in NA intoxication sensitivity, types of NA, and intoxication routes, the dose-response symptomatology established in our study needs to be compared to a sub-cutaneous sarin challenge in mice directly, to confirm NIMP as a valuable sarin analog. However, the intoxication behavior severity score, as determined with our scale, is consistent with the percentage of hippocampal AChE activity inhibition observed at 6 and 24h post NIMP challenge. The most affected animals presented the highest levels of inhibition at both time points. Only the mice exposed to 0.3 LD50 did not show significant cerebral nor blood cholinesterase inhibition compared to control levels. In addition, no intoxication signs were observed in the majority of 0.3 LD50-exposed mice, and less than half of the mice exposed to this dose only showed fasciculation restricted to the injection site. Furthermore, in ECoG recorded animals challenged with 0.3 LD50 of NIMP, no significant modification in brainwave power or distribution was observed; supporting the sub-clinic toxicity at this dose. The three other doses induced significant cerebral AChE activity inhibition, more than 65%, with a culminating 94% inhibition 6 hours after 0.9 LD50 of NIMP exposure. Despite this massive inhibition of AChE activity, no electrical seizure activity was induced by any dose of NIMP, supporting that seizure initiation cannot be explained just by the AChE inhibition levels [START_REF] González | The chemical convulsant diisopropylfluorophosphate (DFP) causes persistent neuropathology in adult male rats independent of seizure activity[END_REF][START_REF] Tonduli | Effects of Huperzine used as pre-treatment against soman-induced seizures[END_REF]. Shortly after NIMP challenge with doses > 0.5 LD50, all frequency ranges of the power spectrum declined. Remarkably, a transient enhancement of delta distribution was observed shortly after NIMP exposure, combined with a compensatory decrease in high theta activity, which lasted several hours and in a dose-dependent manner.

Increase in delta power distribution has been well characterized in different NAexposed animal models, is observed prior to the start of seizure activity, and lasts several hours after the seizure onset [START_REF] Carpentier | Delta activity as an early indicator for soman-induced brain damage: a review[END_REF][START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF][START_REF] Lumley | Dataset of EEG power integral, spontaneous recurrent seizure and behavioral responses following combination drug therapy in soman-exposed rats[END_REF]. In our study, the delta power distribution was significantly increased shortly after the challenge in mice exposed to the two highest doses of NIMP (0.7 and 0.9 LD50), but normalized 6 hours post-intoxication. However, telemetric recordings showed a more sustained increase in delta power distribution for animals exposed to 0.9 LD50 that normalized 48 hours after exposure. The increase in delta power has been linked to the progression of neuropathology after soman induced seizures (Carpentier et al., 2001). Our results emphasize that even a long perturbation (> 5 h) of delta range is not sufficient alone to predict brain histopathology, as no significant neurodegeneration was observed in high sub-lethal doses at any point studied. These results are in line with previous reports on mice exposed to low or mild doses of soman [START_REF] Baille | Acute exposure to a low or mild dose of soman: biochemical, behavioral and histopathological effects[END_REF][START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF]. Consequently, the cholinergic toxicity induced by exposure to sublethal doses of NIMP did not result in neuronal damage, which emphasizes that seizure activity is required to elicit significant neurodegeneration [START_REF] González | The chemical convulsant diisopropylfluorophosphate (DFP) causes persistent neuropathology in adult male rats independent of seizure activity[END_REF].

Therefore, in the case of high sub-lethal exposure, the prevalent distribution of delta rhythms leads to grade 4 EEG abnormalities, which correspond to the unconscious state of animals [START_REF] Guidera | Sevoflurane Induces Coherent Slow-Delta Oscillations in Rats[END_REF].

Nevertheless, ECoG recordings showed long-lasting disruption in low and high theta rhythms following NIMP challenge, including the 0.5 LD50 group. Theta brainwave modification was already reported in mice exposed to a low-dose of soman [START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF]. During the 6 hours post-intoxication, a decrease in theta activity (6-9Hz) is observed, followed by an increase over the 2 next days [START_REF] Crouzier | Disruption of mice sleep stages induced by low doses of organophosphorus compound soman[END_REF]. In this study we have divided the theta rhythm into a low (4-8 Hz) and a high (8-12 Hz) ranges, which, according to us, is a better brainwave representation [START_REF] Buzsáki | Neuronal oscillations in cortical networks[END_REF][START_REF] Buzsaki | Scaling brain size, keeping timing: evolutionary preservation of brain rhythms[END_REF]. This division allowed us to interpret the high theta frequency as the alpha bandwidth found in higher mammal species [START_REF] Buzsaki | Scaling brain size, keeping timing: evolutionary preservation of brain rhythms[END_REF]. In both 0.5 and 0.9 LD50 groups 1-month after NIMP exposure, we observed a significant increase in the low theta band distribution, associated with a compensatory decrease in the high theta rhythm. Similar results were obtained on corresponding bandwidths in rats after exposure to chlorpyrifos, an OP pesticide [START_REF] Timofeeva | EEG spectra, behavioral states and motor activity in rats exposed to acetylcholinesterase inhibitor chlorpyrifos[END_REF]. Theta rhythm is commonly associated with the HF as it predominates in this area, and it is particularly modulated by cholinergic neurons from the medial septum [START_REF] Ma | The Firing of Theta State-Related Septal Cholinergic Neurons Disrupt Hippocampal Ripple Oscillations via Muscarinic Receptors[END_REF].

Considering that in the hippocampus we observed a long lasting AChE activity inhibition after NIMP exposure, it seems consistent that low-theta oscillations are still disrupted one month post-intoxication. Once again, our results underline the similarity between NIMP and sarin, as NIMP is showing the increase in delta and theta frequencies and the decrease in alpha rhythm that were reported on industrial workers accidently exposed to undefined doses of sarin [START_REF] Duffy | Long-term effects of an organophosphate upon the human electroencephalogram[END_REF].

Brainwave power modification could be predictive of cerebral complication, such as an increase of theta rhythm prior to electrical seizure onset [START_REF] Karunakaran | Role of CA3 theta-modulated interneurons during the transition to spontaneous seizures[END_REF]. Although, mice exposed to sub-lethal doses of NIMP did not elicit electrical seizure, the changes in their brainwave activity could be explained by the morphological modifications, i.e., hippocampal swelling, observed 1-week post-exposure. Both delta and theta rhythm abnormalities have been reported in cases of vasogenic edema [START_REF] Fernández-Bouzas | Sources of abnormal EEG activity in the presence of brain lesions[END_REF][START_REF] Fernández-Bouzas | Cerebral blood flow and sources of abnormal EEG activity (VARETA) in neurocysticercosis[END_REF]. Increase in water diffusion in hippocampus has been reported in several magnetic resonance imaging studies, in mice as soon as 3 hours after intoxication with convulsive doses of soman [START_REF] Testylier | Cerebral edema induced in mice by a convulsive dose of soman. Evaluation through diffusion-weighted magnetic resonance imaging and histology[END_REF]. It is still seen increasing 72 hours in rats exposed to seizing dose of DFP [START_REF] Hobson | A magnetic resonance imaging study of early brain injury in a rat model of acute DFP intoxication[END_REF], and is suggested to correlate to neurodegeneration [START_REF] Lee | Quantitative T2 MRI is predictive of neurodegeneration following organophosphate exposure in a rat model[END_REF]. In our study, no neurodegeneration was found and the hippocampal edema was observed in the non-convulsive group (mice exposed to 0.5 LD50 of NIMP). A seizing state could disrupt the blood brain barrier and cause edema [START_REF] Löscher | Structural, Molecular, and Functional Alterations of the Blood-Brain Barrier during Epileptogenesis and Epilepsy: A Cause, Consequence, or Both[END_REF]; however the sub-lethal doses used in our study were not sufficient to induce seizure in mice. So the hippocampal edema observed 7 days post-intoxication could be explained by an increase of astrocyte swelling induced by NF-κB activation as described in astrocyte culture [START_REF] Jayakumar | Activation of NF-kappaB mediates astrocyte swelling and brain edema in traumatic brain injury[END_REF]. A transient increase of NF-κB was associated with the increase of astrocyte reactivity in 0.5 LD50 group, 7 days postintoxication. NF-κB could be rapidly activated in response to cellular aggression and homeostatic changes induced by an increase in pro-inflammatory cytokines (IL-1β and TNF-α), glutamate, and reactive oxygen species [START_REF] Meffert | NF-kappa B functions in synaptic signaling and behavior[END_REF]. Also, sarin surrogates could activate early genes like mitogen activated protein kinase, c-jun Nterminal kinase, and c-fos that modulate the astrocytic response [START_REF] Damodaran | Sarin causes early differential alteration and persistent overexpression in mRNAs coding for glial fibrillary acidic protein (GFAP) and vimentin genes in the central nervous system of rats[END_REF][START_REF] Damodaran | Early differential induction of C-jun in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP)[END_REF][START_REF] Niijima | The effects of sarin-like and soman-like organophosphorus agents on MAPK and JNK in rat brains[END_REF]. Furthermore, a single exposure to 0.5 LD50 of sarin causes a rapid (< 1 h) and persistent (up to 7 days) increase of GFAP in different brain areas of rats [START_REF] Damodaran | Sarin causes early differential alteration and persistent overexpression in mRNAs coding for glial fibrillary acidic protein (GFAP) and vimentin genes in the central nervous system of rats[END_REF]. Compared to our results, this suggests that NF-κB could be another regulating factor of the astrocytic response to CNS injury induced by NA exposure, where the observed increase of TNF-α 24-h post-NIMP exposure could be the triggering event. This hypothesis could be tested using TNF-α inhibitors, and mice exposed to 0.5 LD50 of NIMP. Somehow, the 0.5 LD50 dose of NIMP induced more significant changes than the 0.9 LD50 dose, which could reflect differential microglial and astrocytic phenotype activation states at the studied time points [START_REF] Maupu | Diisopropylfluorophosphate-induced status epilepticus drives complex glial cell phenotypes in adult male mice[END_REF].

During neuroinflammation, microglia are known to be the major source of TNF-α [START_REF] Olmos | Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity[END_REF], consistent with our results showing an increase of IBA-1 labelling 24-h post-NIMP exposure. Although, after exposure to sub-lethal doses of NIMP, mice showed limited neuroinflammation with moderate changes in proinflammatory cytokines (IL-1β, IL-6, VEGF, MIP-1α and TNF-α), which normalized 1 week after intoxication. Only the mice exposed to 0.9 LD50 exhibited a significant persistence of IBA-1 labelling 1-month after NIMP challenge. This is consistent with previous studies showing a transient neuroinflammation after a high dose NA challenge [START_REF] Collombet | Nerve agent intoxication: recent neuropathophysiological findings and subsequent impact on medical management prospects[END_REF]. The remaining neuroinflammation observed at 1-month was suspected to be linked to the delayed neurodegeneration [START_REF] Collombet | Nerve agent intoxication: recent neuropathophysiological findings and subsequent impact on medical management prospects[END_REF], however in our study no histopathological damage was apparent in NIMP-exposed mice, even after long-term observation. Further studies will be needed to understand the mechanism supporting the microglial activation persistence, which could elicit long-term deleterious effects, as it impacts neurogenesis, synaptic pruning [START_REF] Sierra | Surveillance, phagocytosis, and inflammation: how never-resting microglia influence adult hippocampal neurogenesis[END_REF], and promotes neurodegeneration [START_REF] Maqbool | Sustained, neuron-specific IKK/NF-κB activation generates a selective neuroinflammatory response promoting local neurodegeneration with aging[END_REF].

Hippocampal cholinergic innervation plays an important role in cognitive and memory processes, specifically in NORT in which hippocampal ACh efflux, α7 nicotinic cholinergic receptors (nAChRs), and muscarinic cholinergic receptors play crucial roles [START_REF] Jacklin | Evidence for a specific role for muscarinic receptors in crossmodal object recognition in rats[END_REF][START_REF] Li | α7nAchR/NMDAR coupling affects NMDAR function and object recognition[END_REF][START_REF] Stanley | Hippocampal neurotransmitter efflux during one-trial novel object recognition in rats[END_REF]. Considering that 1-month post-NIMP exposure the cholinesterase activity in the hippocampus is still not completely restored, alterations in the cholinergic pathway could explain the performance decrement of both groups of NIMP-exposed mice. Also, it could be interesting to investigate if α7 nAChRs are not downregulated, as they are expressed in microglia and are anti-inflammatory [START_REF] Rahman | Glial mechanisms underlying major depressive disorder: Potential therapeutic opportunities[END_REF]. Poor performance in NORT after chronic DFP in rats has already been reported [START_REF] Terry | Repeated, intermittent exposures to diisopropylfluorophosphate in rats: protracted effects on cholinergic markers, nerve growth factorrelated proteins, and cognitive function[END_REF], but our results emphasize that a single exposure to a sub-lethal dose of NIMP, which could be almost asymptomatic, is enough to disrupt short-term memory. Furthermore, low theta and high theta brainwave distributions are still disrupted 1-month after NIMP exposure in both groups; while in rats, theta oscillations exert a crucial role in coordination with gamma oscillations for the encoding and retrieval of memories during NORT [START_REF] Trimper | Recognition memory and theta-gamma interactions in the hippocampus[END_REF]. Again, our results highlight that in addition to delta rhythm, low and high theta oscillations could be used as biomarkers of sub-lethal NA intoxication and their longterm disruption could be predictive of cognitive complications.

In conclusion, exposure to sub-lethal NA doses represent a high probability of occurrence in the event of a terrorist attack or a military conflict. Our results have emphasized that brainwave distribution is disrupted quickly after NIMP exposure, persists for a long time, and could be used as biomarker of intoxication severity and a helpful tool for the prognosis of possible brain damage. Monitoring electrical brain activity should be implemented in the follow-up of exposed victims, even if they did not show obvious signs of intoxication. Historically, sarin has been the most used chemical weapon and still represents a threat. Here, we confirmed NIMP as a potential surrogate of sarin intoxication in mice and as helpful tool to investigate long-term NA effects. (n=14); 3.14 ± 0.27 for 0.5 LD50 (n=14); 6.45 ± 0.54 for 0.7 LD50 (n=11) and 9.11 ± 0.54 for 0.9 LD50 (n=18) (Kruskal-Wallis tests followed by Dunn's post-tests to compare each dose with surgery and without surgery). (C) Inhibition of AChE activity at 6h (100 ± 6.4 for 0 LD50; 93.44 ± 5.7 for 0.3 LD50; 31.7 ± 5.3 for 0.5 LD50; 13.2 ± 2.5 for 0.7 LD50 and 6.1 ± 0.5 for 0.9 LD50) and 24h (100 ± 8.7 for 0 LD50; 81.7 ± 3.0 for 0.3 LD50; 35.3 ± 5.3 for 0.5 LD50; 15.7 ± 1.5 for 0.7 LD50 and 9.7 ± 1.0 for 0.9 LD50) after NIMP exposure (n=8 per group). Significant differences were determined by one-way ANOVA (F = 59.54 1.21 ± 0.03 for 0.5 LD50 and 1.20 ± 0.04 for 0.9 LD50 and 1-month post-intoxication: 1.0 ± 0.04 for 0 LD50; 0.99 ± 0.04 for 0.5 LD50 and 1.04 ± 0.02 for 0.9 LD50 (n=5 per group).

Significant differences were determined by Kruskal-Wallis tests (p=0.004 and 0.63 for

  PRISM 7 software (GraphPad, U.S.A.). Statistical tests and sample sizes are indicated in figure legends and figures respectively. Results are presented as mean ± SEM. Significance between groups are denoted by * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001.

Figure 1 :

 1 Figure 1: Intoxication severity evaluation. (A) Representation of the experimental

Figure 2 :

 2 Figure 2: Short term cortical activity modifications. (A) Representation of the

  p<0.0001 for 6h and F = 41.49 p<0.0001 for 24h) with Dunnett's post hoc tests to compare to CTL group (p<0.0001). (D) Examples of non-filtered ECoG signal during the baseline (left) and 1h post-0.9LD50 NIMP exposure recording. (E) Power spectrum representations for each tested dose of NIMP over the 24 hours period. In insert, the area under the curve (AUC) is represented (9.4 x10 4 ± 1.9 x10 4 for 0 LD50 (n=13); 9.8 x10 4 ± 1.6 x10 4 for 0.3 LD50 (n=14); 4.5 x10 4 ± 1.0 x10 4 for 0.5 LD50 (n=14); 4.0 x10 4 ± 1.1 x10 4 for 0.7 LD50 (n=11) and 3.3 x10 4 ± 0.9 x10 4 for 0.9 LD50 (n=18)). Significant differences were determined by one-way ANOVA (F = 5.0 p=0.0017) with Dunnett's post hoc tests to compare to CTL group (0 LD50 vs. 0.5 LD50 p=0.04; 0 LD50 vs. 0.7 LD50 p=0.03; 0 LD50 vs. 0.9 LD50 p=0.01). (F) Periodograms representation for each tested dose of NIMP over the 24 hours period. (G) Diagrams of each brainwave relative power changes at different time periods that follow NIMP exposure, during the 24 hours of recording. Significant differences were determined by two-way ANOVA repeated measure (Delta: Fdose = 4.6 p=0.003; Low theta: Fdose = 1.5 p=0.2; High theta: Fdose = 22.2 p=0.003; Low beta: Fdose = 23.9 p=0.001; High beta: Fdose = 24.3 p=0.01) with Dunnett's post hoc tests to compare to CTL group. (H) Diagrams of each brainwave distribution changes at different time periods that follow NIMP exposure, during the 24 hours of recording. Significant differences were determined by two-way ANOVA repeated measure (Delta: Ftime = 22.1 p<0.0001, Fdose = 4.6 p=0.003; Low theta: Ftime = 18.5 p<0.0001, Fdose = 0.7 p=0.6; High theta: Ftime = 33.7 p<0.0001, Fdose = 8.5 p<0.0001) with Dunnett's post hoc tests to compare to CTL group or to baseline.

Figure 3 :

 3 Figure 3: Cortical activity modifications at longer term. (A) Representation of the

Figure 4 :

 4 Figure 4: Neuroanatomical modifications induced by exposition to sub-lethal

Acknowledgments

This work used the imagery and biological analysis platforms of IRBA supported by the Service de Santé des Armées. We particularly thank Xavier Butigieg and Cédric Castellerin for technical support.

Funding sources

This work was supported by the French Ministry of Armed Forces: Direction Générale de l'Armement (DGA) and Service de Santé des Armées (SSA). Funding sources had no involvement in study design, collection, analysis or interpretation of data, or decision to publish.

with CTL values at 24 hours post-intoxication [(Il-6: 1.0 ± 0.10 for 0 LD50; 1.2 ± 0.05 for 0.5 LD50 and 1.13 ± 0.24 for 0.9 LD50); (Il-1β: 1.0 ± 0.09 for 0 LD50; 1.35 ± 0.11 for 0.5 LD50 and 1.02 ± 0.19 for 0.9 LD50); (TNF-α: 1.0 ± 0.19 for 0 LD50; 1.85 ± 0.20 for 0.5 LD50 and 1.27 ± 0.30 for 0.9 LD50); (VEGF: 1.0 ± 0.13 for 0 LD50; 2.05 ± 0.36 for 0.5 LD50 and 1.35 ± 0.33 for 0.9 LD50)] and 7 days post-intoxication [(Il-6: 1.0 ± 0.13 for 0 LD50; 0.87 ± 0.10 for 0.5 LD50 and 1.06 ± 0.13 for 0.9 LD50); (Il-1β: 1.0 ± 0.18 for 0 LD50; 0.75 ± 0.09 for 0.5 LD50 and 0.82 ± 0.08 for 0.9 LD50); (TNF-α:1.0 ± 0.21 for 0 LD50; 0.81 ± 0.11 for 0.5 LD50 and 1.07 ± 0.06 for 0.9 LD50); (VEGF: 1.0 ± 0.26 for 0 LD50; 1.08 ± 0.18 for 0.5 LD50 and 1.49 ± 0.26 for 0.9 LD50)] (n=8 per group). Significant differences were determined by Kruskal-Wallis tests (p=0.03 and 0.04 for TNF-α and VEGF respectively at 24h post-intoxication) followed by Dunn's post-tests to compare to CTL. to CTL at 24 hours: 1.0 ± 0.18 for 0 LD50; 1.62 ± 0.14 for 0.5 LD50 and 1.06 ± 0.26 for 0.9 LD50 and 7 days post-intoxication: 1.0 ± 0.3 for 0 LD50; 1.48 ± 0.37 for 0.5 LD50 and 2.16 ± 0.43 for 0.9 LD50 (n=8 per group). Statistical differences were determined by Kruskal-Wallis test (p=0.12 and 0.25 for 24h and 7D respectively). (C) Diagram of IBA1 labelling optical density percentage normalized to CTL at 24 hours post-intoxication: 1.0 ± 0.20 for 0 LD50; 2.24 ± 0.59 for 0.5 LD50 and 4.39 ± 1.40 for 0.9 LD50; 7 days post-intoxication:

1.0 ± 0.14 for 0 LD50; 2.13 ± 0.37 for 0.5 LD50 and 2.53 ± 1.09 for 0.9 LD50; and 1-month post-intoxication: 1.0 ± 0.07 for 0 LD50; 1.06 ± 0.23 for 0.5 LD50 and 1.67 ± 0.25 for 0.9 LD50 (n=5 per group). Significant differences were determined by Kruskal-Wallis tests Baseline: 72.37 ± 3.88% for 0 LD50 (n=15); 73.37 ± 3.46% for 0.5 LD50 (n=15) and 72.39 ± 3.95 for 0.9 LD50 (n=17); and 1-month post-intoxication: 69.06 ± 3.63% for 0 LD50; 58.23 ± 2.35% for 0.5 LD50 and 54.69 ± 2.31 for 0.9 LD50. Significant differences were determined by two-way ANOVA repeated measure (Ftime = 19.44 p<0.0001, Fdose = 4.2 p=0.10) with Bonferroni's post hoc tests to compare to baseline (marked with #; p=0.002 and p=0.0004 for 0.5 LD50 and 0.9 LD50 respectively) or Dunnet's post hoc tests to compare to CTL group (p=0.06 and p=0.007 for 0.5 LD50 and 0.9 LD50 respectively at 1month post-intoxication).