Cesar Fortes-Lima

Paul Verdu
email: paul.verdu@mnhn.fr

Anthropological Genetics Perspectives on the Transatlantic Slave Trade

Keywords: Population Genetics, Anthropology, Admixture, Slave Trade, Paleogenomics

published or not. The documents may come

Between the 15th and the 19th century, more than 12 million Africans were enslaved and forcibly removed from their homeland to be deported to the Americas and Europe [START_REF] Eltis | Routes to Slavery: Direction, Ethnicity and Mortality in the Transatlantic Slave Trade[END_REF]. The vast majority of them were deported to South America and the Caribbean (around 43% and 52%, respectively), a minority (around 5%)

to North America, and only a reduced fraction (< 1%) to mainland Europe [START_REF] Eltis | Atlas of the Transatlantic Slave Trade[END_REF].

Enslaved Africans were taken from African slaving regions that stretched thousands of miles, from present-day Senegal to Angola and, around the coast, Mozambique and Madagascar. According to historical sources [START_REF] Eltis | Atlas of the Transatlantic Slave Trade[END_REF][START_REF] Eltis | The Cambridge World History of Slavery: Volume 4, AD 1804-AD 2016[END_REF], from 1501 to 1867, enslaved Africans were forcibly embarked mainly from eight coastal regions (Figure 1): 5.7% embarked from Senegambia, 3.2% from Sierra Leone, 2.7% from the Windward Coast, 9.6% from the Gold Coast, 16.1% from the Bight of Benin, 12.3% from the Bight of Biafra, 46.3% from West Central Africa, and 4.1% from Southeast Africa. The West Central African coast was thus the largest slaving and embarkation region throughout most of the TAST, focused mainly South of the Congo river. The Gold Coast, the Bight of Benin, and the Bight of Biafra became increasingly prominent slaving and embarkation regions after the mid-17 th century, as the TAST expanded with Plantation Economy in the Americas (Figure 1).

During more than 300 years, slavers dehumanized their human cargo with a profusion of organized psychological and physical abuses and violence. They forbade slaves to use their name, language, and religion, repeatedly broke-up families, and, with the crossing of the Atlantic Ocean and extensive migrations within the Americas, severed all possible roots between the captives and their original homeland, culture, and people [START_REF] Berlin | Many Thousands Gone: The First Two Centuries of Slavery in North America. Many Thousands Gone: The First Two Centuries of Slavery in North America[END_REF]. As a direct and dreadful consequence, enslaved-African descendants throughout the Americas have little knowledge of their origins in continental Africa; a gap that can only partially be filled with historical sources [START_REF] Eltis | Routes to Slavery: Direction, Ethnicity and Mortality in the Transatlantic Slave Trade[END_REF].

Indeed, historians investigated marine and customs manifests, commercial and tax records and deeds, and other sources to reconstruct TAST routes, possible African ethnic origins of deportees (Figure 1), and, in some rare instances, the genealogies of enslaved-African descendants in the Americas [START_REF] Eltis | Atlas of the Transatlantic Slave Trade[END_REF].

Nevertheless, due to imprecise, missing, or falsified records, the prominence of illegal slave-trade which leaves scarce written traces, and the difficulties to transpose ethnic and linguistic categories used during the TAST era into present-day ethnic categories, large parts of the history of enslaved-African descendants remain obscure [START_REF] Eltis | Routes to Slavery: Direction, Ethnicity and Mortality in the Transatlantic Slave Trade[END_REF]. In this historical context that radically changed the cultural and genetic landscape of the Americas (6,7), molecular and population genetics since the mid-20 th century offered new tools to help reconstruct the migrations and demographic histories of enslaved-African descendant populations.

In this review, we examine population genomics articles published over the past 15 years and focusing on the genetic history of enslaved-African descendant communities in the Americas today. The genetic determination of phenotypes and disease risks have been widely studied in connection to TAST

U N C O R R E C T E D M A N U S C R I P T 4
migrations and admixture in the Americas [START_REF] Mahajan | Trans-ancestry genetic study of type 2 diabetes highlights the power of diverse populations for discovery and translation[END_REF][START_REF] Wojcik | Genetic analyses of diverse populations improves discovery for complex traits[END_REF][START_REF] Adhikari | A GWAS in Latin Americans highlights the convergent evolution of lighter skin pigmentation in Eurasia[END_REF], and the subject of several major reviews [START_REF] Adhikari | The Genetic Diversity of the Americas[END_REF][START_REF] Shriver | Skin pigmentation, biogeographical ancestry and admixture mapping[END_REF][START_REF] Winkler | Admixture mapping comes of age[END_REF].

Instead, we chose to focus primarily here on the complex interactions between genetic inferences about populations' origins, migrations, and admixture histories, and the cultural, anthropological, and historical contexts during and after the TAST era.

Origins of enslaved-African descendants inferred with genetics

Since the 1950s, anthropological genetics research investigated the links between genetic variation patterns observed today across Africa, Europe, and the Americas, and those observed in enslaved-African descendant communities in the Americas, to narrow their possible geographic origins from either continent. To do so, patterns of shared genetic ancestry between present-day enslaved-African descendant communities and worldwide populations were inferred using population genetics methods and, at first, markers such as blood groups, HLA, or uniparentally-inherited mitochondrial DNA and/or Y-chromosome markers [START_REF] Chakraborty | Gene admixture in human populations: Models and predictions[END_REF][START_REF] Fortes-Lima | Revisiting genetic ancestry in African Diaspora communities from Atlantic South America[END_REF][START_REF] Salas | Downloaded from[END_REF].

With the advances of molecular genetics at the genome-wide scale since the 2000s, and with the developments of novel bioinformatics methods, reconstructing the genetic origins of enslaved-African descendants has reached unprecedented resolution [START_REF] Micheletti | Genetic Consequences of the Transatlantic Slave Trade in the Americas[END_REF][START_REF] Bryc | Genome-wide patterns of population structure and admixture in West Africans and African Americans[END_REF][START_REF] Ongaro | The Genomic Impact of European Colonization of the Americas[END_REF]. These studies have shown that virtually all such populations are genetically admixed and share recent genetic ancestries with European, African, and Native American populations today. Furthermore, geneticists have revealed the tremendous variability of genomic admixture patterns across enslaved-African descendant populations, each echoing the specific history of slavery and European colonization they experienced.

As an illustration of a genome-wide study, Patin et al. [START_REF] Patin | Dispersals and genetic adaptation of Bantu-speaking populations in Africa and North America[END_REF] investigated the African genetic origins of US self-reported enslaved-African descendants from three large-scale cohorts totalling 5244 participants from four states. Compared to genetic variation from 1955 individuals from 14 populations living in former slaving regions of Africa's Atlantic shores, they found that US participants had, on average, 75% of African genetic ancestry, and 25% of (mostly) European genetic ancestry [START_REF] Patin | Dispersals and genetic adaptation of Bantu-speaking populations in Africa and North America[END_REF]. Furthermore, the identified African genetic components shared a recent common ancestry, on average, for 13% with Senegambian populations today, 7% with those from the Windward Coast, 50% with those from the Bight of Benin, and up to 30%

with those from present-day Gabon and Angola, thus reflecting the highly diverse origins of African populations having contributed to the gene-pool of enslaved-African descendants in the USA.

Furthermore, population geneticists identified important variation in the African genomic ancestry across Brazil [START_REF] Hünemeier | Niger-Congo speaking populations and the formation of the Brazilian gene pool: mtDNA and Ychromosome data[END_REF][START_REF] Gonçalves | The phylogeography of African Brazilians[END_REF], a country that received the majority (~70%; ~4.8 million) of all enslaved-Africans deported to continental Latin America during the TAST (Figure 1). African ancestries ranged, on average, from 14.7% in the inland South-East of Brazil, and mainly originated from the South-West and South-East of Africa, to 50.8% in the North-East of the country (Figure 2), with West African origins more frequent in this later region [START_REF] Kehdy | Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations[END_REF][START_REF] Gouveia | Origins, Admixture Dynamics, and Homogenization of the African Gene Pool in the Americas[END_REF]. This further illustrated how the genomic heritage of enslaved-African descendants in the Americas can also vary within a country.

U N C O R R E C T E D M A N U S C R I P T 5
With similar population genomics approaches, other studies have identified variable African admixture patterns in numerous enslaved-African descendant populations throughout the Americas (Figure 2), with, for instance, around 89% of African ancestry in enslaved-African descendants from Jamaica [START_REF] Mathias | A continuum of admixture in the Western Hemisphere revealed by the African Diaspora genome[END_REF], 88% in Barbados [START_REF] Martin | Human Demographic History Impacts Genetic Risk Prediction across Diverse Populations[END_REF], 78% in Peru [START_REF] Harris | Evolutionary genomic dynamics of Peruvians before, during, and after the Inca Empire[END_REF], 77% in Choco (Colombia) [START_REF] Conley | A Comparative Analysis of Genetic Ancestry and Admixture in the Colombian Populations of Chocó and Medellín[END_REF][START_REF] Fortes-Lima | Genome-wide Ancestry and Demographic History of African-Descendant Maroon Communities from French Guiana and Suriname[END_REF], 71% in Rio de Janeiro (Brazil) [START_REF] Fortes-Lima | Genome-wide Ancestry and Demographic History of African-Descendant Maroon Communities from French Guiana and Suriname[END_REF], 70% in Saint Vincent [START_REF] Torres | Analysis of biogeographic ancestry reveals complex genetic histories for indigenous communities of St. Vincent and Trinidad[END_REF], and 38% in eastern Cuba (29); each population being also highly variable in relative contributions from each continental African regions.

During the TAST era, enslaved Africans actively mobilized to create systems of resistance against the inhuman repression that accompanied slavery, and escaped captivity, a phenomenon often called marronnage. This is characterized by massive escapes of slaves who then grouped in small and independent communities of free-people, known as Maroon communities, who developed specific linguistic, political, religious, and domestic traditions [START_REF] Price | Maroon Societies: Rebel Slave Communities in the Americas[END_REF][START_REF] Price | Flight to Freedom: African Runaways and Maroons in the Americas. Flight to Freedom: African Runaways and Maroons in the Americas[END_REF]. Genomics investigations of the history of Maroon communities remain overall scarce [START_REF] Brucato | The genetic diversity of three peculiar populations descending from the slave trade: Gm study of Noir Marron from French Guiana[END_REF][START_REF] Brucato | The imprint of the Slave Trade in an African American population: mitochondrial DNA, Y chromosome and HTLV-1 analysis in the Noir Marron of French Guiana[END_REF]. However, Fortes-Lima et al. [START_REF] Fortes-Lima | Genome-wide Ancestry and Demographic History of African-Descendant Maroon Communities from French Guiana and Suriname[END_REF] recently investigated four Noir Marron communities from French Guyana and Surinam. The results highlighted high levels of genomic inbreeding, high (~98%) African genetic ancestry originated mainly from the Bight of Benin (Figure 3A-B), and more precisely from Gbe-speaking populations in Benin [START_REF] Essegbey | Cross-linguistic influence in language creation: Assessing the role of the Gbe languages in the formation of the Creoles of Suriname[END_REF][START_REF] Muysken | Surviving the Middle Passage: The West Africa-Surinam Sprachbund. Surviving the Middle Passage: The West Africa-Surinam Sprachbund[END_REF], and reduced Native American and European ancestries, altogether resulting from more than 350 years of genetic and cultural isolation in the South-American rainforest [START_REF] Price | Les Marrons. Les Marrons[END_REF].This relatively simple admixture pattern, with apparently very few different African source populations, substantially differs from those observed in numerous other enslaved-African descendant populations in the Americas who could not escape bondage and often show genomic contribution from multiple African sources as well as substantial admixture with European and Native American populations [START_REF] Micheletti | Genetic Consequences of the Transatlantic Slave Trade in the Americas[END_REF][START_REF] Ongaro | The Genomic Impact of European Colonization of the Americas[END_REF][START_REF] Gouveia | Origins, Admixture Dynamics, and Homogenization of the African Gene Pool in the Americas[END_REF].

Beyond the African origins of enslaved-African descendant populations, population geneticists also identified numerous populations with significant three-way admixture between African, European, and Native American sources. Interestingly, European source populations have been shown to have contributed differently to the gene-pools of enslaved-African descendant populations [START_REF] Ongaro | The Genomic Impact of European Colonization of the Americas[END_REF], in possible relation with each

European emigration policies and preferred TAST routes [START_REF] Eltis | Routes to Slavery: Direction, Ethnicity and Mortality in the Transatlantic Slave Trade[END_REF]. Enslaved-African descendant populations in Spanish or Portuguese former colonies in the Americas share the majority of their European common genetic ancestry with the Iberian Peninsula rather than with Northern-Western Europe, while the opposite is observed in enslaved-African descendants from the former British colonial empire [START_REF] Micheletti | Genetic Consequences of the Transatlantic Slave Trade in the Americas[END_REF][START_REF] Fortes-Lima | Genome-wide Ancestry and Demographic History of African-Descendant Maroon Communities from French Guiana and Suriname[END_REF][START_REF] Moreno-Estrada | Reconstructing the population genetic history of the Caribbean[END_REF]. Consistently, enslaved-African descendant populations in locations having experienced changes of European empire dominion, such as some Caribbean islands, are sometimes found to bear more variable signatures of admixture from different European regions.

Finally, Native American populations have been dramatically impacted by European colonization, which triggered substantial levels of genetic admixture with African deported populations via joint enslavement and social marginalization, and/or joint resistance and escape from European domination in Maroon communities [START_REF] Mathias | A continuum of admixture in the Western Hemisphere revealed by the African Diaspora genome[END_REF][START_REF] Torres | Analysis of biogeographic ancestry reveals complex genetic histories for indigenous communities of St. Vincent and Trinidad[END_REF]29,[START_REF] Moreno-Estrada | Reconstructing the population genetic history of the Caribbean[END_REF]. Nevertheless, as Native American populations have suffered dramatic demographic reductions during European colonization, population geneticists have faced major challenges to

U N C O R R E C T E D M A N U S C R I P T 6
reconstruct precisely their contribution to the genetic landscape of enslaved-African descendant populations, because genetic datasets from Native American populations relatively unaffected by post-Columbus admixture were largely lacking until recently [START_REF] Posth | Reconstructing the Deep Population History of Central and South America[END_REF][START_REF] Moreno-Mayar | Early human dispersals within the Americas[END_REF][START_REF] Nägele | Genomic insights into the early peopling of the Caribbean[END_REF].

Reconstructing the admixture histories of enslaved-African descendants with genetics

Several population genetics methods have been developed during the past 10 years to infer, from genomic data only, how many recurring or more temporary events of admixture, and their associated timings and respective intensities, have shaped the observed genetic patterns in admixed populations today [START_REF] Fortes-Lima | Complex genetic admixture histories reconstructed with Approximate Bayesian Computations[END_REF][START_REF] Hellenthal | A genetic atlas of human admixture history[END_REF][START_REF] Gravel | Population genetics models of local ancestry[END_REF].

Applied to enslaved-African descendant populations in the Americas, results could then, a posteriori, be compared to historical sources to better understand which major historical events and socio-cultural changes, during or after the TAST era, significantly influenced genomic patterns, and propose possible explanatory mechanisms.

For instance, an unprecedentedly detailed investigation of genomic patterns in enslaved-African descendant communities in the US, analysed together with extensive demographic data in an interdisciplinary framework, highlighted that admixture between European and African descendant populations likely occurred during at least two major periods [START_REF] Baharian | The Great Migration and African-American Genomic Diversity[END_REF]. The first admixture period likely occurred during the Plantation Economy era (~1700s-1850s) mainly in the Southern part of the country, before the US civil war. The second admixture period occurred later, during the early 20 th century, after the massive Southto-North migration of emancipated-slave descendant communities. These results thus showed that slavery and its abolition did not explain, alone, genetic admixture patterns [START_REF] Micheletti | Genetic Consequences of the Transatlantic Slave Trade in the Americas[END_REF][START_REF] Dai | Population Histories of the United States Revealed through Fine-Scale Migration and Haplotype Analysis[END_REF][START_REF] Han | Clustering of 770,000 genomes reveals post-colonial population structure of North America[END_REF]. Indeed, admixture in the US occurred first long before the abolition of slavery, most often as a result of sexual coercion of slave-owners and despite the "Code Noir" and other laws forbidding intermarriages between communities; and roughly two generations after the abolition of slavery, indirectly as a result of migrations and changes in intercommunity relationships, despite the persistence of segregation laws until the late 20 th century.

Furthermore, African and European emigration waves throughout the Americas have been massive long after the TAST, during the late 19 th and throughout the 20 th century [START_REF] Eltis | Routes to Slavery: Direction, Ethnicity and Mortality in the Transatlantic Slave Trade[END_REF][START_REF] Berlin | Many Thousands Gone: The First Two Centuries of Slavery in North America. Many Thousands Gone: The First Two Centuries of Slavery in North America[END_REF]. This led to major social changes among communities and, for instance in Brazil, population geneticists proposed that it likely substantially increased the relative amount of European-derived genomic ancestry in the general population [START_REF] Ongaro | The Genomic Impact of European Colonization of the Americas[END_REF][START_REF] Kehdy | Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations[END_REF][START_REF] Gouveia | Origins, Admixture Dynamics, and Homogenization of the African Gene Pool in the Americas[END_REF]. Altogether, these results highlight the importance of specific socio-cultural relationships among enslaved and non-enslaved descendant communities for shaping genomic diversity patterns of enslaved-African descendant populations, both during and long after the TAST era (Figure 2).

More generally, together with most references cited above, population geneticists identified discrepancies between the TAST demographic history and the expected resulting genetic patterns in enslaved-African descendant populations. Historians have demonstrated that African males represented the vast majority of deported-slaves compared to females, as a consequence of the massive demand for an exhausting-labour force for which males were thought to be better adapted than females (2). Nevertheless, geneticists found very strong biases towards European males reproducing with African females that shaped

U N C O R R E C T E D M A N U S C R I P T
admixture patterns in enslaved-African descendant communities [START_REF] Bryc | Genome-wide patterns of population structure and admixture in West Africans and African Americans[END_REF][START_REF] Fortes-Lima | Genome-wide Ancestry and Demographic History of African-Descendant Maroon Communities from French Guiana and Suriname[END_REF]. Indeed, socio-cultural discriminations, prejudice, and segregation, often enforced legally in particular after the expansion of Plantation Economy, resulted, throughout and after the TAST era, in a strong reproductive isolation between enslaved and non-enslaved communities. In a largely patriarchal and patrilocal context, this resulted in extremely limited signatures of non-slave females having reproduced with slave males, while non-slave males reproducing with slave females, often coercively as demonstrated by historians, could be more often tolerated by the socio-politically dominant European-descendant communities [START_REF] Kennedy | Interracial Intimacies: Sex, Marriage, Identity, and Adoption. Interracial Intimacies: Sex, Marriage, Identity, and Adoption; Vintage Books Edition[END_REF]. While this pattern is qualitatively ubiquitous during the TAST, substantial differences in the intensity of this sex-biased admixture across enslaved-African descendant populations are observed throughout the Americas [START_REF] Adhikari | The Genetic Diversity of the Americas[END_REF][START_REF] Ongaro | The Genomic Impact of European Colonization of the Americas[END_REF][START_REF] Mathias | A continuum of admixture in the Western Hemisphere revealed by the African Diaspora genome[END_REF].

Socio-cultural relationships inherited from the TAST era influence genetic patterns and interpretations

Overall, complex cultural processes resulted in variable forms of reproductive isolation between enslaved and non-enslaved communities during and after the TAST across the Americas. These local barriers to panmixia depended largely on the legal and societal categorization of individuals in either community. In addition, these categories have changed over time when different legislations were established, such as the "Code Noir" in North America, the "Sistema de Castas" in Latin America, and segregation laws during colonial and post-colonial eras after the TAST. Over time, these shifting societal barriers influenced, in turn, identity-constructions and mate choices, further enhancing cultural and reproductive isolation and determining the social status of admixed individuals.

These community constructions are, to some extent, reflected in individual self-report categorization in national population census questionnaires. Among 23 continental American countries, the last census questionnaires record up to four questions about self-reported indigenous, ethnic, linguistic, or racial identity (only four countries use explicitly the word "race" or its literal translation). Apart from French Guyana where no self-categorizations are recorded, the census questionnaires' categories are based on self-reported imprecise skin-color criteria, genealogical ancestry and cultural affinities, linguistic practices, or mixtures of these criteria. 14 countries out of 22 propose a category based on self-reported African descent, seven of which propose the word "Black" (or its translation) as an alternative naming for the same category, and three of 14 which distinguish the "Afro-descendant" category from the "Black" category. Additionally, four countries among the 22 consider only a "Black" category without explicit reference to "Africa".

Furthermore, eight countries out of the 22 propose a separate category for "explicitly" admixed individuals of African descent ("Mulata/o", "Creole" or "Mixed"), two countries have a separate "Brown" category, and three countries gather some or all these labels under the broader "Afro-descendant" category. In this complex categorization patchwork, population geneticists showed that self-perception of ancestry and self-reported identities overlapping national census categories are often at odds with individuals' genomic ancestries and admixture patterns [START_REF] Ruiz-Linares | Admixture in Latin America: geographic structure, phenotypic diversity and self-perception of ancestry based on 7,342 individuals[END_REF][START_REF] Lins | Genetic heterogeneity of self-reported ancestry groups in an admixed Brazilian population[END_REF][START_REF] Lima-Costa | Genomic ancestry and ethnoracial self-classification based on 5,871 community-dwelling Brazilians (The Epigen Initiative)[END_REF]. This is due to the fact that self-constructed cultural identities emerge from multiple familial and societal experiences superimposing self-perceived phenotypic features. This process is

U N C O R R E C T E D M A N U S C R I P T 8
thus much more complex than simple mendelian inheritance which only reflects a very specific (genetic) part of an individual's history, furthermore only very partially translated into complex phenotypic features such as skin-color [START_REF] Race | Ethnicity, and Genetics Working Group (2005) The Use of Racial, Ethnic, and Ancestral Categories in Human Genetics Research[END_REF].

Therefore, it is often difficult to compare average African genomic ancestries across population genetics studies (Figure 2), mainly due to the heterogeneity of inclusion criteria of sampled individuals [START_REF] Micheletti | Genetic Consequences of the Transatlantic Slave Trade in the Americas[END_REF].

Indeed, some studies consider cohorts sampled from the general population with inclusion criteria based on geography, and/or medical or socio-economic status, rather than self-reported ancestral origins or census categories [START_REF] Kehdy | Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations[END_REF]29). Other studies focus instead on groups of individuals from census categories in relation to African descent [START_REF] Gonçalves | The phylogeography of African Brazilians[END_REF][START_REF] Baharian | The Great Migration and African-American Genomic Diversity[END_REF][START_REF] Tishkoff | The Genetic Structure and History of Africans and African Americans[END_REF], shown above to possibly designate different populations with different demographic histories across the Americas. In general, whether participating individuals have known genealogical relationships with enslaved-Africans during the TAST, with post-slavery African-immigrants, with admixed populations, or categorized as "Afro-descendants" based on phenotypic features only, remains too often undiscussed in population genetics studies.

Furthermore, lack of anthropological information about participants' inclusion criteria can introduce possible confounding factors in genome-wide association studies (GWAS) due to cryptic genetic stratification, even when elaborated bio-statistical approaches allow to correct for genetic-relatedness heterogeneities in case and control samples [START_REF] Parra | Implications of correlations between skin color and genetic ancestry for biomedical research[END_REF][START_REF] Via | Recent advances of genetic ancestry testing in biomedical research and direct to consumer testing[END_REF]. Finally, investigations of differential adaptation and natural selection pressures signatures in the genomes of enslaved-African descendant populations may also be affected by confusions in individual categorization, as groups of individuals with, in fact, largely differing demographic and admixture histories may a priori be gathered in a single target population [START_REF] Patin | Dispersals and genetic adaptation of Bantu-speaking populations in Africa and North America[END_REF].

Altogether, the genetic patterns of enslaved-African and non-enslaved descendant communities in the Americas are highly variable across the continent. They depend on the genetic origins of the founding individuals, on the genetic origins of subsequent migration waves into each community and socio-cultural processes of assimilation or rejection of the new migrants, and on admixture processes and inter-community relationships regarding barriers to reproduction and their prevalence in the societies, during and long after the TAST. It is thus crucial to consider the detailed local historical and anthropological contexts to elucidate the evolutionary history of enslaved-African descendant communities.

TAST history research entering the paleogenomics era

Archaeology of the slave-trade has often been described as an "archaeology of the invisible", as, by definition, slaves' material possessions were scarce, numerous sites related to slaves' history (housing, cells or transit centres, etc.) were not primarily built to withstand the erosion of time or have been actively destroyed during history, and as the memory of slaves' burial-sites has often been lost [START_REF] Araujo | Shadows of the Slave Past: Memory, Heritage, and Slavery. Shadows of the Slave Past: Memory, Heritage, and Slavery; Routledge Studies in Cultural History[END_REF][START_REF] Haviser | African sites: archaeology in the Caribbean. African sites: archaeology in the Caribbean[END_REF][START_REF] Blakey | Bioarchaeology of the African Diaspora in the Americas: Its Origins and Scope[END_REF][START_REF] Blakey | Archaeology under the Blinding Light of Race[END_REF]. Nevertheless, bioarchaeological investigations, together with isotopic and paleopathological methods, contributed invaluably to our understanding of the TAST era, questioning the conditions of life of slaves, reconstructing historical migrations, the socio-economic history of slaves amidst slave-societies, and the health status in Maroon or enslaved communities [START_REF] Schroeder | Trans-Atlantic slavery: isotopic evidence for forced migration to Barbados[END_REF].

U N C O R R E C T E D M A N U S C R I P T 9
Analogously to archaeology, paleogenomics, the study of ancient DNA (aDNA), focusing on remains linked to the TAST, often face inextricable difficulties, particularly in tropical environments. Indeed, DNA often preserves poorly under intense seasonal climatic change and reduced protection of skeletal remains, which is often the case for slave burial-sites in the Caribbean and the Americas. Nevertheless, aDNA analyses may yield invaluable information, as it provides direct insights into the genetic reality of populations in the past; contrarily to modern-DNA which only provides incomplete information about the genealogical histories underlying genetic patterns observed today. In this context, the technical improvements of paleogenomics over the last decade brought new promising results about the history of enslaved populations during the TAST [START_REF] Carpenter | Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries[END_REF][START_REF] Schroeder | Genome-wide ancestry of 17th-century enslaved Africans from the Caribbean[END_REF].

A recent study [START_REF] Barquera | Origin and Health Status of First-Generation Africans from Early Colonial Mexico[END_REF] successfully generated aDNA data from remains of three enslaved-Africans buried shortly after the beginning of the colonisation of Mexico City in the 16 th century. The study showed that mitochondrial, Y-chromosome, and autosomal aDNA from these individuals shared a recent common ancestry with different ethnolinguistic groups from different African former slaving regions (Figure 3C-D), despite being buried together. Furthermore, investigating the non-human DNA extracted together with human DNA in these samples, geneticists identified sequences likely belonging to Treponema pallidum pertenue (a bacteria strain responsible for human yaws) and from hepatitis B virus, and matched statistically these sequences with bacterial and virus strains typically found in West African patients, respectively [START_REF] Barquera | Origin and Health Status of First-Generation Africans from Early Colonial Mexico[END_REF][START_REF] Pourkarim | Molecular identification of hepatitis B virus genotypes/subgenotypes: revised classification hurdles and updated resolutions[END_REF]. In another recent study, Sandoval-Velasco et al. ( 64) investigated skeletal remains excavated in large graveyards from St Helena island, midway between the coasts of Angola and Brazil. Between 1840 and 1867, historical records showed that approximately 27,000 enslaved-Africans who escaped at sea were subsequently re-captured by the British Royal Navy and disembarked on St Helena, of whom ~8000 died and were buried on the island [START_REF] Pearson | Liberated African Settlers on St[END_REF]. Successfully obtaining aDNA for 20 individuals, authors found that this genetic material shared common ancestries mainly with several West Central African populations rather than other African regions.

Paleogenomics investigation of the TAST era can thus provide new knowledge about the unknown African origins of deportees, forced migrations across the Americas, admixture histories throughout the TAST, and key information about familial relationships among remains, further revealing familialanthropology and funeral practices in societies with slaves. Finally, as illustrated above, paleo-metagenomics approaches can further unveil the epidemiological status of enslaved communities, and the phylogeographic history of human diseases during this era. This unequivocally demonstrates the future potential of paleogenomics investigation of TAST-related remains to fill major gaps of the history of this era.

Conclusions

Altogether, population genetics has repeatedly shown the diverse African and European origins of enslaved-African descendants throughout the Americas and the Caribbean. These results also show that the genomic make-up of enslaved-African descendant communities reflects the specific histories of marooning and rebellion, subjugation, social segregation, and legal emancipation experienced by these populations during

U N C O R R E C T E D M A N U S C R I P T 10
more than 500 years, hence further demonstrating how the complex history of the TAST has shaped, in the long-term, human genetic landscapes observed today for both enslaved and non-enslaved descendant communities.

Today, genome-wide genotype data can relatively easily be obtained and analysed with increasingly powerful statistical inference methods. It is therefore now crucial for geneticists to investigate in detail the genomic diversity patterns of enslaved-African populations at a very local scale, and explicitly parametrize, in historical, adaptive, and GWAS inferences, the influence of complex anthropological contexts having determined, over time, identity and categorisation constructions, as well as inter-community barriers to admixture. Furthermore, with the advances of local-ancestry mapping approaches, massive efforts should be undertaken to better understand the complex history of genetic admixture between enslaved-African populations and autochthonous American populations, which will also require further sampling at a local scale accompanied with anthropologic, ethnologic and demographic information for the same populations.

Finally, the influence of the TAST on genetic diversity patterns of African populations has seldom been explored [START_REF] Tomás | The peopling of São Tomé (Gulf of Guinea): origins of slave settlers and admixture with the Portuguese[END_REF][START_REF] Beleza | Genetic Architecture of Skin and Eye Color in an African-European Admixed Population[END_REF][START_REF] Verdu | Parallel Trajectories of Genetic and Linguistic Admixture in a Genetically Admixed Creole Population[END_REF], despite extensive historical and demographic work having shown the multifaceted influence of the TAST in continental Africa [START_REF] Lovejoy | The Impact of the Atlantic Slave Trade on Africa: A Review of the Literature1[END_REF][START_REF] Whatley | The Fundamental Impact of the Slave Trade on African Economies[END_REF]. It is imperative that geneticists fill this gap to better understand, not only the TAST itself, but genetic diversity patterns observed within Africa today [START_REF] Schlebusch | Tales of Human Migration, Admixture, and Selection in Africa[END_REF].

Indeed, by triggering massive population movements of communities escaping slavery and strongly influencing pre-existing inter-community socio-economic and cultural relationships, the TAST likely deeply influenced genetic patterns observed today within the continent.

U N C O R R E C T E D M A N U S C R I P T U N C O R R E C T E D M A N U S C R I P T U N C O R R E C

  Atlantic Slave Trade (TAST) was one of the largest forced migrations in recent human history.

Figure 1 .Figure 2 . 18 Figure 3 .

 12183 Figure 1. Census of enslaved Africans who were forced to leave Africa from 1501 to 1875 estimated from historical sources (2). (Top panel), estimated census for each major TAST embarkation region. (Bottom panel), estimated census for each major TAST dis-embarkation region in the Americas and Africa (tables can be found at https://www.slavevoyages.org/estimates/L8KDKZCH).

Downloaded from https://academic.oup.com/hmg/advance-article/doi/10.1093/hmg/ddaa271/6040749 by Museum National d'Histoire Naturelle user on 04 January 2021

Acknowledgements

We thank the editors for the invitation to participate in this special issue, and two external reviewers for useful comments and help improving this manuscript. We would like to thank Jean-Michel Dugoujon, Hannes Schroeder, Jay Haviser, Esteban Parra, Noah A. Rosenberg, and Zachary A. Szpiech for enlightening discussions. We thank Rodrigo Barquera and Thiseas C. Lamnidis for sharing the genotype data of samples from Mexico.

Funding P.V. and C.F-L. were supported in part by the French "Agence Nationale pour la Recherche" (ANR) grant METHIS (ANR-15-CE32-0009-01). C.F-L. was funded in part by the EU Marie Curie Initial Training Network: EUROTAST (MC-ITN FP7/2007-2013, grant no. 290344); the Marcus Borgström Foundation for Genetic Research; the Royal Physiographic Society of Lund (Nilsson-Ehle Endowments); and the Sven and Lilly Lawski's Foundation (Postdoctoral fellowship).

Conflict of interest statement. None declared.


domain: sdv sdv.bc spi

text:
Tiffany Baëtens

Séverine Begard

Emiliano Pallecchi

Vincent Thomy

Steve Arscott
email: steve.arscott@univ-lille.fr

Sophie Halliez
email: sophie.halliez@inserm.fr

The effect of thermal treatment on the neuronal cell biocompatibility of SU-8

Keywords: SU-8, microsystems, microtechnology, neuronal cells, biocompatibility, hardbaking

Future in vitro life science studies that require in situ functionalities-going beyond the common glass petri dish-can potentially be realized by using microsystems technologies to fabricate bio-microelectromechanical systems (BioMEMS) and laboratory-on-a-chip (LOC).

One such material used to construct these microsystems is the commercial photoresist SU-8.

Here, the neuronal biocompatibility of SU-8 is examined as a function of its hard-baking thermal treatment. The SU-8 surfaces were non-patterned large uniform thin films-this allowed the biocompatibility and cell adhesion of the SU-8 to be fully tested. In total, 75 SU-8 samples were fabricated for the study using spin-coating and lithography techniques. We observe that a single hard-baking step of 180°C for 2 hours coupled with a coating (poly-Dlysine + laminin) is enough to detoxify SU-8 and promote primary cortical cell adhesion and survival up to 28 days in vitro (DIV). Therefore, the protocol described here makes SU-8 surfaces compatible with the development of neuronal networks from primary neural cells.

Introduction

The building, functionalization and characterization of complex in vitro neural networks from primary neural cells has benefited from the development of microsystems dedicated to life sciences such as bio-microelectromechanical systems (bio-MEMS) and laboratory-on-a-chip (LOC) [START_REF] Grayson | A BioMEMS Review: MEMS Technology for Physiologically Integrated Devices[END_REF][START_REF] Bashir | BioMEMS: state-of-the-art in detection, opportunities and prospects[END_REF]. This has been achieved by harnessing advantages such as inter alia microscale confinement, environmental control (liquid and gas flow, temperature, surface properties), the parallelization of experiments, and sensors integration [START_REF] Xu | High-throughput three-dimensional chemotactic assays reveal steepness-dependent complexity in neuronal sensation to molecular gradients[END_REF][START_REF] Courte | Reconstruction of directed neuronal networks in a microfluidic device with asymmetric microchannels[END_REF][START_REF] Virlogeux | Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease[END_REF][START_REF] Radotić | Advantageous environment of micro-patterned, high-density complementary metal-oxide-semiconductor electrode array for spiral ganglion neurons cultured in vitro[END_REF]. Despite the many breakthroughs made possible by the application of microtechnologies to life sciences studies, there are still questions concerning the biocompatibility of the specific materials and their associated technological processes when used with biological materials. Indeed, the fabrication of bio-MEMS and LOC requires both the use of structural and functional materials (e.g. glass, silicon, metals, polymers…) and evidently their associated technological processes and associated chemicals (solvents, chemical reagents, strong acids or bases). In the latter case, the chemicals used for manufacturing can be absorbed by the structural materials of the microsystems and can also leave residues on the microsystem which can subsequently interact with living organisms, liquids and gases necessary for cultures. This can occurover very wide time scales from a few minutes to more than several weeks. Given the current relative lack of standardization concerning the building of such bio-MEMS, a systematic study of the biocompatibility of materials (which is performed relatively routinely) and the impact of their associated technological processes (more rarely detailed and studied) is required. Due to the very wide diversity of biological experiments, we would like to underline that fact that it is not possible to define a material or process as being 'universally biocompatible'. Thus, biocompatibility studies must be systematically carried out in the specific context in which the specific bio-MEMS will be used.

In this paper we present a simplified processing protocol to improve the photoresist SU-8's capability to support direct primary neurons network growth up to 28 days in vitro (DIV).

The photoresist SU-8 in bio-MEMS and LOC for neuronal cell studies

SU-8 is a versatile thick film negative photoresist [START_REF] Lorenz | SU-8: a low-cost negative resist for MEMS[END_REF] which has been used for the fabrication of several micro-and nano-technology-based applications, e.g. microelectromechanical systems (MEMS) [START_REF] Zhang | Characterization of the polymerization of SU-8 photoresist and its applications in micro-electro-mechanical systems (MEMS)[END_REF], high frequency waveguides [START_REF] Smith | SU-8 micromachining of millimeter and submillimeter waveguide circuits[END_REF], stretchable technologies [START_REF] Baetens | Metallized SU-8 thin film patterns on stretchable PDMS[END_REF], and microfluidics [START_REF] Carlier | Integrated microfluidics based on multi-layered SU-8 for mass spectrometry analysis[END_REF]. More recently its use in bio-MEMS has considerably expanded [START_REF] Abgrall | SU-8 as a structural material for labs-on-chips and microelectromechanical systems[END_REF][START_REF] Arscott | SU-8 as a material for lab-on-a-chip-based mass spectrometry[END_REF]. In the latter applications, its biocompatibility is an important issue. In this context, we can categorize the use of SU-8 into three different roles: (i) the polymerized SU-8 forms an integral part of the microsystem and the analyte is in contact with the SU-8 [START_REF] Arscott | A planar on-chip micro-nib interface for NanoESI-MS microfluidic applications[END_REF][START_REF] Tuomikoski | Fabrication of enclosed SU-8 tips for electrospray ionization-mass spectrometry[END_REF], (ii) the polymerized SU-8 forms an integral part of the microsystem but the analyte is not in contact with the SU-8 due to an encapsulation layer [START_REF] Herbertson | Electrowetting on superhydrophobic SU-8 patterned surfaces[END_REF] or surface functionalization [START_REF] Blagoi | Functionalization of SU-8 photoresist surfaces with IgG proteins[END_REF], and (iii) the SU-8 is not used as a part of the final system, but is used to make the final systems e.g. a molding step [START_REF] Jo | Three-dimensional microchannel fabrication in polydimethylsiloxane (PDMS) elastomer[END_REF][START_REF] Natarajan | Large-area, high-aspect-ratio SU-8 molds for the fabrication of PDMS microfluidic devices[END_REF]. In the former and latter cases, the issue of biocompatibility comes from (i) the SU-8 itself and (ii) the previous technological steps where contamination can come from the products used for SU-8 processing. SU-8 photoresist is composed of an epoxy resin, bistriarylsulfonium and sulfonium hexafluoroantimonate salts, pyrolytic carbon [START_REF] Peltola | SU-8 based pyrolytic carbon for the electrochemical detection of dopamine[END_REF] and a solvent (either cyclopentanone or gamma-butyrolactone). Its microfabrication processing is welldocumented and involves the use of a developer (1-methoxy-2-propanol acetate) and an isopropanol rinse. It is known that 'hard-baking' at 200°C renders the material less chemically reactive [START_REF] Lorenz | SU-8: a low-cost negative resist for MEMS[END_REF] and modifies its mechanical properties [START_REF] Chung | Effects of temperature on mechanical properties of SU-8 photoresist material[END_REF]. Evidently, SU-8 is often used as part of a larger technological process which can involve other photoresists and their associated processing chemicals e.g. other solvents, acids, and bases.

Being able to use polymerized SU-8 as a structural material for bio-MEMS and LOC dedicated to neuronal development would allow one to extend the scope of what can be presently done. However, when compiling data from the literature, one can see culturing primary neural cells on SU-8 remains relatively controversial. Biocompatibility of SU-8 was previously validated in vitro using mouse fibroblasts in a contactless assay [START_REF] Kotzar | Evaluation of MEMS materials of construction for implantable medical devices[END_REF], human fibroblasts cultured beside SU-8 microprobes [START_REF] Kotzar | Evaluation of MEMS materials of construction for implantable medical devices[END_REF] and grown on oxygen plasma-treated bare SU-8 [START_REF] Cho | Biocompatible SU-8-Based Microprobes for Recording Neural Spike Signals From Regenerated Peripheral Nerve Fibers[END_REF], primary glial cells (peripheral glial cells and astrocytes) cultured beside SU-8 microprobes [START_REF] Kotzar | Evaluation of MEMS materials of construction for implantable medical devices[END_REF] and grown directly on bare nanostructured SU-8 [START_REF] Hennemeyer | Cell proliferation assays on plasma activated SU-8[END_REF], rat glial cell lines grown on bare SU-8 [START_REF] Ereifej | Characterization of astrocyte reactivity and gene expression on biomaterials for neural electrodes[END_REF] and in a contactless assay [START_REF] Nemani | In vitro and in vivo evaluation of SU-8 biocompatibility[END_REF] and rodent and human neuronal cell lines recruited by dielectrophoresis on bare microstructured SU-8 [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF], grown on poly-L-lysinecoated flat or nanostructured SU-8 and even directly grown on bare nanostructured SU-8 [START_REF] Kim | Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth[END_REF][START_REF] Kim | An SU-8-based microprobe with a nanostructured surface enhances neuronal cell attachment and growth[END_REF][START_REF] Ajetunmobi | Characterization of SH-SY5Y human neuroblastoma cell growth over glass and SU-8 substrates: CHARACTERIZATION OF SH-SY5Y HUMAN NEUROBLASTOMA CELL GROWTH[END_REF]. However, a limitation commonly seen in these findings is the experimental duration.

Depending on the neuronal type and the cell density, primary neurons require a period ranging from several days to several weeks to develop into mature networks [START_REF] Ito | Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks[END_REF]. The experiments mentioned beforehand were conducted over at most 7 days. Moreover, if immortalized neuronal cell lines can be induced to differentiate into cells sometimes described as neuron-like cells, these cells are not representative of primary neurons [START_REF] Abdulla | Studies of Neurotoxicity in Cellular Models[END_REF][START_REF] Gordon | General Overview of Neuronal Cell Culture[END_REF] and cannot be used to grow neuronal networks. Primary neuronal cells are generally more sensitive [START_REF] Lepage | On the Use of Neuro-2a Neuroblastoma Cells Versus Intact Neurons in Primary Culture for Neurotoxicity Studies[END_REF], although some notable exceptions have been described [START_REF] Heravi | The primary neuronal cells are more resistant than PC12 cells to α-synuclein toxic aggregates[END_REF]. Therefore, biocompatibility tests performed with neuronal cell lines could be irrelevant for primary neuronal cells. Indeed, biocompatibility of non-hard-baked SU-8 proved to be very poor for primary cortical neurons [START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF] and hippocampal neurons [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF]. Heat treatment [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF][START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF] and hydroxylation [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF] are known to greatly improve the biocompatibility of SU-8 when neuronal cells are not seeded on top of the SU-8 but cultured adjacent to it [START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF] or when dielectrophoresis is applied to recruit neurons on microstructured SU-8 [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF]. It should be noted that primary cortical cells, seeded in a microfluidic-MEA device based on polydimethylsiloxane (PDMS) and SU-8 bonding, have been reported to form a functional neuronal network after two weeks of culture. However, in this configuration, cortical cells are not seeded on SU-8 [START_REF] Ren | A Simple and Reliable PDMS and SU-8 Irreversible Bonding Method and Its Application on a Microfluidic-MEA Device for Neuroscience Research[END_REF]. Dorsal root ganglion (DRG) explants were also cultured in presence of SU-8 microprobes and the latter were observed to sustain the growing of DRGemanating nerve fibers [START_REF] Cho | Biocompatible SU-8-Based Microprobes for Recording Neural Spike Signals From Regenerated Peripheral Nerve Fibers[END_REF]. In the same study, the authors also successfully implanted these microprobes into sciatic nerves of rats-the study lasting for almost one year [START_REF] Cho | Biocompatible SU-8-Based Microprobes for Recording Neural Spike Signals From Regenerated Peripheral Nerve Fibers[END_REF]. However, under these conditions, a substantial degree of maturation was already achieved when the cells came into contact with the SU-8-hence the ability of SU-8 to support fully the adhesion and development of primary neuronal cells is still questionable. Moreover, assays with HeLa cells (human cell line) [START_REF] Stangegaard | Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces[END_REF], the rat adrenal phaeochromocytoma PC-12 cell line [START_REF] Marelli | Flexible and biocompatible microelectrode arrays fabricated by supersonic cluster beam deposition on SU-8[END_REF] and primary neuronal cells [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF][START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF] when seeded directly on top of untreated and bare SU-8 showed poor cell attachment. Plasma activation is reported, which is seen to improve considerably the cell adhesion and their growth [START_REF] Hennemeyer | Cell proliferation assays on plasma activated SU-8[END_REF][START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF]. Methods currently known to improve primary neuronal cell adhesion on SU-8 are heat treatment + sonication +/-parylene coating + poly-D-lysine coating [START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF], , structuration +/-poly-L-lysine coating (cell adhesion is better with poly-L-coating) [START_REF] Kim | Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth[END_REF][START_REF] Kim | An SU-8-based microprobe with a nanostructured surface enhances neuronal cell attachment and growth[END_REF][START_REF] Choi | High Aspect Ratio SU-8 Structures for 3-D Culturing of Neurons[END_REF][START_REF] Wu | Interfacing SH-SY5Y human neuroblastoma cells with SU-8 microstructures[END_REF] and surface functionalization [START_REF] Tao | Surface Modification of SU-8 for Enhanced Biofunctionality and Nonfouling Properties[END_REF]. As previously indicated, applying dielectrophoresis to recruit primary neurons directly on top of microstructured heat-or hydroxylated-treated SU-8 elicit good cell adhesion and viability for 7 days [START_REF] Zhou | Multielectrode array capable of supporting precisely patterned hippocampal neuronal networks[END_REF]. It can be noted that rat cortical cells were seeded and cultured successfully for 20 days on a structured surface consisting of SU-8 tower structures, heated at 110°C for 3 days then heated in water at 45°C for 1 day and coated with poly-L-lysine [START_REF] Choi | High Aspect Ratio SU-8 Structures for 3-D Culturing of Neurons[END_REF]. But all these processes are time-consuming, can be difficult to integrate with other technical requirements, and/or require materials not available in basic biological labs where the cell culture will be ultimately performed. Here, we tested and validated a simplified processing protocol (hard-baking for 2 hours followed by poly-D-lysine and laminin coating) to treat unstructured SU-8 surfaces in order to make it suitable to support the long-term development of neural cells seeded on it.

Material and methods

Sample processing

All technological processes for the fabrication of the samples were performed in an ISO 5/7 cleanroom (IEMN). The samples were then sealed and transferred to the biology laboratory (JPARC). Four types of samples were tested: glass with cover glasses having a diameter of 18 mm (Marienfeld Superior, Germany), tissue culture (TC) treated-polystyrene (Corning, USA), PDMS (Dow Corning, USA), and the photoresist SU-8 (MicroChem, USA). The samples were processed or deposited at the bottom of wells in 12-well cell culture plates (Corning, USA). For TC treated-polystyrene samples, empty well surfaces were used. The PDMS samples were prepared using a commercially available kit (Sylgard 184, Dow Corning) [START_REF] Seghir | Extended PDMS stiffness range for flexible systems[END_REF]. The base and curing agent were mixed to a ratio of 10:1, degassed using a rotary pump, molded directly in wells of cell culture plates, and cured in a box oven (Mpemmert GmbH, Germany) at 70°C for 2 hours.

For the SU-8 samples, we used circular cover glasses having diameter of 18 mm as substrates for the SU-8 deposition. SU-8 '2002' was used in order to obtain a thin thickness of resin. The surfaces were spin coated with SU-8 2002 using a commercial spin-coater (Suss MicroTec Lithography GmbH, Germany). A uniform ~800 nm thick SU-8 films is achieved by spin coating at 2000 rpm, acceleration of 500 rpm s -1 for 12s followed by a pre-exposure bake (prebake) on hotplate at 95°C for 2 minutes-the film thickness was verified using commercial surface profiling (Bruker, USA). Taking the substrate material in account, the samples were optimally exposed [START_REF] Gaudet | A user-friendly guide to the optimum ultraviolet photolithographic exposure and greyscale dose of SU-8 photoresist on common MEMS, microsystems, and microelectronics coatings and materials[END_REF] to UV light (wavelength 365 nm) for 5 s at 10 mW cm -2 in a commercial mask aligner (Suss Microtech, Germany) to promote the polymerization. Next, the samples are placed on hotplate at 95°C for 2 minutes to achieve polymerization-this is the post-exposure base (PEB). 75 SU-8-based samples were spin-coated and processed for the study using this method.

In order to study the behavior of the neuronal cells seededon the SU-8 surfaces, the samples were divided into 3 groups by varying the 'hard-bake temperature' performed in a box oven at 100°C, 180°C and 200°C for 2 hours. Finally, the samples were inserted into cell culture plate. The cell culture plate was then closed and initially sterilized under UV light for 1 hour before transportation to the biology laboratory. The samples were then sterilized for a second time under UV light for 15 minutes prior to coating. Table 1 gives a complete list of all samples used in the study plus their individual processing thermal history.

Cell culture

Each test sample was coated with a mix of Poly-D-lysine (0.5 mg ml -1 , Sigma) and Laminin (10 µg ml -1 , Sigma). Enough volume of coating solution was added per well (around 1 ml) to totally cover the different surfaces even if water-repellent. The coating solution was applied for 2 hours at room temperature then washed using sterile water (Corning) and air-dried.

No additional surface treatment was performed.

Rat primary cortical cells were prepared from 18-days-old Wistar rat embryos-as previously described [START_REF] Galas | The Peptidylprolyl cis/trans -Isomerase Pin1 Modulates Stress-induced Dephosphorylation of Tau in Neurons: IMPLICATION IN A PATHOLOGICAL MECHANISM RELATED TO ALZHEIMER DISEASE[END_REF]. The present experimental research has been performed with the approval of an ethical committee (agreement APAFIS#2264-2015101320441671 from CEEA75, Lille, France) and follows European guidelines for the use of animals. Gestating females (Janvier Labs) were housed in a temperature-controlled (20 -22° C) room maintained on a 12 h day/night cycle with food and water provided ad libitum. The culture medium was made of Neurobasal (Gibco) supplemented with B-27 (Gibco), Antibiotic-Antimycotic (Gibco) and L-glutamine (Gibco). To generate the results presented here, a total of four different cell culture dissociations were performed from four different litters. The number of viable cells in the cell suspensions was determined through trypan blue exclusion test (Gibco). For each sample, 250 000 viable cells were seeded in 1.5 ml culture medium on a total area of 3.5 cm 2 so 71 400 cells/cm 2 . Cultures were kept in a cell incubator (ThermoScientific) at 37°C in a 5% CO2 atmosphere.

Cell viability/metabolic activity assay and cytotoxicity/cell death assay

To assay metabolic activity/number of viable cells, cellular reduction of MTS tetrazolium into formazan was quantified using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) following the manufacturer's instructions. Cells grown on coated glass slides were used as a reference. Briefly, reagent was added in each well (1:5 ratio) and the cells were incubated three hours (at 7-8 DIV and 14 DIV) or two hours (other time points) at 37°C in a cell incubator then 3 x 150 µl per culture well were transferred into a P96 plate (Corning, USA). Absorbance was measured at 490 nm via a microplate spectrophotometer (SpectraMax i3, Molecular Devices) and the SoftMax Pro software (Molecular Devices). The mean result of three technical replicates was considered as n=1 biological replicate. To assay cell death, the release of lactate dehydrogenase (LDH) into the cell culture medium was quantified using the CytoTox 96 Non-radioactive Cytotoxicity Assay (Promega) following the manufacturer's instructions. Maximum LDH release was determined by adding lysis solution (9% Triton X-100) to cells grown on coated glass. Briefly, 3 x 50 µl of culture medium from each culture well were transferred into a P96 plate (Corning, USA). Reagent was added (1:1 ratio) and the cell culture medium samples were incubated during 30 minutes at room temperature. Then 1 volume of stop reaction buffer was added. Absorbance was measured at 490 nm via a microplate spectrophotometer and the SoftMax Pro software. The mean result of three technical replicates was considered as n=1 biological replicate. Significant differences between results obtained for cells grown on glass and results obtained for other substrates were examined using the nonparametric Mann-Whitney and Wilcoxon tests, and p-values <0.05 were considered significant.

Cell culture imaging

Cells were fixed with 4% formaldehyde. Cells were then permeabilised with a blocking buffer (PBS with 1% bovine serum albumin and 0.1% Triton X-100) for 10 minutes at room temperature, and incubated with NeuN (Millipore) and Homer1 (Synaptic System) diluted in blocking buffer overnight at 4°C-followed by Alexa Flour 568-and 488-conjugated secondary antibodies (Invitrogen) for 3 hours at room temperature and dapi (Thermofischer).

Immunolabelling was observed with an inverted confocal microscope (LSM 710, AxioObserver Zeiss) with a ×40 lens. Images were processed with ZEN software (Zeiss). Figure 1 shows 10 µm orthogonal projections from the results.

Results and discussion

With reference to Table 1, three types of SU-8 were characterized in the study. First, SU-8 hard-baked for 2 hours at 100°C, denoted 'S1', second, SU-8 hard-baked for 2 hours at 180°C, denoted 'S2', and third, SU-8 hard-baked for 2 hours at 200°C, denoted 'S3'. Note that we also tested SU-8 which had been hard-baked for a shorter time (15 minutes) at two different temperatures (180°C and 200°C) that induced total cell death after 7 and 14 days respectively in vitro (DIV) although initially the cells adhered to SU-8 (data not shown here).

Metabolic activity assays, that directly reflect the living cell numbers, and cell death assay were performed at 7-8 DIV, 14 DIV, 21-22 DIV and 28 DIV (Fig. 1). Cells grown on glass disks were used as control samples. Additionally, we tested two other substrate materials commonly used to culture neural cells: tissue culture (TC) treated-polystyrene and PDMS. All the results from the different substrates were compared to those obtained using glass.

Primary cortical cells grown on TC treated-polystyrene and PDMS did not exhibit any significant differences compared to primary cortical cells grown on glass except cells grown on PDMS at 21 DIV (see Table 2 and Table 3). Moreover, using optical microscopy, the primary cortical cells appeared to be similar on all these substrates (data not shown). However the metabolic activity and cell death assays revealed, respectively, fewer living cells and increased level of dead cells at almost every stage for type S1 samples (Tables 2 &3). After 21 DIV and 28 DIV, the mean metabolic activity exhibited by cortical cells grown on type S1 samples reached, respectively, 25.8% and 45.9% compared to cortical cells grown on glass, hence inferior to 50%. This led us to conclude that hard baking at 100°C for 2 hours was not sufficient to make SU-8 biocompatible enough to support neuronal cell development.

For the type S2 and S3 samples, metabolic activity and cell death assays revealed fewer living cells at 7 DIV and 21 DIV and increased level of dead cells at 14 DIV and 21 DIV (see Table 2 and Table 3). After 21 DIV and 28 DIV, the mean metabolic activity exhibited by cortical cells grown on type S2 samples reached, respectively, 64.4% and 68.4% compared to cortical cells grown on glass and for cortical cells grown on type S3 samples reached, respectively, 71.5% and 79.4% so largely superior to 50%. However, cortical cells grown on type S3 samples behaved quite differently from cells grown on glass, type S2 samples, or other substrates-they packed together and extend highly fasciculate neurites, a behavior typical of stressed neuronal cells. We also noted a highly variable level of cell death for cells grown on S2 samples at 28 DIV but no significant differences were observed with cells grown on glass at the same stage.

In parallel, we performed immunostainings of cortical cells at 23 DIV when rat cortical cells have been shown to be organized into functional networks-i.e. exhibiting synchronized bursting activity-through microelectrode array recordings (data not shown). Two neuronal proteins were stained: NeuN, a neuronal nuclear protein and Homer, a postsynaptic density scaffold protein involved in the targeting of glutamate receptors. This confirmed the observations made on living cells. Although neuronal cell bodies, visualized through NeuN staining, tend only partially to group together on glass or type S2 samples, neuronal cell bodies were heavily packed on type S3 samples (see Fig. 2). While dendritic arborization, visualized through Homer1 staining, was dense and distributed uniformly around neuronal cell bodies on glass or type S2 samples, dendritic arborization on type S3 samples was highly heterogeneousrevealing uneven neurite outgrowth (see Fig. 2). It is likely that hard-baking SU-8 at 200°C induces both detoxification of the photoresist supported by good results at viability tests and structure alteration that compromises neurite outgrowth but does not induce cell death.

In conclusion, our results suggest that the hard-baking of SU-8 photoresist for 2 hours at 180°C is enough to detoxify SU-8. One can suggest that the modified neuronal biocompatibility of the SU-8 with thermal treatment observed here is associated with the matter content of the SU-8 which is known to change with heating and outgassing [START_REF] Melai | Qualitative and quantitative characterization of outgassing from SU-8[END_REF]. Several species are outgassed from the SU-8 during heating-some or all of these species may be toxic to neurons. Further work is required to verify this. Then poly-D-lysine + laminin coating promotes neuronal cell adhesion on SU-8 although at an inferior level compared to coated-glass. Coupled together hard-baking and traditional coating used in cell biology lead to neuronal survival in a time scale compatible with the development of a functional neural network from primary neural cells. This protocol elicits comparable number of living primary neural cells in long-term cultures with previously published protocols -3 weeks -including hard-baking for three days + sonication + parylene coating + traditional coating used in cell biology (poly-D-lysine) [START_REF] Vernekar | SU-8 2000 rendered cytocompatible for neuronal bioMEMS applications[END_REF] and hard-baking for three days + surface structuration (high aspect ratio towers) + traditional coating used in cell biology (poly-L-lysine) [START_REF] Choi | High Aspect Ratio SU-8 Structures for 3-D Culturing of Neurons[END_REF]. However, we finish by noting that the compatibility of a 180°C hard-bake depends on the temperature sensitivity of the specific technological process employed by the engineer.

Fig. 1 .

 1 Fig. 1. Primary cortical cell biocompatibility of SU-8 hard-baked at different temperatures compared to materials commonly used for cell culture. Comparison of the metabolic activity (reflecting living cell number, a) and the death (ratio of cell death on metabolic activity, b) of primary cortical cells cultured on top of different substrates after 7-8, 14, 21-22 and 28 DIV. Median values are indicated in black. At least n=3 biological replicates for each condition.

Fig. 2 .

 2 Fig. 2. Development of primary cortical cells on SU-8 hard-baked at different temperatures compared to glass. Immunolabelling of primary cortical cells grown on glass (left), SU-8 hardbaked for 2 hours at 180°C (middle) and SU-8 hard-baked for 2 hours at 200°C (right). NeuN is stained in red, homer1, a post-synaptic protein, is stained in green and nuclei are in blue. Scale bars, 20 µm.

Table 1 .

 1 List of samples fabricated and used in the study. Their thermal history and processing are given.

	Sample Ref.	Surface	Number of	Hard-bake	Prebake	PEB	UV exposure
			samples				(mJ cm -2 )
	Glass	Glass	33	n/a
	TC treated-	TC treated-	35	n/a
	Polystyrene	Polystyrene
	PDMS	PDMS	20		70°C/2hrs
	S1	SU-8	18	100°C/2hrs	95°C/2	95°C/2	50
					mins	mins
	S2	SU-8	30	180°C/2hrs	95°C/2	95°C/2	50
					mins	mins

Table 2

 2 Mean metabolic activity (reflecting living cell number) of primary cortical cells cultured on top of different substrates after 7-8, 14, 21-22 and 28 DIV. At least n=3 biological replicates for each condition. Significant differences between results obtained for cells grown on glass and results obtained for other substrates were examined using the nonparametric Mann-Whitney and Wilcoxon tests (*:p<0.05 and ***:p<0.005).

	Sample	Mean metabolic	Mean metabolic	Mean metabolic	Mean metabolic
		activity at 7-8 DIV	activity at 14 DIV	activity at 21-22 DIV	activity at 28 DIV
	Glass	100 ± 8 (n=8)	100 ± 8 (n=9)	100 ± 18 (n=6)	100 ± 21 (n=6)
	(reference)
	TC-treated	120 ± 37 (n=10)	109 ± 24 (n=11)	116 ± 13 (n=7)	136 ± 28 (n=7)
	polystyrene
	PDMS	115 ± 42 (n=6)	124 ± 46 (n=6)	130 ± 13 * (n=4)	132 ± 17 (n=4)
	SU-8 'S1'	71 ± 9 * (n=3)	74 ± 6 *** (n=5)	26 ± 10 * (n=3)	46 ± 4 * (n=3)
	SU-8 'S2'	73 ± 18 * (n=6)	95 ± 43 (n=8)	64 ± 19 * (n=6)	68 ± 51 (n=6)
	SU-8 'S3'	38 ± 19 * (n=3)	80 ± 30 (n=4)	72 ± 11 * (n=3)	79 ± 37 (n=3)

Table 3

 3 Mean mortality (ratio of cell death on metabolic activity) of primary cortical cells cultured on top of different substrates after 7-8, 14, 21-22 and 28 DIV. At least n=3 biological replicates for each condition. Significant differences between results obtained for cells grown on glass and results obtained for other substrates were examined using the nonparametric Mann-Whitney and Wilcoxon tests (*:p<0.05, **:p<0.01 and ***:p<0.005).

	Sample	Mean cell mortality	Mean cell mortality at	Mean cell mortality	Mean cell mortality
		at 7-8 DIV	14 DIV	at 21-22 DIV	at 28 DIV
	Glass	30 ± 13 (n=8)	27 ± 4 (n=9)	22 ± 6 (n=6)	33± 20 (n=6)
	(reference)
	TC-treated	26 ± 7 (n=10)	30 ± 5 (n=11)	22 ± 7 (n=7)	23± 10 (n=7)
	polystyrene
	PDMS	37 ± 12 (n=6)	32 ± 15 (n=6)	19 ± 8 (n=4)	22 ± 9 (n=4)
	SU-8 'S1'	57 ± 7 * (n=3)	34 ± 5 *** (n=5)	86 ± 64 * (n=3)	41 ± 22 (n=3)
	SU-8 'S2'	49 ± 22 (n=6)	37 ± 15 * (n=8)	38 ± 13 * (n=6)	80 ± 74 (n=6)
	SU8 'S3'	31 ± 21 (n=3)	42 ± 16 ** (n=4)	39 ± 6 *(n=3)	44 ± 8 (n=3)

Acknowledgements

The work was partly supported by the French RENATECH network, the LabEx DISTALZ and the Joint Inserm-University Chairs program. We thank Meryem Tardivel of the BICeL-Campus HU Facility for access to systems.

Declaration of competing interest

The authors declare no conflict of interest.


M Cavillon
email: maxime.cavillon@universite-paris-saclay.fr

E Muzi

M Lancry

B Sapaly

D Janner

M Poumellec

Spatially-Controlled Second Harmonic Generation in Lithium Niobate Silicate and Borosilicate Glasses

This work investigates the role of B 2 O 3 addition into a silicate glass matrix on the orientational dependency of second harmonic generation (SHG), induced by LiNbO 3 crystals after femtosecond laser irradiation.

Introduction

Femtosecond (fs) laser direct writing (FLDW) is a powerful and versatile tool to enable a large variety of optical components to be fabricated in transparent materials such as glasses (e.g., Bragg gratings, waveguides, graded index lenses, birefringent optics) [START_REF] Mazur | Femtosecond laser micromachining in transparent materials[END_REF][START_REF] Habel | Femtosecond FBG written through the coating for sensing applications[END_REF]. By controlling the fs-laser parameters (polarization, pulse energy, repetition rate, scanning speed) it becomes possible to induce controlled modifications inside the glass, hence local properties. As an example to this approach, LiNbO 3 crystals, having properties such as piezoelectricity and ferroelectricity, were induced in a 33%Li 2 O -33%Nb 2 O 5 -34%SiO 2 (molar percentage) glass matrix (LNS) [START_REF] Cao | Modifications in lithium niobium silicate glass by femtosecond laser direct writing: morphology, crystallization, and nanostructure[END_REF]. It was shown that the orientation of the polar axis of the LiNbO 3 nanocrystals can be dictated by controlling the writing laser polarization. Because of axial symmetry, the second order susceptibility tensor (χ (2) ) of LiNbO 3 crystal is such that its nonlinear coefficient value along the polar axis (d 33 = 34.4 pm/V) is much greater than the nonlinear coefficients values along the axes perpendicular to it (d 31 = 5.95 pm/V, d 22 =3.07 pm/V). Therefore, a angular modulation of χ (2) properties, such as second harmonic generation (SHG), occurs provided the crystals can be oriented in a non-random fashion.

In this context, the aforementioned work is extended and the role of the glass matrix on the crystal orientability is investigated herein. Furthermore, the partial substitution of SiO 2 with B 2 O 3 is of interest since introduction of B 2 O 3 lowers glass-making temperature. Consequently, addition of B 2 O 3 is expected to modify the kinetics of LiNbO 3 crystallization and the thresholds of the different regimes found for LNS glass. For this purpose, anisotropy of the SHG property is investigated in both static (writing speed = 0 µm/s) and scanning (writing speed ≠ 0µm/s) fslaser irradiation conditions.

Experimental procedure

Four glasses, with compositions given in Table 1, are fabricated using conventional melt-quenching technique, and are shaped into plates of ~1 mm thickness upon cooling. Both top and bottom surfaces of each sample are then polished until optical roughness is reached. Table 1. Glass labels and batch compositions (molar percentage)

Label

Glass batch composition (molar percentage)

LNS34 33%Li 2 O -33%Nb 2 O 5 -34%SiO 2 LNS27B7 33%Li 2 O -33%Nb 2 O 5 -27%SiO 2 -7%B 2 O 3 LNS20B14 33%Li 2 O -33%Nb 2 O 5 -20%SiO 2 -14%B 2 O 3 LNS13B21 33%Li 2 O -33%Nb 2 O 5 -13%SiO 2 -21%B 2 O 3
Each glass sample is thus irradiated using a femtosecond laser (λ = 1030 nm, 300 fs, NA = 0.6) focused shifted by 350 µm below the surface. A first set of experiments consists of static irradiations to investigate the incubation time until SHG is detected online, through the observation of an intense green light characteristic of SHG signal at λ SHG =λ/2=515 nm. In this experiment, laser parameters are varied, including pulse energy (from 0.25 to 2.5 µJ, step of 0.25 µJ) and laser repetition rate (100 kHz, 200 kHz, 340 kHz, and 510 kHz). A second set of experiments is to write lines (fixed frequency of 200 kHz and writing speed of 1 µm/s) using different pulse energies (0.25 µJ to 2.0 µJ, step of 0.25 µJ) and for three different laser polarization configurations: parallel (//), at 45°, and perpendicular (⊥) to the laser writing direction. Following this experiment, the angular dependency (total of 180°) of the SHG intensity for each irradiated line is measured according to the azimuthal angle ( of the polarization of the probing beam.

Results and discussion

Fig. 1a and 1b display the incubation time (in seconds), under static irradiation, to observe intense green light emission, characteristic of SHG. The comparison of the results for two glass samples, LNS34 and LNS20B14, illustrates well the fact that an addition of B 2 O 3 typically promotes fast crystallization, especially at low repetition rates. These data are consistent with differential scanning calorimetry (DSC) measurements, to be discussed at the conference, showing that borosilicate samples (LNSB), relative to the silicate (LNS) one, tend to crystallize at lower temperatures. In Fig. 1c and 1d are reported the normalized SHG intensity, measured for both LNS34 and LNS20B14 samples, in different writing configurations (i.e. //, at 45°, and ⊥) with respect to the writing direction (axis Y in Fig. 1c or 1d), at a fixed pulse energy value of 0.5 µJ. In both samples, the SHG intensity is strongly dependent on the probing polarization azimuthal angle(, and its maximum is found perpendicular to the writing polarization direction. This is consistent with findings in Refs. [START_REF] Cao | Modifications in lithium niobium silicate glass by femtosecond laser direct writing: morphology, crystallization, and nanostructure[END_REF][START_REF] Cao | Femtosecond Laser-Induced Crystallization in Glasses: Growth Dynamics for Orientable Nanostructure and Nanocrystallization[END_REF], where the polar axis (associated with maximum SHG intensity) is aligned perpendicular to the laser polarization (at low pulse energy). At higher pulse energies, the situation is more complex, and the maximum of SHG signal depends on both writing polarization conditions and glass matrix composition. These results will be discussed at the conference.

Conclusions

Lithium niobate silicate and borosilicate glasses are fabricated, and are subsequently irradiated using a femtosecond laser to induce non-centrosymmetric nano-crystals. SHG measurements are performed in order to detect crystallization and the anisotropy in the SHG response reveals a crystal preferential orientation induced by the irradiation. It is shown that substitution of SiO 2 with B 2 O 3 preserves tunability of SHG. The borosilicate glass family is advantageous due to its lower melting temperature. Details with respect to application possibilities will be discussed at the conference.

Fig. 1 :

 1 Fig.1: (a) and (b): time (in seconds) until green light (indicative of SHG) is detected for LNS34 (a) and LNS20B14 (b) samples, using different pulse energies and repetition rates. (c) and (d): SHG intensity as a function of probing polarization angle (0 to 180 °) for LNS34 (c) and LNS20B14 (d) samples. Laser parameters and conditions for (c) and (d): λlaser = 1030 nm (λSHG = 515 nm), 0.5 µJ/pulse, repetition rate: 200 kHz, polarization //, at 45°, and ⊥ to the writing direction. Probing angle is in the YX plane.