Pilar Alda
email: pilaralda@gmail.com

Manon Lounnas

Antonio Alejandro Vázquez

Rolando Ayaqui

Manuel Calvopiña

Maritza Celi-Erazo

Robert T Dillon

Philippe Jarne

Eric Sam Loker

Flavia Caroll

Muñiz Pareja

Jenny Muzzio-Aroca

Alberto Orlando Nárvaez

Oscar Noya

Martini Luiggi

Robles

Richar Rodríguez-Hidalgo

Nelson Uribe

Patrice David

Jean-Pierre Pointier

Sylvie Hurtrez-Boussès

Identifying vectors of fasciolosis: a new molecular tool to simultaneously distinguish three cryptic Galba species

Keywords: Lymnaeidae, Lymnaea, Fossaria, multiplex PCR, microsatellites, infectious disease

We developed a molecular tool that will allow us to simultaneously identify cryptic freshwater snail species (genus Galba) involved in fasciolosis transmission, a worldwide infectious disease among humans and livestock. We designed a multiplex PCR that uses specific primers to amplify microsatellites and differentiate three Galba species (G. cubensis, G. schirazensis, and G. truncatula) based on the size of speciesspecific alleles at microsatellite loci. We tested and validated the accuracy of this new molecular tool by comparing the multiplex PCR results with species identification based on sequences at mitochondrial and nuclear markers. This new method is accurate, inexpensive, simple, rapid, and adaptable to large sets of individuals. It will also be helpful to monitor the invasion of Galba species and therefore to develop managing strategies of species involved in the emergence or re-emergence of fasciolosis.

Introduction

Understanding and preventing infectious diseases requires a good knowledge of vectors involved in parasite dynamics. Fasciolosis is a cosmopolitan disease causing significant economic losses in domestic livestock [START_REF] Rim | Food-borne trematodes: ignored or emerging?[END_REF]. Human cases of fasciolosis have been increasing throughout the world particularly in some regions of South America, suggesting the possibility of re-emergence in this area [START_REF] Esteban | High endemicity of human fascioliasis between Lake Titicaca and La Plaz valley, Bolivia[END_REF][START_REF] Mas-Coma | Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America[END_REF]. The parasites causing this disease are liver flukes (Fasciola spp.). Freshwater mollusks, mainly belonging to the Lymnaeidae family [START_REF] Correa | Bridging gaps in the molecular phylogeny of the Lymnaeidae (Gastropoda : Pulmonata), vectors of Fascioliasis[END_REF][START_REF] Hurtrez-Boussès | Dynamics of hostparasite interactions: the example of population biology of the liver fluke (Fasciola hepatica)[END_REF], serve as intermediate hosts of Fasciola, especially the Galba genus. For example, Galba truncatula and Galba cubensis are well-known vectors of the disease [START_REF] Bargues | Characterisation of Lymnaea cubensis, L. viatrix and L. neotropica n. sp., the main vectors of Fasciola hepatica in Latin America, by analysis of their ribosomal and mitochondrial DNA[END_REF][START_REF] Jabbour-Zahab | Phylogeography and genetic divergence of some lymnaeid snails, intermediate hosts of human and animal fascioliasis with special reference to lymnaeids from the Bolivian Altiplano[END_REF]. Whether Galba schirazensis could also be a vector remains controversial.

A first study suggested that G. schirazensis does not transmit the parasite [START_REF] Bargues | Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: Genotype, phenotype, ecology, worldwide spread, susceptibility, applicability[END_REF], but more recent ones showed that it does [START_REF] Caron | Is Galba schirazensis (Mollusca, Gastropoda) an intermediate host of Fasciola hepatica (Trematoda, Digenea ) in Ecuador?[END_REF][START_REF] Dreyfuss | Differences in the compatibility of infection between the liver flukes Fascioloides magna and Fasciola hepatica in a Colombian population of the snail Galba sp[END_REF].

Populations of Galba are small-shelled freshwater snails mostly originating in the Americas, subsequently invading Europe, Africa, and Asia [START_REF] Bargues | Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: Genotype, phenotype, ecology, worldwide spread, susceptibility, applicability[END_REF][START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF][START_REF] Correa | Bridging gaps in the molecular phylogeny of the Lymnaeidae (Gastropoda : Pulmonata), vectors of Fascioliasis[END_REF]Lounnas et al., 2017a;[START_REF] Meunier | Lack of molluscan host diversity and the transmission of an emerging parasitic disease in Bolivia[END_REF]. Their abilities to survive drought and to reproduce by self-fertilization allow them to disperse over long distances and establish new populations from single individuals [START_REF] Bargues | Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: Genotype, phenotype, ecology, worldwide spread, susceptibility, applicability[END_REF]Lounnas et al., 2017a, 2017b[START_REF] Meunier | Field and experimental evidence of preferential selfing in the freshwater mollusc Lymnaea truncatula (Gastropoda, Pulmonata)[END_REF], 2001). This high invasiveness has probably facilitated the worldwide expansion of fasciolosis.

Absence of reliable morphological traits has, however, led to confusion regarding specific identities of Galba populations worldwide. Six species are considered valid: Galba cousini, Galba cubensis, Galba schirazensis, Galba truncatula, Galba humilis, and Galba viator [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF]. Of these six species only adults of G. cousini display marked differences in shell morphology and internal anatomy [START_REF] Paraense | Lymnaea cousini Jousseaume, 1887, from Ecuador (Gastropoda: Lymnaeidae)[END_REF]. The other five species cannot be accurately distinguished because their shell morphology exhibits greater intraspecific than interspecific variability, and their anatomy is homogeneous [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF][START_REF] Pointier | Freshwater molluscs of Venezuela and their medical and veterinary importance[END_REF][START_REF] Samadi | Morphological studies of Lymnaeid snails from the human fascioliasis endemic zone of Bolivia[END_REF].

Nor can they be distinguished with controlled crosses in the laboratory, as is possible in populations of the genus Physa [START_REF] Dillon | The evolution of reproductive isolation in a simultaneous hermaphrodite, the freshwater snail Physa[END_REF], because Galba populations typically demonstrate high selfing rates [START_REF] Chapuis | High quantitative and no molecular differentiation of a freshwater snail (Galba truncatula) between temporary and permanent water habitats[END_REF]Lounnas et al., 2017aLounnas et al., , 2017b;;[START_REF] Meunier | Field and experimental evidence of preferential selfing in the freshwater mollusc Lymnaea truncatula (Gastropoda, Pulmonata)[END_REF]. Thus, except for G. cousini adults with their distinct morphology, individuals of Galba have in recent years been identified using DNA sequencing technology [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF].

Amplifying and sequencing diagnostic loci can be time-consuming and expensive when sample sizes are large. Here, we suggest a rapid and inexpensive molecular approach to identify cryptic Galba species based on multiplex PCR. Such an approach has already been successfully used to identify a variety of cryptic parasites and their vectors, for example Anopheles mosquitoes, tapeworms, and protozoans diluted within feces or blood [START_REF] Bohórquez | New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm[END_REF][START_REF] Kengne | A multiplex PCR-based method derived from random amplified polymorphic DNA (RAPD) markers for the identification of species of the Anopheles minimus group in Southeast Asia[END_REF][START_REF] Sumbria | Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India[END_REF].

Among lymnaeid species, real-time PCR based on species-specific melting temperatures can be used to identify G. truncatula, G. viator, Pseudosuccinea columella, and Lymnaea diaphana [START_REF] Duffy | Real-time PCR strategy for rapid discrimination among main lymnaeid species from Argentina[END_REF]. Here, we focus on three widely distributed cryptic Galba species, G. cubensis, G. schirazensis, and G. truncatula. We describe a multiplex PCR method using specific primers to amplify microsatellite loci, distinguishing species based on allele size. We were unable to design a multiplex PCR distinguishing the other two cryptic species (G. humilis and G. viator), because specific primers amplifying microsatellites have yet not been described. We test the accuracy of this new molecular method by comparing its results with species identification based on sequences at mitochondrial and nuclear loci.

Material and methods

We based our multiplex PCR method on species-specific primers amplifying microsatellite loci already described for the three targeted cryptic species: G. truncatula [START_REF] Trouvé | W KT781322, KT781323, KT781324 Galba truncatula Morocco ND ND JN614387 Peru Moquegua 17°19'24′′S 70°59'29′′W KU870357 Venezuela El Sapo 08°52[END_REF], G. cubensis (Lounnas et al., 2017b), and G. schirazensis (Lounnas et al., 2017a). We designed 11 primer mixes, each including one speciesspecific primer pair for each of the three species (Table S1). Each mix contained a different combination of primers chosen such that the PCR products from the three species differed in size. We also tested a range of concentrations for each primer: 2, 6, 8, and 10 mM.

We initially developed our primer mixes and concentrations in one negative control and 11 known controls: two G. cubensis, two G. schirazensis, one G. truncatula, two G. cousini, two G. humilis, and two G. viator, all identified by [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF] on the basis of ITS2, ITS1, CO1, and 18S sequences (Table 1). Even though G. cousini is easily distinguishable using shell morphology and reproductive anatomy [START_REF] Paraense | Lymnaea cousini Jousseaume, 1887, from Ecuador (Gastropoda: Lymnaeidae)[END_REF], it was included in the development phase to evaluate the specificity of the multiplex PCR. Each candidate multiplex was tested using samples containing DNA from single species and pooled DNA from multiple species.

We amplified DNA in a total volume of 10 µl containing 5 µl of Taq PCR Master Mix Kit (Qiagen), 1 µl of the primer mix, and 50-100 ng of DNA in an Eppendorf Thermal Cycler. We also tested different temperature of annealing in the PCR amplification: 50, 52, and 54ºC. Finally, we retained the PCR program that resulted in the sharpest bands in agarose gels. It has an initial denaturation step at 95 ºC for 15 minutes; followed by 35 cycles of denaturation at 94 ºC for 30 seconds, annealing at 52 ºC for 90 seconds, and extension at 72 ºC for one minute; the final extension was at 60 ºC for 30 minutes. The amplification products were electrophoretically resolved after 2 hours at 100V in 5% agarose gels and stained with EZ-Vision (Amresco).

By observing the agarose gel, we evaluated each of the 11 primer mixes by three criteria: (1) each primer pair was required to amplify a single locus in the targeted species, (2) primers were not expected to yield PCR amplification in G. cousini, G. humilis, and G. viator, and (3) the PCR products from G. cubensis, G. schirazensis, and G. truncatula were required to differ noticeably in size. Ultimately, we developed a primer mix and concentration that allowed an accurate identification of G. cubensis, G. schirazensis, and G. truncatula (Table 2) in both single-species (Fig. 1) and pooled samples.

We validated our newly-developed multiplex PCR method using DNA extracts of 49 additional Galba previously identified by [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF] and Lounnas et al. (2017aLounnas et al. ( , 2017b) ) based on DNA sequences: 23 G. cubensis, 22 G. schirazensis, and 5

G. truncatula (Table 3). These individuals were removed from sites geographically distant from the sites in which the initial 11 individuals used to develop the method were sampled. DNA-extraction protocols of the individuals here studied are described in [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF] and Lounnas et al. (2017aLounnas et al. ( , 2017b)).

Results

The multiplex PCR that we designed successfully differentiated the individuals of G. cubensis, G. schirazensis, and G. truncatula previously identified by sequencing DNA markers [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF]Lounnas et al., 2017aLounnas et al., , 2017b; Table 1). We identified the species through the size of amplified alleles: DNA bands of each species were accurately separated in the 5% agarose gel (Fig. S1). The loci used in the multiplex PCR have low to intermediate polymorphism preventing overlapping bands among the species on agarose gels (Table 2; Fig. S1). The smallest difference in the size of amplified alleles was observed between some individuals of G. cubensis from Venezuela and Guadeloupe and the individuals of G. schirazensis. Even at this size (27 bp according to Lounnas et al., 2017aLounnas et al., , 2017b)), both species could be accurately identified. We did not find pairs of distinct DNA bands (putatively allelic) at any locus for any individual tested.

Discussion

We designed a multiplex PCR that successfully identifies three cryptic Galba species that are vectors of fasciolosis: G. cubensis, G. schirazensis, and G. truncatula. Until the present study, the only method by which cryptic Galba populations could be identified was by amplifying and sequencing mitochondrial or nuclear genetic markers. This technique is time-consuming because sequencing must usually be outsourced in countries where fascioliasis is widespread and genetics laboratories are few. The multiplex PCR approach developed in this study is: (i) accurate, because it was validated by the results obtained by mitochondrial or nuclear markers; (ii) inexpensive compared to amplifying and sequencing molecular markers; (iii) simple, because a single PCR is sufficient to amplify the species-specific alleles; (iv) rapid, because the results are available in less than a day; and (v) reproducible and adaptable to a large set of individuals (because it is fast and cheap), including adults or juveniles. Our method is, however, insufficiently sensitive to distinguish heterozygotes when the alleles differ by less than 15 bp (Fig. S1). Thus, we do not recommend it to estimate heterozygosity. Researchers wishing to both identify Galba populations and estimate their heterozygosity may wish to supplement the method proposed here with methods that allow distinguishing alleles differing by a single nucleotide, as in the works of [START_REF] Meunier | Field and experimental evidence of preferential selfing in the freshwater mollusc Lymnaea truncatula (Gastropoda, Pulmonata)[END_REF] and Lounnas et al. (2017aLounnas et al. ( , 2017b)).

We expect the multiplex PCR approach proposed here to prove useful as a first approach for the study of Galba populations in unexplored and poorly-known regions of the world. Galba populations from the south of Brazil have been studied on the basis of morphology only, for example, and species could have been misidentified [START_REF] Medeiros | Spatial distribution of Lymnaeidae (Mollusca, Basommatophora), intermediate host of Fasciola hepatica Linnaeus, 1758 (Trematoda, Digenea) in Brazil[END_REF]. Similarly, in North America authors recognize as many as 22 "Fossaria" species on the basis of shell and radula morphology [START_REF] Burch | North American freshwater snails[END_REF][START_REF] Johnson | Conservation status of freshwater gastropods of Canada and the United States[END_REF], most of which are indistinguishable from cryptic Galba species worldwide. [START_REF] Hubendick | Recent Lymnaeidae, their variation, morphology, taxonomy, nomenclature and distribution[END_REF] considered almost all of the nominal North American species to be synonyms of G. humilis, but resolution awaits molecular analysis, and sampling at a comprehensive geographic scale. In Africa, Europe, and Asia, G. truncatula has been cited by many authors for decades (see references in [START_REF] Seddon | Galba truncatula, Attenuate Fossaria[END_REF]. However, most studies identified this species based on shell morphology, and might indeed be dealing with the cryptic, invasive, and widely distributed species G. schirazensis. We have found in some localities Galba populations of mixed species indistinguishable ecologically as well as morphologically (authors' unpublished data). The multiplex PCR developed here would permit to easily distinguish these cryptic species and to avoid misidentifications.

Large-scale studies based on accurate species identification are essential to deepen our understanding of not only the snail, but also the epidemiology of fasciolosis.

Population-genetic studies might reveal their geographic origins, their routes of invasion, and the ecological factors that make some Galba species better invaders than others. The method proposed here could be also helpful to monitor populations once invaded and to develop management strategies with regard to the emergence or re-emergence of fasciolosis. Future efforts will be directed toward extending our techniques to identify G. humilis and G. viator, as well as G. cubensis, G. schirazensis, and G. truncatula.

Table 2. Microsatellite primers used in the multiplex PCR-detection of Galba cubensis (Lounnas et al., 2017b), Galba schirazensis (Lounnas et al., 2017a), and Galba truncatula [START_REF] Trouvé | W KT781322, KT781323, KT781324 Galba truncatula Morocco ND ND JN614387 Peru Moquegua 17°19'24′′S 70°59'29′′W KU870357 Venezuela El Sapo 08°52[END_REF]. We provide the GenBank accession number of primers, the size (in base pairs) of the PCR product (alleles) and the primer concentration. The size of the locus of G. truncatula was retrieved from [START_REF] Chapuis | High quantitative and no molecular differentiation of a freshwater snail (Galba truncatula) between temporary and permanent water habitats[END_REF] [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF]. The GenBank accession numbers from Lounnas et al. 360 (2017b) are those ITS2 sequences (except for KT461809 that is, in fact, a CO1 361 sequence) and the ones from [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF] and Lounnas et al. (2017a)

Table 3 .

 3 Galba individuals (n = 49) used to test and validate the multiplex PCR. Galba 357 cubensis has been molecularly identified byLounnas et al. (2017b), G. schirazensis by 358Lounnas et al. (2017a), and G. truncatula from Peru byLounnas et al. (2017b) and the 359 others by

  Agarose gel electrophoresis of multiplex PCR products of individuals of the six Galba species. These individuals have been genetically identified using ITS1, ITS2, CO1, and 18S sequences by[START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF]. See countries, sites, and GenBank accession numbers of CO1 sequences in Table1.

		Figures captions
		Figure 1.
	362						those of
	363	CO1 sequences. ND: no data.
	364
		Name	Country	Site	Coordinates	Number of	GenBank
		species				individuals	accession
							number
		Galba	Cuba	Contramaestre 20°30'08′′N		KU870347,
		cubensis			76°26'40′′W		KU870348
				Trinidad (Río El	21°43'57′′N		KU870343,
				Junco)	79°33'23′′W		KU870344
			Ecuador	Las Dos	01°56'01′′S		KT461809,
				Puertas	79°34'38′′W		KT461817
				Yaguachi	2°5'34′′S		KT461814
					79°42'30′′W
			Guadeloupe Pinadière	16°19'28′′N		KU870355,
					61°21'54′′W		KU870356
			Peru	Moquegua	17°19'24′′S		KU870349,
					70°59'29′′W		KU870350
				Ocoña	16°25'17′′S		KU870351,
					73°06'56′′W		KU870352
				Río Lurín	12°06'07′′S		KU870353,
					76°47'17′′W		KU870354
			Puerto Rico Canal Salinas	ND		KU870345,
							KU870346
			Venezuela Fincas 4M	10°46'N		KT781217,
				Tucacas	68°24'W		KT781218
				Hato Río de	10°34'48′′N		KT781205
				Agua	62°59'22′′W
				La Linda	10°05'24′′N		KT781202
					67°47'25′′W
				San Mateo	10°12'41′′N		KT781214,
					67°24'52′′W		KT781215
		Galba	Colombia	Finca Jocum	07°06'25" N		KY198253,
		schirazensis		Bucaramanga	73°04'60" W		KY198254
			Ecuador	Hacienda	00°46'18" S		KT781301
				Cienaga	78°37'10" W

Acknowledgments

We thank Nicolás Bonel and anonymous reviewers for their critical review of the manuscript. Fellowships granted by Erasmus Mundus PRECIOSA and Méditerranée Infection supported research stays of PA at the Institute de Recherche pour le Développement, MIVEGEC (Montpellier, France). AV was supported by a grant from IRD (BEST) and ML by a doctoral fellowship from University of Montpellier and a postdoctoral grant from Labex CeMeb. This study was financially supported by University of Montpellier, IRD, and CNRS.

Tables

 Table 1.Galba individuals (n = 11) used to design the multiplex PCR. These individuals have been analyzed and identified by [START_REF] Correa | Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis[END_REF] based on nuclear (ITS1 and ITS2) and mitochondrial (CO1 and 18S) sequences. The GenBank accession numbers provided here are those of the CO1 sequences. Some coordinates were corrected in order to match the specific locality: Frias (Argentina) and Owego (New York, USA).

Species

Supplementary Material

Table

Table S1. Microsatellite primer mixes tested in the 11 individuals of Galba species.

Each mix contained a different combination of primers that were chosen such that the PCR products from the three species differ in size (range given in base pairs into parentheses). Primers have been already described by [START_REF] Trouvé | W KT781322, KT781323, KT781324 Galba truncatula Morocco ND ND JN614387 Peru Moquegua 17°19'24′′S 70°59'29′′W KU870357 Venezuela El Sapo 08°52[END_REF] and Lounnas et al. (2017aLounnas et al. ( , 2017b)). The primer mix showed in row 10 is the one that allowed an accurate identification of Galba cubensis, Galba schirazensis, and Galba truncatula.

Mix Galba cubensis Galba schirazensis Galba truncatula 1 Lc13 (191-200) [START_REF] Chapuis | High quantitative and no molecular differentiation of a freshwater snail (Galba truncatula) between temporary and permanent water habitats[END_REF] and Lounnas et al. (2017aLounnas et al. ( , 2017b)).


Sonia Morgane

Coralie-Anne Thion
email: thion@biologie.ens.fr

Isabelle Mosser

Pauline Ferezou

Sofia Grisel

Donovan Baptista

Florent Low

Sonia Ginhoux

Etienne Garel
email: garel@biologie.ens.fr

Audinat

Fe

´zou, ..., Florent Ginhoux

Sonia Garel

Etienne Audinat
email: etienne.audinat@igf.cnrs.fr

Thion

Coralie-Anne Mosser

Isabelle Fe ´re

Pauline Grisel

Sofia Baptista

Donovan Low

Biphasic impact of prenatal inflammation and macrophage depletion on the wiring of neocortical inhibitory circuits

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

In Brief Thion et al. show that embryonic macrophages and inflammation shape the wiring of inhibitory neurons, in addition to their known roles on excitatory circuits. It highlights that prenatal immune challenges initially trigger hyperinhibition and miswiring, before leading to a reduced inhibitory drive, thereby revealing the importance of examining full developmental trajectories.

INTRODUCTION

Prenatal inflammation and dysfunction of microglia, the brain resident macrophages, have both been associated with the etiology of several neuropsychiatric disorders, including schizophrenia (SZ) and autism spectrum disorders (ASD) [START_REF] Beumer | The immune theory of psychiatric diseases: a key role for activated microglia and circulating monocytes[END_REF][START_REF] Choi | The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring[END_REF][START_REF] Estes | Maternal immune activation: Implications for neuropsychiatric disorders[END_REF][START_REF] Hammond | Microglia and the Brain: Complementary Partners in Development and Disease[END_REF][START_REF] Hanamsagar | Environment matters: microglia function and dysfunction in a changing world[END_REF][START_REF] Kim | bacteria promote neurodevelopmental abnormalities in mouse offspring[END_REF][START_REF] Mattei | Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment[END_REF][START_REF] Patterson | Maternal infection and immune involvement in autism[END_REF][START_REF] Sekar | Schizophrenia risk from complex variation of complement component 4[END_REF][START_REF] Yim | Reversing behavioural abnormalities in mice exposed to maternal inflammation[END_REF][START_REF] Solek | Maternal immune activation in neurodevelopmental disorders[END_REF][START_REF] Takano | Role of Microglia in Autism: Recent Advances[END_REF]. However, how these prenatal immune challenges impact on the developmental trajectory of neocortical circuits remains to be fully examined. On the one hand, microglia invade the brain during early embryogenesis [START_REF] Hoeffel | Fetal monocytes and the origins of tissueresident macrophages[END_REF][START_REF] Prinz | Ontogeny and homeostasis of CNS myeloid cells[END_REF][START_REF] Thion | Microglia and early brain development: An intimate journey[END_REF] and constitute key actors of excitatory synapse density and function during postnatal and adult life [START_REF] Li | Microglia and macrophages in brain homeostasis and disease[END_REF][START_REF] Miyamoto | Microglia contact induces synapse formation in developing somatosensory cortex[END_REF][START_REF] Paolicelli | Function and Dysfunction of Microglia during Brain Development: Consequences for Synapses and Neural Circuits[END_REF][START_REF] Salter | Microglia emerge as central players in brain disease[END_REF][START_REF] Schafer | Microglia Function in Central Nervous System Development and Plasticity[END_REF][START_REF] Wolf | Microglia in Physiology and Disease[END_REF][START_REF] Wu | Microglia: Dynamic Mediators of Synapse Development and Plasticity[END_REF]. On the other hand, models of prenatal inflammation using maternal immune activation (MIA) in rodents have been shown to induce in the adult offspring reduced functional GABAergic transmission from a specific population of interneurons expressing parvalbumin (PV) in both the prefrontal and the somatosensory cortices [START_REF] Canetta | Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons[END_REF][START_REF] Yim | Reversing behavioural abnormalities in mice exposed to maternal inflammation[END_REF]. Inhibitory fast spiking (FS) PV interneurons act as gain modulators in neocortical circuits and play important roles in the plasticity and functional maturation of the neocortical network during development [START_REF] Butt | A role for GABAergic interneuron diversity in circuit development and plasticity of the neonatal cerebral cortex[END_REF][START_REF] Hensch | Critical period plasticity in local cortical circuits[END_REF]. For instance, in sensory cortices, PV interneurons control feedforward inhibition (FFI) in response to external stimuli, which shapes the temporal response in principal neurons, and is essential for sensory perception [START_REF] Chittajallu | Emergence of cortical inhibition by coordinated sensory-driven plasticity at distinct synaptic loci[END_REF][START_REF] Feldmeyer | Inhibitory interneurons and their circuit motifs in the many layers of the barrel cortex[END_REF]. In agreement with their important roles in cortical circuits, increasing evidence points toward PV interneuron dysfunction in neurodevelopmental psychiatric disorders [START_REF] Nelson | Excitatory/Inhibitory Balance and Circuit Homeostasis in Autism Spectrum Disorders[END_REF]. Remarkably, manipulating the activity of PV neurons in adults in the somatosensory or prefrontal cortex is sufficient to rescue some behavioral deficits triggered by MIA [START_REF] Canetta | Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons[END_REF][START_REF] Yim | Reversing behavioural abnormalities in mice exposed to maternal inflammation[END_REF]. We previously found that both MIA and microglia embryonic dysfunction modulate the early positioning of subsets of interneurons in the developing cortical plate with a subsequent mild increase in the density of PV interneurons in the murine somatosensory cortex [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF]. These findings raise the possibility that prenatal immune challenges, including microglia deficits or MIA, which modifies microglial functioning but also a wide range of additional processes [START_REF] Estes | Maternal immune activation: Implications for neuropsychiatric disorders[END_REF][START_REF] Smolders | Controversies and prospects about microglia in maternal immune activation models for neurodevelopmental disorders[END_REF], might interfere with the developmental trajectory of PV interneurons from early stages.

Here, by combining anatomical, electrophysiological, and imaging approaches, we show that transient early macrophage depletion and MIA lead to a reduced inhibitory drive of PV interneurons onto their targets in the layer 4 of the postnatal barrel cortex, consistent with what has been reported in other MIA paradigms. However, this hypo-inhibition was preceded by an unexpected, profound miswiring and hyperconnectivity of PV interneurons in juveniles, at a time point critical for learning and plasticity. These findings reveal a convergence of distinct immune challenges onto well-established actors in cortical networks and highlight a role of microglia in the wiring of inhibitory circuits. Importantly, they reveal an unexpected temporal trajectory, with major importance for understanding pathological brain wiring and designing effective interventional therapies.

RESULTS

Reduced Inhibitory Drive of PV Interneurons onto Principal Cells in Adult Mice

To investigate how prenatal immune challenges impact on inhibitory circuits, we used genetically engineered mice to label PV interneurons (PV cre/+ ;R26 tom/+ ) in two experimental paradigms: (1) MIA triggered by a lipopolysaccharide (LPS) injection at embryonic day (E) 13.5, which mimics a bacterial infection; and (2) early injections of anti-CSF-1R antibodies, which target macrophage precursors in the yolk-sac and lead to a severe depletion of microglia from E7 to E18.5, followed by a repopulation completed by postnatal day (P) 7 [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF][START_REF] Waisman | Homeostasis of Microglia in the Adult Brain: Review of Novel Microglia Depletion Systems[END_REF]. Notably, in addition to microglia, our transient depletion model targets all embryonic macrophages, including meningeal macrophages, perivascular macrophages, and other peripheral populations, which repopulate around E14.5 [START_REF] Hoeffel | C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages[END_REF]. Thus, whereas brain-specific phenotypes are likely due to the depletion of microglia, a contribution of additional populations of macrophages cannot be excluded. We previously reported that both MIA and depletion models cause an increase in inhibitory interneurons entering the cortical plate [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF]. Before probing the connectivity of PV interneurons, we carefully examined the overall barrel cortex architecture using the thalamocortical axonal marker, vGlut2 in coronal and tangential sections of flattened cortices (Figures S1A-S1K). After MIA or in utero macrophage depletion, we found a similar organization of the barrel field (Figures S1A-S1K) and an unchanged number of microglia at P7 compared to controls (Figures S1L and S1M), confirming that the microglial depletion is transient. We next examined both models at P60, in young adults, and found no significant change in the overall density of PV cells within the barrel cortex (Figures 1A,1B,and S2A). To examine functional properties, we recorded unitary inhibitory post-synaptic currents (uIPSC) evoked by PV FS interneurons onto principal excitatory neurons contained within the same barrel at P60-P65 (Figures 1C and1D). In both models, the amplitude of the unitary synaptic responses in principal cells (PC) was almost reduced by half compared to control mice . The short-term dynamics of unitary IPSCs evoked by trains of presynaptic action potentials was not dramatically affected (Figure 1F), although a small but consistent decrease in paired-pulse ratio (PPR), consistent with a small increase in release probability, was observed (data not shown). These severe phenotypes on the amplitude of unitary IPCSs are consistent with previous findings reporting lower synaptic inhibition mediated by PV cells in the prefrontal or somatosensory cortices in the MIA offspring [START_REF] Canetta | Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons[END_REF][START_REF] Yim | Reversing behavioural abnormalities in mice exposed to maternal inflammation[END_REF]. Because microglia have been involved in synaptic formation and elimination [START_REF] Miyamoto | Microglia contact induces synapse formation in developing somatosensory cortex[END_REF][START_REF] Schafer | Microglia Function in Central Nervous System Development and Plasticity[END_REF], we tested whether the sharp decrease in unitary synaptic responses could be explained by a major reduction in the number of synapses. We found a very limited or no decrease in the numbers of synaptic boutons formed by PV interneurons onto the soma of PC in the two experimental conditions (Figure S3). While this reduction could contribute to the observed electrophysiological phenotype, there are likely additional and complex changes in synaptic weight or composition in MIA and depleted offspring. Taken together, our experiments reveal a long-term impact of prenatal inflammation and microglia depletion onto the wiring of PV inhibitory circuits.

Enhanced Inhibitory Drive of PV Interneurons onto Principal Cells in Juveniles

To explore further the developmental trajectory of the PV network, we focused on P20, which corresponds to a time point when neurons express robust levels of PV and to a key period of learning an exploration. At this stage, we detected a mild increase in the density of PV neurons after MIA or transient embryonic depletion of microglia (Figures 2A,2B,and S2A). Next, we recorded unitary inhibitory synaptic connections made by PV FS interneurons onto PC in the same barrel at P20-P25 (Figures 2C and2D). Notably, the firing properties of these FS interneurons was not altered by experimental conditions (Figure 2C and data not shown). In sharp contrast to what we observed in adult mice, the amplitude of unitary synaptic responses was almost twice as large in both MIA and embryonically depleted pups compared to controls (Figures 2C-2F). The short-term dynamics of unitary IPSCs evoked by trains of presynaptic action potentials was not dramatically affected (Figure 2F), although a small but consistent decrease in PPR, consistent with a small increase in release probability, was observed (data not shown). In addition, the densities of synaptic boutons formed by PV interneurons onto the soma of PC was only slightly increased (Figure S3). These observations thus indicate that the observed phenotype is likely due to complex modifications with several contributing factors (Figure S3). Irrespective of the underlying mechanisms, our results show that immune challenges during prenatal life induce, in juveniles, a transient hyper-inhibition of PC by PV interneurons in layer 4.

Miswiring in Juveniles Impairs Propagation of Sensory Information

To examine how modifications of PV interneurons in juveniles might impact onto the functioning of cortical circuits, we first tested their role in gating incoming sensory information arriving from the thalamus. To this aim, we measured FFI in the layer 4 of the barrel cortex (Figure S4). Using thalamocortical slices [START_REF] Agmon | Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro[END_REF] that preserve the thalamic projection to the somatosensory barrel cortex, we could record in PC the monosynaptic excitatory and disynaptic inhibitory currents evoked by thalamic stimulation (Figure S4). These measures constitute a read out of the functional integration of PV interneurons in thalamocortical circuits [START_REF] Feldmeyer | Inhibitory interneurons and their circuit motifs in the many layers of the barrel cortex[END_REF]. We found a trend for enhanced inhibitory currents triggered by thalamic stimulation in PC of both MIA and depleted pups compared to controls, whereas excitatory currents were largely unaffected (Figure S4B). While these experiments are relatively variable and can be biased by the angled sectioning needed to preserve thalamocortical connections, there are consistent with the enhanced inhibitory drive found in paired recordings at P20 (Figure 2). They also suggest, because disynaptic inhibition is more moderate than inhibition triggered by direct stimulation of interneurons (paired recordings), the existence of potential compensation mechanisms in the recruitment of PV interneurons by thalamic inputs. Nevertheless, we observed an overall increase in the inhibition/excitation ratio (GABA/AMPA) after MIA and prenatal depletion (Figure S4C), thus confirming an alteration of layer 4 PV inhibitory circuits in the context of thalamic stimulation.

Axonal arborization of most FS-PV cells is usually confined to the home-barrel, with the exception of minor populations of basket cells [START_REF] Koelbl | A barrelrelated interneuron in layer 4 of rat somatosensory cortex with a high intrabarrel connectivity[END_REF][START_REF] Li | Two functional inhibitory circuits are comprised of a heterogeneous population of fast-spiking cortical interneurons[END_REF][START_REF] Rudy | Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons[END_REF][START_REF] Shigematsu | Gap Junctions Interconnect Different Subtypes of Parvalbumin-Positive Interneurons in Barrels and Septa with Connectivity Unique to Each Subtype[END_REF]. To explore whether such spatial pattern of functional projections of developing PV cells was also affected by MIA or embryonic microglial depletion, we used PV cre/+ ;R26 Chr2/+ mice and measured the inhibitory drive evoked by a local optogenetic activation of PV interneurons on single principal cells belonging to the same versus neighboring barrels (Figures 3A-3F). In controls, in agreement with previous reports [START_REF] Ka ¨tzel | The columnar and laminar organization of inhibitory connections to neocortical excitatory cells[END_REF], optogenetic stimulation of PV cells triggered robust inhibition within the same barrel albeit a very limited horizontal inhibition in adjacent barrels (Figures 3A-3F). In both MIA and embryonically depleted juveniles, the inhibition evoked by the optogenetic stimulation of same barrel was larger than in controls (Figures 3A and3D), consistent with the larger amplitude of intra-barrel unitary synaptic responses observed in paired recordings (Figure 2). In addition, the inhibition onto the adjacent barrel or two barrels away was also much larger in these animals, reaching levels of responses that are obtained in controls by stimulating the barrel containing the recorded neuron (Figures 3B,3C,3E,and 3F). Thus, in addition to a local enhanced inhibition, we detected a stronger horizontal inhibition between adjacent barrels (Figure 3G), revealing a miswiring of inhibitory circuits after prenatal immune challenges.

To further tackle whether these local circuits deficits might impair or modify the flow of sensory information in vivo, we visualized, in real time, cortical activity evoked by whisker stimulation within the barrel field of anesthetized mice by means of voltage sensitive dye imaging (Figure 4A) [START_REF] Ferezou | Visualizing the cortical representation of whisker touch: voltage-sensitive dye imaging in freely moving mice[END_REF]. In control conditions, as well as after MIA or transient embryonic depletion of microglia, a single whisker stimulation evoked first a signal in the corresponding column of the barrel cortex that then propagates horizontally to cover the whole barrel field (Figure 4B; Video S1). Comparing the amplitude of the whisker-evoked signals from a region of interest matching the column corresponding to the stimulated whisker (central region of interest, ROI 1), either at the peak of the response, or at different time points after the stimulation, we observed no significant difference between mice in the control group and the MIA or the depleted group, respectively (data not shown). However, while the latency of the response appeared similar in all groups when measured in this central ROI 1, albeit a bit shifted, MIA or transient embryonic depletion of microglia led to a significant slowdown of the lateral propagation of the signal as compared to control situation (Figures 4B-4E). Thus, horizontal propagation of sensory information in the barrel cortex is impaired by prenatal immune chal-lenges, consistently, with enhanced horizontal inhibition across barrels.

DISCUSSION

Our work reveals a remarkable biphasic impact of two distinct immune challenges on the normal assembly of cortical circuits, thereby providing insights onto how microglia dysfunction or immune risks lead to pathological brain wiring. Specifically, we report that both MIA and embryonic macrophage depletion trigger a transient increase in the number of PV neurons and of their inhibitory drive onto PC in the barrel cortex, before inducing a hypo-inhibition in the adult offspring.

The features and the temporality of the phenotypes observed in both models are remarkably similar, raising the possibility that the defect we characterized in MIA offspring results from an inability of microglia to fulfill their normal physiological roles. This is in sharp contrast with the general assumption that MIA, by perturbing microglial activity, triggers an aberrant excessive response of these immune cells [START_REF] Estes | Maternal immune activation: Implications for neuropsychiatric disorders[END_REF][START_REF] Hirbec | The microglial reaction signature revealed by RNAseq from individual mice[END_REF][START_REF] Mattei | Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment[END_REF][START_REF] Patterson | Maternal infection and immune involvement in autism[END_REF][START_REF] Pont-Lezica | Microglia shape corpus callosum axon tract fasciculation: functional impact of prenatal inflammation[END_REF]. Prenatal depletion of macrophages can indeed result in changes that are opposite to those induced by MIA, such as microglial control of dopaminergic axon outgrowth, suggesting that in this context MIA does trigger an increased microglial activity [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF]. These observations reinforce the notion that microglia exert diverse functions that are precisely regulated and determined by their environment during normal and pathological development [START_REF] Casano | Microglia: multitasking specialists of the brain[END_REF][START_REF] Mosser | Microglia in CNS development: Shaping the brain for the future[END_REF][START_REF] Thion | On place and time: microglia in embryonic and perinatal brain development[END_REF][START_REF] Thion | Microglia and early brain development: An intimate journey[END_REF].

In the barrel cortex, we found that both MIA and macrophage embryonic depletion affect inhibitory circuits from early stages, thereby highlighting the importance of examining the roles of immune cells and cytokines in the development and maturation of GABAergic networks. Notably, we found that both MIA and transient embryonic macrophage depletion alter the inhibitory drive provided by PV interneurons, likely through a complex mechanism that might include changes in synapse numbers and functionalities. Our results in adult offspring are in good agreement with previous studies in other MIA models and with functional analyses in mice. In particular, several studies reported that hypoinhibition from PV neurons constitutes a core functional deficit of neurodevelopmental disorders, impairing the normal functioning of circuits. Accordingly, modulating the activity of these cells is sufficient to improve behavioral deficits in mouse models of neurodevelopmental disorders [START_REF] Canetta | Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons[END_REF][START_REF] Nelson | Excitatory/Inhibitory Balance and Circuit Homeostasis in Autism Spectrum Disorders[END_REF][START_REF] Yim | Reversing behavioural abnormalities in mice exposed to maternal inflammation[END_REF]. Microglia deficits, however, have been mostly associated with the dysregulation of excitatory synapse development and elimination [START_REF] Hoshiko | Deficiency of the microglial receptor CX3CR1 impairs postnatal functional development of thalamocortical synapses in the barrel cortex[END_REF][START_REF] Paolicelli | Synaptic pruning by microglia is necessary for normal brain development[END_REF][START_REF] Schafer | Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner[END_REF][START_REF] Tremblay | Microglial interactions with synapses are modulated by visual experience[END_REF][START_REF] Weinhard | Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction[END_REF][START_REF] Zhan | Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior[END_REF]. Our results underline that examining inhibitory synapses and their developmental trajectories will be essential to grasp the full spectrum of functional deficits associated with microglia and neuroimmune dysfunctions.

In particular, our work reveals a striking biphasic impact of prenatal immune challenges onto PV networks. In juveniles, our slice analyses and in vivo recordings reveal an initial excess inhibition in layer IV, an increased FFI, and a deficit in the horizontal propagation of sensory information within upper layers of the barrel cortex. These results are consistent with the existence of a strong brake on the integration of sensory inputs, at least during this period of increased functional inhibition of the PV network. The transition from juveniles to the adult situation could in theory result from an excessive, misadjusted homeostatic compensation of the large excess of inhibition we observed in juvenile mice. For instance, the excessive numbers of PV interneurons that we observe at P20 are likely eliminated by P60, since interneurons are permanently labeled by the PV cre driver. In addition, the unitary IPSC are drastically reduced in MIA and depleted offspring between P20 and P60, suggesting a profound remodeling of PV interneuron connectivity. While our quantitative analysis of synaptic boutons suggests a consistent reduction in the densities of PV synapses onto the soma of PC in our experimental models between P20 and P60, these modest changes cannot account for the major electrophysiological switch that we have identified. Further experiments will be required to examine potential modifications in the synaptic strength and properties.

Irrespective of the underlying mechanisms, our results have major implications for our understanding of brain wiring. First, they highlight that inhibitory networks have several waves of adaptation to an initial embryonic perturbation, which can be drastically distinct in juveniles and adults. It thus underlines the importance of examining the full developmental trajectory of networks to understand normal physiology as well as to design adapted interventional therapies in children or adolescents. Second, our results show that prenatal immune challenges perturb the wiring of the somatosensory barrel cortex. While many studies report deficits in sensory perception associated with neurodevelopmental disorders, including ASD and SZ patients [START_REF] Leekam | Describing the sensory abnormalities of children and adults with autism[END_REF][START_REF] Markram | The intense world theory -a unifying theory of the neurobiology of autism[END_REF], a limited number of studies focuses on the impairment associated with the circuits involved in sensory perception. Remarkably, it is well established that peripheral perturbations affecting sensory perception are sufficient to induce a large panel of behavioral deficits, including abnormal social interactions [START_REF] Orefice | Peripheral Mechanosensory Neuron Dysfunction Underlies Tactile and Behavioral Deficits in Mouse Models of ASDs[END_REF].

Our finding that the somatosensory cortex is drastically altered early in development, with likely consequences on sensory integration, underlines the importance of examining these structures, in addition to the better characterized alterations in the prefrontal cortex.

In conclusion, our study reveals that defects in cortical inhibition observed in adult offspring after MIA result from a complex set of events that includes an unexpected and large excess of cortical inhibition by PV cells. These observations also strongly support a role for microglia in this impaired development of cortical circuits and shed light on how microglia and prenatal immune challenges can impact the functional wiring of the brain in neuropsychiatric disorders. Both females and males have been used for all experiments presented this study, except for the voltage-sensitive dyes recording that were only performed in males. Embryonic day (E) 0.5 was set as the day of vaginal plug formation on the dam, with postnatal day (P) 0 defined as the day of birth. Mice were housed at the ''Institut de Biologie de l'ENS'' (IBENS), at the ''Centre universitaire des Saints Pe `res'' of Paris Descartes University, Paris, France, and at the Institut des Neurosciences de Paris Saclay'' (NeuroPSI), Gif-sur-Yvette, France. These mice were handled in accordance with European regulations following the recommendations of the local ethics committees. Experiments were performed in conformity with the European (2010/63/UE) and French legislations relative to the protection of animals used for experimental and other scientific purposes (authorization numbers: 2012-0068 delivered by the local ethical committee Charles Darwin for IBENS and #59 for CEEA34.EA.027.11 and CEEA16-032 for Paris Descartes).

METHODS DETAILS

Microglial embryonic depletion and LPS prenatal injections Pregnant females were administered anti-CSF-1R monoclonal antibodies (aCSF-1R, cline AFS98) intraperitoneally at E6.5 and E7.5 (3 mg per day in sterile PBS)(as in [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF]. The efficiency of the procedure was assessed by immunohistochemistry at P0. Pregnant females were administered LPS in sterile PBS (0.12 mg/g mouse; InvivoGen and Sigma) at E13.5 by a single intraperitoneal injection. Control pregnant females were injected at E13.5 with sterile PBS and showed no detectable phenotype. Three different batches of LPS were used and produced similar results.

Immunohistochemistry

Intracardiac perfused and dissected brains were fixed in 4% PFA at 4 C overnight. Immunohistochemistry was performed on 40 mm thick free-floating vibratome sections, as previously described [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF]. Sections were blocked for 2 hours with PBS containing 10% fetal bovine serum and 0.01% Triton X-100, and then incubated overnight with primary antibodies. Sections were rinsed twice in PBS containing 0.01% Triton X-100, followed by several PBS washes, before overnight incubation with secondary antibodies (1/400 in PBS). Hoechst (1/1000) was used for fluorescent nuclear counterstaining.

Image acquisition

To determine cells density, images of the somatosensory neocortex were acquired at 10X with the Leica TCS SP5 confocal microscope. ImageJ and Adobe Photoshop were used for image processing.

Electrophysiology

Slice preparation

Mice aged between 20 and 25 days (juveniles) or between 60 and 65 days (adults) were anesthetized with isoflurane, decapitated, and slices through the somatosensory cortex were cut using a Leica VT1200 vibratome in an oxygenated (5% CO2 and 95% O2) icecold protective extracellular solution containing for the juvenile mice (in mM): 215 sucrose, 2.5 KCl, 1.3 NaH 2 PO 4 , 25.9 NaHCO 3 , 20 Dglucose, 5 sodium pyruvate, 7 MgCl 2 , and 1 CaCl 2 (pH 7.3, 320 mOsm), or for the adult mice (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH 2 PO 4 , 30 NaHCO 3 , 20 HEPES, 2 thiourea, 25 D-glucose, 5 sodium ascorbate, 3 sodium pyruvate, 10 MgCl 2 , and 0.5 CaCl 2 (pH 7.3, 315 mOsm). Thalamocortical slices were cut using the method described by [START_REF] Agmon | Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro[END_REF]. After cutting, slices of young animals were incubated for 30 min at 34 C in regular artificial cerebro-spinal fluid (aCSF; pH 7.3, 310 mOsm) containing (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 20 mM D-glucose, 1 sodium pyruvate, 1 MgCl 2 , and 2 CaCl 2 . For older animals, slices were first incubated in the same protective NMDG-containing extracellular solution for 7 min at 34 C and then incubated at 34 C for 30 min in regular aCSF. The slices were then maintained at room temperature (RT, 22-24 C) for 0.5-6h oxygenated aCSF until the recordings. After the incubation, individual slices selected for best visibility of the barrel field in cortical layer 4 were transferred to a recording chamber perfused with the same solution at 5 ml/min at RT.

Paired recordings

Coronal cortical slices were prepared from PV Cre/+ ;R26 mt/+ juvenile mice, thalamocortical slices were prepared from PV Cre/+ ;R26 mt/+ adult mice. To establish a paired recording, a whole-cell recording from a dtTomato-positive interneuron in layer 4-barrel cortex was first obtained and then sequential whole-cell recordings were made from neighboring stellate cells within 50-100 mm. The presynaptic and postsynaptic cells were patched with the above described K-and Cs-based internal solution, respectively. Presynaptic PV interneurons were held in voltage clamp at À70 mV and induced to fire single action potentials by 4 depolarizing pulses of 50 mV of 1 ms at 20 Hz. Postsynaptic cells were held in voltage-clamp at 0 mV. Paired recordings were performed in presence of the AMPA receptor antagonist (NBQX; 10 mM, Hellobio) and the NMDA receptor antagonist (D-AP5; 50mM, Hellobio).

Optogenetic stimulations

Coronal cortical slices were prepared from PV Cre/+ ; R26 Chr2/+ juvenile mice. Whole-cell patch-clamp recordings were obtained from excitatory principal cells (PC) and Parvalbumin-expressing (PV) fast-spiking interneurons of the layer 4 of the somatosensory barrel cortex in voltage-and current-clamp modes, respectively. For voltage-clamp recordings patch pipettes (3-5 MU) were filled with a solution containing (in mM): 125 CsMeSO 3 , 10 HEPES, 10 EGTA, 8 TEA-Cl, 5 4-AP, 0.4 GTP-Na, 4 ATP-Na 2 , 1 CaCl 2 and 1 MgCl 2 (pH 7.3,290 mOsm). For current-clamp recordings aiming at adjusting parameters of the optogenetic stimulation (see below), pipettes (3-5 MU) were filled with a solution containing in mM: 125 K-Gluconate, 2 MgCl 2 , 10 HEPES, 0.4 Na + -GTP, 4 ATP-Na 2 , 10 Phosphocreatine disodium salt, 5 KCl, 0.5 EGTA (pH 7.3,290 mOsm). Recordings were performed in the presence of the following glutamatergic receptors blockers: 10 mM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX; Hellobio) and 50 mM D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5; Hellobio). IPSCs recorded in layer 4 PC were optogenetically evoked at 0 mV by driving the ChR2 activity in PV interneurons with 470 nm blue light (0.9 mW/mm 2 ) delivered by a LED (Cairn Research, OptoLED) through the 40X water-immersion objective (Olympus). We used a 100 mm diameter light field (i.e. about the size of one barrel) and light stimulations of 5 ms were delivered at 0.05 Hz. In pilot experiments, the light intensity was adjusted to 1.5 times the threshold for eliciting one action potential in FS cells and we verified that this threshold did not differ among the different experimental groups (data not shown). Thalamic stimulations Thalamocortical EPSCs and IPSCs were evoked in principal neurons of the barrels using a stainless steel bipolar microelectrode (100 mm tip diameter, 250 mm intertip distance; Rhodes Medical Instruments) connected to a stimulus isSolation unit (Iso-stim 01D; npi Electronic) and placed in the ventrobasal thalamus or in the internal capsule near the thalamus border. The intensity of stimulation (0.1 ms at 0.05 Hz) was first adjusted according to the protocol of minimal stimulation described in [START_REF] Hoshiko | Deficiency of the microglial receptor CX3CR1 impairs postnatal functional development of thalamocortical synapses in the barrel cortex[END_REF] and then increased 1.5 times to ensure the recruitment of feedforward inhibition. The EPSCs and IPSCs were recorded at À70 mV and 0 mV, respectively.

Voltage-sensitive dye (VSD) imaging VSD imaging of the cortical activity evoked by tactile whisker stimulation was performed on P27-P33 male mice under isoflurane anesthesia (induction: 3%-4%, maintenance: 1%-1.5%). The left barrel cortex was exposed and stained for 1h with the VSD RH1691 (Optical Imaging Ldt, Israel) at 1mg/ml, in Ringer's solution containing [in mM]: 135 NaCl, 5 KCl, 5 HEPES, 1.8 CaCl 2 , 1 MgCl 2 . After removing the unbound dye, the cortex was covered with agarose (0.5%-1%) and a coverslip. Cortical imaging was performed through a tandem-lens fluorescence microscope (SciMedia), equipped with one Leica PlanApo 5x (objective side) and one Leica PlanApo 1x (condensing side), a 100 W halogen lamp gated with an electronic shutter, a 630 nm excitation filter, a 650 nm dichroic mirror, and a long-pass 665 nm emission filter (Figure 4A). The field of view was 2.5 3 2.5 mm, resulting in a pixel resolution of 25 3 25 mm.

The right C2, C3 or C4 macrovibrissae were individually deflected in a pseudo-randomized manner using a multi-whisker stimulator [START_REF] Jacob | The Matrix: a new tool for probing the whisker-to-barrel system with natural stimuli[END_REF]. Whiskers were inserted keeping their natural angle in 27G stainless steel tubes attached to the piezoelectric benders (Noliac, Denmark), leaving 2 mm between the tip of the tube and the whisker base. Each whisker deflection consisted of a 95 mm displacement (measured at the tip of the tube) a 2 ms rising time, a 2 ms plateau and a 2 ms fall. Specific filters were applied to the voltage commands to prevent mechanical ringing of the stimulators. Following the experiments mice were perfused with saline followed by paraformaldehyde (4% in 0.1M phosphate buffer). After an overnight post-fixation in paraformaldehyde, the brains were cut in 100 mm-thick tangential sections that were stained for cytochrome oxidase. Microphotographs of the tangential sections were registered and the barrel maps reconstructed using a method implemented in MATLAB (MathWorks, USA), as previously described [START_REF] Perronnet | An automated workflow for the anatomo-functional mapping of the barrel cortex[END_REF]. The functional VSD data were aligned with the reconstructed barrel maps by using the superficial blood vessels as anatomical landmarks.

QUANTIFICATION AND STATISTICAL ANALYSIS

Image analysis

For barrel characterization, images were converted to greys and inverted before delineating vglut2-positive barrels (thalamocortical) and counting Iba1-positive cells. Hoechst and vglut2 stainings were used to delineate the thickness of cortical layers. The relative fluorescence intensity across the C2 barrel was assessed as described in [START_REF] Toda | Birth regulates the initiation of sensory map formation through serotonin signaling[END_REF]. Dsred-positive cells quantification in PV cre/+ ;R26 tom/+ mice was performed using Bitplane Imaris software. Synapses were automatically detected with the ImageJ software by first applying despeckle followed by a smooth filter, then local maxima were found on the VGAT channel. The local maximas were than visualized on the NeuN channel, and the number of maximas par soma were quantified. Results are represented by the number of synapses par soma.

Electrophysiological data acquisition and analysis

Whole-cell patch-clamp recordings were obtained using either Axopatch 200B or 700B amplifiers (Molecular Devices). Currents and potentials were low-pass filtered at 5 kHz or 6 kHz, collected at a 10 kHz frequency, and analyzed offline using pClamp 10.7 (Molecular Devices) for voltage-clamp recordings, or using a custom written procedure in IGOR Pro 6.0 (Wavemetrics) for current-clamp recordings. For all recordings performed with K-gluconate or Cs-gluconate in the pipette, potentials were corrected for a junction potential of À10 mV. Series resistances (Rs) were not compensated but continuously monitored using 5 or 10 mV hyperpolarizing pulses. Recordings were discarded if Rs increased by > 20% during the whole experiment. For all experiments, the amplitude of EPSCs and IPSCs were quantified from the average of 15-30 consecutives responses. For paired-recording analysis, the peak responses to each stimulation were measured and then averaged over all trials. The paired-pulse ratio was calculated as the average peak amplitude of the second response divided by that of the first one. In current clamp experiments aiming at characterizing basic membrane properties, a series of hyperpolarizing and depolarizing current steps were applied for 1 s in 25-50 pA increments at 5 s intervals. Pairs obtained with presynaptic cells displaying low firing rate or adaptation ratio (< 0.6) or small AHP (< 10 mV) were discarded.

Voltage-sensitive dye data acquisition and analysis Using a CMOS-based camera (MiCam Ultima, SciMedia), sequences of images of 1 s were acquired at 500 Hz, with or without (blank trials) whisker stimulation, alternately, every 20 s. Variations of the light intensity were initially recorded as variations over the resting light intensity (first acquired frame). Acquisition and data preprocessing were done using in-house software (Elphy, G. Sadoc, UNIC-CNRS), further analyses were made using custom written routines in IgorPro (WaveMetrics, USA). Subtraction of a pixel by pixel bestfit double-exponential from the averaged unstimulated sequence was used to correct for photobleaching.

For each averaged whisker evoked response (n = 20 trials), a 2D-Gaussian fit of the evoked signal at 12 ms following the start of the whisker stimulus was used to localize the center of a first region of interest (ROI) defined as a 100 mm-diameter circle. A second, more distal ROI, was defined as a 50 mm-wide ring placed at 575 mm from the center of the first ROI. Profiles of fluorescence were computed from these ROIs and normalized to determine the latency to 90% of the peak. Responses with amplitudes < 0.1% were discarded from the analysis. Signals measured in response to the deflection of the 3 individual whiskers were considered as independent.

Statistical Analysis

All data are presented as mean ± SEM. Non-parametric two-sided Mann-Whitney U-tests were used to compare two distributions. If the normality test was statistically significant, two-sided Student's t tests were used to compare two distributions. Two-way ANOVA with Sidak post hoc test were used to compare groups of data. All graphs and statistical analyses were generated using GraphPad Prism software. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 1 .

 1 Figure 1. Maternal Immune Activation and In Utero Microglia Depletion Do Not Affect the Density of Parvalbumin Interneurons but Decrease Their Synaptic Connections onto Principal Neurons in Layer 4 of the Adult Barrel Cortex (A) Coronal sections through the P60 somatosensory barrel cortex of control, MIA, and microgliadepleted PV Cre/+ ;R26 tom/+ mice revealing no significant modulation of tdTomato-positive interneurons (control: n = 10; MIA: n = 6; depleted: n = 4). Scale bar, 150 mm. (B) Comparison of tdTomato-positive interneurons density within cortical layers between controls, MIA, and depleted P60 mice (control: n = 10, MIA: n = 6, depleted: n = 4). (C) Scheme of the recording configuration depicting dual whole-cell recordings from a fastspiking PV interneuron (FS) targeting a layer 4 principal cell (PC) in the barrel cortex. Inset: representative voltage traces of a FS cell in response to hyperpolarizing and depolarizing current pulses. (D) Representative average traces of inhibitory postsynaptic currents (IPSC 1-4 ) evoked in PC by trains of action potentials elicited in FS interneurons in thalamocortical slices of control, MIA, and microglia-depleted P60 mice. (E) Comparison of the first IPSC (IPSC 1 ) amplitude (control: n cells = 12, 6 mice; MIA: n cells = 8, 4 mice; depleted: n cells = 8, 3 mice). (F) Comparison of the IPSC amplitude evolution during the train between the three experimental conditions. Data are represented as mean ± SEM. Two-sided unpaired Mann-Whitney test was performed to assess differences. *p < 0.05, **p < 0.01. n.s., not significant. See also Figure S2.

Figure 2 .

 2 Figure 2. Maternal Immune Activation and In Utero Microglia Depletion Alter the Density of PV Interneurons and Enhance Their Synaptic Connections onto Principal Neurons in Layer 4 of the Juvenile Barrel Cortex (A) Coronal sections through the barrel cortex of control, MIA, and microglia-depleted P20 PV Cre/+ ;R26 tom/+ mice showing the increased number of dsred-positive interneurons (control: n = 13, MIA: n = 17, depleted: n = 13). Scale bar, 150 mm. (B) Comparison of tdTomato-positive interneurons density within all cortical layers between controls, MIA, and depleted P20 mice (control: n = 13, MIA: n = 17, depleted: n = 13). (C) Scheme of the recording configuration and representative voltage traces of a fast-spiking PV interneuron (FS) (as in Figure 1C). (D) Representative average traces of inhibitory postsynaptic currents (IPSC 1-4 ) evoked in PC by trains of action potentials elicited in FS interneurons in coronal cortical slices of control, MIA and microglia-depleted P20-P25 mice. (E) Comparison of the IPSC 1 amplitude (control: n cells = 31, 13 mice; MIA: n cells = 26, 13 mice; depleted: n cells = 17, 7 mice). (F) Comparison of the IPSC amplitude evolution during the train between the three experimental conditions. Data are represented as mean ± SEM. Two-sided unpaired Mann-Whitney test was performed to assess differences. **p < 0.01, ***p < 0.001. PC, principal cells. See also Figure S2.

Figure 3 .

 3 Figure 3. Maternal Immune Activation and In Utero Microglia Depletion Enhance Inter-Barrel Inhibition in the Cortex of Juvenile Mice (A-F) Representative traces of population IPSCs evoked in single principal cell (PC) by blue light stimulation of ChR2-expressing PV interneurons of the same (A), of the adjacent (B), and of the next adjacent (C) barrels in PV cre/+ ;R26 Chr2/+ P20-P25 mice as illustrated on the drawings. Comparison of the light-evoked IPSCs in the same (D), adjacent (E), and next adjacent (F) barrels in the three experimental conditions (control: n cells = 41, 18 mice; MIA: n cells = 40, 13 mice; depleted: n cells = 22, 5 mice). For the next adjacent barrel, only cells displaying responses in the adjacent barrel were recorded: n cells = 16, 11 mice; MIA: n cells = 34, 13 mice; depleted: n cells = 20, 5 mice). (G) Percentage of PC responding to the optogenetic stimulation of the different barrels in control and prenatal MIA or microglia-depleted mice. Data are represented as mean ± SEM. Two-sided unpaired Mann-Whitney test was performed to assess differences. ***p < 0.001.

Figure 4 .

 4 Figure 4. Maternal Immune Activation and In Utero Microglia Depletion Delay Lateral Spread of Sensory Information In Vivo (A) Scheme of the recording configuration. (B) Spatio-temporal dynamics of membrane potential changes imaged using voltage-sensitive dye (VSD) in anesthetized control, MIA, and depleted animals after a brief deflection of a single whisker. The snapshots display the averaged fluorescence signal (n = 20 trials) for representative cases at six different timings (8, 12, 16, 20, 24, and 28 ms) relative to the time of deflection of the C2 whisker. The barrel map reconstructed from a post hoc cytochrome oxidase staining and overlaid onto the VSD signals is shown as white outlines. C2 deflection evokes a rapid localized depolarization limited to the C2 cortical barrel column and over the next milliseconds, the depolarization spreads across the barrel field. This spread of the sensory responses was assessed by quantifying the fluorescent signals from two different defined areas (region of interest [ROI] 1: green circle at the center of the column matching the stimulated whisker, and ROI 2: pixels included between the two blue circles at 550 and 600 mm radial distance from the center of ROI 1, respectively). Scale bar, 500 mm. (C) Spread of the sensory responses in ROI 1 and ROI 2 in control, MIA, and depleted mice corresponding to (B). (D) Latencies to 90% of the peak response (averaged over 20 stimulations) computed for each ROI in control, MIA and depleted mice (control: n = 6; MIA: n = 6, depleted: n = 5; 3 whiskers stimulated by animal). (E) Differences of latencies between ROI 2 and ROI 1 for control, MIA, and depleted mice. Data are represented as mean ± SEM. Two-sided Student's t test was performed to assess differences, or if the normality test failed, two-sided unpaired Mann-Whitney test was performed. *p < 0.05, ***p < 0.001. n.s., not significant. See also Video S1.

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ACKNOWLEDGMENTS

We are grateful to members of the Garel and Audinat labs for discussions and critical comments on the manuscript. We thank the IBENS Imaging Facility (France BioImaging, supported by ANR-10-INBS-04, ANR-10-LABX-54 MEMO LIFE, and ANR-11-IDEX-000-02 PSL Research University, ''Investments for the future''). We are grateful to C. Auger, A. Delecourt, E. Touzalin, D. Valera, C. Le Moal, A. David, and M. Omnes, for excellent technical assistance. This work was supported by grants from INSERM, CNRS, the ERC Consolidator Grant NImO 616080 to S.G., and by grants from INSERM, the Fondation pour la Recherche Me ´dicale (FRM: DEQ20140329488), European Commission (H2020-MSCA-722053 EU-GliaPhD) to E.A. C.-A.M. was supported by the Re ´gion Île-de-France (DIM Cerveau et Pense ´e) and by the FRM (FDT20170739030). F.G. is an EMBO YIP awardee and is supported by Singapore Immunology Network (SIgN) core funding as well as a Singapore National Research Foundation Senior Investigatorship (NRFI; NRF2016NRF-NRFI001-02).

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS

The authors declare no competing interests.

STAR+METHODS KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to, and will fulfilled by, the Lead Contact Sonia Garel (garel@biologie.ens.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

For visualizing PV interneurons, we crossed PV Cre/+ [START_REF] Hippenmeyer | A developmental switch in the response of DRG neurons to ETS transcription factor signaling[END_REF] and R26 mt/mt mice (ROSA loxP-stop-loxP-tdTomato ) [START_REF] Madisen | A robust and high-throughput Cre reporting and characterization system for the whole mouse brain[END_REF], resulting in the expression of tdTomato in the Parvalbumin-expressing (PV) interneurons in the offspring. For the optogenetic recordings, PV Cre/Cre mice were crossed with R26 Chr2/Chr2 floxed mice (B6; 129S-Gt(ROSA)26Sor