Scott Van Nguyen

Ananda Shankar Bhattacharjee

Abiyad Baig
email: abiyad.baig@gmail.com

Joan Colom
email: joan.colom@nottingham.ac.uk

Paul Barrow

Catherine Schouler

Arshnee Moodley

Rob Lavigne

Robert Atterbury

Biology and Genomics of an Historic Therapeutic Escherichia coli Bacteriophage Collection

Keywords: Escherichia coli, bacteriophage, therapeutics, one-step growth curve, transmission electron microscopy, phylogeny, DNA-dependent RNA polymerase

. The aim of the study was to identify common microbiological and genomic characteristics, which co-relate to the performance of these phages in in vivo study. These features will allow an informed selection of phages for use as therapeutic agents. Transmission electron microscopy showed that six of the nine phages were Podoviridae and the remaining three were Siphoviridae. The four best performing phages in vivo belonged to the Podoviridae family. In vitro, these phages exhibited very short latent and rise periods in our study. In agreement with their microbiological profiles, characterization by genome sequencing showed that all six podoviruses belong to the Autographivirinae subfamily. Of these, four were isolates of the same species (99% identity), whereas two had divergent genomes compared to other podoviruses. The Siphoviridae phages, which were moderate to poor performers in vivo, exhibited longer latent and rise periods in vitro. Two of the three siphoviruses were closely related to each other (99% identity), but all can be associated with the Guernseyvirinae subfamily. Genome sequence comparison of both types of phages showed that a gene encoding for DNA-dependent RNA polymerase was only present in phages with faster replication cycle, which may account for their better performance in vivo. These data define a combination of microbiological, genomic and in vivo characteristics which allow a more rational evaluation of the original in vivo data and pave the way for the selection of phages for future phage therapy trails.

INTRODUCTION

The rapid increase in antimicrobial resistance, is a major cause of concern for both the animal and human health, and threatens to render many clinically important antibiotics ineffective [START_REF] Drulis-Kawa | Expert Round Table on Acceptance and Therapy Re-implementation of Bacteriophage (2016). Silk route to the acceptance and re-implementation of bacteriophage therapy[END_REF]. There is now an urgent need for new antimicrobials and alternative approaches to infection control. Bacteriophages (phages) are one such approach with evidence of antibacterial properties, recently highlighted in 2016 by a joint United Kingdom Department of Health and Wellcome Trust-funded expert panel review of alternatives to antibiotics, and reviewed earlier [START_REF] Drulis-Kawa | Expert Round Table on Acceptance and Therapy Re-implementation of Bacteriophage (2016). Silk route to the acceptance and re-implementation of bacteriophage therapy[END_REF][START_REF] Nobrega | Revisiting phage therapy: new applications for old resources[END_REF].

Phages were discovered in the early twentieth century by [START_REF] Twort | An investigation on the nature of the ultramicroscopic viruses[END_REF] and d'Hérelle (1917). Soon after their discovery d'Herelle and others performed several trials using phages to treat bacterial infections in humans and animals. Although some of these trials were successful, they suffered from a lack of proper controls and the use of phage whose activity and reliability were unknown. This, together with the poor understanding at the time of the mechanism of bacterial pathogenesis, the nature of the interaction between bacteria and phages and the start of the antibiotic era, discredited the idea of using phages as antimicrobials in the West. The clinical use of phage continued in Eastern Europe, although studies published often lacked appropriate control groups and sometimes used phage alongside other antimicrobials, making it difficult to evaluate how much phage contributed to the efficacy of the treatment [START_REF] Barrow | Bacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential[END_REF][START_REF] Summers | Fe ìlix d'H ìerelle and the Origins of Molecular Biology[END_REF][START_REF] Summers | Bacteriophage therapy[END_REF].

Phage therapy continues in Russia and Eastern Europe to this day (Expert Round Table on Acceptance and Therapy Reimplementation of Bacteriophage, 2016). In the West, phage therapy was revived in the 1980s by Smith and Huggins in a series of carefully controlled experiments using systemic Escherichia coli and Enterotoxigenic E. coli (ETEC) infections in mice, calves, pigs, and sheep [START_REF] Smith | The association of the 018, K1 and H7 antigens and the CoIV plasmid of a strain of Escherichia coli with its virulence and immunogenicity[END_REF][START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF][START_REF] Smith | Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs[END_REF]Smith et al., 1987a,b). These studies highlighted that appropriate selection of phages and their application in infections where phage activity is likely to be optimal, can lead to a successful outcome. [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] study reported the isolation of phages which attached specifically to the K1 capsular antigen of E. coli. The capsular polysaccharides (K antigens) are linked to virulence in E. coli [START_REF] Jann | Polysaccharide antigens of E. coli[END_REF], and strains possessing the K1 antigen exhibit increased invasiveness and are associated with septicaemia and meningitis [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF][START_REF] Silver | Polysialic acid capsule of E. coli K1[END_REF]. The K1-specific phages were used to treat and prevent E. coli septicaemia or meningitis in mice [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF]. Some phage-resistant mutants were recovered following the experiments but these were largely K1 negative, and thus avirulent. There were differences in therapeutic efficacy between the nine phages tested. Of the nine, one, phage R, was more effective than any of the antibiotic treatments used, which included several doses of streptomycin. In the present study, we have performed detailed microbiological and genomic characterization of the nine phages originally isolated by Smith and Huggins. Our findings define common phenotypic and genomic characteristics which can be related to the differences in the in vivo performance of these phage. These features could be useful in the selection of phage from closely related phage groups which are most likely to be effective biological control agents.

MATERIALS AND METHODS

Bacterial Isolate and Phage Collection

The host bacterium E. coli O18:K1:H7 ColV + , referred to as MW, and the collection of nine phages used in this study are those isolated by [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF]. Briefly, MW is a prototrophic, non-haemolytic human clinical E. coli isolated from the brain of a baby suffering from meningitis and which produces experimental septicaemia in mice, chickens and colostrum-deprived calves (Smith andHuggins, 1980, 1982). The nine phages were isolated from samples of crude sewage and from pig markets by [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF]. In compliance with the new taxonomic classification for phages [START_REF] Lavigne | Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools[END_REF], the original names of phages have been modified and listed under 'Phage name' (Table 1) with the 'Phage ID' referring to the original name of phages [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF].

Growth Rate of MW E. coli

The mid-exponential phase of MW E. coli was determined by growing the bacteria in LB broth. For this, LB broth was inoculated with an overnight culture of MW E. coli to an OD 600nm of 0.01. The first sample was taken at the point of inoculation and the culture was incubated shaking at 100 rpm at 37 • C. Samples were taken out at hourly intervals for 7 h and finally after 24 h to enumerate the bacteria [START_REF] Miles | The estimation of the bactericidal power of the blood[END_REF]. For this, each sample was serially diluted in Phosphate Buffered Saline (PBS, Sigma-Aldrich) and 0.1 mL of each dilution was spread onto LBA plates which were then incubated overnight at 37 • C. After incubation, the bacterial colonies were counted and CFU/mL calculated.

Propagation of Phages in MW E. coli

All phage were propagated using MW as the host E. coli strain. Phage lysates were produced either in Luria-Bertani (LB) broth or on LB agar (LBA) plates (Sigma-Aldrich). Broth lysates were prepared by inoculating LB with an overnight culture of MW to achieve an optical density of 0.01 (OD 600nm ). The culture was incubated at 37 • C, shaking (100 rpm) for 2 h until the cells reached mid-exponential growth phase (10 8 CFU/mL) before infecting with a phage suspension at a multiplicity of infection (MOI) of 0.1. The culture was further incubated with gentle agitation (100 rpm) overnight at 37 • C. Phage lysates were centrifuged at 5,000 × g for 10 min to pellet the cellular debris. The supernatant containing the phage was filtered using a 0.22 µm filter (Millipore). The phage were enumerated by serially diluting the lysate in SM buffer (50 mM Tris-Cl (pH 7.5), 0.1M NaCl, 8 mM MgSO 4 •7H 2 O and 0.01% gelatin). A 0.1 mL volume of each dilution was added separately to 3 mL of molten LBA containing 0.5% agar and 0.1 mL of an overnight culture of MW. This was poured onto LBA plates containing 1% agar which were then incubated overnight at 37 • C. All phage lysates were stored at 4 • C until required.

Polyethylene Glycol (PEG) Precipitation

A high titre phage suspension (10 10 -10 11 PFU/mL) was produced by PEG precipitation according to the method of [START_REF] Yamamoto | Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification[END_REF]. Briefly, overnight phage lysates were produced in 2L Erlenmeyer flasks. The flasks were removed from the incubator and supplemented with 1 µg/mL each of DNase and RNase (Sigma-Aldrich) before incubating at room temperature for 30 min. To allow the phage particles to dissociate from cellular debris, NaCl was added to a final concentration of 1M and the lysates were incubated on ice for 1 h. The cellular Systematic nomenclature [START_REF] Kropinski | Position paper: the creation of a rational scheme for the nomenclature of viruses of Bacteria and Archaea[END_REF] is used here to avoid confusion with other phage isolates with the same name. ('vB' = Virus of Bacteria; 'Eco' = isolation species E. coli and 'P' or 'S' for podovirus and siphovirus, respectively). * * Phages are arranged in the decreasing order of effectiveness in treatment of MW E. coli infection in mice [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF]. * * *

Number of mice deaths 8 h post intramuscular

treatment of MW E. coli infection [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF]. # 0-3 mice deaths: best performing phages, 4-5 mice deaths: moderate performing phages, >5 mice deaths: poor performing phages.

debris was removed by centrifugation at 11,000 × g (Beckman Coulter Avanti J-E series centrifuge) for 10 min at 4 • C. The phage particles were precipitated by incubating the lysates overnight at 4 • C with PEG 8000 (Sigma-Aldrich) to a final concentration of 10% w/v. The precipitated phage were pelleted by centrifugation at 35,000 × g for 15 min at 4 • C. The phage pellet was resuspended by soaking in SM buffer for 1 h at room temperature. Phage were extracted from PEG and cell debris by adding an equal volume of chloroform to the lysates which were mixed by gentle inversion for 30 s and the aqueous phase containing phage particles (upper layer) was separated by centrifugation at 13,000 × g (Thermo Electron Corporation Heraeus Pico 17 centrifuge) for 20 min.

Phage Purification by Ultracentrifugation

High titre phage preparations were further purified using a caesium chloride (CsCl) gradient as described by [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] and adapted by [START_REF] Hooton | Salmonella Typhimurium-specific bacteriophage WSH19 and the origins of species specificity in the Vi01-like phage family[END_REF]. Briefly, CsCl was mixed with the phage lysate to a final concentration of 0.75 g/ml. The mixture was centrifuged at 240,000 × g for 24 h (15 • C) (Beckman TL 100 series centrifuge). The phage band was extracted by piercing through the ultracentrifuge tube with a sterile 21-gauge hypodermic needle (Medisave). To remove the residual CsCl, the sample was filtered using an Amicon R Ultra -0.5 mL 30 K membrane filter (Millipore) as per the manufacturer instructions.

Transmission Electron Microscopy (TEM)

The sample (3 µL) was applied to a hydrophilic (freshly glowdischarged) carbon-coated Pioloform film coated 300 square mesh copper EM grid (Agar Scientific Ltd.) and left for 2 min to adsorb. Excess sample was removed with a small piece of Whatman No. 1 filter paper, leaving a thin film of sample on the grid surface. The grid was rinsed twice immediately by adding 5 µl distilled de-ionized water and removing excess as described above. One drop of uranyl acetate (1% and 0.22 µm filtered) was applied to the grid and the excess removed immediately. The grid was then allowed to dry. Once dry, the grids were observed on a JEOL JEM-1400 TEM with an accelerating voltage of 100 kV. Digital images were recorded using a SIS Megaview III digital camera with iTEM software.

Phage Adsorption

For each anti-K1 phage, a MW E. coli culture at mid-exponential phase was infected at an MOI of 0.1. The adsorption rate for each phage was determined by taking samples every 2 min for up to 12 min. Bacterial cells with adsorbed phage were removed by centrifugation at 13,000 × g. The supernatant was then filtered using 0.45 µm filter (Millipore) and unbound phage were enumerated on LBA overlay containing 0.1 mL of MW E. coli on LBA plates, incubated overnight at 37 • C. The adsorption period was considered as the time taken to achieve a 90% reduction in free, unbound phage (i.e., 1 log 10 PFU/mL reduction from the initial titre) (Weber-D ąbrowska et al., 2016).

One-Step Growth Curve

A mid-exponential phase MW culture was infected with the respective phage at an MOI of 0.1. The phage particles were allowed to adsorb to the time of 90% adsorption as calculated in the in vitro adsorption assay. The lysate was then diluted to 10 4 PFU/ml in LB broth [START_REF] Hyman | Practical methods for determining phage growth parameters[END_REF]. Samples of the infected culture were collected every 2 min for 20 min for the Podoviridae or every 5 min for up to 60 min for the Siphoviridae, and unbound phages were enumerated on LBA overlays as described above. The burst size was calculated as the difference in the plaque forming unit (PFU) between the end of rise period and the latent period [START_REF] Bolger-Munro | T4 bacteriophage average burst size varies with Escherichia coli B23 cell culture age[END_REF].

Genomic DNA Extraction and Sequencing

DNA extraction of the ultracentrifuged phage preparation was performed using the Wizard R DNA clean-up system (Promega).

Briefly, the phage sample was mixed thoroughly with the DNA Clean-up resin to break open the capsids and release DNA.

The DNA and resin mix was filtered through a mini-column using a syringe barrel. The mini-column was washed with 80% isopropanol and the purified DNA was eluted with pre-warmed deionized water. Genome sequencing of the phage was completed at NU-Omics facility, University of Northumbria. A DNA sequencing library of the phage genomic DNA (50 ng) was created using Illumina Nextera XT DNA preparation kit and libraries were sequenced using Illumina V2 2 bp × 250 bp chemistry on the Illumina MiSeq Platform.

Phage Genome Sequence Analysis

Raw sequence reads in FastQ files were quality-filtered using Cutadapt [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF] and Sickle [START_REF] Joshi | Sickle: A Sliding-Window, Adaptive, Quality-Based Trimming Tool for FastQ Files[END_REF]. Due to massive over sequencing a subset of reads was taken for each phage, to enable manageable assemblies with read depths of approximately 200× (quality filtered FastQ data was randomly subsampled using a Python script 1 ). The sequence reads were de novo assembled using SPAdes (v3.6.2) [START_REF] Bankevich | SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[END_REF] and in each case the phage genomes were resolved as a single contig. To circularize the phage genome, the beginning and the end of each assembled genome was joined with a short string of 'Ns.' The raw sequence reads from the quality filtered FastQ files were re-mapped to the above genome sequence, using Stampy [START_REF] Lunter | Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads[END_REF] and visualized using Samtools and BCFtools [START_REF] Li | 1000 genome project data processing subgroup. The sequence alignment/map (SAM) format and SAMtools[END_REF]. Any missing nucleotide bases replaced the N's, confirming the circularity of the phage genome. The intactness of each phage genome was also confirmed using PHAST [START_REF] Zhou | PHAST: a fast phage search tool[END_REF]. Phage genomes were annotated using Prokka [START_REF] Seemann | Prokka: rapid prokaryotic genome annotation[END_REF], which uses Prodigal to predict coding sequences (CDSs) and also defines the ribosomal and transfer RNA genes (rRNA and tRNA), non-coding RNA and leader signal peptides. The nucleotide sequence of CDSs was used to perform blast searches against the Conserved Domain Database (CDD) (v3.15) of NCBI [START_REF] Marchler-Bauer | CDD: NCBI's conserved domain database[END_REF]. The predicted functionality of the phage genomes was further improved by comparing the functional annotation generated by Prokka, CDD and the best matching published genome in NCBI. Nucleotide and protein comparisons were performed using the 1 https://github.com/fjruizruano/ngs-protocols/blob/master/fastq-pe-random.py command line BLAST+ algorithm [START_REF] Camacho | BLAST+: architecture and applications[END_REF]. The nucleotide and amino acid sequence alignments were produced by ClustalW (CLUSTAL 2.1) [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. The maximum likelihood phylogenetic analysis was performed using the generalized time-reversible (GTR) model to infer nucleotide evolution and Jones-Taylor-Thornton 1992 (JTT +CAT) model for protein evolution, with FastTree [START_REF] Price | FastTree: computing large minimum evolution trees with profiles instead of a distance matrix[END_REF]. The nucleotide BLAST comparison figures were generated using Easyfig [START_REF] Sullivan | Easyfig:a genome comparison visualiser[END_REF]. The color coding for functional annotation was added using Inscape 0.922 . The sequence read data of phage genomes has been submitted to the European Nucleotide Archive (ENA) and the annotated phage genome sequences have been deposited in the NCBI GenBank database (Supplementary Table S1).

RESULTS

Phage Morphology

Transmission electron microscopy showed that six of nine phage studies used by [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] belonged to the Podoviridae family (Table 1), which are known to have short, non-contractile tails [START_REF] Ackermann | Basic phage electron microscopy[END_REF]. Phage vB_EcoP_R (R), the best performing phage reported by Smith and Huggins (Figure 1A), was also a podovirus with an icosahedral head (average diameter 46 ± 5 nm) and a very short tail (mean diameter 10 ± 5 nm). The remaining three phages belonged to the Siphoviridae family (Table 1). Phages from this family have a long flexible non-contractile tail with tail fibers [START_REF] Fokine | Molecular architecture of tailed doublestranded DNA phages[END_REF]. For example, phage vB_EcoS_P (P) (Figure 1B), the poor performing phage in the in vivo study [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] was a Siphoviridae phage of 150 ± 5 nm in size, with icosahedral head (average diameter 49 ± 5 nm) and long tail (average diameter 101 ± 5 nm).

Growth Characteristics

Interestingly, high titre phage lysates (10 9 -10 10 PFU/ml) for the Siphoviridae phages could only be produced using the plate lysate approach, whereas high titre lysates were easily achieved for the six Podoviridae phages from liquid lysates in LB broth. The time taken for the phages to adsorb to MW E. coli varied between 4 and 8 min. The best performing phages from the Smith and Huggins's in vivo study, Podoviridae phages R, vB_EcoP_D (D), vB_EcoP_B (B) and vB_EcoP_C (C), exhibited the shortest latent period (<2 min), a rapid rise period (2-6 min) with a small burst size (19-48 PFU per infected cell). The growth cycle for these four podoviruses was between 10 and 16 min (Table 1). The Podoviridae phages vB_EcoP_F (F) and vB_EcoP_K (K) were moderate performers in the in vivo study [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] andTable 1). Although, phage F had a latent period of <2 min like the best performing phages in vivo (R, D, B and C), it showed a large burst size (213 PFU per infected cell). Finally, phage K had the longest latent period of 8 min followed by a rise period of 8 min and the longest growth cycle of 20 min, compared to the other podoviruses. The three Siphoviridae phages [vB_EcoS_G (G), vB_EcoS_L (L), P] were moderate to poor performers in the in vivo experiment. These phages showed a longer latent (10-35 min) and rise period (10-24 min) and there was wider variation in the burst sizes between these phages, ranging between 3 and 315 PFU per infected cell. The duration of growth cycle in the three Siphoviridae phages was longer (28-53 min), compared with the Podoviridae phages (Table 1).

Genome Sequence Analysis

Illumina MiSeq sequencing generated approximately 150-650 million basepairs (bp) per phage genome. A subsample of this data was used to assemble each phage genome at a read depth of approximately 200× (Supplementary Table S1).

Genome sequence analysis showed that the six Podoviridae phages had genome sizes that ranged between 38025 and 45259 bps, with a G+C content ranging between 45 and 49% and contained 46-61 CDSs. The remaining three Siphoviridae phages had genomes of 41039-41624 bps in size, with a higher G+C content of 51% and their genomes were composed of 62-68 CDSs (Table 1).

To predict the type of termini of nine phages, the amino acid sequence of the large terminase protein from these phages was compared with the terminase protein from published phages with well-studied DNA packaging mechanisms [START_REF] Casjens | Determining DNA packaging strategy by analysis of the termini of the chromosomes in tailedbacteriophage virions[END_REF]. The amino acid sequence of the large terminase subunit from Escherichia virus K1E, K1F, K1G and K1H, was also includes as the best matching published sequence to the Podoviridae and Siphoviridae phages in this study. The phylogenetic analysis showed that the six Podoviridae phages R, B, C, D, K and F were placed in the clade with the phages with short direct terminal repeats (Figure 2). The phages R, B, C, D and K were closely related to the Escherichia virus K1E and the phage F grouped with the Escherichia virus K1F. The Siphoviridae phages along with the Escherichia virus K1G and K1H formed a clade, and were closely related with the phages with different headful packaging strategies (Figure 2). Further comparison of the Podoviridae phages R, B, C, D and K with the Escherichia virus K1E showed that these phages have short direct terminal repeats of 250-289 bp in length with 92-96% sequence identity with the short direct terminal repeats in the Escherichia virus K1E. In the phage F, each short direct terminal repeat was 165 bp in length and showed 88% identity to the short direct terminal repeats in Escherichia virus K1F.

The phylogenetic analysis comparing the Podoviridae phages with selected representative phage species from all genera of the subfamily Autographivirinae showed that the best performing phages in vivo, R, D, C and B grouped together and were closely related to Aeromonas phage phiAS7, an unassigned phage in the subfamily Autographivirinae (Figure 3). Phage K was more closely related to the phage species in the genus Sp6virus of the subfamily Autographivirinae. The Escherichia virus K1E was also included in the genus Sp6virus. Finally, phage F was included in a clade with two unclassified phages of subfamily Autographivirinae of which the best matched phage genome was of Escherichia phage PE3-1, a member of phage representing the T7virus genus (Figure 3).

The BLASTn comparison of the Podoviridae phage genomes using phage R as the reference showed an average of 99% identity with phages B, C and D (Figure 4). The moderately performing Podoviridae phages, K and F had divergent genomic content compared to the best performing Podoviridae phages R, D, C and B. Fourteen genes present in the best performing phage R were divergent from the genome of phage K. These included 10 genes with predicted hypothetical function and the remaining four genes encoding for DNA-dependent RNA polymerase, bacteriophage 1.1 protein, DNA mimic Ocr and a homing endonuclease. Finally, the genomic content of phage F differed most among the other six Podoviridae phages (Figure 4 and Supplementary Table S2). Phylogeny and the nucleotide sequence comparison of the gene encoding DNA-dependent RNA polymerase from the Podoviridae phages R, B, C, D and F showed that the phages R, B, C and D were highly identical to each other (99-100%). Phage F only shared 47% nucleotide sequence identity with the phages, R, B, C and D based on the nucleotide sequence of DNA-dependent RNA polymerase (Supplementary Figures S1,S2).

The phylogenetic analysis comparing the three Siphoviridae phages (P, G and L) with the published genomes from all genera of the subfamily Guernseyvirinae grouped the three phages in one clade, closely related to the two Salmonella phages (Salmonella phage wksl3 and Salmonella phage SS3e) in genus Jerseyvirus (Figure 5). Comparative analysis of the genome of the poor performing Siphoviridae phage P with that of the phages G and L showed that phage P contained nine genes which were divergent from phages G and L. Five of the nine genes had functions related to putative Mte8-like protein, phage tail assembly chaperone, superinfection immunity protein, putative helicase-primase, HNH endonuclease and the remaining four were predicted hypothetical function genes (Figure 6 and Supplementary Table S3).

The gene encoding an endosialidase was present in all nine phages (Figures 4,6). The phylogenetic analysis and comparison of nine phages based on the nucleotide sequence of the endosialidase gene showed that the Podoviridae phages R, B, C, D, K and F were closely related to each other with percentage sequence identity ranging between 92 and 100%. The Siphoviridae phages P, G and L shared >95% endosialidase nucleotide sequence identity with each other and were only distantly related to six Podoviridae phages with the nucleotide sequence identity of 52% (Supplementary Figures S3,S4). [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] tested the performance of nine K1 specific E. coli phages for the treatment of septicaemia, using mice model of infection. In these pioneering phage therapy experiments, the mice were infected by intramuscular or intracerebral route with MW E. coli. The K1 specific phages were administered intramuscularly to treat the infection. Interestingly, not all phages tested were equally effective and showed a gradient of treatment efficiency, noted after 8 h interval between infection and treatment [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] and Table 1). From the best performing phages, phage R was investigated further and was more effective in treatment than multiple doses of either tetracycline, trimethoprim, ampicillin or chloramphenicol. It was also reported that phage R and the other of the more effective phages cleared broth cultures of the K1 positive strain more quickly than those which were less effective [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF].

DISCUSSION

In the present study, we report the microbiological and genomic characteristics of these nine K1 specific E. coli phages and relate these characteristics to their performance in the in vivo study. It was interesting to observe that even though all nine phages were K1-specific, morphologically they belonged to two different phage families (Podoviridae and Siphoviridae) (Figure 1). There were also differences between the podoviruses and siphoviruses based on their growth profiles. The best performing phages in Smith and Huggins's in vivo study belonged to the Podoviridae family, with a faster replication cycle characterized by shorter latent and rise periods. In contrast, the Siphoviridae phages which were moderate to poor performers, exhibited longer latent and rise periods (Table 1). These differences were observed in the bacterial growth media (LB) unlike the report by [START_REF] Bull | Dynamics of success and failure in phage and antibiotic therapy in experimental infections[END_REF] in which the differences in the replication of K1-specific and non-K1 phage could only be observed in mouse sera. This suggests that phenotypic differences between these phages can be observed without the need for any specialist growth media. In a study of T7-like phages, a phage KPO 1 K 2 , capable of infecting Klebsiella pneumoniae isolate B5055, was a Podoviridae phage and was proposed as a potential therapeutic agent against this pathogen. High titres of phage KPO 1 K 2 were observed in the kidney and urinary bladder 6 h after intraperitoneal injection in infected mice. The growth profile of PhiKPO 1 K 2 showed a latent period of 15 min and a burst size of 140 [START_REF] Verma | Characterization of a T7-like lytic bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent[END_REF]. Another study describing the characterization of lytic phages for the treatment of multidrug resistant Klebsiella pneumoniae showed that both Podoviridae and Siphoviridae phages had a similar latent period of 15 min and a burst size of ∼50-60 [START_REF] Kęsik-Szeloch | Characterizing the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae[END_REF]. Although these studies suggest a longer latent period for being the best phage, our study suggests that the Autographivirinae phages that replicate faster in liquid culture, performed better in the mouse infection model. In contrast, the Guernseyvirinae phages that replicated more slowly due to longer time (latent period) required to assemble into complete phage particles performed poorly in vivo. This is also highlighted by the fact that it was difficult to produce high titre preparations of the Siphoviridae phages in the liquid LB media, requiring culture on LBA plates. It remains to be seen whether this is a general phenomenon for other phages or it is just restricted to the phages of these taxonomic groups.

A characteristic of K1 phage genomes is the presence of a gene encoding for endosialidase which accounts for the phage specificity to the K1 capsular antigen of the K1 E. coli [START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] to the published genomes of phage species from all genera of subfamily Guernseyvirinae.

strains [START_REF] Tomlinson | Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide[END_REF][START_REF] Mushtaq | Treatment of experimental Escherichia coli infection with recombinant bacteriophagederived capsule depolymerase[END_REF]. The predicted gene encoding an endosialidase was present in all nine K1 phages confirming the specificity of these phages to the K1 E. coli strain. The phylogenetic placement of the four best performing phages (R, D, B and C) and the moderate performing phage F as closely related to the unassigned and unclassified members of the subfamily Autographivirinae suggests that these phages have divergent genomes compared to the phages in other genera within this subfamily. However, phage K which was a moderate performing phage of all Podoviridae phages had a more resolved position in the phylogeny as being closely related to the phages from genus Sp6virus which includes the Escherichia virus K1E. The three Siphoviridae phages were more closely related to the Salmonella phages in the genus Jerseyvirus which reflects that these phages may also infect Salmonella strains.

The best performance of Podoviridae phages R, D, B, and C in vivo could be associated with a combination of their rapid replication cycle and the presence of the gene encoding for DNAdependent RNA polymerase in the genome of these phages. Although the phage K was phylogenetically closely related to the Escherichia virus K1E, many genes in the genome of this phage were divergent from the Escherichia virus K1E, including the gene encoding for DNA-dependent RNA polymerase suggesting that phage K had diverged from the phages in the genus Sp6virus. In phage T7, the phage-dependent RNA polymerase and the bacteriophage 1.1 protein are the early gene products which play a role in regulating host transcription and protein synthesis [START_REF] Mcallister | Hybridization mapping of restriction fragments from the early region of bacteriophage T7 DNA[END_REF]. The phage T7 RNA polymerase is also directly involved in phage DNA replication [START_REF] Hinkle | Evidence for direct involvement of T7 RNA polymerase bacteriophage DNA replication[END_REF]. If the gene encoding phage-dependent RNA polymerase is functional, it enables a rapid shut-off of host transcription by 5 min after infection, thus allowing a much faster replication cycle of the phages. In the absence of this gene, shut-off of host transcription is delayed and is instead activated by the bacteriophage 1.1 protein after 15 min of infection. In the absence of all early gene products, the host shut-off is observed at least 30 min after the infection [START_REF] Mcallister | Hybridization mapping of restriction fragments from the early region of bacteriophage T7 DNA[END_REF]. The moderate and poor performing phages in this study lacked these genes. The absence of these genes may be responsible for the longer latent period observed and thus the poor performance of these phages in vivo, as more time would be required to shut-off the host transcription. The moderate performance of phage F in vivo may also be linked to its different genomic content compared to the four best performing phages (R, D, B and C) and a large burst size. Similarly, the presence of the gene encoding the superinfection immunity protein in the Siphoviridae phage P, which is a characteristic of prophages [START_REF] Brüssow | Phage ecology[END_REF] may also explain its poor performance in vivo. Selecting phage based on the phenotypic and genomic characteristics may not guarantee success in vivo [START_REF] Tsonos | A cocktail of in vitro efficient phages is not a guarantee for in vivo therapeutic results against avian colibacillosis[END_REF] but if common phenotypic and genomic features can be identified then these can certainly be used to make targeted phage selection and could also help in reducing the number of animals used in experiments.

In the K1-specific phages, the presence of the gene encoding for DNA-dependent RNA polymerase only in the genomes of phages with very fast replication cycle and their best performance in vivo suggests the combination of these phenotypic and genomic characteristics might be used as a marker for selection of suitable phages for phage therapy trails in the future.

MEMBER OF THE ANTIBIOPHAGE CONSORTIUM

University of Nottingham: Robert Atterbury, Paul Barrow, and Abiyad Baig. University of Copenhagen: Jens Peter Christensen, Arshnee Moodley, and Dziuginta Jakociune. INRA: Catherine Schouler. KU Leuven: Rob Lavigne and Jeroen Wagemans.

FIGURE 2 |FIGURE 3 |

 23 FIGURE 2 |The maximum likelihood phylogenetic comparison of the amino acid sequence of the large terminase subunit of nine phages[START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] with the large terminase subunit from selected published tailed phages with well characterized DNA packaging strategy.

FIGURE 4 |

 4 FIGURE 4 | A BLASTn comparison of the Escherichia virus K1E and six Podoviridae phages, constructed with EasyFig.

FIGURE 5 |

 5 FIGURE 5 | The maximum likelihood phylogenetic comparison of the Siphoviridae phages[START_REF] Smith | Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics[END_REF] to the published genomes of phage species from all genera of subfamily Guernseyvirinae.

FIGURE 6 |

 6 FIGURE 6 | A BLASTn comparison of three Siphoviridae phages, constructed with EasyFig.

TABLE 1 |

 1 Biological and genomic characteristics of phages used in this study.

	Number of coding	sequences	(CDSs)
	G+C	content	(%)
	Genome	size	(bp)
	Proposed	taxonomic	classification
	Mean burst size	(PFU per infected	cell ± SD)
	Duration of	growth cycle	(min)
	Duration of	rise period	(min)
	Latent	period	(min)
	Adsorption	time	(min)
	Number of dead	mice * * * and	efficiency #
	Phage	ID * *
	Phage name *		vB_EcoP_R

Frontiers in Microbiology | www.frontiersin.org

August 2017 | Volume 8 | Article 1652

https://inkscape.org/en/ FIGURE 1 | Negative staining transmission electron microscopic images of phages after ultracentrifugation. (A) Podoviridae phage R. (B) Siphoviridae phage P. Frontiers in Microbiology | www.frontiersin.org

ACKNOWLEDGMENTS

We thank Andrew Warry, School of Veterinary Medicine and Science, The University of Nottingham for help with the bioinformatics of phage.

FUNDING

This study was supported by the AntibioPhage project, funded by an Animal Health and Welfare ERA-Net (ANIHWA) grant from the EU FP-Horizon 2020 programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AUTHOR CONTRIBUTIONS

RA, PB, CS, AM, and RL conceived the study. AB, JC, PB, and RA produced the data. AB and JC analyzed the data. AB, JC, PB, CS, AM, RL, and RA wrote the paper.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01652/full#supplementary-material

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ph.D Jodi L Pawluski
email: j.pawluski@gmail.com

Rafaella Paravatou

Alan Even

Gael Cobraiville

Marianne Fillet

Nikolaos Kokras

Christina Dalla

Thierry D Charlier

Effect of sertraline on central serotonin and hippocampal plasticity in pregnant and non-pregnant rats

Keywords: SSRI, perinatal depression, pregnancy, female, neurogenesis, maternal brain, peripartum, hippocampus

Introduction

Prescription rates for selective serotonin reuptake inhibitor medications (SSRIs) during pregnancy are between 4-10% in developed countries studied to date [START_REF] Benard-Laribiere | Patterns of antidepressant use during pregnancy: a nationwide population-based cohort study[END_REF][START_REF] Liang | Detecting Patterns of Prescription Drug Use During Pregnancy and Lactation with Visualization Techniques[END_REF]. SSRIs are prescribed to treat the growing number of women diagnosed with peripartum mood and anxiety disorders despite the fact that we know very little about how these medications affect the maternal brain [START_REF] Drury | The Neurobiological Impact of Postpartum Maternal Depression: Prevention and Intervention Approaches[END_REF][START_REF] Gavin | Perinatal depression: a systematic review of prevalence and incidence[END_REF][START_REF] Moses-Kolko | In search of neural endophenotypes of postpartum psychopathology and disrupted maternal caregiving[END_REF]Pawluski et al., 2019a;Pawluski et al., 2019b;[START_REF] Pawluski | The Neurobiology of Postpartum Anxiety and Depression[END_REF]. SSRIs and their metabolites cross the placenta [START_REF] Angelotta | Treating Depression during Pregnancy: Are We Asking the Right Questions?[END_REF][START_REF] Oberlander | Sustained neurobehavioral effects of exposure to SSRI antidepressants during development: molecular to clinical evidence[END_REF][START_REF] Pohland | Placental transfer and fetal distribution of fluoxetine in the rat[END_REF]) raising questions about the safety for the fetus of using these medications [START_REF] Field | Prenatal depression and selective serotonin reuptake inhibitors[END_REF]Gemmel et al., 2018a;[START_REF] Glover | Of rodents and humans: A comparative review of the neurobehavioral effects of early life SSRI exposure in preclinical and clinical research[END_REF][START_REF] Homberg | New perspectives on the neurodevelopmental effects of SSRIs[END_REF][START_REF] Hutchison | Perinatal selective serotonin reuptake inhibitor (SSRI) effects on body weight at birth and beyond: A review of animal and human studies[END_REF]. Much research has focused on how these medications may affect the fetus and developing child but much less is known about how these medications affect the mother [START_REF] Lonstein | The dynamic serotonin system of the maternal brain[END_REF][START_REF] Pawluski | Pregnancy: a final frontier in mental health research[END_REF]Pawluski et al., 2019b).

Recent work in peripartum women shows that SSRIs significantly increase serum brain derived neurotrophic factor (BDNF) levels suggesting a link between SSRIs and brain plasticity (Pawluski et al., 2019a). Direct effects of SSRIs on the brain plasticity in the peripartum female exists only in animal models, has only investigated effects during the postpartum period, and focuses on the effects of the SSRIs fluoxetine and escitalopram (Gemmel et al., 2018b;Gemmel et al., 2016c;Gobinath et al., 2018a;Gobinath et al., 2018b;[START_REF] Haim | The effects of gestational stress and Selective Serotonin reuptake inhibitor antidepressant treatment on structural plasticity in the postpartum brain--A translational model for postpartum depression[END_REF]Pawluski et al., 2012b;[START_REF] Workman | Parity modifies the effects of fluoxetine and corticosterone on behavior, stress reactivity, and hippocampal neurogenesis[END_REF]. Serum SSRI levels are known to change markedly during gestation, particularly during the late stages of gestation when the activity of cytochrome P450 enzymes, key players in drug metabolism, is reduced (Kokras et al, 2019b). To date no research has investigated sertraline (Zoloft®), one of the most commonly prescribed SSRIs during pregnancy [START_REF] Liang | Detecting Patterns of Prescription Drug Use During Pregnancy and Lactation with Visualization Techniques[END_REF], on the brain when pregnant. A recent study of 6 pregnant women prescribed sertraline showed a decrease in the mean dose-corrected plasma concentration of sertraline during the last half of gestation with a return to early gestational levels at 12 weeks postpartum (Kokras et al., 2019b;[START_REF] Sit | Changes in antidepressant metabolism and dosing across pregnancy and early postpartum[END_REF]. How changes in sertraline metabolism affect the maternal brain during pregnancy remains to be determined.

During the postpartum period SSRIs have an effect on the maternal brain. Gemmel et al (2016) show that fluoxetine treatment decreases serotonin turnover in the hippocampus of the R e v i s e d m a n u s c r i p t 2 dam and prevents the effect of gestational stress on serotonin turnover in the maternal prefrontal cortex (Gemmel et al., 2016c). Further research in this area has focused on postpartum SSRI effects on the hippocampus of the mother in part due to the role of the hippocampal neuroplasticity in mental illness and the actions of SSRIs in alleviating depressive-like and anxiety-like behaviors (Gemmel et al., 2018b;[START_REF] Haim | The effects of gestational stress and Selective Serotonin reuptake inhibitor antidepressant treatment on structural plasticity in the postpartum brain--A translational model for postpartum depression[END_REF][START_REF] Workman | Parity modifies the effects of fluoxetine and corticosterone on behavior, stress reactivity, and hippocampal neurogenesis[END_REF]. When administered perinatally from gestation day 10 to postpartum day 28 fluoxetine (Gemmel et al., 2018b), and the serotonin-norepinephrine reuptake inhibitor venlafaxine [START_REF] Belovicova | Effects of venlafaxine and chronic unpredictable stress on behavior and hippocampal neurogenesis of rat dams[END_REF], increase the number of immature neurons in the hippocampus of the rat dam. The morphology of immature neurons in the granule cell layer (GCL) is also affected by SSRI treatment with 3 weeks of postpartum fluoxetine administration reducing the percent of cells in the intermediate stage and increasing the percent of cells in the postmitotic stage of development but with no effect on the overall number of immature neurons in the dam postpartum (Gemmel et al., 2016c;[START_REF] Gobinath | Maternal postpartum corticosterone and fluoxetine differentially affect adult male and female offspring on anxiety-like behavior, stress reactivity, and hippocampal neurogenesis[END_REF]Pawluski et al., 2012a;[START_REF] Workman | Parity modifies the effects of fluoxetine and corticosterone on behavior, stress reactivity, and hippocampal neurogenesis[END_REF]. Recent research has also reported that the discontinuation of sertraline treatment at birth had no effect on hippocampal neurogenesis in the rat dam 3 weeks after giving birth [START_REF] Kott | Elevated stress hormone levels and antidepressant treatment starting before pregnancy affect maternal care and litter characteristics in an animal model of depression[END_REF]. Thus there are effects of SSRI treatment postpartum on the morphology of new neurons in the absence of overall differences in rates of neurogenesis. Whether these SSRI effects on hippocampal neurogenesis differ during pregnancy remain to be determined.

Apart from the effects of SSRIs on the hippocampus during the postpartum period, pregnancy and motherhood are times of marked plasticity in the female brain [START_REF] Barba-Muller | Brain plasticity in pregnancy and the postpartum period: links to maternal caregiving and mental health[END_REF][START_REF] Hoekzema | Pregnancy leads to long-lasting changes in human brain structure[END_REF][START_REF] Kim | The maternal brain and its plasticity in humans[END_REF]Kinsley and Lambert, 2006;[START_REF] Leuner | Parenting and plasticity[END_REF][START_REF] Levy | Plasticity of the parental brain: a case for neurogenesis[END_REF][START_REF] Pawluski | Neuroplasticity in the maternal hippocampus: Relation to cognition and effects of repeated stress[END_REF]. In the hippocampus of the female, motherhood and lactation are associated with a decrease in cell proliferation, neurogenesis and significant dendritic remodeling [START_REF] Brus | Interactions with the young down-regulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers[END_REF][START_REF] Darnaudery | Early motherhood in rats is associated with a modification of hippocampal function[END_REF][START_REF] Leuner | Maternal experience inhibits the production of immature neurons in the hippocampus during the postpartum period through elevations in adrenal steroids[END_REF][START_REF] Pawluski | Reproductive experience alters hippocampal neurogenesis during the postpartum period in the dam[END_REF]. Although less well studied, pregnancy is associated with a decrease in cell proliferation and dendritic remodeling in the hippocampus (Kinsley et al., 2006;[START_REF] Pawluski | Pregnancy decreases oestrogen receptor alpha expression and pyknosis, but not cell proliferation or survival, in the hippocampus[END_REF][START_REF] Pawluski | Effects of stress early in gestation on hippocampal neurogenesis and glucocorticoid receptor density in pregnant rats[END_REF]Pawluski et al., 2012d;Pawluski et al., 2011). Even in humans recent research shows that hippocampal volume changes during pregnancy and this change lasts into the postpartum [START_REF] Hoekzema | Pregnancy leads to long-lasting changes in human brain structure[END_REF]. The heightened restructuring of the hippocampus in the female during pregnancy and postpartum suggests this brain area may be particularly R e v i s e d m a n u s c r i p t 3 sensitive to, or play a significant role in, peripartum mood and anxiety disorders and subsequent SSRI treatment.

The primary aims of the current study were to determine how sertraline, one of the most commonly used SSRIs during pregnancy [START_REF] Benard-Laribiere | Patterns of antidepressant use during pregnancy: a nationwide population-based cohort study[END_REF][START_REF] Liang | Detecting Patterns of Prescription Drug Use During Pregnancy and Lactation with Visualization Techniques[END_REF], 1) affects the serotonergic system of the hippocampus and prefrontal cortex (PFC) and 2) affects measures of neuroplasticity (cell proliferation, immature neurons, and synaptic protein levels) in the hippocampus when compared to age-matched non-pregnant females. Two doses of sertraline (2.5mg/kg/day and 10mg/kg/day) were administered via a biscuit and vehicle (0mg/kg/day) was administered as a control to pregnant females for the last half of pregnancy (10 days) and nonpregnant age-matched virgin females. Serum levels of sertraline and norsertraline were determined. It was expected that sertraline dose and reproductive state (pregnant vs nonpregnant) would significantly alter measures of hippocampal plasticity in the female brain and that these effects would be related to changes in central serotonergic measures. Increasing our understanding of how SSRIs affect the maternal brain during pregnancy will aid in developing safe and effective peripartum treatments for both mother and fetus.

2.

Material and Methods

Animals.

Eighteen pregnant (gestational day (GD) 10) (289-353g) and 18 non-pregnant (virgin) female (224-254g) Sprague-Dawley rats were obtained from Janvier Laboratories, France, and singly housed upon arrival at the animal facility. GD19, and at matched points in non-pregnant females, animals were deeply anesthetized with pentobarbital and decapitated. Trunk blood samples were taken and brains were extracted. In pregnant females the size of the litter, sex of the fetuses, anogenital distance and number of fetal anomalies were determined.

2.2.

Sertraline administration. Sertraline (Fagron, Belgium) or vehicle was administered via a biscuit as previously described (Gemmel et al., 2018b;[START_REF] Pawluski | Fluoxetine dose and administration method differentially affect hippocampal plasticity in adult female rats[END_REF] were monitored to ensure that biscuits were eaten. Biscuits were consistently eaten by all females within 5 minutes.

2.3

Serum sertraline and norsertraline levels. To determine serum levels of sertraline and its active metabolite norsertraline blood samples were taken on ice, stored at 4°C overnight and centrifuged at 10,000 g for 10 minutes. Serum was collected and stored at -80°C until further use.

Serum sample preparation.

Serum samples frozen at -80°C were thawed at room temperature, vortexed and centrifuged during 5 min at 13400 rpm. mode of the transitions at m/z 306.1→274.9 for sertraline, m/z 292.1→274.9 for norsertraline, and m/z 298.1→280.9 for norsertraline 13 C6 (internal standard). ChemStation (Agilent Technologies) was used for instrument control. MS detection parameters were set by TrapControl (Bruker Daltonik GmbH, Bremen, Germany). Raw MS data were processed using Data Analysis software (Bruker Daltonik GmbH).

Brain collection.

On gestation day 19 and at matched points in non-pregnant female animals were deeply anesthetized with pentobarbital and rapidly decapitated. Brains were rapidly dissected with the right hemisphere flash-frozen for high-performance liquid chromatography with electrochemical detection (HPLC-ED) techniques and the left hemisphere immersion fixed in 4% paraformaldehyde for 48h, and then placed in saturated 30% sucrose solution for a week.

We are aware that pentobarbital can have an effect on brain serotonin levels particularly 60 minutes post pentobarbital injection [START_REF] Kuschinsky | The effect of pentobarbital on brain 5-HT metabolism in mice[END_REF]. In our study all animals were treated in the same fashion and were decapitated within 5 minutes of injection. We are confident that any group differences that exist are primarily a result of sertraline treatment or reproductive state and not pentobarbital. Immersion fixed brains were stored at -80°C until coronally sliced in R e v i s e d m a n u s c r i p t 6 40 micron sections by cryostat (Leica Biosystems, Wetzlar, Germany) in series of 12. Tissue sections were transferred to a glycol based tissue antifreeze solution and stored at -15°C.

HPLC-ED analysis.

Monoamine levels in the hippocampus and PFC were measured using high performance liquid chromatography with electrochemical detection (HPLC-ED). Dissected brain tissue was weighed, homogenized and deproteinized in 0.1N perchloric acid solution (Applichem, Darmstadt, Germany) containing 7.9mM Na2S2O5 and 1.3mM Na2EDTA (Riedelde Haën AG, Seelze, Germany), centrifuged at 20,000g for 45min at 4°C. The supernatant was stored at -80°C until analysis. The analysis was performed using a GBC LC1150 HPLC pump (GBC Scientific Equipment, Braeside, Victoria, Australia) coupled with a BAS-LC-4C

(Bioanalytical Systems Inc., West Lafayette, IN) electrochemical detector, as previously described [START_REF] Kokras | Sex-related differential response to clomipramine treatment in a rat model of depression[END_REF][START_REF] Kyratsas | Experimental evidence for sildenafil's action in the central nervous system: dopamine and serotonin changes in the medial preoptic area and nucleus accumbens during sexual arousal[END_REF][START_REF] Novais | Neudesin is involved in anxiety behavior: structural and neurochemical correlates[END_REF], with the working electrode of the electrochemical detector set up at þ800mV. Reverse phase ion pairing chromatography was used to assay serotonin (5HT) and its metabolite 5-hydroxyindoleacetic (5HIAA). The mobile phase consisted of a 50mM phosphate buffer regulated at pH 3.0, Samples were quantified by comparison of the area under the curve against known external reference standards using a PC compatible HPLC software package (Chromatography Station for Windows ver.17 Data Apex Ltd). In addition, the 5HT turnover rate was calculated separately for each chromatograph as the ratio of 5HIAA/5HT. Turnover rates estimate the serotonergic activity better than individual neurotransmitter and metabolite tissue levels as they reflect 5HT

release and/or metabolic activity [START_REF] Bessinis | Sex-dependent neurochemical effects of environmental enrichment in the visual system[END_REF][START_REF] Dalla | Sex differences in the effects of two stress paradigms on dopaminergic neurotransmission[END_REF][START_REF] Kokras | Sex-related differential response to clomipramine treatment in a rat model of depression[END_REF][START_REF] Mikail | Sertraline behavioral response associates closer and dose-dependently with cortical rather than hippocampal serotonergic activity in the rat forced swim stress[END_REF]. Prior to analysis outliers 2.5 standard deviations above the mean were removed. This occurred two times for 5HT levels in the hippocampus; once in non-pregnant vehicle group and once in 2.5 mg/kg/day pregnant group.

Immunohistochemistry. Immersion fixed brain halves were coronally sliced in 40 micron

sections by cryostat (Leica Biosystems, Wetzlar, Germany) in a series of 12. Brain tissue was used for immunohistochemical analysis of cell proliferation (anti-Ki67) and immature neurons R e v i s e d m a n u s c r i p t

(anti-doublecortin (DCX)) in the dentate gyrus (DG) of the hippocampus and analysis of synaptophysin density in CA3, and DG as previously described (Gemmel et al., 2016c). For all immunohistochemical protocols, tissue sections were rinsed between steps with TBS and TBS plus 0.01% Triton X-100 (TBST). Tissue was incubated in 0.6% H 2 O 2 for 30 minutes at room temperature, blocked in 5% donkey serum in TBST for 30 minutes at room temperature, and then incubated at 4°C in primary rabbit anti-Ki67 (1:200 Abcam ab1666, USA), primary goat anti-doublecortin (1:300, Santa Cruz Biotechnology, USA), mouse anti-synaptophysin (1:500, Sigma s5768, France) in 2% donkey serum and TBS overnight. Sections were incubated for 2h at room temperature in appropriate secondary antibody (1:400 donkey anti-rabbit, DAKO EO432; donkey anti-goat 1:500 705-065-003 Jackson ImmunoResearch Laboratories;1:200 horse antimouse DAKO EO433). Further processing of the tissue was completed using the avidin-biotin complex (ABC Elite kit; 1:1000; Vector laboratories, France) and DAB (3,3-diaminobenzidine;

Vector laboratories, France). Sections were mounted on Superfrost Plus slides (Fischer Scientific, Europe), dried, dehydrated, and cover-slipped with Permount (Fisher Scientific, Europe).

Quantification.

The number of Ki67-and DCX-immunoreactive (-ir) cells were counted under a 40x objective, as described [START_REF] Rayen | Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-dependent effects on hippocampal plasticity[END_REF][START_REF] Rayen | Fluoxetine during development reverses the effects of prenatal stress on depressive-like behavior and hippocampal neurogenesis in adolescence[END_REF] in the granule cell layer (GCL) and subgranular zone (SGZ) of the dorsal and ventral hippocampus on every 6 th section throughout the left hemisphere of the brain. The dorsal region was considered up to -4.92 and the ventral region was considered after this. Cells were considered Ki67-ir or DCX-ir if they were darkly stained, had oval or circular cell bodies, and in the area of interest (GCL/SGZ).

Total estimates of cell numbers were calculated by multiplying the counts by 12. The number of hippocampal sections counted throughout the hippocampus were recorded to ensure that a similar number of sections were analyzed in each female.

DCX-ir cell morphology was quantified and characterized based in dorsal and ventral hippocampal regions. A total of fifty DCX-ir cells were randomly assessed, twenty-five cells per dorsal or ventral region and categorized as either proliferative (no process or short process), intermediate (medium process with no branching) or post-mitotic neurons (neurite branching, see full description in [START_REF] Dechartres | Glyphosate and glyphosate-based herbicide exposure during the peripartum period affects maternal brain plasticity, maternal behaviour and microbiome[END_REF][START_REF] Plumpe | Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation[END_REF][START_REF] Workman | Parity modifies the effects of fluoxetine and corticosterone on behavior, stress reactivity, and hippocampal neurogenesis[END_REF]. DCX-ir cells for morphology characterization were selected from a minimum of 3 dorsal and 3 ventral For optical densities of synaptophysin-ir, four sections of the dorsal hippocampus between stereotaxic coordinates -2.64 mm to -4.92 mm bregma were analyzed as previously described (Gemmel et al., 2016b;[START_REF] Rayen | Developmental exposure to SSRIs, in addition to maternal stress, has long-term sex-dependent effects on hippocampal plasticity[END_REF]. Photomicrographs were taken for two areas within the CA3 and the DG. Immunoreactivity for all sections was examined under a 40x objective using an Olympus AX70 TRF and a DP71 digital camera with Cell^F software (Olympus). ImageJ64 software (Wayne Rasband, NIH, Bethesda MD, USA) was used for quantification of optical densities. The relative optical density was defined as the difference in optical density (grey level), after calibration, between the area of interest and the background, which was an equivalent area adjacent to the area of interest with minimal staining. All quantification was carried out by a researcher blind to the conditions. 1B) in late pregnant females compared to age-matched non-pregnant female rats. There was also a significant dose effect on serum sertraline levels (F(1, 19)=5.6440, p=.02819) with rats receiving the high dose (10mg/kg/day) having higher serum levels at sacrifice. This effect of reproductive state on serum levels of sertraline and norsertraline was also evident from the pooled serum samples taken from tail nicks on GD14 (Table 1) where pregnant females had higher mean levels of sertraline and norsertraline particularly at the high sertraline dose (10mg/kg) compared to non-pregnant females (Table 1). Serum norsertraline levels at perfusion on GD19 did not show significant dose effects (F(1, 19)=4.1187, p=.057). There were no other significant main effects or significant interactions (p> 0.158).

Statistical

3.2 5HT levels in the hippocampus and PFC were affected by sertraline and pregnancy. In the hippocampus, sertraline treatment at both doses significantly decreased 5HIAA levels (p's <0.003:main effect of dose F(2, 30)=17.320, p=.00001: Figure 2B) and sertraline treatment at 10mg/kg dose significantly decreased 5HIAA/5HT ratios compared to vehicle (p=0.009: main effect F(2, 30)=5.0962, p=.01244; Figure 2C). There was a significant main effect of reproductive state on 5HIAA (F(1, 30)=5.3195, p=.02818) with pregnant females having less 5HIAA. The reproductive state x dose interaction effect for 5HT levels was not significant (F(2, 28)=3.2233, p=.05498: Figure 2A).

In the PFC, sertraline at the high dose significantly increased 5HT (high vs low p=0.009; main effect of dose F(2, 30)=5.7225, p=.00785; Figure 2D), significantly decreased 5HIAA

(high vs vehicle p=0.0005; main effect of dose F(2, 30)=9.1731, p=.00078; Figure 2E) and lowered 5HIAA/5HT ratios (high vs low and vehicle p's < 0.005: main effect of dose F(2, 30)=10.308, p=.00039; Figure 2F). There was also a significant main effect of reproductive state on 5HT (F(1, 30)=4.6989, p=.03825) and 5HIAA/5HT(F(1, 30)=7.4165, p=.01067) with pregnant females having more 5HT and a smaller 5HIAA/5HT ratio in the PFC (Figure 2D,F).

There were no other significant main effects or interaction effects on 5HT measures in the hippocampus or PFC (p's > 0.06).

R e v i s e d

m a n u s c r i p t 10 Serum levels of sertraline and norsertraline were negatively correlated with 5HIAA levels and the ratio of 5HIAA/5HT in both the hippocampus and the PFC of pregnant and non-pregnant females (see Table 2 for list of correlations and p values).

3.3 Measures of hippocampal neuroplasticity are affected by sertraline and pregnancy.

3.3.1 Synaptophysin-ir is affected by sertraline in non-pregnant rats and by pregnancy.

Sertraline at low and high doses increased synaptophysin-ir in the DG of non-pregnant rats but not pregnant rats (p< 0.01: DG interaction effect F(2, 30)=3.3386, p=.04908; Figure 3B). In the CA3 region, the highest dose of sertraline increased synaptophysin-ir in non-pregnant females (p<0.03: CA3 interaction effect F(2, 30)=3.3525, p=.04852; Figure 3C) but not in pregnant females. Pregnancy increased synaptophysin-ir density compared to vehicle non-pregnant females in both the DG and CA3 regions (p's <0.03; Figures 3B and3C). There were also significant main effects of reproductive state on synaptophysin-ir density in the DG (F(2, 30)=12.162, p=.00014) and CA3 (F(1, 30)=7.0857, p=.01236). There were main effects of dose in the DG (F(2, 30)=12.162, p=.00014) and CA3 (F(2, 30)=4.9952, p=.01341) with higher doses of sertraline resulting in greater density of synaptophysin-ir cells. There were no other significant main effects or interaction effects on synaptophysin density.

Hippocampal cell proliferation and neurogenesis is reduced in late pregnancy

. There were no significant differences in the number of sections counted per brain (p> 0.2) thus estimates of total cell counts were used. Overall, pregnant females had significantly fewer Ki67-ir cells (main effect F(1, 26)=26.568, p=.00002; Figure 4A) and DCX-ir cells (main effect F(1, 30)=13.279, p=.00101; Figure 4B) in the GCL/SGZ of the hippocampus. There were significantly more Ki67ir cells and DCX-ir cells in the dorsal than in the ventral hippocampus (Ki67-ir; F(1, 26)=69.412, p=.000001: DCX-ir; F(1, 30)=14.243, p=.00071). There were no significant main effects of sertraline treatment or other significant interactions (p> 0.4). Figure 5A). In the dorsal region of the hippocampus the cell type x reproductive state x dose interaction was not significant (F(4, 60)=2.4267, p=.05759). In both the dorsal and ventral regions of the hippocampus there were significant main effects of cell type (dorsal -F(2, 60)=15.415, p=.000001; ventral -F(2, 60)=24.204, p=.000001; Figure 5B) with significantly more intermediate cells than proliferative or postmitotic cells (p's < 0.00008).

Sertraline levels are generally negatively correlated with hippocampal measures.

There were significant correlations between serum sertraline levels and synaptophysin-ir density in the DG (r=0.4622, p=0.026; Figure 6A), number of DCX-ir (r=-0.6438, p=0.001; Figure 6B), and number of Ki67-ir cells (r=-0.5791, p=0.006; Figure 6C). Serum norsertraline levels were also significantly negatively correlated with number of Ki67-ir cells (r=-0.5886, p=0.005). Further analysis of correlations per condition (pregnant/non-pregnant) revealed that in non-pregnant females only there were significant negative correlations between serum sertraline or norsertraline levels and number of Ki67-ir cells in the GCL/SGZ (sertraline -r=-0.6290, p=0.038: norsertraline -r=-0.6136, p=0.045; Figure 6C). There were no other significant main effects or interaction effects.

Weight increased during pregnancy.

As expected pregnant females had a greater change in body weight gain during the study than non-pregnant females (F(1, 30)=188.47, p=.000001:

Table 3). There was no effect of sertraline treatment on change in weight from the beginning to the end of the study (p=0.45).

Fetal characteristics were not affected by sertraline treatment.

There was no significant effect of sertraline on number of fetuses (p=0.9), the number of female (p=.23) or male (p=0.06) fetuses, number of obvious fetal abnormalities (p=0.521) or anogenital distance per litter of male (p=0.400) or female (p=0.579) fetuses (Table 3).

Discussion

Up to 10% of women are prescribed SSRIs while pregnant with sertraline being one of the most popular SSRIs prescribed to date [START_REF] Andrade | Use of antidepressant medications during pregnancy: a multisite study[END_REF] the female brain during pregnancy. In fact treatment and dosing strategies of antidepressant medications are often based on data from healthy male volunteers and little consideration has been given to the impact of sex/gender or the complex physiology of pregnancy and their unique effects on mental illness [START_REF] Feghali | Pharmacokinetics of drugs in pregnancy[END_REF]Kokras et al., 2019a;[START_REF] Pawluski | Pregnancy: a final frontier in mental health research[END_REF]. Findings from this study show that sertraline has a significant impact on the female brain with differences in serum medication levels, the serotonergic system in the hippocampus and PFC, and measures of neuroplasticity in the hippocampus. Importantly, pregnancy is a significant factor contributing to the effects of sertraline on the female brain.

The main function of sertraline, as with all SSRIs, is as a serotonin reuptake inhibitor acting on the serotonin transporter to increase serotonin levels in the synaptic cleft. Sertraline and its metabolite norsertraline are evident in the bloodstream and previous studies in pregnant women report a decrease in the mean dose-corrected plasma concentration of sertraline during the last half of gestation with a return to early gestational levels at 12 weeks postpartum [START_REF] Freeman | Pharmacokinetics of sertraline across pregnancy and postpartum[END_REF]Kokras et al., 2019b;[START_REF] Sit | Changes in antidepressant metabolism and dosing across pregnancy and early postpartum[END_REF]. Reduced levels of sertraline and increased levels of its main metabolite are attributed mainly to pregnancy-related changes in the activity of the cytochrome P450 family of enzymes. In our study, sertraline and norsertraline levels decreased from GD14 to GD19 in non-pregnant rats and the metabolic ratio (norsertraline/sertraline) slightly increased from 1.2 to 1.6 on average between GD14 and GD19.

This suggests that sertraline treatment did not reach steady state pharmacokinetics during the 10 days of treatment as evidenced by the differences between GD14 and GD19. Interestingly in pregnant rats, the metabolic ratio was on average 0.9-1.0 on GD14 and GD19 and for both doses

(2.5mg/kg and 10mg/kg). This clearly demonstrates that the activity of CYP P450 was reduced in pregnant rats. Moreover, blood levels of sertraline and its metabolite were higher in pregnant rats in comparison to non-pregnant rats, and surprisingly sertraline and norsertraline levels increased from GD14 to GD19 in females treated with the low dose, whereas they decreased from GD14 to GD19 in females treated with the high dose. This further strengthens the hypothesis that steady state pharmacokinetics were not achieved in the timeframe of the study. Future research is needed to determine the precise change of sertraline/norsertraline levels during pregnancy in humans and rodent models. From our study it becomes clear the pregnancy affects the metabolism of sertraline. The steroid hormone profile of pregnancy can indeed affect the activity of the CYP P450 enzymes and it differs between humans and rodents [START_REF] Pawluski | Effects of steroid hormones on neurogenesis in the hippocampus of the adult female rodent during the estrous cycle, pregnancy, lactation and aging[END_REF]. For example, near the end of gestation in the rat estradiol begins to increase whereas estradiol increases more remarkably throughout pregnancy in humans. Changes in estrogens can both increase and decrease CYP450 isozymes (for example decrease 1A2 and induce 2D6) [START_REF] Jeong | Altered drug metabolism during pregnancy: hormonal regulation of drug-metabolizing enzymes[END_REF][START_REF] Kokras | Sex differences in pharmacokinetics of antidepressants[END_REF]Kokras et al., 2019b) and because sertraline is metabolized by many CYP isozymes, it could be that the responsible isozymes for sertraline metabolism are differentially affected during the rat and the human pregnancy.

When we investigated the 5HT turnover levels in the brain of pregnant and non-pregnant female rats, as a function of sertraline dose, a higher dose of sertraline (10mg/kg/day) decreased 5HT turnover in the hippocampus and PFC of pregnant and non-pregnant females. 5HT turnover was significantly reduced in the PFC of pregnant females in comparison to non-pregnant female rats. This suggests that even though serum drug levels are significantly higher in pregnant females, the effects on 5HT in the brain is not as markedly affected and these effects are brain region specific (PFC differs from hippocampus). Interestingly, when looking at behavioral outcomes, recent work in male rats shows that sertraline treatment affects forced swim test performance associated with changes in 5HT levels in the PFC [START_REF] Mikail | Sertraline behavioral response associates closer and dose-dependently with cortical rather than hippocampal serotonergic activity in the rat forced swim stress[END_REF].

Furthermore, when tested on the same paradigm, immobility levels of female rats were more responsive to lower sertraline doses than males. This suggests that the effects of sertraline in mental health are partially brain region and sex/gender-dependent [START_REF] Dalla | Sex differences in animal models of depression and antidepressant response[END_REF]Kokras et al., 2015). Whether similar effects are evident in females across reproductive stages is the focus of further research by our group.

We also found that serum levels of sertraline were associated with a decrease in cell proliferation and the number of immature neurons, as well as an increase in synaptophysin density in the DG of female rat, particularly in non-pregnant females. The negative association between serum sertraline levels and the measures of hippocampal neurogenesis (cell proliferation and immature neurons) are particularly pronounced in non-pregnant females. This association in non-pregnant females is in contrast with a growing body of literature showing that serotonin and SSRIs can act to increase hippocampal neurogenesis in male rats [START_REF] Kraus | Serotonin and neuroplasticity -Links between molecular, functional and structural pathophysiology in depression[END_REF][START_REF] Malberg | Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus[END_REF][START_REF] Santarelli | Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants[END_REF]. However, others have also reported minimal effects of SSRIs R e v i s e d m a n u s c r i p t 14 on hippocampal neurogenesis in the female. It has been shown that while 14 days of the SSRI fluoxetine can increase hippocampal cell proliferation in adult male rats there are no effects in adult female rats [START_REF] Hodes | Prozac during puberty: distinctive effects on neurogenesis as a function of age and sex[END_REF]. We have also reported that 14 days of fluoxetine treatment has minimal effects on hippocampal neurogenesis in virgin females with only serum fluoxetine levels of 50ng/ml, administered with a minipump, increasing cell proliferation while higher or lower serum levels did not have the same effect [START_REF] Pawluski | Fluoxetine dose and administration method differentially affect hippocampal plasticity in adult female rats[END_REF]. In the present study we did not find a main effect of sertraline dose on measures of neurogenesis in the hippocampus of pregnant or non-pregnant females. It may be that this 10 day treatment with sertraline was not long enough to show alterations in hippocampal neurogenesis. However, previous research

shows that the SSRI fluoxetine significantly increases hippocampal cell proliferation after 7 days of treatment to male rats [START_REF] Micheli | Depression and adult neurogenesis: Positive effects of the antidepressant fluoxetine and of physical exercise[END_REF]. In human cell culture derived from hippocampus, 3-10 days of treatment with sertraline was enough to increase neurogenesis [START_REF] Anacker | Antidepressants increase human hippocampal neurogenesis by activating the glucocorticoid receptor[END_REF]. Thus it is likely that SSRI duration, type and dose of treatment, sex/gender and reproductive state alter the effects of antidepressant medications on neuroplasticity in the hippocampus.

In the present study sertraline increased synaptophysin density in the DG and CA3 of non-pregnant females with a high dose showing promise in increasing synaptophysin density in the DG of pregnant females. Synaptophysin is a membrane glycoprotein found in presynaptic vesicles of neurons. An increase in synaptophysin density after sertraline treatment may be indicative of increased synaptic plasticity and function. Thus while there are no effects of 10 days of sertraline treatment on hippocampal cell proliferation or neurogenesis there is an effect on synaptic structure which may translate into behavioral outcomes. This increase of synaptophysin density in the DG after sertraline treatment to non-pregnant females is in contrast with our previous work using fluoxetine. Previously we reported that fluoxetine treatment for 14 days significantly decreased synaptophysin density in the DG of non-pregnant females [START_REF] Pawluski | Fluoxetine dose and administration method differentially affect hippocampal plasticity in adult female rats[END_REF]. Although not previously investigated during pregnancy, fluoxetine treatment for 3 weeks postpartum has minimal effects on synaptophysin density in the DG of rat dams (Gemmel et al., 2016a). However, this SSRI treatment does prevent the increase in synaptophysin density in the cingulate cortex after gestational stress in the mother (Gemmel et al., 2016a). This suggests that the actions of SSRIs on the brain depend on the type of SSRI, reproductive status as well as brain region investigated.

R e v i s e d m a n u s c r i p t

Given the marked physiological changes during pregnancy it is not surprising that pregnancy and motherhood have significant effects on the female brain (for review see [START_REF] Barba-Muller | Brain plasticity in pregnancy and the postpartum period: links to maternal caregiving and mental health[END_REF][START_REF] Leuner | Parenting and plasticity[END_REF][START_REF] Pawluski | Neuroplasticity in the maternal hippocampus: Relation to cognition and effects of repeated stress[END_REF]. During late pregnancy and the early postpartum period there is a marked decrease in hippocampal neurogenesis in the maternal brain [START_REF] Darnaudery | Early motherhood in rats is associated with a modification of hippocampal function[END_REF][START_REF] Leuner | Maternal experience inhibits the production of immature neurons in the hippocampus during the postpartum period through elevations in adrenal steroids[END_REF][START_REF] Pawluski | Effects of stress early in gestation on hippocampal neurogenesis and glucocorticoid receptor density in pregnant rats[END_REF][START_REF] Pawluski | Reproductive experience alters hippocampal neurogenesis during the postpartum period in the dam[END_REF] as well as changes in dendritic morphology and spine densities of pyramidal neurons (Kinsley et al., 2006;Pawluski et al., 2012d). We expand this work and show that in addition to significantly lower rates of cell proliferation in the hippocampus and fewer immature hippocampal neurons pregnant females have significantly elevated levels of synaptophysin in the CA3 and DG compared to non-pregnant females. Thus, even though there are lower rates of neurogenesis during late pregnancy it may be that synaptic communication is strengthened. We also found that while pregnant a female rat shows differences in the morphological stages of immature neurons compared to non-pregnant females, particularly in the dorsal hippocampus.

Pregnant females have more intermediate immature neurons than proliferative cells and nonpregnant females have more intermediated immature neurons compared to both proliferative and postmitotic cells. This suggests there may be a higher turnover of immature neurons or more new cell death in the hippocampus of late-pregnant females. Taken together the hippocampus during pregnancy shows significant remodeling, and perhaps fine-tuning, of mature neurons via changes in synapse formation and communication.

Limitations and future directions

This is a first study demonstrating the complexities of SSRI neuropharmacology in the hippocampus during pregnancy. This work has generated more questions than answers and there are a number of directions for future research. For example, future research should aim to expand these findings by 1) investigating changes in other brain areas such as the PFC which is known to play a role in peripartum mental illness and maternal behaviors (Gemmel et al., 2016c;[START_REF] Haim | The effects of gestational stress and Selective Serotonin reuptake inhibitor antidepressant treatment on structural plasticity in the postpartum brain--A translational model for postpartum depression[END_REF]; 2) using a longer treatment regimen of the SSRI as previous research has shown that 4 weeks of postpartum fluoxetine treatment can alter neurogenesis in the maternal hippocampus (Gemmel et al., 2018b); 3) using a model of maternal depression or repeated stress to understand the actions of SSRI in this more clinically relevant context (Gemmel et al., 2016c;Pawluski et al., 2012c;Pawluski et al., 2012d); and 4) investigating the role of SSRI treatment on R e v i s e d m a n u s c r i p t 16 behavioral outcomes related to mood and anxiety measures as well as maternal care-giving patterns (Pawluski et al., 2019b;Pawluski et al., 2011).

Although we have much to learn about how and when SSRIs are effective in treating peripartum mental illness, when looking specifically at sertraline only one study investigated how sertraline treatment during gestation affects maternal behaviors in the postpartum period [START_REF] Kott | Elevated stress hormone levels and antidepressant treatment starting before pregnancy affect maternal care and litter characteristics in an animal model of depression[END_REF]. This work showed that with the cessation of sertraline treatment there were minimal effects of the SSRI on maternal affective-like behaviors or hippocampal neurogenesis in the dam during the postpartum. However the delay between gestational sertraline treatment and testing may have contributed to the lack of findings. In adult virgin rats sertraline can diminish depressive-like behaviors [START_REF] Kokras | Forced swim test: What about females?[END_REF][START_REF] Mikail | Sertraline behavioral response associates closer and dose-dependently with cortical rather than hippocampal serotonergic activity in the rat forced swim stress[END_REF] and this effect is associated with changes in 5HT in the PFC. Thus, a key first step for further research in pregnant females is to understand how sertraline may act to counteract stress effects in the hippocampus as well as the PFC.

Conclusions

In the present study we report that the effects of sertraline treat on the hippocampus of pregnant and non-pregnant females differs as a function of reproductive state as well as dose. We found that although serum drug levels were elevated in late pregnant females the rates of serotonin turnover in the hippocampus and PFC were similar in pregnant and non-pregnant females. However, we found that sertraline increased synaptophysin density and was associated with decreased cell proliferation rates in the hippocampus of non-pregnant females only.

Pregnancy alone resulted in significant reductions in hippocampal neurogenesis and increased synaptophysin density, regardless of sertraline treatment. In conclusion, this work highlights the need to consider how reproductive state affects the central actions of antidepressant medication treatment for peripartum mood and anxiety disorders. (ug/g wet tissue). In the hippocampus B)sertraline treatment at both doses significantly decreased 5HIAA levels (p's <0.003) and C) sertraline treatment at 10mg/kg dose significantly decreased 5HIAA/5HT ratio compared to vehicle (p=0.009). In the PFC D) sertraline at the high doses significantly increased 5HT (high vs low p=0.009), E) significantly decreased 5HIAA (high vs vehicle p=0.0005) and F) significantly lowered 5HIAA/5HT ratios ( high vs low and vehicle p's < 0.005). * denotes significance p <0.05. a denotes main effect of 2.5 and 10 mg/kg dose p < 0.05. b denotes main effect of 10mg dose p <0.05.

  containing 5-octylsulfate sodium salt (ion pairing reagent) at a concentration of 300mg/L and Na2EDTA at a concentration of 20mg/L (Riedelde Haën AG); acetonitrile (Merck, Darmstadt, Germany) was added at a 8-15% concentration. The reference standards were prepared in 0.1N perchloric acid containing 7.9mM Na2S2O5 and 1.3mM Na2EDTA. The column used was an Aquasil C18 HPLC Column, 1502.1mm, 5mm Particle Size (Thermo Electron Corporation, UK).

  percentage of these cell types per animal was calculated as a function of the total cells investigated per region.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Mean (+SEM) levels of A) 5HT, B) 5HIAA and C) 5HIAA/5HT in the hippocampus and D) 5HT, E) 5HIAA and F) 5HIAA/5HT in the prefrontal cortex (PFC) (B). (n = 5-6/group).

Figure 3 .

 3 Figure 3. A) Photomicrograph of synaptophysin-ir in the dentate gyrus (DG) and CA3 of the hippocampus. Mean (+SEM) synaptophysin-ir density (OD) in the B) DG and C) CA3 region of the hippocampus. (n=6/group). 'a' denotes non-pregnant vehicle significantly different from all other groups (p's < 0.01);'b' denotes non-pregnant vehicle significnatly difference from 10mg/kg non-pregnant group (p's < 0.03 );* denotes significance p<0.05.

Figure 4 .Figure 6 .

 46 Figure 4. Photomicrograph of A) Ki67-ir cells and B) DCX-ir cells in the GCL/SGZ of the dentate gyrus of non-pregnant and pregnant females under 40x objective (scale bar=20um). Mean (+SEM) total number of C) Ki67-ir cells and D) DCX-ir cells in the GCL/SGZ. (n=5-6/group). * denotes significance p<0.05. DCX=doublecortin

  Fig 1

.2 LC-chip-MS/MS analysis 2.3.2.1 LC-chip conditions. Chromatographic

	5
	interface. Chromatographic separation was performed on a Ultra High Capacity chip including a
	500 nL trapping column and a 150 mm x 75 μm analytical column, both packed with a Zorbax
	80SB 5 μm C 18 phase (Agilent Technologies). The mobile phase was composed of H 2 O/FA
	(100:0.1, v/v) (A) and ACN/H 2 O/FA (90:10:0.1, v/v/v) (B). The analysis was performed using a
	gradient starting at 21% B that linearly ramped up to 76% B in 7 min at a flow rate of 400
	To 50 µL of each serum nL/min. Column was then rinsed with 90% B for 2 min. All the experiments were carried out m with a 5 µL sample injection volume. 2.3.2.2 MS/MS detection. MS detection was performed using a 6340 Ion Trap equipped with a nanoESI source operating in positive mode (Agilent Technologies, Waldbronn, Germany). Skimmer, capillary exit, octopoles and lenses voltages were optimized by means of the Smart Ramp tool included in TrapControl software in order to maximize precursor ion intensity for both analytes and internal standard. Capillary voltage was set on 1800 V and the endplate offset on 500 V. Nitrogen was used as drying gas at a flow rate of 4 L/min and the source temperature a n u s c r i p t was set at 325°C. Quantification was obtained using the MRM (multiple reaction monitoring)
	sample was added 100 µL of 5% orthophosphoric acid, 50 µL of formic acid 0.1% and 50 µL of internal standard solution (50 ng/mL) in a 96-well polypropylene plate. An Oasis µElution MCX 96 well plate was used to clean up the samples for the analysis. The strong cation-exchanger polymeric sorbent was first conditioned by 300 µL methanol followed by 300 µL water. The entire sample was then transferred in the corresponding well of the extraction plate and drawn through the sorbent under vacuum. The plate was washed with 200 µL of 2% formic acid followed by 200 µL methanol. The extracts were eluted with 2 × 50 µL of methanol/ammonium hydroxide (95:5, v/v). After elution, 400 µL of 2% formic acid was added to each well. A calibration curve in serum was made with standard concentrations of 5, 20, 50, 200 and 500 ng/ml. R e v i s e d
	2.3separation was achieved on a 1200 series LC-chip
	system consisting of a nanoflow pump, a capillary pump, a wellplate sampler and a LC-chip/MS

Serum levels of sertraline and norsertraline were increased in pregnant females

  Bonferroni test. Pearson correlations were analyzed between serum drug levels, measures of 5HT in the hippocampus and PFC, and measures of plasticity in the hippocampus. Significance was sertraline and its metabolite, norsertraline, there were significantly higher levels of sertraline (F(1, 19)=50.096, p=.000001: Figure1A) and norsertraline (F(1, 19)=24.969, p=.00008: Figure

	m a n u s c r i p t
	set at p<0.05. 3. Results 3.1 R e v i s e d

Analysis. Data were analyzed using the Statistica 13 software (Dell Inc.).

Analysis of variance tests (ANOVAs) were conducted on serum levels of sertraline and norsertraline with reproductive state (pregnant/non-pregnant) and treatment dose (2.5mg/kg, 10mg/kg) dose as factors. ANOVAs were conducted on neurotransmitter levels (5HT, 5HIAA, 5HIAA/5HT) and synaptophysin-ir density with reproductive state (pregnant/non-pregnant) and treatment dose (0mg/kg, 2.5mg/kg, 10mg/kg) as factors. For measures of Ki67-ir and DCX-ir cells, repeated measures ANOVAs were conducted for cell counts in the dorsal and ventral regions of the hippocampus with condition (pregnant/non-pregnant) and treatment dose (0mg/kg, 2.5mg/kg, 10mg/kg) as factors. DCX-ir cell types (stages of cell maturity) were analyzed using repeated measures ANOVAs with cell type (proliferative, intermediate, postmitotic) as the within-subjects factor. Significant interaction effects were analyzed in more detail by a . When comparing serum levels at sacrifice of the low (2.5mg/kg/day) and high (10mg/kg/day) doses of

  [START_REF] Liang | Detecting Patterns of Prescription Drug Use During Pregnancy and Lactation with Visualization Techniques[END_REF]. Virtually nothing is known about how sertraline or SSRIs affect

	al., 2018a;
	m a n u s c r i p t
	R e v i s e d

;

[START_REF] Benard-Laribiere | Patterns of antidepressant use during pregnancy: a nationwide population-based cohort study[END_REF] Gemmel et

Table 1 .

 1 ) in pregnant and non-pregnant females. Samples were pooled (3 females per group pooled) for analysis to provide an indication of any change in drug levels.

		Sertraline	Sertraline	Norsertraline	Norsertraline
		(ng/ml)	(ng/ml)	(ng/ml)	(ng/ml)
		Morning	Afternoon	Morning	Afternoon
	Non-pregnant
	2.5mg/kg	10.30±0.36	12.49±4.57	14.00 ±3.94	13.74 ±3.13
	10mg/kg	11.46 ± 0.19	16.42 ± 0.44	16.31 ± 1.01	20.52 ± 1.62
	Pregnant
	2.5mg/kg	10.8 ± 6.60	14.79 ± 2.06	11.33 ± 3.15	13.52 ± 3.07
	10mg/kg	31.18 ± 17.16	36.70 ± 26.50	34.59 ± 9.62	35.28 ± 12.87
	Serum levels of sertraline and norsertraline during the morning (9-10am) and afternoon (2-
	3:30pm

Table 3 .

 3

			Non-pregnant			Pregnant
		0mg/kg	2.5mg/kg	10mg/kg	0mg/kg	2.5mg/kg	10mg/kg
	Weight change (%)	16.7 ± 1.1	14.7 ± 1.9	16.1 ± 0.8	37.0 ± 1.3	37.1 ± 1.7	33.9 ± 3.1
	Fetal Characteristics
	No. of males	-	-	-	6.8±0.9	7.3±0.7	4.3±1.0
	No. of females	-	-	-	6.3±1.0	5.5±0.9	8.0±1.1
	Anogenital distance	-	-	-	4.1±0.03	4.1±0.1	4.3±0.2
	males (mm)
	Anogenital distance	-	-	-	2.2±0.08	2.3±0.1	2.2±0.1
	females (mm)
	Fetal anomalies	-	-	-	0.0±0.0	0.3±0.2	0.7±0.7

Mean (±SEM) weight change (%) of non-pregnant and pregnant females and fetal characteristics in pregnant females. There were no significant effects.

Acknowledgements:

We would like to thank Ikinaya Mandhui, Mado Gueguen, and Julie Dechartes for the help with this project.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. JLP and TDC were funded by the Regione Bretagne (SAD).