Primary Cultures of Rat Mesencephalon
All animal work was conducted according to relevant national and international guidelines and approved by the Animal Studies Committee of ANLIS Carlos G Malbran (Resolución 20/4/2004). Rat embryos were recovered at day 14 from timed pregant Wistar rats, and the ventral midbrain was dissected, mechanically dissociated, and plated on polyethylenimine (1 mg/ml)-coated culture wells, in DMEM/F12 medium, supplemented with 5% horse serum (US) and 2.5%
fetal calf serum (FCS), at a density of 1×106
cells per 6-mm-diameter well. After 2 days in culture FCS content was reduced to 0.5% to prevent astrocyte proliferation; medium was changed daily. Cultures are maintained for 7–11 days in vitro. Dopaminergic neurons can be identified after fixation by immunohistochemistry for tyrosine hydroxylase (TH). This is the accepted standard methodology to prepare dopaminergic neurons in culture, in part because at this developmental age, dopaminergic neurons account for 10–20% of the total number of neurons, express specific phenotypic markers and functional glutamate receptors, and release and uptake dopamine 
Replication of viruses under liquid medium and plaque determinations are performed in MDCK cells (passage 59) obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, GA).
We identified two common isolates from community samples of Buenos Aires from the Instituto Malbrán, with antigenic configurations H3N2 and H1N1. The results of characterization with ferret antisera are: A/New Caledonia/20/99-like (H1N1) (A/NC-L/99), and A/Sydney/5/97-like (H3N2) (A/Sy-L/97). Isolates were amplified in MDCK cells and frozen in lots of 100 cryotubes. We used a well characterized neurovirulent strain obtained from the CDC (Atlanta, GA) (A/WSN/33) as a positive control for toxicity to dopaminergic neurons 
and behavioral abnormalities in the offspring of infected pregnant mice 
Titration of plaque forming units (PFU) was performed in MDCK cells and normalized for each lot to:
Lot 1 A/Sy-L/97 (H3N2) 3,000 PFU/ml.
Lot 1 A/NC-L/99 (H1N1) 3,000 PFU/ml.
Lot 1 A/WSN/33 (H1N1) 3,000 PFU/ml.
Influenza a Genome Amplification
Viral RNA was purified from selected samples by QIAamp®Viral RNA Mini Kit.
Reverse Transcription-polymerase Chain Reaction (RT-PCR)
RT PCR was used to amplify regions of the protein coding domains of viral RNA segment 7 to obtain finally an amplicon of 560 bp, using the QIAGEN®One Step RT-PCR Kit.
Influenza a Antigen Detection
The antigen detection was performed by Indirect immunofluorescent assay (IFA) using the standard antibody for surveillance recommended by CDC (Atlanta, GA), namely mouse monoclonal anti influenza a nucleoprotein (immunized with Influenza A/Puerto Rico/8/34 (H1N1) and A/Bangkok/1/79 (H3N2) viruses), from spleen cells from BALB/c mice fused with cells of the P3 Ag8.653 mouse myeloma cell line (AbD Serotec, USA).
In vitro Infection
For neurotropism experiments we inoculated 50 µl of viral suspension (150 PFU/well) in primary cultures of rat mesencephalon, and examined the presence of viral infected cells at 24 h, 48 h and 72 h (after fixation with formalin), by indirect immunofluorescence with monoclonal anti-InfV A antibodies (CDC, Atlanta, GA) followed by fluorescein-conjugated anti mouse (Light Diagnostics) (). 50% Tissue Culture Infective Dose (TCID50) was calculated for each viral strain using the Reed and Muench Calculator 
(Brett D. Lindenbach, 2008).
Indirect immunofluorescence for Influenza virus in primary mesencephalic cultures.
In vivo Infection
BALB-C mice were obtained from the Bioterio Central of the Instituto Nacional de Producción de Biológicos. Because infection entails discomfort to the animals, we minimized it by ensuring adequate hydration, temperature control, and cage conditions according to strict guidelines (Instituto Malbrán/CDC). Intranasal inoculation with 30 µl (3.000 PFU/ml of InfV, 90 PFU/mouse) strains or vehicle (PBS, pH 7.4) was performed at embrionic day 9–11 on the assumption that viremia peaks (and most likely invades CNS) three days later (and therefore ~ embryonic day 14). Post-infection all animals were individually kept in filtered cages with ad lib access to food and water, at 25°C. Seroconversion was established by inhibition of hemagglutination of turkey red blood cells at day 14 following infection. We calculated the 50% infective dosing by measuring change in lung weights post infection in lots of 5 animals for each strain and for each of four dilutions. ID50 (expressed as dilutions of a normalized hemagglutinin unit) for each strain were: A/Sy-L/97
1.41E-02, and A/WSN/33
2.49E-02. We did not find lethal infections at any of the used viral loads and therefore LD50 could not be estimated for any strain. Institutional and ethical committee approval for this protocols was obtained at both centers. ID50 was calculated for each viral strain using the Reed and Muench Calculator 
, Brett D. Lindenbach, 2008).
Pups from infected mothers were examined in a behavioral test battery including elevated plus maze, open field, novel object, and spontaneous activity before puberty (30 days) or in adulthood (90 days). In the latter we also carried out an object recognition task (90 days). Behavioral tests were carried out in an experimental room evenly illuminated provided with white noise. Animals were handled for 5 min daily for 3 days prior to testing. Sessions were recorded and later analyzed using a computerized video-tracking system (Ethovision XT, version 5, Noldus Information Technology, Wageningen, Netherlands) or ethological observation software (JWatcher V1.0).
Elevated plus maze
Elevated plus maze testing was carried out in a standard mice apparatus elevated 37.5 cm from the floor. At designated times animals were placed onto the centrdal platform facing an open arm and allowed to freely explore the maze for 5 min. After each session the apparatus was cleaned. An arm entry was counted when all four paws were placed into an arm.
Animals were placed in the center of a standard open field apparatus and total distance moved, number of rearings and time spent in the central area were recorded for 20 minutes. A mouse was considered to be into the central area (arbitrarily defined as a square of 30×30 cm) when its four paws were in it. The apparatus was cleaned between sessions.
Upon completion of the 20-min open field session, a novel object (a metal cup), was placed in approximately 20 cm away from the mouse and the latency to contact the object and time spent in contact with it were recorded for 10 min.
Two days after the open field and novel object tests, the spontaneous locomotor activity was monitored using a webcam and quantified using a simple criteria for number of squares crossed per unit time (24 h).
Object recognition task
For 3 consecutive days prior to this task mice were handled once a day for 5 min and placed 10 min in the open field to allow habituation. On the fourth day each mouse was observed during two 5 min trials separated by an interval of 1 h during which animals were returned to their cage. In the sample trial (T1) mice were faced with two identical objects placed in a symmetrical position and the time exploring each object was recorded. In the retention trial (T2) mice one of the two objects was replaced by a novel, non-familiar object and the time exploring each was recorded. Sets composed of three copies of the same object were used to prevent odor cues and all combinations and location of objects were used to prevent bias due to preference for a particular object or location. Exploration time was computed when the snout pointed to the object at a distance ≤2 cm.
After behavioral testing mice were sacrificed and fixed, brain sections (15 µm) were obtained in stereological series (every 8th section), and processed for histopathological analysis that included gross morphology with Nissl staining, stereological quantitation of immunohistochemistry for dopaminergic neurons and reactive astroglia in the substantia nigra and ventral tegmental area, and electron microscopy of dopaminergic neurons stained with tyrosine hydroxylase. Post-fixed brains stored in 1× PBS containing 0.05% sodium azide were embedded in 3% low-melting agarose, sectioned cornally (40 µm) on a vibratome (Leica VT1200) and stored in cryoprotectant (30% glycerol, 30% ethylene glycol in 1× PBS) at −20°C until processing. Immunohistochemistry was performed on every 6th section. For immunohistochemistry sections were washed in PBS endogenous peroxidases were blocked by incubation in 3% H2O2, and blocking of non-specific binding was achieved by incubating in PBS containing 0.25% (w/v) Triton-X 100 and 10% normal serum. Sections were incubated overnight at 4°C with one of the following primary antibodies: Tyrosine Hydroxylase (TH, Pelfreeze Biologicals, Rogers, AR; or Boehringer GmbH, Mannheim, Germany), Nuclear Factor kappa B (NFkBp65, Abcam, Cambridge, MA), Anti-GFAP antibody - Astrocyte Marker (ab4674, Abcam, Cambridge, MA) and Anti-InfV A antibodies provided by the Instituto Malbrán (Buenos Aires, Argentina). For microglia activation we used lectin staining (IsoB4) and antibody staining (ED-1). Following washing, sections were then incubated with appropriate biotinylated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hr followed by incubation with avidin-biotin-peroxidase complex (ABC Elite Kit; Vector Laboratories) detection system with diaminobenzidine (Ultratech HRP Streptavidin-biotin Universal Detection System and DAB Chromogen Kit, Inmunotech Co, Marseille, France). For immunofluorescence labeling, sections were stained with streptavidin conjugated Alexa Fluor 568 (working solution 1–5 µg/ml; Molecular Probes, Eugene, OR). For detection and quantification of apoptosis (programmed cell death) at single cell level, we used a protocol based on labeling of DNA strand breaks (TUNEL technology) carried out following manufacturer’s recommendations (DeadEnd™ colorimetric TUNEL System, Promega, Madison, WI).
Embryonic brains 3 days after maternal infection were fixed with 3% v/v glutaraldehyde in PBS overnight, washed, postfixed in 1.5% w/v osmium tetroxide in the same buffer for 2 hs, contrasted with 2% w/v acuose uranyl acetate, and embedded in Fluka Poly-Bed 812 (Sigma-Aldrich, St Louis, MO). Ultrathin sections (90–100 nm) were collected in cupper grids and stained with uranyl acetate and Reynolds solution, and imaged using a Philips EM300 transmission electron microscope.
All experiments were carried out in triplicates. For stereology, cells were counted using every 8th consecutive section, 400 µm apart from each other, throughout the entire ventral mesencephalon (substantia nigra compacta and ventral tegmental area) using a Nikon 80i Eclipse microscope (Tokyo, Japan) equiped with Stereologer software (SRC, Tampa, FL) and a motorized stage. Digital photographs were taken using a Zeiss camera with AxioVision software (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Digital images were then processed with ImageJ (NIH). The cross sectional area of each section was determined with Stereologer and the data were expressed as number of cells per hemibrain. Statistical analyses were performed with SPSS software (IBM Corporation, Somers, NY). Data are presented as mean ± standard error of the mean (SEM). Comparisons among groups were performed using a one or two way ANOVA as indicated, followed by a Kruskal–Wallis post hoc test as appropriate. For significant differences α was set at 0.05. For multivariate analysis of behavioral performance, discriminant analysis was performed using prenatal exposure as the classification criterion (SPSS, IBM Corporation, Somers, NY).