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Hyaluronic Acid Derived from Other Streptococci Supports Streptococcus pneumoniae In Vitro Biofilm Formation

We investigate the role of hyaluronic acid (HA) on S. pneumoniae in vitro biofilm formation and evaluate gene expressions of virulence and/or biofilm related genes. Biofilms were grown in medium supplied with HA derived from capsule of Streptococcus equi. The biomasses of biofilms were detected by crystal-violet (CV) microtiter plate assay, and the morphology was viewed under scanning electron microscope (SEM). The gene expressions were assessed by relative quantitative RT-PCR. The results showed that the HA support pneumococcal growth in planktonic form and within biofilms. The CV-microtiter plate assay detected significantly increased biofilm growth in medium containing HA. The SEM analysis revealed thick and organized biofilms in positive control and HA supplemented medium. The nanA, nanB, bgaA, strH, luxS, hysA, ugl, and PST-EIIA encoding genes were significantly upregulated in the planktonic cells grown in presence of HA, while the lytA and comA genes were downregulated. Similarly the luxS, hysA, ugl, and PST-EIIA encoding genes were significantly upregulated by more than 2-folds in HA biofilms. The results of this study indicate that the HA derived from capsule of S. equi supports pneumococcal growth in planktonic state and within biofilms and upregulated virulence and biofilm related genes.

Introduction
Streptococcus pneumoniae causes otitis media, pneumonia, meningitis, and sepsis, particularly in young children and the elderly [1,2]. S. pneumoniae colonizes the human nasopharynx during the first months of life, where it can persist as part of the commensal flora. Pneumococcal colonization and disease are often associated with biofilm formation [3,4]. Various reports have documented the presence of biofilm in the lungs of animals infected with S. pneumoniae [5] and in human biopsy specimens of the sinus and middle ear mucosa and the adenoids [6][7][8].
S. pneumoniae depends on carbohydrates for its growth; however, there is a low concentration of free carbohydrate in the nasopharynx, a niche where initial colonization takes place [9]. Instead, carbohydrates are found in the form of glycans and glycosaminoglycan modifications on human lipids and proteins. The most common terminal carbohydrate present on N-and O-linked glycans is sialic acid [10], which is cleaved by pneumococcal neuraminidase NanA. Previous studies have reported the upregulation of virulence related genes and increased biofilm growth in presence of glycoconjugates [11,12].
Hyaluronic acid (HA), a glycosaminoglycan, is a key component of the extracellular matrix that is present on the apical surface of the epithelial cells [13]. Several bacterial species, including S. pyogenes and S. equi, which colonize the upper respiratory tract, express a hyaluronic acid capsule [14]. The HA derived from these bacterial capsules has essentially the same structure as that isolated from humans. Previous studies have reported that S. pneumoniae can utilize human and bacterial HA as a carbon source and demonstrated that hyaluronate lyase, a putative PTS transporter (PTS-EIIA), and ugl genes are required for pneumococcal growth on human HA [15][16][17]. However little is known about the role of bacterial derived HA on pneumococcal biofilms formation and expression of virulence and pathogenesis related gene. Therefore, we investigate the role of HA on S. pneumoniae in vitro biofilm formation and evaluate gene expressions of virulence and/or biofilm related genes.

Bacterial Strain and Growth
Conditions. S. pneumoniae D-39 (NCTC 7466) an encapsulated, serotype 2 pathogenic strain was obtained from the Health Protection Agency Culture Collections (HPA, Salisbury, UK). Bacteria were routinely grown in tryptic soy broth (TSB) or on blood agar plates supplemented with 5% v/v sheep blood at 37 ∘ C in an atmosphere of 5% CO 2 . Yeast extract medium (YE) was prepared with 10 g/L yeast extract (Becton Dickinson), 5 g/L NaCl, and 3% wt/vol of 0.5 moles K 2 HPO 4 [12]. S. equi derived hyaluronic acid was purchased from Sigma (Sigma 53747, MO, USA).

Effect of HA on Planktonic Cell Growth.
To determine the effect of HA on planktonic cell growth, S. pneumoniae D39 strain was grown in YE medium or YE medium supplied with either HY or glucose (positive control). The pneumococcal colony was grown in TSB medium up to log phase, pelleted by centrifugation, and washed with phosphate buffer saline (PBS), and diluted (1 : 100) cell suspension was prepared in YE medium. Hyaluronic acid and glucose 0.2% (wt/vol) were added, and cells were incubated at 37 ∘ C in 5% CO 2 . The cell growth (0-48 hrs) was detected by measuring the optical density (OD) at 600 nm.
For the gene expression study, the planktonic cells were grown up to log phase in corresponding medium, that is, in YE medium alone or in presence of either 0.2% HY or Glu, and immediately processed for RNA extraction. The RNA extracted from cells grown in YE medium without supplements was used as standard condition for relative quantification of gene expression.

Effect of HA on
In Vitro Biofilm Growth. The effect of HA on pneumococcal biofilm growth was studied in YE medium and YE medium supplemented with 0.2% HA. Biofilms grown in YE medium supplied with 0.2% glucose was used as positive control. In vitro biofilm formation was carried out in 96-well (flat-bottom) polystyrene tissue culture plate (BD falcon, Sparks, MD, USA) in static model by a procedure previously described [18,19]. The cell suspension was prepared as given above and inoculated 200 L in 96well microtiter plate. The plates were incubated at 37 ∘ C in 5% CO 2 for various time points (6,12,18,24, and 30 hrs). After incubation, medium was discarded and plates were gently washed three times with 200 L sterile PBS. Thereafter, plates were air-dried and stained with 50 L crystal violet (CV) (0.1%) for 15 min. Excess stain was decanted off, and plates were washed three times with sterile distilled water. The biofilm was dissolved in 200 L of 95% ethanol, and OD at 570 nm was measured in an automatic spectrophotometer.
All experiments were performed in triplicate, and the average was calculated. The experiments were repeated three times.
For relative quantification of gene expression of pneumococcal biofilms, biofilms were grown for 24 and 30 hrs in a 24-well tissue culture plate by the procedure described early. The adherent cells of two wells were scraped and immediately processed for RNA extraction. The RNA extracted from biofilm cells grown in YE medium (without supplements) was used as a standard for relative quantification of gene expression.

Scanning Electron Microscopic Analysis of Pneumococcal
Biofilm. For SEM analysis, biofilms were grown in a 24-well tissue culture plate by the procedure described early and incubated at 37 ∘ C for 24 hrs in 5% CO 2 . After incubation, the medium was removed and the plate was gently washed two times with sterile PBS to remove planktonic cells. The SEM analysis was performed using a variable pressure field emission scanning electron microscope (VP-FE-SEM, SUPRA-55VP, Carl Zeiss, Germany). The samples were fixed with 4% formalin solution, dehydrated in a gradual ethanol/distilled water mixture from 50 to 100% in steps, and dried for 2 days. The morphologies of samples were coated with platinum (Pt) and observed with VP-FE-SEM.

RNA Extraction and cDNA Synthesis.
For RNA extraction, biofilm cells were scraped with a sterile scraper, washed twice with sterile PBS, and immediately processed for RNA extraction. For planktonic cell RNA, 5 mL log-phase grown cells were washed twice and used for RNA extraction. Total RNA was extracted by using "RNeasy Total RNA Isolation System Kit" (Qiagen, MD, USA) according to the manufacturer's instructions with few modifications: on column RNAase-free DNAse (Qiagen, MD, USA) treatment was performed for 10 min at 20-25 ∘ C. The concentration and quality of RNA were assessed with a spectrophotometer. cDNA synthesis was carried out by using the "ImProm-II Reverse Transcriptase Kit" (Promega, WI, USA) according to the manufacturer's instructions. Tailing of RNA with a random hexamer primer was performed at 70 ∘ C for 5 min, annealed at 25 ∘ C for 5 min, extended at 37 ∘ C for 1 hr, and inactivated of samples at 70 ∘ C for 15 min.

Relative Quantification of Gene Expression by Real-Time PCR.
Quantitative real time PCR was performed as previously described (20) with a light cycler apparatus by using the SYBR Green I Master Mix (Roche Applied Science, IN, USA). Primers were designed by standard procedures from nucleotide sequence of S. pneumoniae R6 strain and checked for corresponding sequence in D39 strain (Table 1). A set of 11 genes to be monitored (involve in pneumococcal biofilm formation, HA metabolism, and autolysis) were decided after reviewing the literature. Primers used for gyrB (spr1099) and comA genes (spr0043) were as previously described [20]. Real-time PCR was carried out in a total volume of 20 L, consisting of 10 L 2x SYBR Green PCR Master Mix (Roche Applied Science, IN, USA), 2 pmol of forward and reverse primers, and 2 L cDNA. PCR conditions included initial denaturation at 95 ∘ C for 10 min, followed by 45 cycles of denaturation (95 ∘ C for 10 s), annealing (56 ∘ C for 15 s), extension (72 ∘ C for 15 s), and final extension (72 ∘ C for 5 min) followed by melting curve analysis from 60 to 95 ∘ C. Negative controls containing sterile water instead of RNA were run concomitantly to confirm that the samples were free from contamination. To verify the absence of contaminating genomic DNA, each RT-PCR experiment included a no reverse transcriptase control. The relative gene expression was analyzed by using the 2 −ΔΔCT method [21], and the reference gene was gyrB. The planktonic cells grown in YE medium were used as the standard condition for the planktonic cell gene expression study. The biofilms grown in YE medium were used as the standard condition for the gene expression of cell grown within biofilms. The experiments were repeated two times in quadruplicate.

Statistical Analysis.
Statistical analysis was performed using 2-tailed Student's test and one-way analysis of variance (ANOVA), and a difference of < 0.05 was considered significant.

HA Support Pneumococcal
Growth. Experiments performed in YE medium or in YE medium supplied either with HA or Glu showed that HA supports the growth of S. pneumoniae D-39 strain (Figure 1). In positive control (glucose) the bacterial growth was high, and bacteria attained  control. The OD 600 of positive control decreased sharply at decline phase, indicating higher rate of cell lysis. However the OD 600 of HA containing medium decreased slowly in comparison to positive control. These results indicate that probably the cell lysis process was slow in presence of HA.

HA Support Pneumococcal Biofilm
Growth. The CV microplate assay detected increased biofilm growth in medium containing HA and Glu (positive control) in compare to YE medium (Figure 2). At 6 and 12 hours of incubation, there was significant ( < 0.05) increase in biofilms growth in Glu medium compared to YE and HY media. However, at 18, 24, and 30 hrs of incubation, significantly increased biofilms were detected in HA and Glu media ( < 0.05). The biofilm biomasses of the positive control (Glu) decreased at 18, 24, and 30 hrs, in time-dependent manner. In contrast to the positive control, the biomasses of HY biofilm significantly increased at 18, 24, and 30 hours. This may be due to higher cell growth and thereafter higher cell lysis in Glu biofilms compared HY biofilms. The result indicates that HA supports in vitro biofilm growth of S. pneumoniae D39.

Scanning Electron Microscopic Analysis of Pneumococcal
Biofilm. Scanning electron microscopic images of S. pneumoniae biofilms grown in YE medium or in YE medium either supplied with HA or Glu are shown in Figure 3. Pneumococcal biofilm growth in HA medium is thick with organised structures. The adherent cells are interconnected to each other and/or bound the cells to the intercellular matrix (Figure 3(a)). No dead cells were visible in the HA biofilms. The biofilms in positive control samples (Glu) were thick and organised, with fully grown cells, and few dead cells are visible (Figure 3(b)). No organized biofilms growth was detected in YE medium without supplements. In YE medium very few dead cells were detected, which were scattered and attached to the bottom of the tissue culture plate (Figure 3(c)).

Relative Quantification of Gene Expression. The fold changes in gene expression of planktonic cells grown in
presence of HA or Glu in comparison to cell grown in YE medium are shown in Table 2.

Discussion
S. pneumoniae can modify N-linked glycans, O-linked glycans, and glycosaminoglycans present in the human airway, and on cell surface of other streptococci. Utilization of these glycoconjugates provides a carbon source for growth, biofilm formation, competition with other bacteria within the airway, and exposing receptors for adherence. Previous studies have reported that pneumococci can utilize HA derived from human and bacteria for its growth. We studied the effect of HA on pneumococci growth in planktonic form and within in vitro biofilms and evaluate the changes in gene expression of virulence and/or biofilm related genes.
Our results demonstrated that HA supports pneumococcal growth in planktonic and within biofilms and upregulates the gene expression of virulence and biofilm related genes. In previous study, Marion et al. [16] reported that the S. pneumoniae 1121 strain can utilize S. progenes HA capsule for its growth and colonization. Here, our results demonstrated that S. pneumoniae D-39 strain can effectively utilize S. equi    [22]. The modifications of capsule may make the other streptococci vulnerable to phagocytic killing by host cells, thereby decreasing interspecies competitions [23][24][25]. Thus it can be assumed that in in vivo, at niche, a site where multiple species are present, the utilization of HA capsule by pneumococci contributes to colonization both through niche competition and acquisition of carbohydrates. The growth of pneumococci in the presence of HA was low with longer lag phase, and the doubling time of bacteria was more than the positive control (glucose). The reduced growth efficiency of glycoconjugates was observed for Nlinked glycans [12,16,26]. At decline phase, the rate of decrease of OD 600 of HA culture was slow in comparison to cell grown in glucose medium (control). This might be due to slow cell lysis in presence of HA. To look further inside, we analysed the gene expression of autolysis related gene (lytA). Autolysis is mainly dependent on the activity of the muramidase (LytA) and on cell-wall composition [27,28]. We detected downexpression of lytA gene and competence related genes (comA) in cells grown in HA medium. The autolysis and competence in S. pneumoniae are linked to luxS gene, which is upregulated in this study in cells grown with HA. Romao et al. [29] reported that functional luxS was found to be related on the one hand to downregulation of competence and on the other hand to attenuation of autolysis in cultures entering stationary phase.
Interestingly, we detected upregulation of nanA, nanB, bgaA, and strH genes in presence of HA. The pneumococcal exoglycosidases encoding genes (nanA, nanB, bgaA, and strH) are virulence related genes that have been upregulated in presence of glycoconjugates [12,26]. The upregulation of exoglycosidases encoding genes in presence of HA indicates that probably HA stimulates the expressions of these genes due to change in carbohydrate source. In S. pneumoniae, regulation of carbohydrate-specific catabolic pathways as well as modulation of virulence factors by catabolite control protein A (CcpA) has been reported [30,31], and the alteration in carbohydrate source and metabolic pathway leads to an increase in expression of these virulence factors [32,33]. No significant changes in neuraminidases encoding gene in glucose medium are in agreement with previous study that reported that glucose represses neuraminidase expression [34].
The upregulation of hysA, ugl, and PTS-EIIA in presence of S. equi capsule HA indicates that similar set of genes are required for the utilization of bacterial and human derived HA [16]. Marion et al. [16] found that the hyaluronate lyase (hyl or hysA in R6), a putative PTS transporter (PTS-EIIA), and ugl are required for pneumococcal growth on human HA. The surface-associated hyaluronate lyase (hyl) produced by S. pneumoniae is a virulence factor that degrades HA into disaccharide units [15]. These disaccharides are transported by putative PTS transporter and further cleaved by glucuronyl hydrolases (ugl) [17].
HA derived from capsule of S. equi supports in vitro pneumococcal biofilm formation. Our relative quantification results showed upregulation of luxS, nanA, hysA, ugl, and PTS-EIIA in HA biofilms. The role of luxS and nanA in pneumococcal biofilms has been reported earlier [11,19]. A previous report demonstrated the upexpression of PTS-EIIA and ugl during infection of the lung, indicating that utilization of HA may also contribute to bacterial growth during disease [35]. Probably in in vivo the pneumococcal hyaluronate lyase aids bacterial invasion and this localized invasion contributes to bacterial colonization in addition to degrading HA for nutrition [14,36].
In conclusion, this study indicates that the HA derived from capsule of S. equi supports pneumococcal growth in planktonic and within biofilms and upregulated virulence and biofilm related genes. This study will enhance our knowledge of pneumococcal pathogenicity.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Psychology Medicine

text:
Maternal Obesity Caused by Overnutrition Exposure Leads to Reversal Learning Deficits and Striatal Disturbance in Rats

Maternal obesity caused by overnutrition during pregnancy increases susceptibility to metabolic risks in adulthood, such as obesity, insulin resistance, and type 2 diabetes; however, whether and how it affects the cognitive system associated with the brain remains elusive. Here, we report that pregnant obesity induced by exposure to excessive high fatty or highly palatable food specifically impaired reversal learning, a kind of adaptive behavior, while leaving serum metabolic metrics intact in the offspring of rats, suggesting a much earlier functional and structural defects possibly occurred in the central nervous system than in the metabolic system in the offspring born in unfavorable intrauterine nutritional environment. Mechanically, we found that above mentioned cognitive inflexibility might be associated with significant striatal disturbance including impaired dopamine homeostasis and disrupted leptin signaling in the adult offspring. These collective data add a novel perspective of understanding the adverse postnatal sequelae in central nervous system induced by developmental programming and the related molecular mechanism through which priming of risk for developmental disorders may occur during early life.

Introduction
Current data from epidemiological as well as in vivo animal studies have contributed to the concept of developmental programming, whereby an unfavorable prenatal environment is believed to be associated with long-term metabolic consequences [1,2]. A recent longitudinal study revealed that maternal obesity not only predisposed the fetus to develop obesity, insulin resistance and type 2 diabetes [3], but also adversely affected IQ performance, emotional problems and core symptoms of attention deficit and hyperactivity disorder (ADHD) in school-aged children [4][5][6]. Although the postnatal sequelae in the central nervous system induced by development programming has been noticed, current literature [4][5][6] based on the human studies are limited and inconsistent, from which it is difficult to accurately identify the etiology and related mechanisms linking maternal obesity to offspring's cognition, thus clinically posing obstacle in guiding the pregnant woman to achieve a healthy weight. Besides, the adverse neurodevelopmental outcomes in offspring could be, at least partly, caused by the predisposed overweight that usually develops later in the offspring's life [3]. Therefore, whether and how maternal obesity directly affects the cognitive system remains to be established.
Cognitive flexibility is an essential ability of adaptive behaviors while cognitive rigidity is a behavioral symptom related to impulsivity and compulsivity, which is often detected in many developmental disorders, including ADHD, autism, Torette syndrome, Rett syndrome and schizophrenia [7][8][9][10]. As a kind of behavioral paradigm to evaluate the cognitive flexibility, reversal learning is defined as the ability to modulate responses to a previously non-reinforced stimulus, which indexes vulnerability for disorders characterized by impulsivity such as proclivity for initial substance abuse and compulsive aspects for the dependence [11]. In contrast to reversal learning which pertains to a specific exemplar, attentional set-shifting, another paradigm of cognitive flexibility, refers to the ability to adapt behavior flexibly following feedback, but pertains to broader stimulus dimensions (e.g. from lines to shapes, from texture to odor) [12]. Recently, it has been reported that core symptoms of ADHD, such as attention deficit and hyperactivity, are readily detected in the children born to the obese mothers [3], suggesting a stunted cognitive flexibility including reversal learning and attentional set-shifting likely associated with maternal obesity, since these disrupted behaviors are frequently co-developed in the ADHD patients through some shared mechanisms, although the direct causality and the underlying mechanisms still remain to be examined.
The present study was designed to investigate the impact of maternal obesity caused by overnutrition pre-conception and during pregnancy on the abilities of reversal learning and attentional set-shifting in rats and determine the potential mechanisms underlying the bio-communication between mother and offspring.

Ethics statement
All experiments of research were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine, China.

Care and maintenance of animals
Wistar rats were purchased from Shanghai Laboratory Animal Center, and maintained under controlled temperature with food and water provided ad libitum. Thirty 9-week-old female and 10 male were housed in individual cages under controlled temperature at 22uC, with a 12:12-h light-dark cycle (light on 7:00 AM), and were given commercial standard pellets for 1 week for adaption. Three groups of females were then formed according to the Ctrl, HF or HP diet (n = 10 in each group). After 6 weeks, females were caged in collective cages for mating and returned into individual cages after 14 days. At the time of birth, the male pups were kept for further investigation. After weaning, three groups of male pups were fed on Ctrl diet and allowed to free access to food and water. For the subject representativeness as well as devoid of behavioral variability, one to two male offsprings with the median weight(s) among the littermates from each mother (n = 9 for Ctrl group, n = 10 for HF group and n = 11 for HP group) were picked up for the further investigation on the following metabolic and behavioral examination.

Experiment 1: Body weights of dams and offspring
Dams were measured at three time points (6 weeks before mating, at the time of mating and after the delivery of their offspring) (n = 10 in each group, Fig. 1A). In addition, the pups were weighted at birth and the age of 10 weeks (n = 9 for Ctrl group, n = 10 for HF group, and n = 11 for HP group, Fig. 1B-C).

Experiment 2: Plasma parameter determination
The 10 weeks-old adult offspring (n = 9 for Ctrl group, n = 10 for HF group, and n = 11 for HP group, Fig. 1D-F) were fasted overnight and received intraperitoneal injection of recombinant rat leptin (1 mg/kg) (Huijia Technology, Xiamen, China, cat #: CYT-592) or saline. After 30 minutes, rats were killed by decapitation after cervical elongation. Blood was collected on heparin (10 IU/ml) and hypothalamus was quickly removed. The hypothalamus was immediately frozen into liquid nitrogen. Plasma glucose level was measured by enzymatic assay (Sigma-Aldrich, St. Louis, MO, USA). Insulin and leptin were assayed by radioimmunoassay using commercial diagnostic kits (LINCO Research, St. Louis, MO, USA). The other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Experiment 3: Visual Discrimination and Serial Reversal Learning (VDSRL)
Apparatus. Operant conditioning chambers (30 cm 624 cm 630 cm; Med Associates, St Albans, VT, USA) were used to carry out behavioral tests, each chamber enclosed within a soundattenuating wooden box which fitted with a fan for ventilation and masking of extraneous noise. In each chamber, two retractable levers were located on either side of a centrally positioned food magazine, into which an external pellet dispenser could deliver 45 mg sucrose pellets (Noyes dustless pellets; Sandown Scientific, Middlesex, UK). A light-emitting diode (LED) was positioned centrally above each lever. Besides, there were a magazine light and a house light. Magazine entry was detected by an infrared photocell beam located horizontally across the entrance. The software Med-PC-IV from Med Associates was used for apparatus control and behavioral recording.
Training and testing. A schematic representation of the behavioral training and testing protocol is shown in Fig. 2A, the protocol was adapted from Castañé et al. [13].
Pre-training: All rats (n = 9 for Ctrl group, n = 10 for HF group, and n = 11 for HP group, Fig. 2A-C, Fig. 3A) were initially familiarized with the testing apparatus for 30 min. During this time, the house light was turned on and pellets were presented. After the habituation, training took place on each lever separately, firstly under a fixed ratio 1 (FR1) schedule to a criterion of 50 presses in 15 min, then under a fixed ratio 3 (FR3) schedule to a criterion of 150 presses in 15 min for each lever for 2 consecutive days. The FR3 schedule was used to avoid the possibility of reinforcing single, accidental presses on the correct lever. The presentation order of left and right lever was counterbalanced across subjects.
Acquisition of spatial discrimination: After the acquisition of a two-lever discrimination task as previously described [13], animals were trained to nose-poke the central magazine in order to trigger presentation of the retractable levers. Then, only three lever presses on one of these levers resulted in reward. Each session lasted 30 min and consisted of a maximum of seven 10-trial blocks. Each trial began with the switching on the house light. In the limited hold period of 20 s, the rat had to nose-poke in the magazine in order to provide the presentation of the two-levers which initiated a 10-s response interval. No response in either the limited hold period or the response interval resulted in the return to the inter-trial interval (ITI) state until the next trial was scheduled to begin, while the trial was recorded as an omission. Once the rat pressed one of the levers three times, both levers were retracted and the house light was turned off. Each rat had one training session per day and was trained until reaching a criterion of nine correct trials in one block of 10 trials (binomial distribution p,0.01, likelihood of attaining criterion in a 10-trial block). The completion of nine correct choices out of ten also implicated the end of the session. Once the criterion was reached, the initial discrimination phase was considered complete, and the animal was returned to the home cage. If the criterion was not achieved, this phase was repeated the next day till criterion achievement. Two days after acquisition, rats were retested on discrimination. Once the criterion was achieved, the reversal phase started.
Serial reversal learning task: During this phase, rats were trained to serially reverse an instrumental spatial discrimination in a between-session design. Three reversals were given, whereby in each reversal the previously correct lever became incorrect and the previously incorrect lever became correct (reversals 1-3). Each reversal session lasted 30 min and consisted of a maximum of seven 10-trial blocks. The learning criterion was the same as in the discrimination phase (nine correct trials in a 10-trial block). Animals usually required more than one session to reach criterion on the reversals. Thus, they received multiple, separate training sessions, the data of which were summed together. Between successive reversals, animals were given a session of up to seven 10trial blocks where they were required to show retention of the previous reversal phase by reaching the nine of 10 correct criterion in one 10-trial block.
Behavioral measures: The main measures of the rat's ability to learn the discriminations/reversals were: (1) the number of trials to criterion and (2) the number of error to criterion (i.e. incorrect trials). Additional measures recorded for each trial were (3) the type of error, (4) the latency to collect the reward, and (5) the probability of shifting after an error or a correct response [14]. The type of errors was analyzed as described previously [15][16][17]. Briefly, sessions were classified as perseverative (where responding to the previously correct stimulus was significantly above chance), or learning (where responding to the newly correct stimulus was at, or above chance). The test was calculated by each rat's daily session considering the number of errors and the number of trials data. As an example, $44 errors in a 70 trials session indicated perseveration, between 43 and 27 errors was chance performance, and #26 errors showed accurate responding to the newly correct lever. Errors during perseverative sessions were considered perseverative errors and so on [15][16][17].

Experiment 4: Food consumption
Food consumption of rats was tested as previously described [13]. Briefly, rats (n = 9 for Ctrl group, n = 10 for HF group, and n = 11 for HP group, Fig. 3B) were individually housed in standard rat cages where food pellets were freely available in a food cup. The amount of pellets consumed was calculated within 30 minutes.   The rewarded and unrewarded lever is indicated by the ''+'' and ''2'', respectively. The rewarded lever was counterbalanced across rats. (B-C) Total trials (B) and errors (C) to criterion during acquisition of spatial discrimination (Acq), retention of discrimination (Ret-D) as well as during the reversal phases (reversal 1-3: Rev1-3; retention of reversal 1-3: Ret-R1-3). (D) Perseverative and learning errors accumulated across reversals (perseverative errors accumulated: P-Accum;learning errors accumulated:L-Acuum) and during 3 reversals (perseverative errors of reversal 1-3: P-Ret1-3; learning errors of reversal 1-3: L-Ret1-3) were shown. A series of three reversals were performed by male offspring born to dams fed with the Ctrl diet, HF diet, and HP diet. Between successive reversals, animals were given a session to test retention of the previous reversal phase (Ret-R1-3). (E) Mean probabilities of rats shifting their responding to the other stimulus after making either an incorrect choice (and therefore not receiving reward) or a correct choice and receiving reward. n = 9 for Ctrl group, n = 10 for HF group and n = 11 for HP group. *p,0.05; **p,0.001. doi:10.1371/journal.pone.0078876.g002 Experiment 6: Attentional set-shifting task (ASST) Apparatus. Attentional set-shifting task (ASST) procedures were conducted according to Izquierdo et al. [18]. Rats (n = 9 for Ctrl group, n = 10 for HF group, and n = 11 for HP group, Fig. 4A-C) were trained in a Plexiglas arena that measured 36 cm (height) 645 cm (width) 668 cm (length). The box was divided equally into thirds so that each compartment was about 23 cm long. The front of the apparatus was further divided into two separate sections where the bowls were contained separately, to avoid animals having access to both bowls simultaneously. In addition, access to each compartment (and bowl) in the front of the box could be restricted by an opaque, removable divider. The back third of the apparatus (inter-trial chamber) was separated from the other compartments with a removable divider, and rats were placed in that chamber at the beginning of each trial. Access to the inter-trial chamber was blocked once a trial began.
Food rewards were buried half-way down ceramic bowls (4 cm tall, internal diameter is 8 cm). Rats were trained on successive days to make discriminations based on two dimensions: media of varying textures (e.g., vermiculite, confetti, gravel), or scents (e.g., paprika, thyme or oregano). Scents could be mixed interchangeably with media so that combinations of the two dimensions were possible, but pairs of scents or media were kept constant (e.g., cumin was always presented with cinnamon; vermiculite was always presented with gravel).
Training and testing. Food restriction and habituation: During food restriction, rats were fed about 13-17 g of food per day, and weight was monitored daily to maintain a target weight of 85% normal weight. To habituate the animals to the feeding Figure 3. Feeding behaviors and locomotor activity of offspring in or after the VDSRL task. (A) Latencies to collect the food during acquisition (Acq), retention of spatial discrimination (Ret-D), reversal phase (Rev1-3), and retention of reversal 1-3 (Ret-R1-3) in the VDSRL task. A series of three reversals (reversal 1-3: Rev1-3) were performed. Between successive reversals, animals were given a session to test retention of the previous reversal phase (Ret-R1-3). *p,0.05; **p,0.001. (B) Food comsuption during a 30-min test session after the VDSRL task (n = 10 in each group). (C) Number of beam breaks during a 60-min test session after the VDSRL task (n = 9 for Ctrl group, n = 10 for HF group and n = 11 for HP group.). doi:10.1371/journal.pone.0078876.g003 bowls, and to familiarize them with digging for rewards, food was given in small ceramic bowls in the home-cage for several days before training. Rats were returned to free feeding once testing was complete, and they were kept on food deprivation for no more than 20 days. After deprivation, rats were habituated to the arena for 5 min before training. To familiarize the animals with digging for a food reward, animals were presented with two baited unscented bowls filled with home cage bedding. Once the animal had retrieved the food reward, the animal was moved back to the inter-trial chamber and the bowls were re-baited. The divider was then lifted, allowing the animal access to the bowls. All subsequent trials were conducted in this same manner. Once the animal was reliably digging for a food reward, training on the task was begun. A schematic representation of the behavioral training and testing protocol is shown in Fig. 4A, the protocol was modified as described previously [18].
Training: During the training phase, rats were trained on two simple discriminations (SDs), one scent-based (thyme vs paprika), and the second media-based (vermiculite vs plastic beads). All rats were trained on the same discriminations, in the same order. Criterion completion of training was achieved when the rat made a correct discrimination for six consecutive trials. Rats that did not learn to dig for food reward were returned to their home-cage and a second attempt was made one day later. More than 90% of rats completed the training within two attempts.
Testing: The day after training testing was delivered. A trial was initiated by raising the dividers, giving the rat access to two bowls, only one of which was baited. For each phase, the rat was given four discovery trials, whereby the rat was allowed to dig in both bowls to retrieve the food reward. Errors were recorded during the discovery trials, but did not count toward trials to criterion [18]. On subsequent trials, if the rat dug in the unbaited bowl, an error was recorded, and the trial was terminated. In a single-test session, rats were given the following discrimination phases to learn: (1) scent-based discrimination (SD, nutmeg (+) vs cloves); (2) compound discrimination (CD), where media (paper squares vs shredded paper) dimension was introduced, while scent was still rewarded, irrespective of medium (nutmeg (+) /paper squares and nutmeg (+) / shredded paper); (3) CD-reversal (CDr), where previously unrewarded scent was now rewarded, irrespective of medium (cloves (+) /paper squares and cloves (+) /shredded paper); (4) intradimensional shift (ID), where animal must still attended to scent and correctly discriminated the rewarded scent, while novel scents (cinnamon (+) vs cumin) and media (foam triangles vs straws) were introduced; (5) ID-reversal (IDr), where previously unrewarded scent was now rewarded, irrespective of medium (cumin (+) /foam triangles and cumin (+) /straws); (6) extra-dimensional shift (ED), where the rat was trained to attend to medium cues (1/4 foam shells (+) vs crushed foam) and ignore scent cues (celery seed vs sumac); (7) ED-reversal (EDr), where previously unrewarded medium was now rewarded, irrespective of scent (crushed foam (+) / celery seed and crushed foam (+) /sumac). During each phase, the rats were tested until they had achieved a criterion of six consecutive correct choices. Two measures were used to quantify performance in each phase: (1) trials to criterion (the number of trials taken to reach criterion), and (2) the number of errors made in reaching criterion. The order of the discriminations and the exemplar pairings were always the same, but the pairs of exemplars were counterbalanced between groups. Typically, rats completed all phases of testing within a single day.

Experiment 7: [ 125 I]RTI-55 binding to dopamine transporter (DAT)
After behavioral tests, rats (n = 9 for Ctrl group, n = 10 for HF group, and n = 11 for HP group, Fig. 5A-B) were anesthetized with an overdose of sodium pentobarbital (250 mg/kg, i.p.), decapitated, and their brains were removed and frozen at 220uC by immersion in isopentane. A measure of 20 mm-thick coronal sections were cut on a cryostat at the level of the anterior striatum (AP coordinates +1.7 mm to +0.8 mm, according to Paxinos and Watson [19]), thaw-mounted on Vectabond-treated glass slides and stored at 220uC for autoradiography use. Warmed slides removed from the 220uC freezer were preincubated in a solution of assay buffer (10 mM Na 3 PO 4 , 120 mM NaCl, 100 mM sucrose) containing 100 nM fluoxetine for 5 min to remove endogenous ligands that could interfere with subsequent radioactive ligand binding. As [ 125 I]RTI-55 bound with high affinity to both DAT and serotonin transporter (SERT), 100 nM fluoxetine was included in both the preincubation and incubation media to block the radioligand binding to SERT. After preincubation, the sections were incubated in a solution of assay buffer containing 25 pM [ 125 I]RTI-55 and 100 nM fluoxetine for 2 h. The sections were then rinsed twice for 2 min each at 41uC in assay buffer, then once for 10 s in 41uC distilled water. The rinsed slides were then rapidly dried under a stream of heated air. The dried slides and [ 14 C]-containing autoradiographic standards were opposed to Hyperfilm MP (GE Healthcare) for 48 h before development. Quantification of [ 125 I]RTI-55 binding was done using a medical image analyzer (Shenteng Ltd, Shanghai, China). Image densities were converted to [ 125 I]RTI-55 binding levels using a calibration curve based on images of the standard slides packed with each film. Regional densities of RTI binding were obtained by outlining the desired structures on their respective [ 125 I]RTI-55 images. Values obtained represented the average of measurements taken from both hemispheres in a total of four sections per animal. For analysis, the image of striatum (see Fig. 5A) was first subdivided into caudate-putamen (CP) and nucleus accumbens septi (NAc). The CP was then subdivided into dorsal (dCP) and ventral (vCP) parts, which were separately quantified for [125I]RTI-55 binding.

Experiment 8: Western blot analysis
Samples were prepared as previously described [20]. Briefly, frozen hypothalami were homogenized in lysis buffer: 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1mM EDTA, 0.5% nonidet-P40, 1% Triton X-100, protease inhibitor cocktail (0.35 mg/ml PMSF, 2 mg/ml leupeptin and 2 mg/ml aprotinin), and phosphatase inhibitor cocktail (10 mM sodium fluoride, 1 mM sodium orthovanadate, 20 mM sodium b-glycerophosphate, and 10 mM benzamidine). After lysis in ice for 90 min, insoluble materials were removed by centrifugation (15,000 rpm at 4uC for 45 min), and protein concentrations of the resulting lysates were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins (50 mg) were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. Blots were blocked with 5% nonfat milk and then incubated in the presence of appropriate primary antibodies (anti-phosphorylated STAT-3 or anti-total STAT-3 from Cell Signaling Technology; Danvers, MA, USA) and secondary antibodies. Following nitrocellulose membrane washing, targeted proteins (about 92 kDa) were revealed using enhanced chemiluminescence reagents (Super-Signal, West Femto, Pierce, Rockford, IL, USA). The intensity of bands was quantified by using medical image analysis software 2.0 (Shenteng Information Technology, Shanghai, China) (Fig. 6A). In each group, the phosphorylated STAT-3 (p-STAT-3) levels were normalized to total STAT-3. The p-STAT-3/total-STAT-3 ratio was measured in saline injected rats and in leptin injected rats. For clearly comparison, all the p-STAT-3/t-STAT-3 ratios were normalized to the mean value that obtained from the Ctrl group (Fig. 6B). The sensitivity towards leptin was defined as an elevation of mean p-STAT-3/t-STAT-3 ratio in leptin-injected compared to saline-injected rats (Fig. 6C).

Statistics
Data were analyzed using the SAS software package (Version 11.8). One-way ANOVA or repeated measures ANOVA were applied to detect intergroup differences. If significant interactions were found, they were further explored through post hoc comparisons (Newman-Keuls) to establish simple effects. Values were expressed as the mean 6 S.E.M., *P,0.05, and **P,0.001 was considered statistically significant.

Effect of maternal diet on dam, baby and adult offspring
In order to confirm that the overnutrition-induced maternal obese models were successfully set up, body weight of dam was measured at the following time points. At 6 weeks before mating, no difference was found for dam body weight among HF and HP diet groups as well as their counterparts (Fig. 1A, F (2, 27) = 0.03, P = 0.97); After HF and HP diet, the dam body weight of both HF and HP groups were higher than Ctrl group at the time of mating (Fig. 1A, F (2, 27) = 3.97, P = 0.03) and after the delivery of their offspring (Fig. 1A, F (2, 27) = 6.82, P = 0.004), indicating that maternal obese model was successfully set up. Next whether maternal obese during pregnancy affected the basal physiological parameter of offspring was investigated. Weight of the pups at birth (Fig. 1B, F (2,27) = 0.14, P = 0.87) and the age of 10 weeks (Fig. 1C, F (2, 27) = 3.047, P = 0.07) was similar among three different groups. Serum parameters, measured in three groups of adult offspring aged 10 weeks after overnight food deprivation, were showed in Fig. 1D-F. The offspring born to dams under either HP or HF diet displayed comparable levels of serum glucose (F (2, 27) = 2.20, P = 0.13), insulin (F (2, 27) = 2.64, P = 0.08) and leptin (F (2, 27) = 0.76, P = 0.47) to those of control offspring, indicating basic physiological parameters were not significantly affected by the maternal obesity and prenatal overnutrition.

Visual discrimination and serial reversal learning
In visual discrimination task ( Fig. 2A), three groups of offspring rats did not differ in the number of trials (Fig. 2B, F ( Next we investigated the reversal learning of offspring. A repeated measures ANOVA was conducted for testing the differences in the number of trials or errors to criterion, which revealed significant differences in main effects of maternal diet on the number of trials or errors to criterion (Fig. 2B, F ( among three groups, but no significant difference between maternal HF and HP diets (P.0.05). We found significant interaction of maternal diet 6 reversal in trials (F (4, 54) = 3.65, P,0.001), of maternal diet 6 reversal in errors (F (4, 54) = 5.51, P,0.001), and an obvious main effect of reversal (F (2, 54) = 33.32, P,0.001 for trials; F (2, 54) = 45.75, P,0.001 for errors), which indicated experience might enhance reversal performance in all three groups of rats. Besides, the performance deficits (also indicated by more trials and errors to criterion) caused by maternal diets were selectively observed in the early phase of reversals (Rev1) (Fig. 2B, F ( When we further explored the type of errors (perseverative and learning) as described in the methodology, we found there were significant difference in main effects of maternal diet (F (2,27) = 52.52, P,0.001), reversal (F (2, 54) = 1002.45, P,0.001) and interaction (F (4, 54) = 15.89, P,0.001) for the number of perseverative errors across all reversals (Fig. 2D), but no significant differences in main effects of maternal diet (F (2,27) = 0.58, P = 0.57), reversal (F (2, 54) = 0.41, P = 0.53) and interaction (F (4, 54) = 0.09, P = 0.92) for the number of learning errors (Fig. 2D). Besides, the difference in perseverative errors caused by maternal diets didn't display in Rev2 (F (2, 27) = 1.38, P = 0.27) and Rev3 (F (2,27) = 0.25, P = 0.78) but exclusively in Rev1 (F (2,27) = 99.92, P,0.001) (Fig. 2D), where post hoc analysis revealed significant difference between Ctrl and the other groups (P,0.001), but not within HF and HP group (P = 0.13) (Fig. 2D).
Additional analyses were undertaken to investigate whether maternal obesity caused by different diet led to any reversal differences in response to positive and negative feedback. Fig. 2E displayed the probability of shifting after an error or a correct response in three groups across all reversals. Two way ANOVA analysis of arcsine-transformed probability data from three groups of rats revealed a significant main effect of maternal diet (F (2, 54) = 44.06, P,0.001) and interaction in maternal diet 6 feedback (F (2, 54) = 47.54, P,0.001), collapsed across all three reversals. Simple main effects of maternal diet for each feedback type revealed maternal diet caused no difference in the probability of shifting after an correct response (shift given reward, F (2, 27) = 1.22, P = 0.31), but significant difference in the probability of shifting after an incorrect response (shift given no reward, F (2,27) = 67.99, P,0.001), consistent with their marked perseverative responding to the previously rewarded, but now incorrect stimulus. Subsequent post hoc analysis of the ''shift given no reward'' data revealed no significant difference between HF and HP groups (P = 0.92).
As shown in Fig. 3A, during the discrimination or reversal phases there were no any differences between groups in the latency to collect the food reward (P value more than 0.05 for all comparisons). Besides, there were no differences on the amount of food consumed by offspring born to dam fed on different diet in a 30 min test where pellets were freely available (Fig. 3B, F (2,27) = 0.96, P = 0.40). Both HP and HF groups showed similar levels of spontaneous locomotor activity (ambulatory movement) as control subjects, as measured by the number of beam breaks during the 60min test session (Fig. 3C, F (2,27) = 0.68, P = 0.51). These data indicated again that three group rats had similar performance in most behaviors.

Attentional Set-Shifting Task
During training, the rats readily learned to discriminate foodbaited bowls based on either scents or media (Fig. 4A). Testing was conducted over seven phases as shown in Fig. 4A. A repeated measure ANOVA was conducted to investigate the differences in trials-to-criteria and errors made for three groups. For the trials-tocriterion measure, the main effects of maternal diet (F (2,27) = 58.80, P,0.001), phase (F (6, 162) = 16.36, P,0.001) and the phase 6 maternal diet interaction (F (12, 162) = 10.92, P,0.001) were significant across all the phases (Fig. 4B). For the errors measure, the effects of maternal diet (F (2,27) = 46.98, P,0.001) and phase (F (6, 162) = 51.32, P,0.001) were significant but not interaction (F (12,162) = 0.06, P = 0.94) (Fig. 4C). Subsequent post hoc tests revealed that HF group and HP group required more trialsto-criterion and made more errors than did Ctrl group for the phases of CDr (F (2,27)  However, when comparing the effect of maternal diet on the attentional set-shifting of offspring, indicated by ED-ID difference scores, no differences were detected in 3 groups (F (2,27) = 0.27, P = 0.77 for trials-to-criterion Fig. 4B; F (2, 27) = 0.90, P = 0.42 for errors, Fig. 4C). As shown in Fig. 4B-C, our findings showed that all the offspring rats in 3 groups required more trials to reach criteria, and committed more errors for the discrimination requiring an extradimentional shift, comparing with intradimentional shift (paired sample t-tests. P values ,0.001 for trials-tocriterion and errors). But, the ED-ID difference scores didn't significantly differ in 3 groups, indicating that maternal obesity induced by overnutrition didn't impact the attentional set-shifting abilities of offspring.

Altered dopamine homeostasis upon maternal obesity
In order to explore the potential molecular mechanisms, we investigated whether expression of dopamine (DA) transporter (DAT) in striatum might be influenced by the maternal obesity due to overnutrition, as imbalanced striatal DA homeostasis was reported to be associated with reversal learning deficit, especially in early phase [21]. In Fig. 5A-B, analysis of [ 125 I]RTI-55 binding to DAT revealed that maternal HF and HP diets produced a significant reduction of DAT within CP (F (2, 27) = 14.23, P,0.001) of their offspring, whereas NAc DAT was unaffected (F (2, 27) = 0.51, P = 0.60). Further analysis revealed that this depletion of DAT occurred in CP of both dCP (F (2,27) = 68.97, P,0.001) and vCP (F (2,27) = 114.94, P,0.001). However, no regional DAT binding difference was found within HF and HP group (P = 0.23).

Leptin-dependent STAT-3 phosphorylation in the hypothalamus
To gain additional mechanistic insights on the cause of altered striatal DA homeostasis upon maternal overnutrition, we have turn to examine the consequent changes of central leptin signaling, a pathway frequently affected in obesity. The hypothalamus, especially the lateral hypothalamic area acts in concert with the ventral tegmental area and other components of the mesolimbic DA system to shape diverse aspects of animal behaviors [22,23]. Mechanistically, the defected leptin signaling in hypothalamus associated with obesity probably confers partially at least for the altered striatal DA homeostasis upon maternal overnutrition. To assess the central leptin signaling in hypothalamus, the phosphorylation levels of STAT-3 under basal conditions and in response to a bolus of leptin in three groups of the offsprings were compared. Ten week-old rats of three groups were starved overnight and divided into three groups that received saline or leptin (i.p.), respectively. In each group, STAT-3 phosphorylation levels were normalized to total STAT-3. Under basal conditions, the levels of pSTAT3 in both HF and HP groups were significantly lower compared to control group (Fig. 6A-B), implying the disrupted leptin signaling upon maternal obesity, since the circulating levels of leptin in the offspring born to dams under either HP or HF were comparable to that from control offsprings (Fig. 1F). Similarly, under the condition in response to leptin, the levels of pSTAT3 in these groups (i.e. HF and HP) were also substantially reduced ( Fig. 6A-B). Moreover, the quantification of leptin sensitivity by assessment of the elevation of p-STAT-3/t-STAT-3 ratio in leptininjected compared to saline-injected rats (Fig. 6C) showed that the significant increase (,68%) of STAT-3 phosphorylation observed in the adult offspring born to normally fed dams was totally abolished in those born to HF or HP dams (F (2,27) = 68.46, P,0.001), while no significant group difference was found between HF and HP group when post hoc analysis was further explored (P = 0.58) (Fig. 6A-C). Together, a defected central leptin signaling indeed occurred in offspring that exposed maternal obesity caused by overnutrition, which probably underlies the aforementioned deficiency in reversal learning and striatal DA disturbance.

Discussion
Maternal obesity is an increasing public health concern in developed countries and the developing world. In the United Kingdom, about 20% women of childbearing age are obese; in the United States this figure is up to 34% [20]. Epidemiological studies reveal that maternal obesity is associated with adverse outcomes for not only mothers but also children [24]. Neonates born to obese mothers are posited at higher risk of congenital anomalies, fetal macrosomia, and neonatal unit admission [24,25]. Long-term consequences for these offspring include an increased risk of obesity in their later life [24,26]. Besides, recent human studies have revealed that, in addition to obesity, type 2 diabetes, insulin resistance, and hyperleptinemia are also associated with pregnant obesity, which usually causes developmental programming through poor bio-communication between mother and fetus [27].
Furthermore, maternal obesity during pregnancy is recently reported to greatly affect central nervous system of offspring as well since the adverse neurodevelopmental outcomes such as low IQ performances and core symptoms of ADHD are observed in the children born to obese mothers [28,29]. Corresponding to the report on the human investigation, animal studies have also suggested that maternal obesity indeed negatively affect the development and function of central nervous system in offspring [4][5][6]. For example, it was found that maternal obesity impaired spatial learning performance in the mouse offspring through the disruption of production of brain-derived neurotrophic factor (BDNF) in hippocampus [5]. However, further understanding of other behavioral impacts and related mechanisms are restricted.
For example, both unfavorable nutritional status and poor physical activity during pregnancy may cause maternal obesity and subsequent developmental disorders in offspring through some shared or individualized mechanisms, which are not elucidated. Besides, it has reported that different dietary macronutrient intake, such as high fat diet or sucrose-rich diet, during pregnancy may play the incomparable influence on offspring [30,31], indicating further studies are required. Moreover, current human studies can't exclude the possibilities that the observed cognitive disadvantages might be a result of predisposed obesity developing in adult offspring, since offspring born to obese dams are more vulnerable to adulthood obesity which usually causes a disturbance in central neuronal system through defective insulin regulation. Thus, further investigation under appropriate body weight control of offspring need to be conducted.
In the present study, we found out for the first time that the rats born to obese dams were greatly impaired in reversal learning, as assessed by both visual discrimination reversal learning (VDSRL) and attentional set-shifting (ASST), however no significant abnormal serum metabolic parameters were detected simultaneously. The effect on VDSRL was highly specific, as it was contained within the early stage of reversal, particularly when animals committed more perseverative errors of shifting after given no reward rather than reward. With repeated practice offspring born to obese dams recovered from the impairments and reached criterion at a comparable rate with control rats in the late stage of reversal. Notably, there were no differences in acquisition and retention of discrimination phase as well as retention of spatial reversals across three groups, indicative of unaffected basal ability of learning and memory. In consistent with the findings in VDSRL, the reversal deficit was also observed in ASST, where the rats born to obese dams displayed slower learning abilities during reversal CDr, IDr and EDr, while left the set-shifting ability intact in ID/ED shift (represented by ED-ID difference scores). Considering the increased errors found in the reversal learning paradigms possibly confounded by other behaviors, such as locomotor activity and food motivation, we conducted tests for measurement of ambulatory movements and latency to collect food pellets. Our findings indicated that all the offspring exhibited comparable food intake, body weight, as well as basal ambulatory movements, indicating performance deficits observed in the offspring born to obese dams indeed attributed to a failure to environmental adaptivity.
Additionally, these reversal impairments were found in offspring whose dams fed on HP diet or HF diet, and displayed no significant difference within two groups, indicating that the postnatal reversal abnormalities induced by maternal overnutrition are likely irrespective of whatever kind of diet intake during pregnancy. In order to verify whether the observed reversal deficits were dependent on their predisposed obesity in the later life, we fed the offspring on the standard chow after weaning and kept their body weight in appropriation control before the behavioral tests were finished. Our results revealed that disturbed reversal learning of offspring was indeed independent of their body weight, indicating maternal obesity during pregnancy was the main cause, instead of predisposed obesity in offspring.
Anatomically, reversal learning involves orbitofrontal cortex (OFC) and dorsal striatum [32][33][34][35][36][37]. In our studies, impairments in reversal learning strongly suggested that there might be a dysregulation of OFC-striatum pathway in offspring born to the obese dam. More specifically, the abnormal response was selectively found to a previously rewarded but now incorrect stimulus rather than a previously no-rewarded but now correct stimulus, arguing a more likely involvement of striatum rather than OFC, since striatum is particularly sensitive to negative outcomes while the OFC sensitive to both negative and positive outcomes [14]. At present, it has been long considered that OFC-striatum pathway subjects to neuromodulatory inputs by DA, serotonin (5-HT), and noradrenaline (NA) [32][33][34][35][36][37]. In line with that, we found reduced DA uptake function in both dorsal and ventral divisions of the CP, with ventral CP in a greater extent of deficiency, indicating compromised striatal circuitry caused by abnormal DA clearance might underlie the maladaptive responding reported above. Also, the present interpretation linking striatum DA disturbance to the specific reversal learning deficits accords well with a recent report that medial striatal lesions in monkeys produced perseverative impairments during reversal learning [14]. Furthermore, a recent microdialysis study demonstrated a specific role of DA in the early but not late stage of reversal learning [21], which fit well with the observed selective behavioral impairments in VDSRL that only occurred in the early but not late phases. Specially, the activated DA efflux has been demonstrated increased in rats performing the first reversal compared with controls performing the previously acquired discrimination, but was absent during a third reversal [21], supporting the particular involvement of DA activity for newly formed positive response-reward associations during reversal learning. By contrast, NA was considered to play less important role in reversal learning, as NA efflux was only weakly activated during most phases of reversal learning [21]. Recently, a group of studies suggested that improved reversal learning could be caused by increased NA efflux through administration of a2receptor antagonist such as idazoxan [38] and atipamezole [39], supporting a part for NA in cognitive flexibility. However, this conclusion would be argued by the fact that all a2-receptor antagonists, including idazoxan and atipamezole, increase cortical DA efflux in addition to NA efflux [40,41]. Collectively, we prefer to the view that the disturbed DA activity most likely underlie here identified cognitive rigidity upon maternal overnutrition, although the possible involvement of NA action are not absolutely excluded.
In an attempt to understand mechanisms underlying the abnormalities in striatal DA clearance and reversal learning caused by maternal obesity, we investigated the regulation of neuronal leptin signaling, a critical link between maternal environmental factors and the developing physiology of the infant. Functionally, neuronal leptin contributes to preventing the storage of excess adipose tissue by feedback to the hypothalamus to reduce food intake and increase energy expenditure [42]. However, in most human with obesity, system leptin levels are often responsively elevated due to the leptin resistance [43]. Recent researches reveal that leptin signaling is exquisitely sensitive to maternal diet during prenatal and early postnatal life, which possibly plays a regulatory influence on striatal DA function [27]. It had been reported that the highest levels of leptin expression were found in the hypothalamus. Specially, recent report indicated that in the young age, at least prior to about 8 week of age, hypothalamus was the principal site of leptin-mediated regulation, with increasing age, the functions of other neuronal networks gradually increased [44]. Thus, it was proposed that hypothalamic leptin signaling was critical for establishing essential baselines in young animals that are maintained through the actions of distinct signaling networks in mature animals. Considering all the offspring rats under our investigation were as young as about 10 weeks old, we assumed hypothalamus could probably remain as the key brain region for regulation of leptin signaling. Research from rodent models had illuminated the underpinnings of leptin's ability to modulate emotion and neurobehavioral responses through the modulation of DA by leptin receptors [23]. Anatomically, leptin receptors are located in many brain regions that influence dopaminergic neurotransmission including areas of DA synthesis and projection sites such as striatum and nucleus accumbens, and via leptin receptor expressing neurons of the lateral hypothalamus that modulate dopaminergic neurons in the ventral tegmental area. In that regards, the defected leptin signaling in hypothalamus upon maternal overnutrition probably confers in part at least for the altered striatal DA homeostasis in the offspring.
Depending on the anatomical target, central leptin signaling was considered to participate into regulating many DAdependent behaviors [22], such as substance abuse, locomotor activity, enforced leaning, food intake, sleep and adaptive behaviors. Thus, it could be speculated reversal learning, as one of adaptive behaviors associated with DA activity might be potentially regulated by leptin signaling, which however needs further elucidation. In our studies, we examined final body weight, circulating leptin level and assessed the hypothalamic leptin sensitivity by measuring leptin-dependent STAT-3 phosphorylation. We found that offspring born to obese dams fed on HP or HF diet normally exhibited body weight and related plasma parameters like in serum leptin and insulin levels, were quite similar to those of control rats. However, among three groups of adult male rats born to HF, HP and control dams, only control group displayed an increased phosphorylation level of STAT-3 in the hypothalamus in response to leptin challenge, implying that offspring born to the obese dams brought an early defective leptin signaling, which might account for abnormal striatal DA modulation and consequent impaired adaptive behaviors observed in the former experiments. It was worthy to note that some other potential routs such as phosphoinositide 3-kinase-protein kinase B (Akt)-extracellular signal-regulated kinase (PI3K-Akt-ERK) pathway may also bear the defective leptin signaling, which warrant the further investigation. Considering that defective leptin signaling is also frequently found as a shared etiology in obese patients with physical inactivity, we might hypothesize that here identified reversal learning deficits caused by maternal overnutrition probably could be applied to pregnant obesity caused by physical inactivity as well, which needs further investigation in the future.
In summary, our data demonstrate that maternal obesity during pregnancy, caused by either HF or HP food, disrupts the reversal learning through an altered leptin signaling in offspring. Such disrupted leptin signaling may contribute to the disturbance in DA homeostasis in striatum and consequently cause above mentioned behavioral abnormalities. In line with other reports in human and animal studies, our findings add new level of evidence that maternal obesity cause the long-term impairments in cognitive flexibility. Our findings reinforce the importance of healthy weight achievement during pregnancy and provide cues for understanding the related mechanistic processes by which priming of risk for developmental disorders may occur during early life.


extfieldsofstudy:
Materials Science
Medicine

text:
Single-cell lysis for visual analysis by electron microscopy.

The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.

Introduction
The aim of systems biology is to understand the emergence of biological functions from interaction networks (Westerhoff, 2011). This requires knowledge of the intracellular players and their interconnections, for which an inventory of the individual components of the system, i.e. the transcriptome, the proteome, the metabolome and, finally, the interactome, has to be assembled. Such an inventory will strongly vary from cell to cell, as the stochastic nature of biological processes leads to ''biological noise'' (Eldar and Elowitz, 2010;Raj and van Oudenaarden, 2008). This makes the study of individual systems, e.g., single cells, a necessity (Wang and Bodovitz, 2010).
Genome sequencing (Zong et al., 2012) and expression profiling (Flatz et al., 2011) are far advanced, and amplification techniques are ready to be applied to single cells (Kalisky et al., 2011). The analysis of the metabolism of a biological system profits from the experience and advances of analytical chemistry (Fiehn, 2001); for example, mass spectrometry (MS) can be used to identify metabolites with single-cell sensitivity (Amantonico et al., 2008). Moreover, excellent imaging techniques, such as light-and electron microscopy (EM) or X-ray diffraction imaging, are available for structural analyses.
However, proteomic studies at the single-cell level are hampered by the low expression level of many proteins and the lack of amplification techniques. Although powerful and valuable techniques, such as MS (Picotti et al., 2009) and cryo-electron tomography (cryo-ET) (Henderson et al., 2007;Medalia et al., 2002;Nickell et al., 2006), are applied for single-cell proteomic studies, such studies still remain a challenging task, especially for eukaryotic cells (Bantscheff et al., 2007;Diebolder et al., 2012;Mader et al., 2010). Thus, adjuvant techniques utilizing novel or hybrid approaches are beneficial to further untangle the complexity of single-cell protein networks.
A combination of microfluidics and TEM was suggested as an alternative and complementary approach to investigate the protein content of single eukaryotic cells (Engel, 2009(Engel, , 2010. The idea is to physically lyse single cells and spread the entire sample onto EM grids for structural analysis by transmission electron microscopy (TEM), or mass measurements by scanning TEM (STEM). This ''lyse and spread'' approach provides access to EM imaging at a higher signal-to-noise ratio (SNR) than when in the cellular background, and enables a more straightforward correlation of structural information with mass data. A prerequisite of this envisaged approach is a targeted lysis of individual cells and an efficient preparation of their lysate for TEM analysis.
A variety of different techniques for single-cell lysis exists today, and most of them have been implemented in microfluidic systems (Brown and Audet, 2008). Many of these systems utilize the principle of electroporation (Fox et al., 2006;Movahed and Li, 2011) to lyse detached or suspended cells in flow-through configurations, whereas only a few report on electrical lysis of ''standard'' adherent eukaryotic cells in cultures (Han et al., 2003;Nashimoto et al., 2007). However, despite their popularity none of these systems has been utilized to prepare samples of a single-cell lysate for electron microscopy.
Here, we present a system for the electrical lysis of individual adherent eukaryotic cells and subsequent preparation of minute sample volumes for negative-stain TEM. The setup includes a custom-designed microcapillary electrode ( Fig. 1), which targets and lyses individual cells observed in a light microscope. Immediately after lysis, the cell-fragments are aspirated into the microcapillary, deposited on an EM grid and negatively stained. This method offers the potential for an alternative approach to analyze proteins and protein complexes from individual eukaryotic cells.

Instrument setup
The principle and basic design of the system developed for the electrical lysis of single cells is shown in Fig. 1 A. The system is designed for use with an inverted optical microscope (OM; Zeiss Axiovert 40C). The microscope is equipped with a custom-built stage that has a customized mounting frame on the objective guide. The latter accommodates an indium tin oxide (ITO)-coated glass slide (ground electrode and sample platform) and can be moved manually in the xy-plane. Miniaturized Petri dishes on the surface of the glass slide (see below) allow cell cultures to be grown. A tapered gold-coated microcapillary that serves as second electrode can be positioned in close proximity above the glass slide. The upper un-tapered end of the microcapillary is inserted in a steel adapter and electrically connected with silver paint (Fig. 1B). The insulating holder of the adapter is attached to a stepper motor (PI M-126.PD2, Physik Instrumente, Germany) mounted on top of an xy-platform on the microscope stage. This platform allows the microcapillary tip to be centered above the objective lens of the OM. The stepper motor moves the capillary in the z-direction. The other end of the steel adapter holding the capillary is connected via a PEEK (poly(ether-ether-ketone)) tube (inner diameter (ID) 250 lm) to a pressure reservoir ( Fig. 1). The tube is intercepted by a solenoid valve (LVFA0550310H, The Lee Company, USA) that is controlled by the computer through an NI USB-6009 module. A pressure controller (PCNC-0001-00, Seyonic, Switzerland) is used to apply positive or negative pressure to the system via the pressure reservoir. A function generator (33220A, Agilent, Switzerland) delivers a voltage signal, which is amplified 20 times by a linear voltage amplifier (F20A, FLC Electronic AB, Sweden). The output of the linear amplifier is electrically connected to the capillary, and the conductive glass slide is grounded. All electronic components of the system are controlled by a LabVIEW-based, custommade software (Supp. Fig. 1). A camera (GC750 GigE, Prosilica, USA), mounted on the microscope, enables live-cell imaging and video recording. The next version of the control software will be available as an open-source plug-in for the openBEB (open biological experiment browser) system (www.openBEB.org). single-cell lysis setup, which is mounted on an inverted optical microscope. A camera allows for live-cell imaging. The stepper motor approaches the gold-coated microcapillary to the ITO-coated glass slide, where individual cells can be targeted. The function generator sends a voltage pulse to the capillary tip to lyse the cell. Meanwhile, the pressure controller builds up a negative pressure on the closed solenoid valve. Upon cell lysis, the valve is opened for a defined period of time, and the cell lysate is aspirated into the capillary. (B) Lysis microcapillary and cell culture slide. The upper end of the gold-coated microcapillary is inserted into a steel adapter and electrically connected with silver paste. The other end of the steel adapter is attached to a piece of PEEK tubing that connects the microcapillary to the pressure controller via a pressure reservoir. The microcapillary is positioned above a grounded, ITO-coated, glass slide with a PDMS ring on its surface. Slide and ring form a mini Petri dish that can be filled with cell culture medium (red).

Microcapillaries
Fused-silica (FS) microcapillaries (outer diameter (OD) 350 lm, ID 250 lm, Polymicro) were cut to a length of 10-15 cm. The polymer coating in the middle was removed over a length of 1-2 cm, using hot chromic-sulfuric acid (102499, Merck, Switzerland). Afterwards, the microcapillaries were rinsed thoroughly with double distilled H 2 O (ddH 2 O), followed by isopropanol and subsequently pulled with a laser-based micropipette puller (Sutter Instruments, P2000) to obtain a taper with a 30-50 lm ID at the tip. The tapered microcapillaries were plasma-cleaned before the sputter-deposition of a 2 nm Ti adhesive layer, followed by a 50-100 nm Au layer on their outer surface to increase conductivity. Coating was successively performed from two opposite sides at an angle of 45 degrees to obtain a closed conductive layer around the microcapillaries.

Miniaturized Petri dishes on conducting glass slides
Commercially available ITO-coated glass slides (Diamond Coatings UK, 8-12 X/square) were used as a conductive substrate for cell culturing. These slides have a silver electrode at each end for electrical connections. They were washed and sonicated in detergent solution (1% Alconox, Alconox Inc., USA), rinsed with ddH 2 O and stored in ethanol until use.
PDMS (poly(dimethylsiloxane), Dow Corning SYLGARD 184) rings with an ID of 1 cm and a height of 2-3 mm, were fabricated and reversibly bonded onto glass slides to form sample wells with a volume of about 250 ll. These miniaturized Petri dishes allowed cells to be grown and kept in a physiological buffer solution during the experiment. An ITO glass slide with a PDMS ring filled with cell culture medium is shown in Fig. 1B.

Cell culture
Adherent baby hamster kidney fibroblasts (BHK21; ECACC 85011433) were cultured in polystyrene T75-flasks containing 30 ml DHI-5 medium (see below) at 37°C and 5% carbon dioxide. To split the cells, the medium was removed, and the flask was washed with 10 ml of 37°C warm PBS w/o calcium and magnesium (Dulbecco's Phosphate Buffered Saline, D8537, Sigma, Switzerland). To detach the cells, 3 ml of trypsin-EDTA solution (0.05% Trypsin, 0.53 mM EDTA; 25300-054, Invitrogen, Switzerland) were added, and the cells were incubated at 37°C for 5 min. The detached cells were diluted with 7 ml of 37°C warm DHI-5 medium and homogenized using a pipette. 0.5 ml of the homogenized cell suspension and 30 ml of fresh media were returned to the flask for further cultivation. The rest of the cell suspension was used for experiments or disposed. A miniaturized Petri dish was filled with approximately 250 ll of medium, and 1-2 ll of cell suspension were added. The cells were incubated on the glass slide base of this dish for 1-2 days at 37°C and 5% CO 2 .

Cell lysis
Live BHK21 cells were lysed in situ in PBS, HEPES or ISB buffer. During the experiments, the conductive glass slide hosting the cell culture was electrically grounded, and the microcapillary was connected to a function generator. The miniaturized Petri dish, formed by a PDMS ring and the glass slide, was positioned on the OM stage. An individual cell was then selected and centered in the field of view with the help of the microscope. Next, the tip of the goldcoated microcapillary was immersed in the buffer solution, brought in close proximity to the conductive surface of the glass slide (distance %20 lm) using the stepper motor and aligned directly above the targeted cell by moving the microscope stage. A camera mounted on the microscope allowed for live-imaging of the individual cells and their surroundings during the following lysis procedure. After aligning the microcapillary tip above a targeted cell, a burst of five to ten DC square pulses with amplitudes of 6-10 V and a frequency of 10 kHz (pulse duration: 50 ls) was applied to the gold-coated microcapillary using the function generator. This generated a strong electric field (3-5 kV/cm) across the selected cell and resulted in cell lysis. Note, for the compensation of electrode aging, higher voltages were applied in rare cases (maximally 20 V). Right after the burst, the solenoid valve was opened toward the partially evacuated pressure reservoir (Fig. 1A) and remained open for a defined period of time, aspirating sub-microliter volumes (200-400 nl) of lysate and buffer into the microcapillary. The amount of sample aspirated was controlled by the pressure difference (À50 to À600 mbar) and the opening time of the solenoid valve (50-400 ms). To transfer the sample, an EM grid was clamped by tweezers and centered under the microcapillary. The tip of the microcapillary was approached close to the surface of the grid by the stepper motor. The aspirated sample was then dispensed directly onto the carbon film of the grid by applying a positive pressure to the microcapillary.
Single-cell lysis and aspiration experiments were also performed with fluorescently stained HEK293 cells (see Supplementary material and Supplementary movie 1 and 2).

Electron microscopy
Loaded sample grids were incubated for 60 s-20 min in a humidity chamber to prevent desiccation. Grids with adsorption times of longer than 3 min were washed on four drops of ddH 2 O and blotted once at the end and air-dried. All samples were negatively stained with two 5 ll drops of 2% uranyl acetate (UA) or 2% ammonium molybdate (AM) and imaged in a Phillips CM10 electron microscope operated at 80 kV. The images were recorded on a 2 k Â 2 k CCD camera (Olympus SIS, Münster, Germany).

Enzyme activity assay
Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (8.5 mg/ml, A2554, Sigma, Switzerland) was diluted to a concentration of 4.25 lg/ml in PBS. 3 ll of the sample was placed onto an ITO coated glass slide, and the microcapillary electrode was placed in the drop, about 20 lm above the glass slide. Different voltages (0 V, 4-18 V in 2 V steps and 22 V), each with a burst count of 5 and a single pulse length of 50 ls were applied. 2 ll were removed from the treated sample droplet, diluted to 8.5 ng/ ml in PBS, and the enzymatic activity of HRP was analyzed by enhanced chemiluminescence (ECL) on a dot plot (Amersham ECL Prime Western Blotting Detection Reagent; RPN2232, GE Healthcare, UK).

Results
The single-cell lysis setup (Fig. 1) allows for live-cell imaging, lysis of single adherent eukaryotic cells, rapid aspiration of the lysate, and transfer of the sample onto EM grids. This is demonstrated using adherent BHK21 cells.
The adherent cells were individually lysed in situ in buffer solution, while the process was monitored by means of optical micros-copy. To achieve lysis, a gold-coated microcapillary tip was positioned in close proximity to and directly above the selected BHK21 cell growing on the ITO coated glass slide inside of a miniaturized Petri dish (Fig. 1B). The distance between the capillary tip and the conductive surface of the glass slide was approximately 20 lm; there was no direct cell contact. As documented by light microscopy, the electric field, generated across the cell by a burst of five to ten 50 ls DC square pulses with amplitudes of 6-10 V applied to the microcapillary electrode, was sufficient to reproducibly and efficiently lyse the BHK21 cells in PBS or HEPES buffer ( Fig. 2A-C). Electrical pulses alone were sufficient to achieve lysis as indicated by a necrotic morphology of the targeted cells, nevertheless, cells were not completely disrupted, and cell fragments stayed in place if not aspirated ( Fig. 2A and Supp. Fig. 2A). Moreover, aspiration alone was not sufficient to lyse, disrupt or detach cells.
As demonstrated, only the targeted cell is lysed and neighboring cells remain unaffected, due to the small electrode dimensions and when conductive buffers are used ( Fig. 2A-C and Supp. Fig. 2 A-D). This findings are supported by finite-element analysis (FEA) results, which show that a high electric field is only generated in very close proximity to the cell, between the microcapillary tip and the ITO support (Fig. 3A). Further, adaptation of the electrical parameters to obtain a higher peak-voltage and longer pulse duration allowed the cells to be lysed in low-conductivity, isotonic sucrose buffers, but in this case neighboring cells were also affected (Supp. Fig. 2 E and F).
FEA was also used to estimate the maximal temperature increase by Joule heating due to five 50 ls DC square pulses with amplitudes of 10 V. This estimation predicts a maximal local temperature of 34°C for a time span of less than 1 ms (Fig. 3 B and Supp. Fig. 3 and 4). The average cell temperature stays below 30°C throughout the overall time span used for simulation. To experimentally study the effects of the short temperature increase, the enzymatic activity of horseradish peroxidase (HRP) after applying electrical pulses was quantified. HRP is known to be irreversibly impaired by exposition to increased temperatures ( (Chattopadhyay and Mazumdar, 2000) and Supp. Fig. 5). Fig. 3C shows the normalized activity of HRP after exposing 3 ll droplets to electrical pulses of different voltages. The effect was measured by quantification of the chemoluminescent reaction catalyzed by HRP. Our results show, that the activity is not significantly decreased below 10 V, but effects are clearly seen around 14 V and higher. Similar experiments performed with synthetic filamentous actin did not reveal signs of protein aggregation or structural damage of the filaments below 10 V (Supp. Fig. 6). Immediately after the applied burst, lysate and surrounding buffer were aspirated into the microcapillary to prevent diffusion of the cellular content.
Subsequent to cell lysis and aspiration, the sub-microliter volumes (200-400 nl) were loaded onto EM grids, negatively stained and inspected by TEM.
TEM images of buffer solution, aspirated from the miniaturized Petri dish in the proximity of a cell after the washing steps, but before a cell lysis event, showed a background of predominantly small particles as expected (Fig. 2D); these are most likely residues from the growth medium that were not removed by the buffer washes (see Section 2). TEM images of the sample aspirated from the immediate proximity of an individual adherent BHK cell directly after lysis, reveal cellular content (Fig. 2E-G,  Fig. 4). Membrane patches, filaments and other structures with distinctive shapes can be recognized and correlated to the specific cell using the light microscopy information. Further examples of prominent structures observed in the lysate of single cells are shown in Fig. 4. Although the control experiments documented the presence of some (predominantly small) particles in the bare buffer solution (Fig. 2D), distinctive structures similar to those found in the lysate (Fig. 2E-G and Fig. 4) were not detected.

Discussion
We present a setup for the controlled lysis of single, adherent eukaryotic cells and a subsequent visual inspection of cellular components by negative-stain TEM. The developed instrument enables (i) live-cell monitoring before and during cell lysis by optical microscopy, (ii) precise selection of individual cells, (iii) fast lysis (<500 ms) without prior disturbance of the cell and (iv) immediate aspiration of the cellular components into a microcapillary for further treatment.
The ITO coating on the glass slide that forms the base of these dishes, features the required electrical and optical properties and serves as ground electrode. In contrast to gold, ITO is suitable for fluorescence microscopy, allows live-cell imaging, and can be combined with various functionalization options to support cell growth if needed (Luo et al., 2008;Shah et al., 2009). The ability to carry out live-cell imaging prior to cell lysis, not only allows studying biological processes, but also enables lysis to be initiated at a defined time-point by means of an optical feedback; for example, a target cell in a specific state can be identified and selected by fluorescence signals of genetically labeled proteins.
Microfluidic chips with fixed electrodes often suffer from bubble formation due to electrolysis. In the flexible-electrode setup described here, sample aspiration and downstream processing were not affected by electrolysis, and there were no signs of bubbles inside the capillary. Furthermore, microfluidic chips with embedded electrodes depend on cells being in suspension and are not well suited to study adherent eukaryotic cells, which must be detached before lysis.
Biological processes involving proteins elapse within time frames of milliseconds to days (Souchelnytskyi, 2005). This makes the total lysis time an important parameter for cellular dynamics studies. As the method presented here combines fast electrical lysis (61 ms) with rapid sample loading (6400 ms), the described technique offers a temporal resolution that is suitable to study dynamic processes in protein networks of single cells.
The complete detachment and disruption of the cells, observed after sample aspiration ( Fig. 2B and C and Supp. Fig. 2B-F), was most likely due to the additional shear forces occurring during the aspiration process. The combination of electrical pulses and mechanical forces, allows applying relatively mild conditions for electrical lysis (low pulse amplitudes and short pulses) in comparison to those reported previously (Han et al., 2003;Nashimoto et al., 2007).
The preservation of the native protein structure and supramolecular assemblies is crucial for the subsequent visual analysis in EM. FEA predicts elevated temperatures on time scales 61 ms ( Fig. 3B and supp. Fig. 4) and indicates that thermal protein denaturation is unlikely to occur. All our experimental data corroborate these findings: Quantitative assays using HRP did not show a significant degradation of enzyme activity at the conditions used to lyse individual cells (Fig. 3C). Furthermore, experiments with synthetic actin filaments only showed structural effects at voltages exceeding the ones used for lysis (Supp. Fig. 6).
Negative-stain TEM revealed a variety of cellular components, such as membrane patches, filaments and other particles (Figs. 2 and 4), and indicates the structural preservation of the observed cellular components. No signs of protein aggregation were observed (compare to supp. Fig. 6 D). Due to the unique and distinct shape of several particles, assumptions about their identity can be made directly from the raw images, e.g., for the vault organelles (Kedersha et al., 1990). Future template matching algorithms must include scoring algorithms for quantitative analysis (Beck et al., 2009(Beck et al., , 2011Best et al., 2007) or complementary, labeling procedures (Giss et al., manuscript in preparation) can be employed. Although the aspects of protein structure and protein complex preservation have to be further consolidated and the recovery of sample constituents has to be improved in the future, the TEM images clearly demonstrate the potential of this method to prepare the lysate of a single cell for a visual analysis in negative-stain TEM. Furthermore, single cell lysis of adherent eukaryotic cells is also of great interest for other analysis methods, such as mass spectrometry (Aebersold and Mann, 2003) or reverse-phase protein arrays (RPPA) (Dernick et al., 2011). The application of our lysis method for the detection of actin by RPPA is shown in Supplementary  Fig. 7.
The ''lyse and spread'' approach for visual analysis of eukaryotic cells presented here is envisaged to complement classical methods, such as cryo-ET or mass spectroscopy. In comparison to cryo-ET of whole vitrified cells or cellular sections, the here presented approach does not provide information about the 3D arrangement of the proteome. In addition, the excellent preservation capabilities of vitrification have to be replaced by mild and physiological conditions during sample preparation and eventually supported by crosslinking procedures. On the other hand, the ''lyse and spread'' method, focusing particularly on the cytosolic fraction, has several advantages: (i) Adherent eukaryotic cells can be studied; (ii) the proteins can be prepared by negative staining to obtain a higher SNR; and (iii) the physical segmentation renders the cell components directly accessible for separation and labeling experiments (Giss et al., in preparation). In contrast to mass spectroscopy, mass  (Kedersha et al., 1990). (B) Helical structures that have the shape and dimensions of actin filaments (Aebi et al., 1986). (C) Examples of other less abundant structures observed in the lysate of individual cells. These resemble (top) the CplXAP complex (Ortega et al., 2004) and (bottom) nucleoprotein filaments (Sehorn et al., 2004). (D) More detailed view of the frequently observed ring-like structures shown in F. These have variable dimensions and resemble top views of Hsp60 (De Carlo et al., 2008) and the 20s proteasome (Cascio et al., 2002); the rectangular structure in the top image resembles a side view of one of the latter complexes. determination by STEM is less accurate and the visual analysis by TEM is less high-throughput amenable. However, the single molecule detection limit of the EM allows low-abundance proteins and protein complexes to be studied. Moreover, EM provides additional structural information and facilitates the study of large protein assemblies.
The presented single cell lysis method is only a first step toward the global approach described above as ''lyse and spread visual proteomics''. Currently, a limiting step is the low transfer efficiency of the cell lysate onto the EM grid, involving several blotting and washing steps. Therefore, the combination with methods exhibiting higher transfer efficiency will be beneficial in order to obtain a more complete access to the proteome. For example, the use of a microfluidic sample-conditioning device, combined with lossless micropatterning of the sample onto an EM grid, could be a suitable solution as shown by Kemmerling et al. (2012).

Conclusion and outlook
The system presented here allows single-cell lysis to be performed in less than a millisecond and to aspirate the cellular components into a microcapillary within a few hundred milliseconds for further preparation and subsequent analysis. The combination of this setup with an optical system facilitates live imaging and the evaluation of lysis and sample loading. The system is compatible with standard adherent-cell culturing methods and tools, and can potentially also be applied to biological tissues. Moreover, the system can be easily adapted to different sample carriers, which facilitates the use with additional or alternative analysis techniques such as reverse-phase protein arrays (Supp. Fig. 7).
In combination with a lossless sample deposition onto the EM grids and optional procedures for crosslinking, protein separation and labeling this method for single cell lysis offers the potential for targeted and quantitative visual proteomics studies on eukaryotic single cells.