s2fieldsofstudy: Biology Psychology

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Differential Stress-Induced Neuronal Activation Patterns in Mouse Lines Selectively Bred for High, Normal or Low Anxiety

There is evidence for a disturbed perception and processing of emotional information in pathological anxiety. Using a rat model of trait anxiety generated by selective breeding, we previously revealed differences in challenge-induced neuronal activation in fear/anxiety-related brain areas between high (HAB) and low (LAB) anxiety rats. To confirm whether findings generalize to other species, we used the corresponding HAB/LAB mouse model and investigated c-Fos responses to elevated open arm exposure. Moreover, for the first time we included normal anxiety mice (NAB) for comparison. The results confirm that HAB mice show hyperanxious behavior compared to their LAB counterparts, with NAB mice displaying an intermediate anxiety phenotype. Open arm challenge revealed altered c-Fos response in prefrontal-cortical, limbic and hypothalamic areas in HAB mice as compared to LAB mice, and this was similar to the differences observed previously in the HAB/LAB rat lines. In mice, however, additional differential c-Fos response was observed in subregions of the amygdala, hypothalamus, nucleus accumbens, midbrain and pons. Most of these differences were also seen between HAB and NAB mice, indicating that it is predominately the HAB line showing altered neuronal processing. Hypothalamic hypoactivation detected in LAB versus NAB mice may be associated with their low-anxiety/high-novelty-seeking phenotype. The detection of similarly disturbed activation patterns in a key set of anxiety-related brain areas in two independent models reflecting psychopathological states of trait anxiety confirms the notion that the altered brain activation in HAB animals is indeed characteristic of enhanced (pathological) anxiety, providing information for potential targets of therapeutic intervention.

Introduction
Pathological fear and anxiety and its physiological expression can be conceptualized as representing a continuum, ranging from persistent anxiety not attributable to specific factors to exaggerated responses to a perceived threat or a bias towards interpreting ambiguous situations as threatening [1]. Dysfunctional excitability in neurons of the anxiety/fear circuitry is speculated to be a common abnormality in anxiety disorders [1]. While it is well established that certain brain areas are involved in the perception and processing of acute fear (for review, see [2][3][4]), much less is known about the regional nature of changes in proposed anxiety circuitries (for review, see [5]) predisposing individuals to be hyperanxious. One approach toward obtaining a better understanding of these mechanisms is to use psychogenetically selected rodent lines, developed from a common foundation population that exhibit consistent and robust differences in the selection criterion. Using this strategy, various rat lines have been generated that differ in certain aspects of emotionality including anxiety, such as the Maudsley Reactive and Nonreactive strain, Roman high and low avoidance rat lines, Tsukuba strains, and high/low anxiety-related behavior (HAB/LAB) rat lines (for review, see [6][7][8][9]). To our knowledge, corresponding mouse models selectively bred for extremes in anxiety are not available, with one exception (see below).
In HAB/LAB rats, systematic immediate early gene expression studies succeeded in mapping differences in neuronal activity patterns underlying behavioral responses to a variety of aversive situations [10][11][12][13][14]. So far, no such information is available in mice. Recently, HAB and LAB mouse lines have been established by selective and bidirectional breeding for high (HAB) and low (LAB) anxiety-related behavior measured on the elevated plus-maze (EPM) [15,16]. Compared to LAB mice, HAB mice were more anxious and showed increased risk assessment behavior in a number of tests, including the EPM test, open-arm exposure test, light/dark avoidance test and ultrasound vocalization test. Moreover, unselected CD1 ''normal'' anxiety-related behavior (NAB; for definition, see Material and Methods) mice, as well as HAB/LAB F1 intercrosses, displayed intermediate behavioral scores in most of the tasks performed [16].
In the present study, we aimed for the first time to investigate in mice whether genetically determined differences in anxiety-related behavior as well as risk assessment would be reflected by differential stress-induced c-Fos expression as a reliable marker of neuronal activation in key brain areas of anxiety circuitries previously described in rats (for review, see [14]). Placement of HAB, NAB and LAB mice on an open arm (OA) of the EPM was chosen as a mild anxiety-based stressor. The investigation of c-Fos in NAB mice provides an additional advantage, as the intermediate phenotype of these animals may act as a reference of changes in brain activity, suitable to determine whether HAB or LAB mice reveal neuronal alterations. By confirming similarly affected neuronal populations in an additional model of a different species, the general significance of the previous findings would be strengthened, suggesting the difference in trait anxiety presumably being the cause. At the same time, this cross-species replication would provide further information as to possible effects of genetic drift, giving rise to genetic differences that are unrelated to the selected phenotype. Differences in c-Fos activation, in other words, could reflect both the selection pressure and drift-related phenomena.

Animals
Ethics statement. The study described here was designed to minimize animal suffering and number of animals used, and was approved by the local Ethical Committees on Animal Care and Use.
All animals tested were bred in the animal facilities of the Max Plank Institute of Psychiatry in Munich (Germany) as described previously [16]. Briefly, .250 animals from .25 litters of outbred Swiss CD1 mice purchased from Charles River were used as a starting point for selective and bidirectional breeding for anxietyrelated behavior on the EPM at the age of seven weeks, with at least six families routinely maintained within each selected line. Males and females that spent either the least or most time on the open arms of the EPM were mated to establish the HAB and LAB mouse lines. This intra-line approach was chosen to make sure that the HAB/LAB lines show a maximum divergence in the selected trait, while maintaining a high degree of similarity in nonselected traits. All experiments in the present study were carried out on inbred adult male HAB (n = 14), LAB (n = 13) and NAB (n = 13) mice (22-25g body weight; 13-14 weeks of age). The animals were routinely tested at an age of 7 weeks in Munich with HAB and LAB mice spending less than 10% and more than 50% of their time, respectively, on the open arms of the EPM. This was also the selection criterion. NAB mice are bred for ''normal'' (i.e. intermediate) anxiety-related behavior. They were selected from a group of CD1 mice maintained in the laboratory while HAB and LAB lines were being selected. As .80% of CD1 mice spent between 25% and 35% of their time on the open arms of the EPM, this range was chosen for the selection of NAB mice without any overlapping either with HAB or with LAB animals. While CD1 mice in the parental generation were used as NAB controls by Krömer et al. [16] and Kessler et al. [15], we then decided to start inbreeding them in parallel with HAB and LAB mice to further reduce variables that are unrelated to anxiety, such as slight differences in body weight between outbred vs. inbred animals. In the present study, HAB and LAB mice of generations 18-22 and NAB mice of generations 1-3 were used. Importantly, in a wide variety of tests and parameters, the intermediate scores of (bred) NAB, (purchased) CD1, and HAB/LAB F1 controls were found to be similar if not identical ( [16]; unpublished results). We generated six independent families within the HAB, LAB and NAB lines using a within-family selection design.
In Innsbruck, HAB, NAB and LAB animals were housed under standard laboratory conditions (12:12 h light/dark cycle with lights on at 7:00; 21uC; 50% humidity; pelleted food; and water ad libitum) for 6 weeks in groups of 3-4 litter mates per cage. At least 24 h before the experiment, animals were taken in their home cages to the experimental rooms for habituation. The behavioral test was carried out during the light phase of the cycle (between 8:30 and 12:30 a.m.).

OA exposure
Mice (HAB: n = 9, LAB: n = 8, NAB: n = 8) were placed in the middle of the OA (5065 cm) of an EPM (facing the proximal compartment). The maze was elevated 73 cm above the floor and illuminated by a light intensity of 100 lux. Access to the neutral zone and the closed arms of the maze was prevented by a bar that made it impossible for the mouse to leave the OA. The arm was thoroughly cleaned with water before the introduction of each mouse and divided into a distal, a middle and a proximal zone. The behavior of the mice during the 5-min testing period was analyzed by an automatic videotracking system (Videomot 2.0, TSE, Bad Homburg, Germany). In addition, the test session was videotaped for later analysis of the head-dip behavior by an experienced observer using the Eventlog 1.0 (EMCO Software). The behavioral parameters scored included the number of entries into the distal zone of the OA, the time spent in the distal zone of the OA, total distance traveled, number of head dips below the surface of the OA, the time spent head dipping and the latency until the first head dip. Immediately after behavioral testing, animals were returned to their home cages. Animals assigned to the control (basal) group (n = 5 for all three lines) were not exposed to the OA and were taken directly from their home cages for further analysis.

c-Fos immunohistochemistry
The maximum level of c-Fos protein can be detected between 1 and 3 h following an acute challenge, then it gradually disappears from the cell nucleus [17][18][19]. Therefore, 2 h after the onset of the OA exposure test, animals were deeply anesthetized with an overdose of sodium pentobarbital (200 mg/kg) and transcardially perfused with 100 ml of 0.9% saline followed by 100 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffered solution (PBS, pH 7.4). Mice not exposed to the test paradigm were treated identically immediately after removal from their cages in the experimental room. Brains were then removed and postfixed at 4uC overnight in 4% paraformaldehyde in PBS. Coronal sections (100 mm) were cut with a Vibratome (Ted-Pella, Inc., Redding, CA, USA) and collected in Immunobuffer. C-Fos immunoreactivity was performed in three runs for i) the rostral (from Bregma +1.94mm to +0.14mm), ii) the middle (from Bregma 20.82mm to 21.46mm) and iii) the caudal part (from Bregma 23.88mm to 25.40mm) of the mouse brain. Within a given run, sections of all groups were processed simultaneously in order to avoid batch effects. The sections were processed as described previously [20]. Briefly, sections were incubated for 72 h in a polyclonal rabbit anti-c-Fos primary antibody (sc-52, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted (1:20000) in immunobuffer (pH 7.4). The sections were then rinsed and placed in a biotinylated goat anti-rabbit secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA) for 24 h. An avidin-biotinhorseradish peroxidase procedure (Vectastatin ABC Kit, Vector laboratories, Burlingame, USA) with 3,39-diaminobenzidine (DAB, Sigma, Germany) as chromogen was used to visualize c-Fos positive cells. The incubation time with the DAB-solution was 10 min for all sections. The chromogen reaction was initiated by the addition of the H 2 O 2 solution (0.004%) and terminated after 7 min (colour change to brown) by adding Tris buffer (50mM). The staining procedure generally yielded low background staining and differential staining intensities of c-Fos positive cells. A cell was considered as c-Fos-labeled (c-Fos positive), if the brown-black DAB-stained nucleus was unambiguously darker than background staining, which included all cells from low to high intensities of staining. The lighting of the microscope was optimized for the best visibility of c-Fos labeled cells and kept constant for all sections. The c-Fos quantification was performed at different levels of the brain in 59 different structures, which (amongst others) are known to show stress-induced increase in c-Fos expression [13,21,22] . Many of these regions have been implicated in the anxiety circuitry (for review, see [5,23,24]). The anatomical localization of c-Fos-positive cells was aided by use of adjacent Nissl stained sections and the illustrations in a stereotaxic atlas [25]. The anterior-posterior levels of sections included for detailed analysis and associated structures are shown in Figure 1. The number of c-Fos-positive cells was quantified bilaterally in a tissue area of 100 mm6100 mm. This was performed for the whole experiment by one and the same well-experienced observer, who was blind to the experimental groups. Generally, cell counting was performed maximally for 3 h per day in the time period from 9 to 12 a.m.

Statistical Analysis
Statistical analysis using the Kolmogorov-Smirnov test and Shapiro-Wilk test (Software: Statistica 7.1, Statsoft Inc.,USA) revealed parametric (normal) distribution for the behavioral data and the c-Fos data for most brain regions. Therefore, overall statistical analysis of behavioral data was performed using the 1way ANOVA followed by Fischer LSD post hoc analysis. The number of c-Fos positive cells was analysed by using the 2-way ANOVA followed by (if there was a significant line x stress interaction in ANOVA) Fischer LSD post hoc analysis to detect statistically significant differences between the groups. Correlations between the parameter distal arm entries and the number of c-Fos cells were performed using the Spearman's rank correlation coefficient test. The level of significance was set at P,0.05. All values were expressed as mean6SEM.

OA behavior
ANOVA analysis revealed significant differences between the three mouse lines for the parameters ''distal time'' (F(2,24) = 5.759, P = 0.010), ''distal entries'' (F(2,24) = 13.880, P,0.001), ''distance traveled'' (F(2,24) = 48.895, P,0.001), ''time spent head dipping'' (F(2,24) = 14.810, P,0.001), ''number of head dips'' (F(2,24) = 28.715, P,0.001) and ''latency until first head dip'' (F(2,24) = 6.413, P = 0.006). During OA exposure, the Fischer exact revealed that the time spent in the distal zone and the number of entries into the distal zone of the OA were significantly lower in HAB than in LAB mice (see Figure 2). NAB animals displayed intermediate anxiety-related behavior, although the difference for the parameter ''time spent head dipping'' failed to reach statistical significance from HAB and LAB lines ( Figure 2a). Risk assessment behavior as indicated by head dips below the surface of the OA [26] was higher in LAB and NAB mice compared with their HAB counterparts. In HAB animals the number of head dips and the duration of head dipping were significantly lower, while the latency until the first head dip was significantly higher as compared with NAB and LAB mice. Head-dip behavior did not differ between NAB and LAB mice ( Figure 2b).

Line Differences in OA-induced c-Fos expression
An overview of the 59 brain areas in which c-Fos expression was quantified is given in Figure 1. Mean numbers6SEM of cells expressing c-Fos in these brain regions are shown in Table 1.
Basal c-Fos expression. In mice of the basal groups which were not exposed to the OA, the number of cells expressing c-Fos was low in most areas examined. Moderate numbers of c-Fospositive cells were, however, detected in some cortical, thalamic, and hypothalamic areas. No differences in basal c-Fos expression were observed among HAB, NAB and LAB mice (Table 1).
c-Fos expression after OA exposure. OA exposure induced c-Fos expression in a variety of brain areas, with moderate to pronounced increases in areas involved in stress responses (see Table 1, 2-way ANOVA analysis for the factor stress), including different cortical areas, limbic areas such as subregions of the amygdala, the bed nucleus of the stria terminalis, areas of the hippocampal formation, the lateral septum, the nucleus accumbens, as well as the thalamic and hypothalamic nuclei, parts of the periaqueductal gray (PAG) and diverse brainstem nuclei. Only in a few areas did OA stress fail to induce a significant increase in c-Fos expression, including in the medial orbital and granular insular cortices, the medial and lateral globus pallidus, the caudate putamen, the anterodorsal thalamic nucleus, the central nucleus of the amygdala and the medial parabrachial nucleus.
2-way ANOVA analysis revealed a significant line x stress interaction for the number of c-Fos positive cells in 18 out of 59 brain areas investigated, including the cingulate cortex, the nucleus accumbens (core, shell), the lateral septum (ventral, intermediate), the paraventricular hypothalamic nucleus (PVN), the medial preoptic area, the lateral hypothalamic area, the dorsomedial hypothalamic nucleus, the ventromedial hypothalamic nucleus, the anterior hypothalamic nucleus, the medial nucleus of the amygdala, the lateral nucleus of the amygdala, the anterior cortical amygdala, the dentate gyrus, the caudal periaqueductal gray (ventrolateral, dorsolateral) and the locus coeruleus (LC). For details of statistics (F and P values) see Table 1.
In all 18 brain areas, post hoc analysis revealed a differential OAinduced c-Fos response between HAB and LAB mice. The c-Fos response to OA exposure was increased in HAB as compared with LAB mice, in 16 areas, including the shell and the core region of the nucleus accumbens, the ventral and intermediate part of the lateral septum, the PVN, the lateral and anterior hypothalamic area, the dorsomedial and ventromedial hypothalamic nucleus, the medial preoptic area, the medial, lateral and anterior cortical amygdala, the caudal PAG (ventrolateral, dorsolateral) and the LC (Figure 3, Figure 4, Figure 5). Conversely, a lower number of c-Fos positive cells in HAB than in LAB animals was found in 2 areas, the cingulate cortex and the dentate gyrus of the hippocampus (Figure 4, Figure 5).
In 15 of these 18 areas a similar difference in the neuronal activation pattern was seen, when NAB were compared with HAB mice. The exception was the PVN, the anterior hypothalamic area and the caudal periaqueductal gray (ventrolateral), where the statistically significant difference in the c-Fos response was lost when HAB were compared with NAB animals. Thus, the neuronal activation response of NAB and LAB mice was very similar in most of these areas, but LAB mice displayed a lower c-Fos response compared with NABs in the PVN, the anterior hypothalamic area and the medial preoptic area (Figure 4). In all other brain areas, OA-induced c-Fos expression in NAB mice did not differ from HAB or LAB mice. Since we used a number of comparisons (59 for each brain area), it should be noted that there is the theoretical possibility of false positive results, although the probability of this is very low.
Correlation analysis between distal arm entries and c-Fos expression. Correlation analysis between OA distal arm entries and c-Fos expression after OA exposure was performed for the 18 brain areas where HAB and LAB mice showed significant differences in c-Fos response (see above). The Spearman test revealed significant negative correlation for the PVN, the lateral hypothalamic area, the dorsomedial hypothalamic nucleus, the  Franklin and Paxinos (1997). Squares indicate the placement of grids for counting of c-Fos positive cells. Asterisks indicate the regions in which HAB mice showed changes in OA-induced c-Fos expression as compared to LABs. AcB, nucleus (n.) accumbens; AcBc, n. accumbens core; AcBsh, n. accumbens shell; ACo, anterior cortical n. of the amygdala; AD, anterodorsal thalamic n.; AH, anterior hypothalamic area; Arc, arcuate hypothalamic nucleus; BlA, basolateral n. of the amygdala; BNST, bed n. of the stria terminalis; CA1, CA1 field of the hippocampus; CA3, CA3 field of the hippocampus; CeA, central n. of the amygdala; Cg 1, cingulate ctx (area1); Cg 2, cingulate ctx (area2); Cl, Claustrum; CPu, caudate putamen; cPAGdl, caudal dorsolateral periaqueductal gray; cPAGdm, caudal dorsomedial periaqueductal gray; cPAGl, caudal lateral periaqueductal gray; cPAGvl, caudal ventrolateral ventromedial hypothalamic nucleus, the anterior hypothalamic area, the medial preoptic area, the lateral septum (ventral), the medial nucleus of the amygdala, the lateral nucleus of the amygdala and the LC, indicating that higher anxiety behavior (fewer distal entries) is correlated with enhanced c-Fos response in these areas. Along these lines, a positive correlation was found for the dentate gyrus. None of the other brain regions correlated significantly with the distal arm entries during OA exposure. Details of the correlation analysis (including R and P values) are given in Table 2.

Discussion
This study demonstrates that HAB mice display clear signs of a hyperanxious phenotype in the OA exposure test as compared to LAB mice, while NAB animals show intermediate anxiety-related behavior. It furthermore confirms in large part the OA-induced brain activity pattern previously found in the corresponding HAB/ LAB rat model (see [13]). The striking new finding of the present HAB/LAB mouse study is that this mild anxiogenic stimulus invoked differences in neuronal activation patterns in additional brain areas, including subregions of the amygdala, the hypothalamus, the nucleus accumbens, the midbrain and the pons, and that the OA-induced neuronal activation profile in NAB mice resembled in large part that of LAB mice. Therefore it is suggested that it is predominantly the HAB line that shows altered processing of mild anxiety-provoking stimuli, thus substantiating the search for correlates of anxiety-related phenomena particularly in this line [7].

Line differences in OA behavior
The behavioral results of the present study confirm previous observations demonstrating that HAB mice are more anxious [16]  MPA, medial preoptic area; MPO, medial preoptic n.; MPB, medial parabrachial n.; PE, periventricular n; Pir, piriform ctx; PLCo, posterolateral cortical n. of the amygdala; PrL, prelimbic ctx; PV, paraventricular thalamic n.; PVA, paraventricular thalamic n. (anterior); PVN, paraventricular hypothalamic n.; rPAGdl, rostral dorsolateral periaqueductal gray; rPAGdm, rostral dorsomedial periaqueductal gray; rPAGl, rostral lateral periaqueductal gray; RSA, retrosplenial agranular ctx; RSG, retrosplenial granular ctx; S1J, primary somatosensory cortex, jaw region; VMH, ventromedial hypothalamic n. doi:10.1371/journal.pone.0005346.g001 Interestingly, in the present study we noted reduced ''total distance traveled'' of HAB animals compared with both LAB and NAB mice. While the distance traveled, which is rather low due to the small size of the OA, may be considered to reflect locomotor activity, decreased activity is also considered to be a form of anxiety in which a ''behavioral inhibition system'' is activated [3]. Therefore, immobility (behavioral inhibition) per se is proposed to be an indirect sign of intense anxiety/fear states in rodents. This is also reflected by the fact that HAB animals [16] and rodents in general showing high levels of anxiety-and depression-like behavior display reduced general locomotor activity [28][29][30][31] and vice versa [32,33]. Moreover, while traveling similar distances, NAB mice also clearly differed from LAB mice in anxiety-related parameters, further supporting the critical role of anxiety (rather than locomotor effects) in the behavioral divergence among HAB, NAB and LAB animals as observed in the OA exposure test.

Differential c-Fos expression in response to OA exposure in HAB and LAB mice
The development of anxiety-related symptoms is closely related to stress coping [14,[34][35][36][37][38]. Stress-induced neuronal activation is thought to delineate neuronal stress circuitries in rats and mice [22,[39][40][41][42][43]. Exposure to the OA of an EPM is considered as a mild stressor inducing higher fear/anxiety than exposure to the closed arm [44][45][46]. Indeed, the present study revealed that mice stressed by OA exposure, compared to non-stressed (basal) mice, showed enhanced c-Fos expression in widespread brain regions related to fear/anxiety.
While the OA-induced c-Fos response in HAB compared to LAB rats was increased in 5 out of 8 differentially modulated brain areas (the lateral septum [ventral part], the PVN [parvocellular part], the medial preoptic area, the anterior hypothalamic area and the lateral hypothalamic area), attenuated c-Fos response was found in the cingulate cortex as well as in the dentate gyrus and the CA3 region of the hippocampus [13]. Remarkably, in the present study we were able to confirm these findings in the corresponding mouse model. In all but one of these regions, HAB and LAB mice displayed the same differences in neuronal activation after exposure to the OA. Only in the CA3 region of   the hippocampus did the mouse lines display no differences. As extensively discussed by Salome et al. [13], disturbances in these brain areas and associated systems may contribute to behavioral and neuroendocrine responses typical of high trait anxiety. The present study in mice, however, identified a number of additional brain areas showing increased c-Fos response in HAB versus LAB mice not seen in the rat experiment. These included various limbic areas (subregions of the amygdala, the dorsomedial and ventromedial hypothalamic nucleus, the nucleus accumbens [core and shell], the lateral septum) as well as PAG subregions and the LC. Supporting the relationship between anxiety-like behavior and c-Fos response in particular brain areas, we were able to demonstrate a significant correlation between distal arm entries and c-Fos response in specific amygdaloid and hypothalamic regions, in the lateral septum ventral, in the LC, and in the dentate gyrus. In the rat model, line differences in c-Fos response in these more widespread regions were observed only when stronger challenges were used, such as social defeat, airjet, FG-7142 injection or forced swimming (for review, see [14]). Hence, the HAB/LAB mouse model may be particularly sensitive in reflecting phenotype-specific neuronal activation. However, another explanation could be that the additional differences not common to the two species are not related to differences in trait anxiety. Future studies using anxiolytic drugs and additional challenges not related to the EPM may help to clarify this issue. While the HAB/NAB/ LAB lines are of the same CD1 background, we cannot completely rule out the possibility of a different time course in c-Fos responses between the lines. This may represent a potential limitation of the present study. Nevertheless this seems unlikely given that all three lines are of the same CD1 background.
A central finding of the present study is the evidence of a hypersensitive amygdala and a hyposensitive prefrontal cortex (cingulate cortex) in HAB mice. These areas are homologous with important fear-/anxiety-related regions of the human brain [47][48][49], thus resembling the situation in stressor-exposed posttraumatic stress disorder patients with poor top-down control of the amygdala by structures such as the medial prefrontal cortex [50,51]. In the present study, OA exposure stress enhanced c-Fos expression in the cingulate cortex in all three lines, but the maximum level of activation was reduced in HAB mice compared with LAB and NAB mice. This effect was not very pronounced. However, since cingulate cortex hypoactivation was also found in HAB rats after exposure to various stressors (for review, see [14]), we believe that this response might be a general feature of HAB rats and HAB mice mediating high anxiety levels during stress exposure. Given that the amygdala is a central relay station transferring fear-/anxiety-related information to other brain areas (such as the PAG, the brain stem, and the hypothalamus) for the processing, expression and integration of anxiety-related emotions (e.g. [52][53][54]), it is likely that hyperactivation in the amygdala is involved in the mediation of the increased anxiety-related behavior of HAB mice. The association of fear-and anxietyrelated phenomena has recently been highlighted [55][56][57][58].
The PAG is well known to integrate limbic and emotional inputs with a repertoire of behavioral and autonomic responses [59][60][61], and the LC is also well established as being related to anxiety disorders [34,62]; in addition, the HPA axis and sympatoadrenal system [62][63][64] produce physiological and behavioral responses to stressful stimuli (for review, see [65]).
Thus, hyperactivation of these areas as observed in HAB mice may be a general feature of highly anxious rodents (see also [14]).

Activation patterns in NAB compared to HAB and LAB mice
To resemble human studies, in which differences in neuronal activity between anxiety patients and healthy subjects are assessed, it is of relevance to include an additional group with ''normal'' anxiety in animal studies. Thus, for the first time, we here aimed also to map immediate early gene expression in NAB mice and to compare the c-Fos response with that in HAB and LAB mice. In 15 out of the identified 18 key brain areas showing differences in the neuronal activation pattern between HAB and LAB animals, similar differences were seen between HAB and NAB mice. Hence, with a few exceptions (see below), the neuronal activation responses of NAB and LAB animals were largely similar, indicating that in particular the increased anxiety-related phenotype of HAB mice may be associated with altered neuronal processing within specific brain areas.
LAB compared to NAB mice showed differential OA-induced c-Fos responses in some hypothalamic sites, namely the PVN, the anterior hypothalamic area and the medial preoptic area. This activation pattern may be associated with the non-anxiety phenotype of LAB vs NAB mice, and/or with enhanced novelty seeking of these animals (see [66][67][68][69]). Since, however, not much information is available concerning brain areas critically involved in extreme nonanxiety, such a conclusion remains speculative at present.
In our intra-line approach, selection pressure was exerted on anxiety-related behavior only, while a high degree of similarity was maintained in non-selected traits. This, however, does not necessarily mean that any difference detected between HAB and LAB animals, including that in regional c-Fos expression, is causally related to anxiety or anxiety-linked phenomena. Confounds include, for example, random genetic drift across time, giving rise to genetic differences that are unrelated to the selected phenotype. While drift-related risk cannot be entirely avoided, we tried to reduce it by: (i) running independent families within HAB, NAB and LAB lines [16]; (ii) replicating key findings of c-Fos expression in both mice and rats, using similar inbreeding protocols (this study and [13]); and (iii) showing that, in a pharmacological validation approach, paroxetine treatment atten- Figure 5. Quantitative analysis of c-Fos immunoreactivity in HAB, NAB and LAB mice under basal conditions and after OA exposure. Depicted are those areas (septal, hippocampal, amygdalar and hind brain areas) for which the Fischer LSD post hoc test revealed statistically significant differences in OA-stress-induced c-Fos response in HAB, NAB and LAB mice. Each column indicates the mean6SEM number of c-Fos positive cells in a tissue area of 0.01mm 2 (total c-Fos expression was quantified in the dentate gyrus). Basal groups: n = 5, OA-exposure: HAB: n = 9, NAB: n = 8, LAB: n = 8; *p,0.05, **p,0.01 vs HAB OA-group; # p,0.05, ## p,0.01 vs corresponding basal group; + p,0.05, ++ p,0.01 vs LAB OA-group; doi:10.1371/journal.pone.0005346.g005 uated both c-Fos expression and depression-like behaviour [11], further confirming an association between neuronal excitability and the behavioral phenotype beyond genetic drift. Future efforts will focus on testing further how strongly c-Fos and anxiety-related behavior are functionally related, including short-term selection and F2 panel associations. Only convergent information from multiple approaches will give rise to a more objective assessment of drift-related compared to other risks.
In conclusion, the data presented here demonstrate that differential c-Fos responses to an unpleasant emotional challenge are found in specific limbic, cortical, hypothalamic and hindbrain areas of HAB vs LAB mice. Remarkably, the key areas differentially activated after OA exposure in the HAB vs. LAB rat lines [13] could be confirmed in this corresponding mouse model, supporting the notion that the altered brain activation pattern in HAB animals may be a generalized feature, being indeed characteristic of enhanced trait anxiety. Similar brain areas were found to display altered activation processing also in anxiety disorder patients (see references in [14]), underlining the translational value of the present findings. The c-Fos response pattern in NAB mice, which displayed intermediate behavioral scores, was similar to that of LAB mice, both showing clear-cut differences to that of HAB animals, suggesting that it is mainly the HAB mouse line which may show altered neuronal activation at least upon OA exposure. This activation pattern typical of the high anxiety-related phenotype, however, may either indicate exaggerated activation of pathways mediating anxiety or represent dysfunctional adaptive responses which normally serve to suppress anxiety.


extfieldsofstudy:
Materials Science
Medicine

text:
Important Factors for the Three-Dimensional Reconstruction of Neuronal Structures from Serial Ultrathin Sections

Quantitative analysis of anatomical synaptic connectivity in microcircuits depends upon accurate three-dimensional (3D) reconstructions of synaptic ultrastructure using electron microscopy of serial ultrathin sections. Here we address two pitfalls in current methodology that lead to inaccurate reconstructions and compromise conclusions drawn from the data. The first pitfall is inaccurate determination of ultrathin section thickness, which negatively affects the 3D shape of reconstructions and therefore impairs quantitative measurement of synaptic structures. Secondly, current methodology significantly underestimates the number of synaptic junctions, with only two-thirds or less of genuine synaptic contacts being identified in dendrites that radiate within the plane of section. Here we propose a new methodology utilizing precise optical measurements of section thickness and successive observations of synaptic elements across serial ultrathin sections that corrects for these limitations to allow accurate 3D reconstruction of synaptic ultrastructure. We use this methodology to reveal that parvalbumin-expressing cortical interneurons have a much higher synaptic density than previously shown. This result suggests that this technique will be useful for re-examining synaptic connectivity of other cell types.

INTRODUCTION
A deep understanding of cortical microcircuitry requires accurate quantitative measurements of morphological parameters. Such measurements are reliably made from three-dimensional (3D) reconstructions of tissue structure using electron-microscopic (EM) images of successive ultrathin sections (Fiala et al., 2002;Holtmaat et al., 2006;Karube et al., 2004;Kubota and Kawaguchi, 2000;Kubota et al., 2007;White et al., 1994). Because the generation of reconstructions is laborious and time-consuming, more effi cient methods have recently been introduced that automate the reconstruction process and facilitate structural measurements (Denk and Horstmann, 2004;Harris et al., 2006;Knott et al., 2008;Micheva and Smith, 2007). However, two pitfalls in current methodology hinder accurate reconstruction of tissue morphology. The fi rst is accurate determination of the thickness of ultrathin sections, any error in which will signifi cantly affect the shape of reconstructed structures and the quantitative measurements that result. The commonly used method to estimate section thickness is the minimal folds method (Small, 1968) which measures the width of ridges in the tissue formed when small folds of tissue adhere to themselves and protrude above the plane of the section. Section thickness is assumed to be one-half the width of the ridges. However, this method generates results that vary according to the condition of the resin, the type of tissue embedded, and the height of the fold. Therefore, the accuracy of these measurements is not always certain. Further uncertainty may result from distortion of ultra thin resin after folding. The second pitfall in serial reconstructions is accurate identifi cation of synaptic contacts. The classic characteristics the line of the rhinal fi ssure. Tissue sections were put in glass tubes containing 15% sucrose in 0.1 M PB for 1 h, and then in 25% sucrose and 10% glycerol in PB for 2 h, frozen in liquid nitrogen, and then thawed at room temperature. The sections were then incubated in 0.1 M PB containing 1% sodium borohydrate for 30 min and in 0.05 M Tris-buffered saline (TBS) containing 1% H 2 O 2 for 30 min before incubation with primary antiserum against parvalbumin developed in mouse (1:4000, P-3171, Sigma-Aldrich, Saint Louis, MO, USA) diluted in TBS containing 10% normal goat serum and 2% bovine serum albumin overnight at 4°C. The sections were then incubated in biotin-conjugated secondary antiserum (1:200, BA-2000, Vector Laboratories, Burlingame, CA, USA) followed by avidin-biotin-peroxidase complex (VECTASTAIN Elite ABC Kit PK-6100, Vector Laboratories), and staining with diaminobenzidine (DAB) with nickel (0.02% DAB, 0.3% nickel in 0.05 M Tris-HCl buffer) as the chromogen for the peroxidase reaction (0.01%). The stained sections were post-fi xed for 40 min in 1.5% potassium ferrocyanide and 1% osmium tetroxide, followed by 1 h in 1% osmium tetroxide alone and dehydrated in graded dilutions of ethanol with 1% uranyl acetate added at the 70% ethanol dehydration state. Sections were fl at-embedded on silicon-coated glass slides in Epon. Following re-embedding of samples of tissue, they were serially re-sectioned at thickness settings between 30 and 90 nm using an ultramicrotome (Reichert Ultracut S, Leica Microsystems, Wetzlar, Germany).

ESTIMATION OF SECTION THICKNESS
The 90 nm thick sections were mounted on a formvar-coated single-slot copper grid. The minimal folds were photographed at ×100,000 magnifi cation and the width of the folds were measured following calibration by a diffraction grating replica (# 607, Ted Pella Inc., Redding, CA, USA). Other 30, 70 and 90 nm thick sections were placed on a clean glass slide and the thickness estimated with a color 3D laser confocal microscope (VK-9750; Keyence, Osaka, Japan), which uses a laser (408 nm) to directly measure section thickness with an accuracy of 1 nm 1 .

RECONSTRUCTION OF RED BLOOD CELLS
Red blood cells often remain in blood vessels following perfusionfi xation. After light microscopic photography, the tissue containing red blood cells was re-sectioned at 90 nm perpendicularly to the original plane of section and cells were reconstructed from the serial ultrathin sections. The EM images of the red blood cells were captured using a CCD camera and three-dimensionally reconstructed using the 3D reconstruction software Visilog (Noesis, France).

POSTEMBEDDING GABA STAINING AND RECONSTRUCTION OF PARVALBUMIN-POSITIVE DENDRITES
Serial sections cut at a setting of 90 nm thickness of parvalbuminpositive tissue were mounted on formvar-coated grids. To discriminate between inhibitory and excitatory presynaptic terminals, we applied postembedding GABA immunohistochemistry to the serial ultrathin sections. The ultrathin sections were washed with TBS containing 0.1% triton-X (TX) and incubated with rabbit antiserum against GABA (1:5000; Sigma-Aldrich, A-2052) in TBS containing 0.1% TX overnight, then incubated with 15 nmcolloidal gold conjugated anti-rabbit IgG (1:100; BBInternational #GAR15, Cardiff, UK) overnight in TBS containing 0.1% TX, and stained with 1% aqueous uranyl acetate followed by lead citrate. Quantitatively synaptic boutons could be divided into two classes on the basis of gold particle densities (Figure 2A). Particle densities were greatly different in GABA-negative and GABA-positive terminals: 0.57 ± 1.66/µm 2 (n = 35) and 43.9 ± 17.4/µm 2 (n = 18), respectively. In serial ultrathin sections, given presynaptic GABAnegative or GABA-positive boutons always showed similar colloidal gold density in multiple sections. GABAergic terminals were defi ned as terminals with a gold-particle density above 26 particle/ µm 2 , while terminals with a gold particles lower than 10 particle/ µm 2 were assumed to be glutamatergic. The labeled dendrites and associated structures were photographed and digitized from EM negatives using a scanner (GT-9800F, Epson, Suwa, Japan). The structures were reconstructed using the 3D reconstruction software, Reconstruct 2 .

ELECTRON MICROSCOPIC TOMOGRAPHY
The synaptic structures on the parvalbumin-positive dendrite were analyzed by tomography. The EM images were taken at 10,000× by tilting the sections between −60° and +60° in 2-degree steps using Hitachi H 7650 electron microscope equipped with a 1 MB CCD camera (1024 × 1024 pixels). The synapse images were processed by the tomography software EMIP (Hitachi High-Technologies Corp., Tokyo, Japan). Z-slices (estimated thickness, 1.75 nm) were obtained from the 70 nm thick sections.

ESTIMATION OF THE THICKNESS OF ULTRA THIN SECTIONS
We fi rst estimated the thickness of the sections by the minimal folds method. The estimation of section thickness is half the width of minimal folds in the ultrathin section, which occur when a section adheres to itself in a low ridge. The minimum folding can be confi rmed by the existence of a black line at the center of the ridge ( Figure 1A). The estimated section thickness by the minimal folds method was 20.4 ± 1.0 nm (n = 6) for the section cut at 30 nm set by the microtome, 29.9 ± 1.2 nm (n = 5) at 40 nm, 26.9 ± 2.5 nm (n = 6) at 50 nm, 42.4 ± 1.7 nm (n = 3) at 60 nm, 40.7 ± 1.2 nm (n = 6) at 70 nm, 49.8 ± 1.7 nm (n = 3) at 90 nm, and 62.7 ± 2.9 nm (n = 4) at 95 nm ( Figure 1B). The thickness obtained by the minimal folds method linearly increased, but the slope was smaller than unity ( Figure 1B).
On the other hand, optical measurements by the color laser 3D profi le microscope reported the thickness to be 32.5 ± 2.1 nm (n = 2) at 30 nm, 71.0 ± 4.5 nm (n = 5) at 70 nm, and 89.0 ± 2 nm (n = 4) at 90 nm ( Figures 1B-E). The section thickness measured by the optical method linearly increased, and the slope was close to unity.
To validate the reliability of the minimal folds method and optical laser measurements, we compared the measured diameter of red Important factors for 3D reconstruction

FIGURE 1 | Thickness estimation of ultrathin sections for 3D reconstruction. (A)
Minimal folds of an ultrathin section cut by a microtome set to cut 95 nm sections. Width of the minimal folds, twice the section thickness, was 130 nm, much less than expected. Note the black vertical line at the center of the fold, which is the adhered, folded membrane perpendicular to the plane of a section. (B) Correlation between section thicknesses set by the microtome and those measured by a color laser 3D microscope or the minimal folds method. The thicknesses measured by the optical method were more similar to those set by the microtome than those measured by the minimal folds method, which were 50-80% of the set section thickness. (C, D) Threedimensional view of ultrathin section (90 nm thickness, pink) on glass slide (beige) obtained by the laser scanning microscope. The border of the section and slide surface is clearly identifi ed in larger magnifi cation (C). (E) The pseudocolor image representing height of the ultrathin section (orange) and glass slide (blue). Note that the colors are uniformly distributed on the surface of the section and glass slide, indicating the surface fl atness. Thickness of the ultrathin section was estimated from the difference between average heights of the two areas. Rectangles (about 50 µm 2 ), indicate areas used for evaluating the average height. (F) Light microscopic photograph of red blood cells.
(G, H) The 3D images of the red blood cells reconstructed assuming 50 nm (G) and 90 nm (H) in section thickness. The view of the upper images is the same as in the light microscopic photograph in (F); the view of the lower images is "side-on" . The red blood cells reconstructed assuming a 90 nm section thickness were more similar in shape and size to those in the light microscopic photograph than were cells reconstructed assuming a 50 nm section thickness.

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May 2009 | Volume 3 | Article 4 | 4 Kubota et al. Important factors for 3D reconstruction blood cells in reconstructed ultrathin sections with photographs of the same cells taken by light microscopy. Measurements from photographs (taken using a 100× oil-immersion objective lens, 1.4 N.A.; Olympus, Tokyo, Japan) indicated the vertical and horizontal diameters of the red blood cell in the right side of Figure 1F were 6.15 and 5.73 µm, respectively (corresponding to yellow reconstructions shown in Figures 1G,H). The tissue was cut perpendicular to the plane of section for EM observation with the microtome thickness set to 90 nm. Assuming an actual 90 nm thickness per section, the reconstructed diameters of the red blood cell were 6.20 µm (about 101% of LM image) and 5.73 µm (about 100% of LM image) in the vertical and horizontal directions, respectively (yellow red blood cell in Figure 1H). On the other hand, when using the 50 nm section thickness estimated by the minimal folds method, the vertical and horizontal diameters were only 3.41 µm (about 55% of LM image) and 5.73 µm (about 100% of LM image), respectively (yellow red blood cell in Figure 1G). Further, the reconstructed morphology of the red blood cell was much less elliptical when using a 90 nm section thickness. Therefore, we conclude that the minimal folds methods underestimate the thickness of ultra thin sections, and therefore recommend that thickness measurements of sample sections be obtained by the optical method.

IDENTIFICATION OF SYNAPTIC CONTACTS
We used the nickel-DAB method for pre-embedding parvalbumin immunohistochemistry so as to clearly observe postsynaptic density (PSD) structure. Immuno-precipitation of nickel-DAB selectively stains intracellular structures such as microtubules and PSD, and the distribution of the precipitation under the EM is much sparser than when DAB-only methods are used. The resulting images allow clear identifi cation of PSD structures unobscured by dense staining of the cytoplasm (Figure 2). It is relatively simple to identify synaptic contacts when the plane of the synaptic cleft is vertical or steeply angled to the plane of section (Figures 2A-C). On the other hand, it is much more diffi cult to identify synaptic contacts when the cleft plane is parallel to, or at a low angle with respect to, the plane of section of the slice. Since the dendrites upon which synapses make contact often radiate in multiple planes, it is a frequent occurrence that some reconstructed dendrites extend parallel to the plane of section (Figures 2A-F). We have observed that cortical parvalbumin-positive dendrites receive many synapses on their dendritic shafts (unpublished observation), as has been observed in hippocampal parvalbumin-positive dendrites (Gulyas et al., 1999). When these neurons are reconstructed and examined for synaptic contacts using traditional methodologies for identifying synaptic junctions, the dendritic surfaces parallel to the plane of section were found to have very few synaptic contacts compared to those dendritic surfaces at right angles to the plane of section, suggesting a failure to identify synaptic junctions occurring parallel to the plane of section (Figures 2G,J). In an attempt to better identify these synapses, we looked for progressive sequences of synaptic characteristics in serial 70 nm thick sections (Figures 2D-F). A better example of such a progressive sequence done with a tangential section of a parvalbumin-positive dendrite without GABA postembedding immunohistochemistry is shown in Figure 3. The electron dense nickel-DAB immuno precipitate was found lightly on the intracellular structures in the cytoplasm of the parvalbumin positive dendrite, and it still prevented detailed observation of the ultra structure of synaptic contact. Therefore, we avoided using the stained portion of the dendrite. Instead, we followed the dendrite into the middle of the section to fi nd the positive dendrites lost the stain density for this analysis. We found the objects related to synapse formation in correct synaptic sequence in the serial 70 nm thick sections. In the fi rst and second sections, there were many synaptic vesicles in the presynaptic bouton (Figures 3A,B). In the next section, we found many roundshaped electron dense structures of ∼35 nm diameter (Figure 3C), but only few synaptic vesicles. These round-shaped electron dense structures likely to correspond to the "presynaptic grid" (Peters et al., 1991). In the fourth section, electron dense fl occulate material was seen, which probably indicates a PSD (Figure 3D). In the fi fth section, we observed a thin snippet of PSD within the cytoplasm of the postsynaptic dendrite ( Figure 3E). Wider fi eld of this synaptic structure in serial sections were shown in Figures 3F-J to see the surround structure. The other synapse in the serial tangential sections also showed the similar progressive sequence (Figures 3K-O). We therefore believe the contact, identifi ed in these progressive sequences of serial sections, is a synaptic contact.
To confi rm the presence of a synaptic contact, we applied tomographic analysis to observe structural changes occurring within individual 70 nm thick sections and detected gradual structural changes in the following order: (1) many synapse vesicles (Figures 4 and 5); (2) presynaptic grids ( Figure 5); (3) synaptic cleft structures (Figures 5 and 6); (4) PSDs (Figures 5-7); and (5) cytoplasm of postsynaptic dendrite or spine (Figure 7). Structures following this sequence are indicative of a synaptic contact (Supplementary Material available online), regardless of the target structure (spine, dendrite, or soma). Tomography analysis confi rmed that we can identify synapses even when synaptic membranes lie in the plane of the slice section or intersect at very oblique angles.
Overall, we found about one-third to one-half of synapses onto shafts were not identifi ed using traditional standards for synaptic contacts when the cleft plane is parallel to, or at a low angle with respect to, the plane of section of the slice. For instance, the parvalbumin-positive dendrite (7.6 µm in length) shown in Figures 2G-L had 17 synapses (5 GABA-positive and 12 GABAnegative synapses) identifi ed using classic synaptic indicators, all of which were located on the surface perpendicular to the plane of sectioning (Figures 2G,J). On the other hand, 19 synapses (3 GABA-positive and 16 GABA-negative synapses) were found on the upper and lower surfaces of the dendrite parallel to the plane of section using the progressive identifi cation of synaptic markers in serial sections proposed in this study (Figures 2H,I,K,L). With these added synapses, we reveal that the dendrite is densely covered by synaptic boutons on almost all surfaces (Figures 2I,L). Analysis of fi ve parvalbumin-positive dendrites including the dendrite in Figure 2

DISCUSSION
We propose two new methodologies that improve the accuracy of 3D reconstruction of neuronal ultrastructure and facilitate accurate synapse identifi cation. We use these methodologies to reveal that parvalbumin-positive cortical interneurons have a much higher synaptic density than previously appreciated from data using classic methodologies for synaptic identifi cation. Accurate measurement of section thickness is a critical fi rst step for precise 3D reconstruction of synaptic structures. Our data indicate that the most frequently used method for measuring section thickness, the small fold method, produces signifi cant errors that impair subsequent reconstruction of synaptic structures. An alternative method measures section thickness using optical interferometers (Nomarski) (De Groot, 1988). However, this technique has not been widely used because of the complexity of the instrument and its diffi culty of use and because it may depends upon the user's subjective visual impression. A third way to estimate section thickness is the cylindrical diameter method, which depends on the measured width of elongated mitochondria found parallel to the plane of section (Fiala and Harris, 2001). Based on the number of sections containing longitudinally located mitochondria and the width of the mitochondria, one can estimate the thickness of the sections, assuming the cross section of the mitochondria are round. However, mitochondria may not be truly cylindrical and/or the upper and bottom end sections may contain the mitochondria only in part of its thickness, and therefore this method may not provide an accurate estimation of thickness (Knott et al., 2008).
Our data indicate the color 3D laser confocal microscope provides a more robust measure of section thickness than any of these other classic methodologies. It also has the advantage of providing a surface image of any object with 1 nm resolution in height and width measurement. Here we demonstrate that it can be used for accurate measurement of ultra thin sections of resin embedded tissue. Our present results also indicate that ultrathin sections with the desired thickness can easily be made from the ultramicrotome with a high degree of certainty.
There are other aspects that might limit accurate 3D reconstruction of synaptic structures, such as distortion of shape during ultrathin sectioning or damage of ultrathin sections under electron beam. However, we found that reconstructed red blood cells were quite similar in shape and size, indicating that careful processing of tissue can limit damage to the tissue and preserve accurate reconstructions. One must also consider tissue shrinkage during fi xation and embedding. Our previous measurements indicated that, after fi xation, dehydration, and embedding in Epon, the tissue shrank to 90% of original size (Karube et al., 2004). Shrinkage was not corrected for in the present analysis.
Underestimating the number of synapses onto dendritic shafts radiating parallel to the plane of section is signifi cant problem for quantitative stereology. Our experience processing serial sections for 3D reconstruction analysis suggests this underestimation is 25% or more. In a previous study, we tested whether all axonal boutons identifi ed at with light microscopy are associated with synaptic junctions (Karube et al., 2004). Consecutive investigation of light microscopically identifi ed boutons (59 boutons) of two FS cells with EM found that 46 boutons (78%) showed clear synaptic junc-tions, with another 12 boutons (20%) having synaptic vesicles but no identifi able junction, and one bouton (2%) contained only a mitochondrion ( Figure 1F in Karube et al., 2004). It is likely that the missing synaptic junctions for those boutons with synaptic vesicles were lying in the plane of section. We also found similar ambiguity when identifying synapses onto spine heads. About 75% of cortical spines (94 out of 125) contacted by symmetrical synapses from axon terminals of nonpyramidal cells were found to also receive an asymmetrical synapse (Figure 4 in our previous paper, Kubota et al., 2007). The remaining 25% of GABA-receptive spine heads did not have any additional asymmetrical synaptic contact. Since we now know that all synapses on neuronal surface are not identifi ed when using only the classical synapse indicators, it is possible that additional asymmetrical contacts onto these 25% of spines were obscured based on their orientation within the plane of section of the ultrathin slice.
The present study clearly demonstrates that progressive sequences of synaptic characteristics in serial ultrathin sections can identify synaptic contacts even when they occur parallel to the plane of section. We confi rmed this using EM tomography by observing the structural changes occurring within individual 70 nm thick sections. We can now postulate that synaptic junctions can be identifi ed by the progressive sequence of synaptic indicators, as well as by classical methodologies. This may improve the quality and quantity of morphological data because tomography analysis is time consuming and the technology required is not widely distributed.
Together, our data demonstrate that more accurate measurement of section thickness (using a laser measurements or a well-calibrated microtome) and careful identifi cation of synaptic contacts occurring tangential to the plane of section, generate more accurate reconstructions and therefore more robust data with which to understand synaptic connectivity in neuronal circuits.


extfieldsofstudy: Chemistry Medicine

text:
Differential Bacterial Surface Display of Peptides by the Transmembrane Domain of OmpA

Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor β-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from ‘aberrant’ to normal, and predict that some β-barrels will not display any significant heat-modifiability at all.

Introduction
Bacterial surface display is widely used to screen peptide libraries for e.g. epitope mapping and selection of high affinity binders [1]. As differences in display efficiency among different clones could cause unwanted biases during the selection process, knowledge of the capabilities, as well as the limitations of the particular display scaffold used, is considered to be of importance [2].
One such scaffold is the OmpA protein in Escherichia coli. OmpA is an integral outer membrane protein (OMP) embedded in the bacterial outer membrane (OM) as a b-barrel. It contains four surface exposed loops in which peptides can be inserted that subsequently are displayed on the cell surface [3]. Several reports exist in which the OmpA protein has been used as a bacterial surface display system, for applications such as peptide library screening [4,5] use in a novel selection strategy [6], use as live vaccines [7] or to sequestrate cadmium for bioremediation [8].
The full-length, processed OmpA protein (325 residues) consists of two domains, a N-terminal transmembrane (TM) domain of 170 residues, connected via a short 19-residue Ala-Pro rich hinge region to a C-terminal periplasmic domain of 136 residues [9]. The periplasmic domain plays a structural role by tethering the OM to the peptidoglycan cell wall layer. However, genetically truncated OmpA-171 consisting of only the TM domain assembles into the outer membrane as efficiently as the full-length protein [10]. For a comprehensive review on OmpA structure and function see [11].
Our goal was to display an epitope on the bacterial cell surface that could be recognized by commercially available antibodies and used as a handle in biophysical force experiments (to be published elsewhere). OmpA was chosen because it is very abundant (typically about 10 5 copies/cell (Koebnik et al. 2000)) and widely studied [12,13]. For our biophysical application, adverse effects of peptide-insertion on protein levels or OM insertion were unwanted. Because epitope-sized insertions in loop 2 or 4 of OmpA have been shown to be tolerated without negative effects [3], this served as an additional reason to choose OmpA. Thus, we have inserted the epitope tags 3xFLAG and 2xmyc into loop 2 and 3 of the transmembrane domain of OmpA (here also referred to as OmpA-177), and studied their stability and outer membrane incorporation in vivo. As the cell wall anchoring by the periplasmic domain was unwanted in these experiments, the TM domain of OmpA was initially used instead of the full-length protein.
We show that these engineered OmpA TM domain variants can be incorporated into the OM and that the epitopes are successfully displayed on the cell surface. To our surprise however both are present at roughly 10-fold reduced levels compared to the b-barrel without epitope due to degradation. Since to our knowledge, all in vivo loop insertions to date have been made in full-length OmpA, we investigated a possible stabilizing role of the periplasmic domain. Our data demonstrate that the periplasmic domain did not stabilize the 3xFLAG insertion variants. These results suggest an incompatibility of 3xFLAG and 2xmyc tags with the OM biogenesis pathway of OmpA. In the end, we created an OmpA insertion variant that met our requirements by truncating a full length OmpA containing an SA-1 peptide identified in a search for streptavidin binding peptides [5]. The results obtained with this variant demonstrate that it is possible to display a peptide in the OmpA b-barrel without negative effects on protein levels or OM insertion. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitope, as both 2xmyc (20-mer) and 3xFLAG (22-mer) are highly charged (50% and 70% charged residues, respectively) whereas the SA-1 peptide (15-mer) is moderately charged, containing only 2 charged residues.
In addition, we report that the high-level expression of epitopecontaining OmpA constructs had a strong, transient, negative effect on the cellular growth rate that was strain dependent. In a MC4100 (a standard E. coli laboratory strain) derivative, growth rate was transiently impaired, whereas in strain MC1061 as well as a DOmpA MC1061 derivative no detrimental effects were observed. This suggests that when over-expressing outer membrane proteins, MC1061 is a strain of choice.
To test for outer membrane incorporation, we make use of OmpA's so-called heat modifiability [14]. In its folded form, OmpA migrates to a different position in SDS-PAGE compared to its (heat denatured) unfolded form [15]. Whereas for full length OmpA, the folded form migrates faster through the gel, for the transmembrane domain of OmpA it is the other way around [10]. This was termed 'aberrant heat modifiability' [10]. We show that small epitope insertions in the transmembrane domain restore normal heat modifiability, and this observation predicts that some b-barrels might not exhibit any heat modifiability at all.

Materials and Methods
Bacterial strains and growth conditions E. coli strains (Table 1) were grown at 37uC in TY medium containing 1% Bacto trypton, 0.5% Bacto yeast extract, 0.5% NaCl and 3 mM NaOH. Expression of the constructs was induced by adding up to 1 mM IPTG or 0.02% L-arabinose, depending on the plasmid vector. Antibiotics were ampicillin (100 mg/ml) or Chloramphenicol (25 mg/ml). LMC500 (MC4100 lysA) was made chemically competent using the calcium chloride method. MC1061 and its derivative MC1061 DOmpA were transformed using electroporation.

Constructs
All constructs (Table 1) were cloned into a pTrc99A vector (Pharmacia Biotech, USA), a pBR322 derivative plasmid, of which the trc promoter was modified with a down mutation to reduce expression levels [16]. All DNA manipulation, analysis and bacterial transformations were performed according to standard protocols (Sambrook et al., 1989). All PCR fragments were sequenced, either at Baseclear (Leiden) or at the AMC DNA sequencing facility (Amsterdam Medical Centre). Primers were ordered from MWG or Biolegio, and Advantage DNA polymerase (Clontech) or pfuTurbo DNA polymerase (Stratagene) was used for the PCR reactions. The cloning steps performed to obtain the plasmids are described in the Materials and Methods S1.

Preparation of cell lysates
Fresh overnight cultures grown at 37uC were diluted 1000x into 50-100 ml fresh TY medium and cultured at 37uC. Growth was monitored by measurement of the optical density at 600 nm with a spectrophotometer (Perkin-Ellmers). IPTG was added at around an OD600 of 0.1, and when the cells reached an OD600 of 1.0, they were transferred to a 50 ml Falcon tube and put on ice. The cells were then collected by centrifugation for 15 min at 4000 rpm in a tabletop centrifuge at 4uC (Eppendorf). The supernatant was carefully removed, and the cells were resuspended in ice-cold sonication buffer (10 mM Tris-HCl buffer, pH 7.9, supplemented with 1 mM EDTA and 1 tablet of Roche Protease Inhibitor Cocktail), at a concentration corresponding to an OD600 of 250. This cell suspension was transferred to a 2 ml Eppendorf tube, and sonicated on ice with a tip sonicator (Branson) in 4-5 10-second bursts with 10 second cooling in between each burst. Debris and intact cells were pelleted in a 4uC cooled centrifuge at 2700 x g for 2 min. The supernatant was transferred to a 1.5 ml Eppendorf tube and frozen at 220uC as total cell lysate.

Fractionation of cell lysates
After thawing, the cell lysate was diluted to 4 ml (corresponding to an OD600 of 12.5), and 100 ml of this was saved as ''total cell lysate''. The samples were pelleted at 45000 rpm (corresponding to 200.000 x g) for 45 min in an ultracentrifuge (Beckman-Coulter). After centrifugation, 500 ml was saved as ''supernatant''. The membrane pellet was resuspended in 100 ml sonication buffer and frozen at 220uC.

SDS-PAGE and Western blotting
For SDS-PAGE, samples were mixed with sample buffer (end concentration: 62.5 mM Tris pH 6.8, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol) and either heated to 99uC for 5 min or heated to 50uC for 15 min and electrophoresed on 15% polyacrylamide slabs. Anti-FLAG and anti-myc monoclonal antibodies used for the immunoblots were obtained from Sigma and Roche, respectively. The polyclonal anti-OmpA antibody was a kind gift from A. Driessen (University of Groningen, Netherlands). The bands were detected using the ECL+ chemiluminescence kit (Amersham) and scanning with a STORM 860 fluorescence imager. Densitometry was performed using ImageJ (http://rsb. info.nih.gov/ij/). The mean pixel value of a rectangular region was calculated close but outside a band of interest to calculate the mean background pixel value. The same selection rectangle was positioned to include the band of interest, and again a mean pixel value is calculated. Subtraction then gives a band intensity value. All band comparisons were performed using the same selection rectangle.

Fluorescent labeling of fixed cells
Cells were fixed in 2.8% formaldehyde (FA) and 0.04% glutaraldehyde (GA) in growth medium for 15 min at room temperature, then washed and resuspended in PBS (140 mM NaCl, 27 mM KCl, 10 mM Na 2 HPO 4 ?2H 2 O, 2 mM KH 2 PO 4 pH 7.2). Cell concentration was adjusted to an OD600 of 0.6 and samples were incubated in 75 ml PBS containing 30 mg/ml BSA to block non-specific sites on the cell surface for 30 min at 37uC. Then antibodies were added, either anti-FLAG (M2, Sigma) or anti-myc (9E10, Roche) at an end concentration of 20 mg/ml, and samples were incubated at 37uC for 30 min. The cells were washed 3 times with 2 volumes of PBS containing 30 mg/ml BSA, and then incubated in 1 volume with Donkey-anti-Mouse-Cy3 conjugate (Jackson ImmunoResearch) at 10 mg/ml end concentration for 30 min at 37uC, washed 3 times with 2 volumes PBS and imaged.

Fluorescent labeling of living cells
Cells were put on ice, and an amount of cells equivalent to 1 ml OD600 of 0.3 (around 2?10 8 cells) was taken for labeling. Cells were collected in all cases by centrifugation at 20.000 x g for 5 min at 4uC. The pellet was resuspended in 75 ml PBS at room temperature (RT) with 0.1% BSA. The cells are left at RT for 10 min to block aspecific sites on the cell surface. Then either biotinylated anti-FLAG (Sigma) was added (50 mg/ml) (FLAG constructs), or streptavidin-Alexa 488 (Molecular Probes) was added directly (40 mg/ml) (SA-1 constructs). Cells were incubated at RT for 30 min. The cells were spun down and washed twice with 0.5 ml PBS, and resuspended in 150 ml PBS. For the cells labeled with biotinylated FLAG, streptavidin-Alexa 546 (Molecular Probes) was added (5 mg/ml), and samples were incubated for 30 min at RT. Then, PBS (0.85 ml) was added and the cells were pelleted. After a second wash with 0.5 ml PBS, the cells were fixed in 1 ml PBS with 2.8% formaldehyde and 0.042% glutaraldehyde, washed in 1 volume of PBS and resuspended in 0.1 volume PBS. The cells were either imaged directly or stored at 4uC over night before imaging.

Fluorescence Microscopy
Cells were immobilized on 1% agarose in water slabs-coated object glasses as described by [17] and photographed with a CoolSnap fx (Photometrics) CCD camera mounted on an Olympus BX-60 fluorescence microscope through a UPLANFl 100x/1.3 oil objective (Japan). Images were taken using the public domain program Object-Image2.19 by Norbert Vischer (University of Amsterdam, http://simon.bio.uva.nl/object-image.html), which is based on NIH Image by Wayne Rasband. In all experiments the cells were first photographed in the phase contrast mode. Then a fluorescence image was taken using either a green excitation/red emission (U-MNG, ex. 530-550 nm), or a blue excitation/green emission filter cube (U-MNB or EGFP, ex. 470-490 nm).

Design of loop insertions
A topology model of the transmembrane domain of OmpA is shown in Figure 1. For locations in loop 2 and loop 4 (after Y63, G70 and I153 respectively), it has been shown that small (up to 21 residue) peptides can be inserted without any reduction in protein levels [3,18] and membrane incorporation (after G70 and I153, [3]). For loop 2, reported inserted peptides are listed in Table 2. Initially, we used only the OmpA TM domain, but later also the periplasmic domain was added. 3xFLAG and 2xmyc peptides were chosen as epitope tags (Table 2). We will refer to them as FLAG and myc from now on. High-affinity monoclonal antibodies are commercially available for these epitopes. SWISS-Model [19] was used to predict OmpA folding after peptide insertion. First, a continuous model was generated of the first 176 residues, based on the published crystal structures of OmpA-171 ( Figure S1A). Then, models were generated of loop insertions after different residues in the protein, and the resulting (static) loop conformations were examined for their propensity to extend away from the membrane normal axis. Models were generated of insertions in loop 2, 3 and 4. Finally, loop 3 was chosen, since the computer-generated model of an inserted FLAG peptide after N109 predicted the largest distance away from the surface ( Figure S1C).
To be able to compare the performance of our constructs with results reported in literature, we also constructed loop 2 insertions with the FLAG and myc epitopes. For loop 2, insertions after G65, G70, Y72 and Q75 were modeled. In the end G70 was chosen, because (a) as mentioned, it was shown that at this location, a 21residue peptide could be inserted without any negative effects on membrane insertion [3], and (b) modeling with SWISS-Model of these four locations predicted that at G70, the loop would be extended away from the surface more than at the other three positions in loop 2 ( Figure S1B).
During the course of this work, a loop 1 peptide insertion (SA-1 tag) in full-length OmpA was described in the literature [5]. The position of this insertion is also indicated in Figure 1 (after N26). As mentioned in the introduction, the SA-1 peptide tag (listed in Table 2) binds streptavidin directly with high affinity. Since this peptide was neutrally charged and could be conveniently labeled with fluorescent streptavidin, we decided to compare this loop insertion variant with our constructs. Therefore, it was cloned into our expression vector, both as a truncated OmpA consisting only of the TM domain and as a full-length protein, and assayed in the same way as the FLAG/myc insertions.

Growth of cells expressing OmpA-177 loop insertion proteins
The constructs were tested for expression in LMC500 (MC4100 lysA), a well characterized laboratory strain [20,21], MC1061 DOmpA [5], an OmpA knockout strain, and in its parental strain MC1061 [22]. To test to what extent the proteins could be expressed without affecting the growth rate, the growth was followed by measuring the optical density before and after addition of the inducer IPTG. Growth curves from a typical experiment are shown in Figure 2. In this experiment, cells carrying an empty vector were compared with cells expressing an OmpA-177 protein with a FLAG insertion either in loop 2 or loop 3. Without IPTG induction, all growth curves appear identical. Approximately 15 minutes after addition of IPTG however, the OmpA-177 protein with a FLAG insertion in loop 2 shows a lag phase where growth stops for approximately 30 min, after which growth continues normally. In contrast, for the growth curve of the OmpA-177 variant containing a FLAG insertion in loop 3, no lag phase is  [40]). Black arrowheads indicate positions where peptides have been inserted: after N26 [5] and this study, Y63 [18], G70 [3] and this study, N109 this study, and I153 [3,41]. Residues present in bstrands are indicated with squares. Other residues are presented as circles. doi:10.1371/journal.pone.0006739.g001 observed after induction with IPTG. Instead, the growth curve is similar to the growth curve of uninduced cells. The induction experiment was performed for all the OmpA TM domain variants and the results are listed in Table 3. It can be concluded that in strain LMC500, induction of the loop 2 (FLAG/ myc), and to a lesser extent, the loop 1 (SA-1), but not the loop 3 (FLAG/myc) insertions caused a lag phase of 30-60 min after which growth continued normally. Surprisingly, this lag phase was absent both in MC1061 and in MC1061DOmpA. Induction with 0.3 mM IPTG of the loop 2 (FLAG), or loop 1 (SA-1) in these strains had no effect on the growth rate at all. Because in LMC500 and MC1061, similar protein levels were detected on immunoblots, it appears that strain MC1061 and its derivative MC1061DOmpA are more able to cope with the perturbation caused by the induction (see also discussion).

Expression of OmpA-177 loop insertion proteins
Samples, harvested at the end of the induction experiment described above were analyzed by SDS-PAGE and immunoblotting. For the FLAG and myc constructs, a blot is shown in Figure  S2. Based on the immunoblots it was clear that all four variants (either FLAG or myc in Loop 2 or 3) were expressed, no degradation bands were observed, and all ran at a similar height that was retarded with respect to their calculated molecular weight (approx. 3-4 kDa). This was also observed for full-length constructs that carried a FLAG insertion (see below). The reduced mobility on gel was attributed to the high amount of negative charge in the FLAG and myc peptides. Unexpectedly, unprocessed (precursor) proOmpA-177 was also detected in uninduced samples as well as in induced samples expressing either FLAG or myc in loop 3 (see also Figure S3). The difference in protein levels between induced and uninduced samples was a factor 2-5 fold (all protein levels were determined from immunoblot by densitometry using ImageJ, data not shown). Without induction, for FLAG as well as myc, about 25% of the loop 3 constructs detected were not processed.
Induced OmpA-177 without loop insertion was directly visible on a Coomassie Brilliant Blue (Coomassie) stained SDS-PAGE (see Figure 3) and could be detected on immunoblot using a polyclonal antibody against OmpA, but the band was very weak compared to endogenous (full-length) OmpA (data not shown). From this, we conclude that this polyclonal antibody primarily recognizes the periplasmic domain of OmpA. Also, an attempt was made to detect the streptavidin binding peptide SA-1 construct on blot using a Streptavidin-HRP conjugate, but only the endogenous cytoplasmic biotinylated biotin carrier protein BCCP was detected (data not shown). Apparently, streptavidin does not bind sufficiently strong to the denatured conformation of SA-1.
To be able to directly compare the amounts of protein in the membrane, membrane fractions were isolated of cells expressing the various OmpA TM domain variants ( Figure 3). Induction of both OmpA-177 and OmpA-177 SA-1 constructs resulted in strong bands at roughly their expected height (calculated MW of OmpA-177 is 19.3 kDa and of OmpA-177 SA-1 is 21.1 kDa). Surprisingly, no bands could be identified that corresponded to the FLAG or myc constructs.
To determine how much of the expressed construct is present in the envelope fractions put on gel, we compared soluble (S) to membrane (M) fractions. Although not visible on a Coomassie stained gel, immunoblots of the fractions showed that the FLAG and myc constructs were present, and fractionated predominantly to the membrane fraction ( Figure S3). We conclude that these constructs are present in the cell at greatly reduced amounts (less than 10% as judged from the Coomassie stained gel) compared to wild type OmpA-177 or the OmpA-177-SA1. This could either be   due to a reduced synthesis rate (not expected for FLAG and myc tags), due an increased degradation rate (see also below) or both.

Role of the periplasmic domain
To our knowledge, all OmpA loop insertions reported in the literature have been made in full-length OmpA. To establish whether the periplasmic domain might play a role in preventing a reduction in protein levels after FLAG or myc insertion in the transmembrane domain, full-length constructs were made for the FLAG epitope in loop 2 and 3, and the SA-1 insertion in loop 1. These constructs were transformed to LMC500 and MC1061 DOmpA and were subjected to the same induction experiment as for the OmpA TM domain variants. Without induction, all fulllength constructs grew normally in both genetic backgrounds. After induction, the growth rate remained unaffected for all fulllength OmpA loop insertion constructs in the MC1061 DOmpA background whereas expression in LMC500 resulted in growth curves similar to those obtained for the OmpA-177 constructs (only tested for the loop 3 FLAG insertion).
The full-length constructs were detected both on Coomassie stained gel ( Figure 4A) as well as on immunoblot using a polyclonal antibody that recognizes the periplasmic domain of OmpA ( Figure 4B, Figure S4). As with the FLAG OmpA-177 proteins, full-length OmpA FLAG constructs had a higher apparent molecular weight than calculated. Comparing soluble to membrane fractions using immunoblots yielded similar results as for the OmpA-177 variants, with the majority of each construct fractionating to the membrane fraction, except loop 3, which was divided over the soluble and membrane fraction ( Figure S4).
Again, the FLAG constructs appear greatly reduced compared to both the OmpA without insertion and the OmpA with an SA-1 insertion, all expressed from plasmid. The intensities of the anti-OmpA bands from Figure S4 were quantified using ImageJ and plotted as bar graphs in Figure 5. Together with Figure 4A, it can be concluded that full-length OmpA constructs with FLAG insertions either in loop 2 or loop 3, are present at approximately 5-10% compared to full-length OmpA without insertion or the full-length OmpA carrying a SA-1 insertion, respectively.
Apart from intact construct, the immunoblots probed with anti-OmpA antibody revealed a strong band around 17 kDa (the expected size of the periplasmic domain) that fractionates to the soluble fraction ( Figure 4B). This band was absent from samples containing induced full-length OmpA without insertion and weakly detected in induced full-length OmpA with the SA-1 insertion (data not shown). From this we conclude that the degradation band is specific and that it is related to the reduced protein levels of the FLAG constructs. In addition, the FLAG constructs were detected using anti-FLAG antibody ( Figure S4 and not shown). The 17 kDa degradation band did not react with anti-FLAG (the smallest degradation band detected with anti-FLAG ran around 27 kDa). Apparently, the periplasmic domain of OmpA was cleaved from the TM domain and the latter was to a large extent degraded.
To test whether the 17 kDa degradation band was present in periplasmic aggregates, we fractionated the cells into soluble periplasmic contents and insoluble pellet by osmotic shock (See Figure S6 and Materials and Methods S1). We find that the degradation band was not aggregated but present in the soluble periplasmic fraction, consistent with the idea that the FLAG/myc OmpA variants are to a large extent degraded by periplasmic proteases.
Taken together, it can be concluded that both the full-length and the OmpA-177 constructs with a FLAG insertion are present in greatly reduced amounts, compared to without insertion or with a SA-1 insertion. Therefore, addition of the periplasmic domain does not improve protein levels of the FLAG insertion. Furthermore, the reduction in protein levels is, at least partly, caused by degradation, as observed on immunoblots.

OM incorporation of truncate and full-length OmpA constructs
To study whether the OmpA variants, present in the membrane fraction, have obtained their native form, we examined their heatmodifiability. The native form of the OmpA TM domain is a compact b-barrel that has a particularly tight fold with a half life of 30 min when heated to 72uC in 2% SDS [23]. When heated in SDS at lower temperatures (e.g. 50uC) the b-barrel becomes soluble without unfolding and migrates faster through the gel (30 kDa) relative to its denatured form, which runs at the expected molecular weight (35 kDa). This effect is called heat modifiability [14] and it is a general property of b-barrels. Various techniques have been used to confirm that the 30 kDa form corresponds to the native fold of OmpA (for references consult [24]. It is generally assumed that in vivo, the native form of OmpA is generated only after proper insertion into the outer membrane (Ried, 1994).
Cell membranes containing IPTG-induced OmpA-177 or OmpA-177 SA-1 proteins, expressed in LMC500 or MC1061 DOmpA were either heated in sample buffer for 5 min at 99uC or for 15 min at 50uC, then applied on gel, separated by SDS-PAGE and stained with Coomassie ( Figure 6). Note that both OmpC and OmpF do not become soluble at 50uC and are therefore only visible in samples heated at 99uC [10]. As expected, the endogenous OmpA of LMC500 is fully heat-modifiable. Furthermore, the OmpA-177 protein, but not the OmpA-177 containing the SA-1 insertion, shows the aberrant heat-modifiability already observed in the literature for OmpA-171 [10], where the folded protein migrates slower than the unfolded protein (see also discussion). Finally, the OmpA-177 SA-1 protein is also fully heatmodifiable. We conclude that the majority, if not all, of both constructs have reached their native form.
For the FLAG and myc constructs, because of their low levels in the cell, immunoblotting was used to visualize their heatmodifiability (Figure 7). The OmpA TM domain constructs with FLAG or myc are all predominantly heat-modifiable ( Figure 7A, indicated ''mature''). As expected, the loop 3 precursor bands (indicated ''unprocessed''), present in the membrane fraction, are not at all heat-modifiable. Also a full-length OmpA construct carrying a loop 2 FLAG insertion was found to be fully heatmodifiable ( Figure 7B). We conclude that all FLAG and myc constructs are at least partially heat-modifiable, and thus can be properly incorporated in the outer membrane.

Surface display of loop insertions: fluorescent labeling of cells
To determine the accessibility on the cell surface of the inserted antigenic peptides, both fixed and living cells were labeled. Cells carrying the FLAG and myc in either loop 2 or 3 of OmpA-177  were induced with 0.3 mM IPTG, fixed and stained with monoclonal anti-FLAG or monoclonal anti-myc. As a negative control, the primary antibody was left out, and no fluorescence was observed for any of these samples. All four constructs were detected on the bacterial cell surface ( Figure S5). Loop 2 insertions show more staining at the poles, whereas loop 3 insertions are more homogeneous.
Living cells were labeled using a biotinylated variant of the anti-FLAG antibody. Labeling of living cells was performed because it was found that fluorescent streptavidin (needed for the SA-1 peptide) penetrated fixed cells to show a nucleoid-like staining (data not shown). The cells could be fixed after labeling to preserve the staining.
Results of labeled, uninduced cells carrying either OmpA-177 FLAG (loop 3) or the full-length OmpA-FLAG (loop 3) showed that both have comparable levels of antibody-accessible FLAG epitope on their surface ( Figure 8A, B). This provides further evidence that adding the periplasmic domain does not result in increased stability of the protein. The limited increase in fluorescence after induction correlates with a modest increase of intact protein, and a larger increase of the 17 kDa degradation band as detected on immunoblot (data not shown).
Finally, living LMC500 cells expressing OmpA-177-SA1 were directly labeled with fluorescent streptavidin, fixed and imaged ( Figure 8C). To be able to compare with and without induction directly, a short exposure time (150 ms) was chosen. Without induction staining was homogeneous along the perimeter of the cells. After induction (and after recovery from the lag phase that occurs in LMC500, see earlier), fluorescence increased markedly, and during labeling, strong streptavidin-mediated cross-linking between cells occurred, leading to clumps. Overall, we conclude that all the constructs are detected on the cell surface, but for  FLAG, and likely also myc, the increase after induction is reduced due to degradation, whereas for SA-1, both efficient labeling and a strong effect of induction on the surface display is observed.

Discussion
In this work we have characterized peptide insertions in the OmpA protein, both in TM domain constructs (OmpA-177) and full-length constructs. As insertions we used the popular FLAG and myc epitopes, and a streptavidin binding peptide SA-1. It has been demonstrated that similar-sized peptides can be inserted in loop 2 of OmpA without reduction in protein levels or membrane incorporation [3,18]. Unexpectedly, introducing a FLAG or myc peptide at this location in the OmpA protein reduced the protein levels with approximately 90% (Figure 3, Figure 5). However, the majority of the intact protein was inserted properly in the OM, as judged by its heat-modifiability ( Figure 7A). In contrast, insertion of the SA-1 peptide in loop 1 did not reduce protein levels, similar to the reported insertions in loop 2 ( Table 2). These results have been summarized in Table 4.
It could be argued that the observed differences are due to overexpression of the proteins. However, our expression vector (a weakened pTrc99A [16]) produces less than 5?10 3 proteins in the absence of IPTG [25], and already at these low expression levels the marked difference in protein level between OmpA with a FLAG insertion and OmpA without insertion is observed ( Figure 5). This suggests that introducing the FLAG epitope leads to an intrinsically reduced protein level, independent of the induction level.
What could be the reason of the observed reduction in protein levels of the FLAG and myc constructs? Both epitopes are highly charged (Table 2). In addition, both the FLAG and myc epitopes are effectively negatively charged, and therefore might interact unfavorably with the negatively charged LPS in the OM, or with the YaeT/BamA protein complex responsible for OM insertion [26]. We speculate that a reduced rate of OM incorporation might cause a buildup of unincorporated (misfolded) OmpA proteins, whose subsequent degradation would explain the reduced cellular levels of the FLAG and myc constructs reported here.
Although the number of tested insertions thus far is small, one might speculate that the abundant charge of the inserted epitopes is the cause of the degradation and consequently, of the reduced display levels. Negative effects on OM incorporation after insertion of highly charged peptides have also been reported for outer membrane protein PhoE [27]. It was found that an 20-mer insertion containing 10 positively charged residues, as well as a 28mer insertion containing 6 negative and 2 positively charged residues were not correctly incorporated in the OM [27], whereas a PhoE variant with a VP1 epitope inserted at the same location was incorporated in the OM at normal protein levels [28,29]. It might thus be worth testing more systematically the effect of charged peptide insertions on OMP incorporation in systems that are used for display and/or screening purposes.
One may subsequently ask to what extent differences in display levels affect the application of the OmpA scaffold in, for instance, random library screening using FACS. Whether variations in display efficiency affect a particular screening method is determined by the detection sensitivity and specificity of that method. Interestingly, the FLAG anti-FLAG M2 epitope/ antibody combination has been subjected to epitope mapping with MACS and FACS using the related OmpX display system, and the consensus recognition sequence DYKDxD [30,31] was successfully recovered [4]. This indicates that selection for a sequence containing four charged residues in a surface-displayed library of random 15-mers is possible. Unfortunately, whether the isolated clones had reduced display levels was not determined. It will be interesting to assess if peptides with more (mostly negative) charge such as 3xFLAG or 2xmyc can also be selected for with FACS, and to what extent the various selection methods are affected by display biases.
Unexpectedly, in exponentially growing cells without IPTG, about 25% of the loop 3 insertions are not processed. Since OmpA is mostly post-translationally translocated to the periplasm via the Sec system [32], in these cases perhaps some tertiary structure forms in the cytoplasm that delays, or interferes with, its translocation [33]. However, induction of constructs with FLAG in loop 2 or loop 3 results in similar amounts of processed OmpA ( Figure S2), and in similar amounts of FLAG epitope detected on the bacterial surface ( Figure S5). This suggests that after processing and release into the periplasm, both proteins behave in a similar way, and we conclude that the adverse effects of the FLAG and myc epitopes on protein levels do not appear to be loop specific.
Surprisingly, induction of either (FLAG or myc) loop 2 constructs or the OmpA-177-SA1 in LMC500 (MC4100 lysA) affects growth rate profoundly in a transient manner. It is difficult to understand that loop 2 FLAG/myc and loop 1 SA-1 constructs cause a similar effect on growth rate in LMC500, since their amounts differ 10-fold. Perhaps the cell regulates the amount of proteins in the OM that are tolerated (little in the case of FLAG/ myc, a lot in the case of SA-1). When IPTG induction disturbs this balance, the observed lag period of 30 min might reflect a period in which the cell adapts and restore this balance, after which the cells continue growth.
Accumulation of misfolded OMPs in the cell envelope causes the activation of the s E controlled extracytoplasmic stress response [34] that down-regulates OMP expression ( [35]). Indirect evidence for s E activation upon IPTG induction of our OmpA loop insertion variants comes from the membrane fractions shown in Figures 3 and 4, where OMP expression (OmpC/F and OmpA) is consistently down-regulated after IPTG induction, but only for loop insertion variants.
In the different genetic background of strain MC1061 and its derivative MC1061 DOmpA, expression levels were similar to LMC500, but the growth rate was unaffected upon IPTG induction (data not shown). It has been shown that the s E transcription factor is also controlled by intracellular ppGpp levels [36]. MC1061 has the spoT1 mutation, that abolishes the ppGppase activity of SpoT and results in increased levels of (p)ppGpp (Cashel, 1996). This could offer an explanation for the observed robustness of MC1061 towards overexpression of the OmpA loop insertion constructs. If MC1061 is better able to cope with folding stress in the periplasm (e.g. by having its stress response genes already expressed, or in higher levels), the balance can be restored immediately, without disturbing the growth rate. Whatever the molecular mechanism may be, our results indicate that MC1061 is a strain of choice when over-expressing engineered outer membrane proteins such as OmpA.
Finally, our results provide insight on the aberrant heatmodifiability observed for 8-stranded b-barrels such as OmpA-171, OmpA-177 and NspA ( [10,37] and this study). Heatmodifiability has been termed ''aberrant'' when the unfolded form migrates faster through the gel compared to the folded form. Surprisingly, for the OmpA-177 insertion variants (SA-1, FLAG or myc) heat-modifiability is ''normal'' again ( Figure 6). Comparing the mobility of OmpA-177 to OmpA-177-SA-1, we find that the folded barrels run at almost similar height, as if the extra residues of SA-1 were absent, whereas after boiling, OmpA-177-SA-1 is retarded with respect to OmpA-177 with an amount corresponding to their difference in molecular mass. In other words, the additional residues inserted in the exterior loop have a stronger impact on the unfolded form than on the folded form. These results suggest that as more and more residues are added to the OmpA-171 TM domain, the relative positions between the folded and unfolded domains first decrease until they are equal, before increasing again to appear as ''normal'' heat-modifiability. This predicts that for some rare OMPs, it would seem as if they would not have any heat-modifiability at all.

Conclusions
Most bacterial display studies focus on either identifying novel scaffolds or novel applications. In this work, a thorough analysis was performed of constructs that contain similar-sized peptides inserted into the OmpA display scaffold. The effect of their expression on E. coli growth, as well as their relative amount, subcellular localization, folding state and surface accessibility has been determined. In doing so, we believe that several useful observations have been made that are relevant for the fields of surface display, (outer) membrane protein over-expression and purification as well as for applications that make use of bacteria as antigendisplaying whole-cell adsorbents.
First, we conclude that OmpA displays not all small peptides equally efficiently, since the highly charged and hydrophilic 3xFLAG and 2xmyc epitopes reduce protein levels 10-fold due to degradation by periplasmic proteases. Second, the growth curves obtained after inducing high-level expression of the OmpA epitope variants in two standard laboratory strains MC4100 (LysA) and MC1061 suggest that strain MC1061 is a strain of choice when expressing OMPs in large amounts in E. coli. Finally, our observation that the heat modifiability of the OmpA TM domain can be changed from 'aberrant' to normal by insertion of small epitopes predicts that some b-barrels might not exhibit significant heat-modifiability at all.