SEARCH

SEARCH BY CITATION

Keywords:

  • Amygdala;
  • fear;
  • hippocampus;
  • inbred strains;
  • mouse models;
  • Nfkbia;
  • post-traumatic stress disorder;
  • prodynorphin;
  • stress-responsive genes;
  • Tsc22d3

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Post-traumatic stress disorder (PTSD) is an anxiety disorder that develops in predisposed individuals following a terrifying event. Studies on isogenic animal populations might explain susceptibility to PTSD by revealing associations between the molecular and behavioural consequences of traumatic stress. Our study employed four inbred mouse strains to search for differences in post-stress response to a 1.5-mA electric foot shock. One day to 6 weeks after the foot shock anxiety, depression and addiction-like phenotypes were assessed. In addition, expression levels of selected stress-related genes were analysed in hippocampus and amygdala. C57BL/6J mice exhibited up-regulation in the expression of Tsc22d3, Nfkbia, Plat and Crhr1 genes in both brain regions. These alterations were associated with an increase of sensitized fear and depressive-like behaviour over time. Traumatic stress induced expression of Tsc22d3, Nfkbia, Plat and Fkbp5 genes and developed social withdrawal in DBA/2J mice. In 129P3/J strain, exposure to stress produced the up-regulation of Tsc22d3 and Nfkbia genes and enhanced sensitivity to the rewarding properties of morphine. Whereas, SWR/J mice displayed increase only in Pdyn expression in the amygdala and had the lowest conditioned fear. Our results reveal a complex genetic background of phenotypic variation in response to stress and indicate the SWR/J strain as a valuable model of stress resistance. We found potential links between the alterations in expression of Tsc22d3, Nfkbia and Pdyn, and different aspects of susceptibility to stress.

Post-traumatic stress disorder (PTSD) is a debilitating anxiety disorder that occurs in certain individuals in response to an extreme stress (Nemeroff et al. 2006; Olff et al. 2005; Yehuda & LeDoux 2007). A key question is, why do some people develop PTSD while others appear to experience only few negative effects following a potentially traumatic event.

Clinical studies provide essential findings, but research on human patients is constrained. Therefore, a number of animal models of PTSD has been proposed (Adamec et al. 2008; Cohen et al. 2011; Mikics et al. 2008; Philbert et al. 2011). We have used an inescapable shock paradigm as a mouse model of PTSD (Siegmund & Wotjak 2007b) due to its ability to produce long-lasting effects and employed four inbred mouse strains (C57BL/6J, DBA/2J, SWR/J and 129P3/J). Their variable behavioural phenotypes (Korostynski et al. 2006; Solecki et al. 2009), like dissimilarities in activity level (Paulus et al. 1999; Tang et al. 2002), impulsivity (Gubner et al. 2010) or anxiety-like behaviour (Milner & Crabbe 2008), may indicate their different susceptibility to trauma-related disorders.

There is evidence that stress can alter molecular mechanisms that lead to long-lasting plastic changes in brain regions, such as amygdala and hippocampus (Fenoglio et al. 2006; McEwen et al. 2011; Pittenger & Duman 2008; Radley et al. 2011). Interactions between genetic susceptibility and environment can determine risk of anxiety disorders, but little is known about specific factors underlying such disorders. Several genes have been previously linked to trauma-induced pathologies. Fkbp5 gene is a regulator of glucocorticoid receptor sensitivity and hypothalamic-pituitary-adrenal (HPA) axis reactivity that may influence coping strategies after a stressor (Binder 2009; Touma et al. 2011). Corticotrophin-releasing hormone type 1 receptor gene (Crhr1) has been reported to play a role in moderating the effect of early-life trauma on anxiety responses in adulthood (Amstadter et al. 2011; Wang et al. 2012) and in modulating stress-induced dendritic remodelling (Wang et al. 2011). Prodynorphin (Pdyn)-derived peptides (dynorphins, alpha-neo-endorphin and beta-neo-endorphin) were reported to modulate anxiety-like behaviours (Wittmann et al. 2009) and dynorphins are supposed to play a role in the formation and extinction of fear memories (Bilkei-Gorzo et al. 2012). Tsc22d3 gene is another potential molecular factor for the stress-related disorders due to its dependence on HPA axis activation and putative involvement in dendritic spines alterations (Ayroldi & Riccardi 2009; Piechota et al. 2010; Yang et al. 2008). Other genes were chosen on the basis of their hypothetical role in triggering plastic changes in the brain. Nf-κB signalling is involved in processes such as memory and neuronal remodelling (Mattson et al. 2000; Romano et al. 2006; Yeh et al. 2004), while the tissue plasminogen activator (Plat) gene is supposed to play a role in hippocampus-dependent learning and stress-induced anxiety (Pang et al. 2004; Pawlak et al. 2003).

By combining phenotypic, genetic and molecular factors, we aimed to identify the stress-susceptible/stress-resilient mouse strains and gene expression patterns potentially characteristic for a specific stress response.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Animals

All tests were performed on inbred strains: C57BL/6J, DBA/2J, SWR/J and 129P3/J (Jackson Laboratories, Bar Harbor, ME, USA). Mice (males, 21–28 g, 8–9 weeks) were housed in groups of five to seven per cage, in a temperature- and humidity-controlled room under a 12:12-h light/dark cycle (light on from 0800 to 2000 h) with free access to food and water. All experimental procedures were approved by the local Bioethics Commission at the Institute of Pharmacology, Polish Academy of Sciences (Krakow, Poland).

Experimental schedule and groups

For the assessment of individual foot shock sensitivity, the behavioural tests and gene expression analysis of independent cohorts of animals were used. Experimental animals (n = 24–30 per strain), employed in the mouse model of PTSD (Siegmund & Wotjak 2007b), had received an electric foot shock and were randomly assigned to the three experimental subgroups (n = 8–10 per strain). To prevent habituation, each subgroup was tested for conditioned (CF) and sensitized fear (SF) separately, on the 2nd, 14th or 28th experimental day. Control animals (n = 8–10 per strain) went through the same experimental procedure as the day-28 group but without receiving any foot shock. Thereafter, the experimental animals were re-grouped and randomly distributed for procedures of social interaction test (SIT), forced swimming test (FST) and conditioned place preference (CPP) tests (Fig. 1a). Analyses of the expression of candidate genes by quantitative polymerase chain reaction (qPCR) were performed on animals (n = 4–8 per each experimental and control group) sacrificed 1 and 4 h after the foot shock (Fig. 1b). Our preliminary study conducted 0.5, 1, 2 and 4 h after the foot shock revealed evident differences in gene expression at chosen timepoints.

image

Figure 1. Schedules of behavioural tests (a) and samples collection for qPCR analysis (b). Independent cohorts of animals were used for behavioural test and qPCR analysis as well as for day 1, 14 and 28 of CF and SF tests.

Download figure to PowerPoint

Foot shock sensitivity

In order to assess animals' individual pain threshold, the electric foot shock current was gradually increased by hand from 0 mA, until the animal showed the first sign of pain – jumping or vocalising (Siegmund et al. 2005). At this moment, the current was immediately switched off and the respective current intensity was taken as a measure of the animal's pain threshold.

Shock application

Mice received a 2-second 1.5-mA electric foot shock 198 seconds after being placed in the conditioning chamber (Med Associates, St. Albans, VT, USA) and 30 seconds later returned to their home cages.

Test of CF and SF

For experimental procedure (Kamprath & Wotjak 2004) two different contexts were used: a shock chamber with a metal grid floor for CF and a plexiglas chamber with a loudspeaker as a new context for the test of sensitised fear (SF). Both tests were assessed in the same animals: in the morning of the day following shock application, mice were re-exposed to the shock chamber and freezing behaviour (immobility, except for respiratory movements) was measured for 3 min. In the afternoon, the mice were exposed to the new context for 3 min and then freezing to a 3-min tone presentation (80 dB, 9 kHz) was scored. CF and SF tests were performed alternately – in the morning and the afternoon, on additional groups of animals in the 14th and 28th experimental days. The shock chamber was cleaned after each trial with 70% ethanol, whereas the plexiglas chamber was cleaned with water.

Social interaction test

The trials were performed under red light, with bedding being changed after each trial. Additional eight adult mice per each strain were involved as partners for interaction. The procedure consisted of 3-min acclimatization to the cage (35 × 20 × 18 cm) and 4-min interaction with the partner (Siegmund & Wotjak 2007b). Social interaction (sniffing, close following and attacking) and avoidance (escaping to the opposite part of the cage) were measured.

Forced swimming test

The mouse was placed for 6 min in a glass cylinder (height: 28 cm, diameter: 15 cm), containing water (28 ± 1°C) up to 20 cm. The water was changed after each trial. Floating (absence of movement lasting more than 2 seconds), swimming and struggling (attempting to climb the walls of the cylinder) were measured (Siegmund & Wotjak 2007b). Afterwards, the animal was allowed to dry in a container with lignin. Behavioural items in FST and SIT tests were scored using the EthoLog v.2.2.5 programme (Ottoni 2000).

Morphine-induced CPP

Each CPP apparatus (Med Associates, St. Albans, VT, USA) consisted of three compartments with different visual and tactile cues. The CPP procedure (Solecki et al. 2009) began with an acclimatization to the apparatus (5 min) on day 0. During the pre-conditioning test (day 1), mice were allowed to walk freely in the apparatus for 20 min. The less preferred of the two lateral compartments was paired with morphine (Polfa, Warsaw, Poland) and the other with vehicle (0.9% NaCl; Polfa) administration. During the conditioning test (days 2–7), the mice were randomly assigned to treatment groups, injected with morphine (10 mg/kg) or saline (saline: day 2, 4 and 6; morphine: day 3, 5 and 7) and immediately confined to the respective compartment for 40 min. The post-conditioning test (day 8) was similar to the pre-conditioning one. The difference (delta) between the times spent in the drug- and vehicle-paired compartments during the post-conditioning test was considered to be a measure of CPP (CPP score)(Tzschentke 2007).

Tissue collection and RNA isolation

The animals were sacrificed 1 and 4 h after the foot shock. Brains were removed from their skulls and tissue samples were collected, including the amygdala, due to its critical role in the acquisition and extinction of fear memory (Mahan & Ressler 2011; Maren 1996) and the hippocampus, which is involved in contextual fear (Kaouane et al. 2012). The samples of whole hippocampus and amygdala were bilaterally dissected from the brain and subsequently pooled. The samples were placed in individual tubes with the tissue storage reagent RNAlater (Qiagen Inc., Valencia, CA, USA) and stored at −70°C until RNA isolation. Samples were thawed at room temperature and homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA isolation was performed in accordance with the manufacturer's protocol. The total RNA concentration was measured using a NanoDrop ND-1000 Spectrometer (NanoDrop Technologies Inc., Montchanin, DE, USA). RNA quality was determined by chip-based capillary electrophoresis using Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). Reverse transcription (RT) was performed using Omniscript reverse transcriptase (Qiagen Inc.) at 37°C for 60 min. Reverse transcriptase reactions were carried out in the presence of an RNase inhibitor (rRNAsin; Promega, Madison, WI, USA) and oligo(dT)12–18 primer (Invitrogen).

Quantitative PCR

The qPCR reactions were performed using Assay-On-Demand TaqMan probes: Fkbp5 (Mm00487401_m1), Tsc22d3 (Mm00726417_s1), Nfkbia (Mm00477798_m1), Plat (Mm00476931_m1), Pdyn (Mm00457573_m1), Crhr1 (Mn00432670_m1), Hprt (Mm00446968_m1) (Applied Biosystems, Carlsbad, CA, USA) and run on the CFX96 Touch Real-Time PCR (BioRad, Hercules, CA, USA). cDNAs were diluted 1:10 with H2O, and approximately 50 ng of the cDNA synthesized from the total RNA template from each animal was used for each reaction. To reduce contaminating genomic DNA contribution, primers were designed to span exon junctions. In addition, control reactions without the RT enzyme were performed for each assay. Amplification efficiency for each assay was determined by running a standard dilution curve. The expression of the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) transcript was quantified at a stable level between the strains and after the treatment to control for variation in cDNA amounts. The cycle threshold values were calculated automatically by CFX Manager v.2.1 software with default parameters. RNA abundance was calculated as 2−(threshold cycle).

Data analysis

Foot shock sensitivity was examined with one-way analysis of variance (anova) followed by Neuman-Keuls post hoc using strain as a factor (C57BL/6J; DBA/2J; 129P3/J; SWR/J). The CF and SF tests were analysed using two-way anova followed by Neuman-Keuls post hoc test with strain (C57BL/6J; DBA/2J; 129P3/J; SWR/J) and treatment (control; day 1; 14; 28) or day (day 1; 14; 28) and test (CF; new context; SF) as factors. The remaining behavioural data were analysed using two-way anova followed by Neuman-Keuls post hoc test with strain (C57BL/6J; DBA/2J; SWR/J; 129P3/J) and treatment (control; shocked group) as factors. qPCR data were analysed using two-way anova followed by Neuman-Keuls post hoc test using brain region (amygdala and hippocampus) and treatment (control; 1 or 4 h after shock) as factors. Detailed statistical analysis is presented in Table S1–S4. The statistical threshold was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Sensitivity to foot shock

Test for foot shock sensitivity was used in order to assess whether pain perception differs between the selected strains. One-way anova revealed that 129P3/J strain had higher pain threshold than the remaining three strains that did not differ (F3,51) = 59,217; P < 0.001) (Fig. 2).

image

Figure 2. Foot shock sensitivity in four inbred mice strains. Data are presented as the mean ± SEM of current intensity at which the animals are jumping or vocalizing. N = 13–14. Significant differences between 129P3/J and the three remaining strains was marked with ‘*’ (***P < 0.001).

Download figure to PowerPoint

Test of CF and SF

The test of CF was used to measure fear responses to trauma-related cues. The test of SF assessed hyperarousal by measuring freezing in response to a neutral sound. Two-way anovas revealed a significant effect of strain × treatment interaction on CF (F9,121 = 2.25; P < 0.05) and SF (F9,121 = 2.73; P < 0.01). C57BL/6J, DBA/2J and 129P3/J differed significantly (P < 0.001) from their control groups already 1 day after foot shock (Fig. 3a–c), whereas the SWR/J strain developed the lowest ongoing CF response and differed significantly (P < 0.05) from control only 28 days after foot shock (Fig. 3d). All shocked mice froze more in response to a neutral sound than the control ones did (P < 0.001). Additionally, the C57BL/6J strain displayed increased freezing over time (day 1 < day 28, P < 0.001). There were no significant differences between control groups in both tests. Importantly, mice of all strains froze significantly more to the tone than during the pre-tone period (two-way anova followed by Neuman-Keuls post hoc separately for each strain; data presented in Fig. 3 and Table S4). This supports the concept that freezing to the neutral sound (SF) may reflect non-associative memory processes.

image

Figure 3. Conditioned fear (CF), freezing to pre-tone period (new context) and sensitized fear (SF) in four inbred mouse strains. Freezing responses to shock chamber (CF), pre-tone period (new context) and tone period (SF) were analysed in the same animals during one time period (day 1, 14 or 28) belonging to three independent subgroups of each inbred mouse strain. Data are presented as the mean ± SEM of time of freezing; N = 8–10. Control groups did not differ between strains. Significant differences in comparison to controls were marked with ‘*’ (***P < 0.001, *P < 0.05). Significant difference within strain C57BL/6J in comparison to day 1 group was marked with ‘&’ (&&&P < 0.001). Significant differences between ‘new context’ and two remaining tests were marked with ‘$’ ($$$P < 0.001, $$P < 0.01).

Download figure to PowerPoint

Social interaction test

The SIT was employed in order to measure detachment from others (one aspect of numbing of the general responsiveness). Analysis of interaction in four inbred mouse strains using a two-way anova showed a significant effect for strain (F3,56 = 5.51, P < 0.01). Statistical analysis of avoidance revealed a significant strain × treatment interaction effect (F3,56 = 3.81, P < 0.05). The electric foot shock intensified the avoidance in the DBA/2J strain (P < 0.001 vs. control). Control groups did not differ among the four inbred strains (Fig. 4b).

image

Figure 4. The SIT in four inbred mouse strains. Thirty days after shock application, mice were analysed for social interaction (a) and avoidance (b). Data are presented as the mean ± SEM of the time of interaction/avoidance [seconds]; N = 8. Significant differences in comparison to control were marked with ‘*’ (***P < 0.001).

Download figure to PowerPoint

Forced swimming test

The FST was performed to analyse coping strategies and depression-like behaviour (another aspect of numbing of the general responsiveness). A significant effect of strain × treatment interaction (F3,56 = 7.85, P < 0.001) was observed for floating. Shock increased floating in C57BL/6J and SWR/J mice (Fig. 5a). Two-way analyses of variance anova revealed the effect of strain as the only significant effect for both struggling (F3,56 = 11.66, P < 0.001) and swimming (F3,56 = 13.82, P < 0.001) (Fig. 5b,c).

image

Figure 5. The FST in four inbred mouse strains. Thirty-one days after shock application, floating (a), struggling (b) and swimming (c) in the FST test were measured. Data are presented as the mean ± SEM of time of floating/struggling/swimming; N = 8. Significant differences in comparison to control were marked with ‘*’ (**P < 0.01).

Download figure to PowerPoint

Morphine-induced CPP

In order to examine how an aversive encounter affects the rewarding properties of opioids, a morphine-induced CPP was employed. A two-way anova showed a significant effect for strain × treatment interaction on the CPP score (F3,81 = 4.06, P < 0.01). The exposure to traumatic stress increased sensitivity to the rewarding properties of morphine in 129P3/J mice. The control groups from all strains did not differ (Fig. 6).

image

Figure 6. Morphine-induced CPP in four inbred mouse strains. Thirty-two days after the foot shock, mice were tested for morphine-induced CPP. The difference (delta) between times spent in the drug- and vehicle-paired compartment during the post-conditioning test was considered to be the measure of CPP (CPP score). Data are presented as the mean ± SEM; N = 6–22. Significant differences in comparison to control were marked with ‘*’ (**P < 0.01).

Download figure to PowerPoint

Stress-induced regulation of the mRNA level of selected genes in the amygdala and hippocampus

The qPCR method was used to evaluate the influence of the foot shock application on the mRNA expression of selected genes. Our analysis found increased levels of Nfkbia and Tsc22d3 transcripts 1 h after foot shock among three strains: C57BL/6J, DBA/2J and 129P3/J. Moreover, we observed significant up-regulation of Plat in both brain regions in C57BL/6J and only in amygdala in DBA/2J. Increased expression of Fkbp5 was found in DBA/2J and Crhr1 in C57BL/6J. In the SWR/J strain, strong up-regulation of Pdyn in amygdala was detected. Four hours after foot shock, all the alterations in mRNA abundance levels returned to the basal level (Fig. S1). The expression level of Hprt1 was stable among the strains and after the treatment. For results and statistical analysis see Fig. 7 and Table S1 and S2.

image

Figure 7. Stress-induced regulation of the expression of selected genes by quantitative PCR (qPCR) in amygdala and hippocampus. Animals were sacrificed 1 h after shock application. Results are presented separately for each strain (a, b, c and d) as the fold change over the control group with standard errors (n = 4–8). Significant treatment effects were marked with ‘#’(###P < 0.001, ##P < 0.01, #P < 0.05), significant treatment-region interaction effects were marked with ‘$’ ($ P < 0.05) and‘*’ indicates significant up-regulation of specific gene in specific brain region vs. control group (**P < 0.01, *P < 0.05).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Psychiatric diseases are affected by complex interactions between genes and diverse environmental factors that modify expression of these genes. One of such factors is stress. Inter-strain dissimilarity in PTSD-like symptoms examined in this study might reflect the individual differences in susceptibility to PTSD in humans.

Our results of CF and SF are consistent with other studies showing fear responses in the C57BL/6J strain (Radulovic et al. 1998; Schimanski et al. 2007; Siegmund & Wotjak 2007b). In accordance with previous papers (Belzung et al. 2001; Cohen et al. 2008), we suggest that a strong stressor is able to produce the same level of fear in C57BL/6J and DBA/2J mice. A constant level of fear in the 129P3/J mice can be explained by the fact that this strain exhibits poor fear extinction (Camp et al. 2009) and no habituation in anxiety-related behaviour (Salomons et al. 2010). The most intriguing observation from our experiments is the presence of an irregular pattern of associative and non-associative fear responses in SWR/J mice. There are explanations for such results. First, hyperarousal does not depend on trauma-related contextual memory, and therefore may lead to a different level of freezing (Siegmund & Wotjak 2007a). Moreover, SWR/J mice may exhibit a more ‘active’ response to stress by escaping or attacking, rather than freezing to trauma-related cue. We also conclude that low level of CF within SWR/J strain may not result from the perception of pain (i.e. the electric foot shock) as its foot shock sensitivity was comparable to those displayed by C57BL/6J and DBA/2J.

According to manifestations of numbing of general responsiveness after trauma, DBA/2J was the only strain that markedly increased avoidance. These results are comparable to previous data showing avoiding interaction in DBA/2J (Moy et al. 2008; Uchiumi et al. 2008). Low avoidance and high social interaction observed in SWR/J mice confirm their strong activity and may correspond to sociability and tendency to attacking described previously (Moy et al. 2008). Increased floating in the FST in the C57BL/6J strain suggests that these mice are more susceptible to post-shock depressive-like behaviour. However, in SWR/J mice increased floating may not be related to depression due to the co-occurring high level of struggling. We suggest that SWR/J use both passive and active coping strategies with stress.

Finally, we explored the comorbidity of PTSD and addiction, which is often observed in humans (Chilcoat & Breslau 1998; Jacobsen et al. 2001). Recent evidence suggests that uncontrollable stress (inescapable shock) increases the effect of morphine-induced CPP (Rozeske et al. 2009). Low morphine-induced CPP before and after stress in SWR/J mice confirmed our hypothesis that this strain might be resilient to the long-term behavioural consequences of trauma. As it has been previously reported, the 129P3/J strain has low morphine tolerance (Kest et al. 2002) and low physical dependence (Kest et al. 2002). Strong morphine preference after foot shock suggests that this strain can be used in models of susceptibility to drug addiction after stress.

By using various behavioural tests, we were able to obtain a model that corresponds to Diagnostic and Statistical Manual of Mental Disorders, fourth edition, text revision (DSM-IV-TR) criteria and reveals a wide profile of PTSD-like symptoms. Our study may provide information about molecular mechanisms that initiate long-term behavioural symptoms by revealing alterations in gene expression, discussed below.

We analysed genes potentially involved in different stress-related mechanisms. In our model we observed moderate increases in the Fkbp5 and Crhr1 mRNA levels only in individual mouse strains – Fkbp5 in DBA/2J and Crhr1 in C57BL/6J strain. Alterations in expression of those two genes can correspond to the specific symptoms of PTSD, i.e. up-regulation of Fkbp5 may be related to the increase in post-traumatic avoidance typical for the DBA/2J strain. Accordingly, the link between genetic variation in Fkbp5 gene and harm avoidance in humans was previously found (Shibuya et al. 2010). We also suggest that the increase of Crhr1 in C57BL/6J may correspond to accumulation of SF. It has been recently shown that Crhr1-deficient mice have reduced SF (Thoeringer et al. 2012).

We found comparable increases in the expression of Tsc22d3 and Nfkbia in C57BL/6J, DBA/2J and 129P3/J strains. Tsc22d3 is a marker of glucocorticoid action and has already been considered in the context of its neuronal function (Ayroldi & Riccardi 2009; Korostynski et al. 2007; Yachi et al. 2007). Our recent studies suggest that Tsc22d3 knockdown provokes changes in spine morphology (Piechota et al. 2010). Therefore, based on alterations in expression ofTsc22d3 we hypothesize there to be an involvement of this gene in vulnerability to long-lasting consequences of traumatic stress. It was also shown that the NF-κBpathway mediates cortical neurons remodelling and dendrite branching through interaction with Notch signalling (Bonini et al. 2011). Our results, in combination with current literature, suggest that the NF-κB pathway may act as a central integrator of the stress response with cell plasticity (Memet 2006). A rapid increase in the expression of the NF-κB protein inhibitor, probably in response to NF-κB activation, may further support this hypothesis. Plat gene, up-regulated in C57BL/6J and DBA/2J mice, has been previously associated with experience-induced synaptic plasticity (Shiosaka 2004; Skrzypiec et al. 2008). The stress-induced up-regulation of Tsc22d3, Nfkbia and Plat genes in the amygdala and hippocampus may indicate activation of the three independent molecular pathways. Interestingly, prodynorphin, previously described as a marker of stress (Knoll & Carlezon 2010), was up-regulated in amygdala only in the SWR/J strain. However, unaffected behaviour and transcription of the remaining genes suggest that the SWR/J strain may be resilient to traumatic stress. We hypothesized that in case of SWR/J mice Pdyn expression is a marker of stress resistance. Reduction of Pdyn expression in amygdala has been previously found in patients with major depression (Hurd 2002). Mice lacking dynorphin had an enhanced cue-dependent fear conditioning and delayed extinction (Bilkei-Gorzo et al. 2012). Thus, it is possible that Pdyn up-regulation in response to a traumatic event may protect against development of stress-related psychiatric disorders.

We are convinced that three strains (C57BL/6J, DBA/2J and 129P3/J) are more vulnerable to traumatic stress than the SWR/J strain is. The data previously published (Cohen et al. 2008) indicated that C57BL/6J displayed a greater corticosterone response to stress than other inbred strains studied and had greater persistence of hippocampal long-term potentiation (Matsuyama et al. 1997). Both C57BL/6J and DBA/2J revealed the most pronounced changes in shock-induced gene expression and they appear to provide an adequate model of PTSD behavioural symptoms – particularly, C57BL/6J for modelling of hyperarousal and depressive-like behaviour, and DBA/2J for modelling of social withdrawal.

To the best of our knowledge, we are the first group to investigate SWR/J mice in an animal model of PTSD and reveal their lack of susceptibility to trauma, especially in two clusters of symptoms: re-experiencing (weak fear responses to trauma-related cues) and avoidance/numbing of general responsiveness (lack of social withdrawal and flexibility in coping strategies with stress). Although poor retention of CF may be considered in the context of stress vulnerability (Acheson et al. 2011), fear response to trauma-related internal or external cues is one of the criteria required for PTSD diagnosis (Nemeroff et al. 2006; Siegmund & Wotjak 2006; Yehuda & LeDoux 2007). The absence of PTSD-like behavioural symptoms and the lack of changes in expression of HPA axis activity-dependent molecular markers make the SWR/J strain potentially ‘stress-proof’. Considering that the pre-existing factors are able to influence not only vulnerability, but also resilience-related traits, we identified the SWR/J strain as a valuable model of stress resistance.

To summarize, our data supports the idea that genetic factors are important for phenotypic variation in response to stress. We propose candidate genes (Tsc22d3, Nfkbia and Plat) that may play a dynamic role in the development of long-lasting consequences of traumatic event. Conversely, we hypothesize that up-regulation of the Pdyn transcript in response to stress may provide resistance to anxiety disorders. In order to generate new strategies for the diagnosis and efficient therapy of PTSD, future research should be focused on objective parameters of the disease, such as changes in transcription of genes and interactions between diverse signalling pathways.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
  • Acheson, D.T., Gresack, J.E. & Risbrough, V.B. (2011) Hippocampal dysfunction effects on context memory: possible etiology for posttraumatic stress disorder. Neuropharmacology 62, 674685.
  • Adamec, R., Holmes, A. & Blundell, J. (2008) Vulnerability to lasting anxiogenic effects of brief exposure to predator stimuli: sex, serotonin and other factors-relevance to PTSD. Neurosci Biobehav Rev 32, 12871292.
  • Amstadter, A.B., Nugent, N.R., Yang, B.Z., Miller, A., Siburian, R., Moorjani, P., Haddad, S., Basu, A., Fagerness, J., Saxe, G., Smoller, J.W. & Koenen, K.C. (2011) Corticotrophin-releasing hormone type 1 receptor gene (CRHR1) variants predict posttraumatic stress disorder onset and course in pediatric injury patients. Dis Markers 30, 8999.
  • Ayroldi, E. & Riccardi, C. (2009) Glucocorticoid-induced leucine zipper (GILZ): a new important mediator of glucocorticoid action. FASEB J 23, 36493658.
  • Belzung, C., El Hage, W., Moindrot, N. & Griebel, G. (2001) Behavioral and neurochemical changes following predatory stress in mice. Neuropharmacology 41, 400408.
  • Bilkei-Gorzo, A., Erk, S., Schurmann, B., Mauer, D., Michel, K., Boecker, H., Scheef, L., Walter, H. & Zimmer, A. (2012) Dynorphins regulate fear memory: from mice to men. J Neurosci 32, 93359343.
  • Binder, E.B. (2009) The role of FKBP5, a co-chaperone of the glucocorticoid receptor in the pathogenesis and therapy of affective and anxiety disorders. Psychoneuroendocrinology 34 (Suppl. 1), S186S195.
  • Bonini, S.A., Ferrari-Toninelli, G., Uberti, D., Montinaro, M., Buizza, L., Lanni, C., Grilli, M. & Memo, M. (2011) Nuclear factor κB-dependent neurite remodeling is mediated by notch pathway. J Neurosci 31, 1169711705.
  • Camp, M., Norcross, M., Whittle, N., Feyder, M., D'Hanis, W., Yilmazer-Hanke, D., Singewald, N. & Holmes, A. (2009) Impaired Pavlovian fear extinction is a common phenotype across genetic lineages of the 129 inbred mouse strain. Genes Brain Behav 8, 744752.
  • Chilcoat, H.D. & Breslau, N. (1998) Posttraumatic stress disorder and drug disorders: testing causal pathways. Arch Gen Psychiatry 55, 913917.
  • Cohen, H., Geva, A.B., Matar, M.A., Zohar, J. & Kaplan, Z. (2008) Post-traumatic stress behavioural responses in inbred mouse strains: can genetic predisposition explain phenotypic vulnerability? Int J Neuropsychopharmacol 11, 331349.
  • Cohen, H., Kozlovsky, N., Alona, C., Matar, M.A. & Joseph, Z. (2011). Animal model for PTSD: from clinical concept to translational research. Neuropharmacology 62, 715724.
  • Fenoglio, K.A., Chen, Y. & Baram, T.Z. (2006) Neuroplasticity of the hypothalamic-pituitary-adrenal axis early in life requires recurrent recruitment of stress-regulating brain regions. J Neurosci 26, 24342442.
  • Gubner, N.R., Wilhelm, C.J., Phillips, T.J. & Mitchell, S.H. (2010) Strain differences in behavioral inhibition in a Go/No-go task demonstrated using 15 inbred mouse strains. Alcohol Clin Exp Res 34, 13531362.
  • Hurd, Y.L. (2002) Subjects with major depression or bipolar disorder show reduction of prodynorphin mRNA expression in discrete nuclei of the amygdaloid complex. Mol Psychiatry 7, 7581.
  • Jacobsen, L.K., Southwick, S.M. & Kosten, T.R. (2001) Substance use disorders in patients with posttraumatic stress disorder: a review of the literature. Am J Psychiatry 158, 11841190.
  • Kamprath, K. & Wotjak, C.T. (2004) Nonassociative learning processes determine expression and extinction of conditioned fear in mice. Learn Mem 11, 770786.
  • Kaouane, N., Porte, Y., Vallee, M., Brayda-Bruno, L., Mons, N., Calandreau, L., Marighetto, A., Piazza, P.V. & Desmedt, A. (2012) Glucocorticoids can induce PTSD-like memory impairments in mice. Science 335, 15101513.
  • Kest, B., Hopkins, E., Palmese, C.A., Adler, M. & Mogil, J.S. (2002a) Genetic variation in morphine analgesic tolerance: a survey of 11 inbred mouse strains. Pharmacol Biochem Behav 73, 821828.
  • Kest, B., Palmese, C.A., Hopkins, E., Adler, M., Juni, A. & Mogil, J.S. (2002b) Naloxone-precipitated withdrawal jumping in 11 inbred mouse strains: evidence for common genetic mechanisms in acute and chronic morphine physical dependence. Neuroscience 115, 463469.
  • Knoll, A.T. & Carlezon, W.A. Jr. (2010) Dynorphin, stress, and depression. Brain Res 1314, 5673.
  • Korostynski, M., Kaminska-Chowaniec, D., Piechota, M. & Przewlocki, R. (2006) Gene expression profiling in the striatum of inbred mouse strains with distinct opioid-related phenotypes. BMC Genomics 7, 146.
  • Korostynski, M., Piechota, M., Kaminska, D., Solecki, W. & Przewlocki, R. (2007) Morphine effects on striatal transcriptome in mice. Genome Biol 8, R128.
  • Mahan, A.L. & Ressler, K.J. (2011). Fear conditioning, synaptic plasticity and the amygdala: implications for posttraumatic stress disorder. Trends Neurosci.
  • Maren, S. (1996) Synaptic transmission and plasticity in the amygdala. An emerging physiology of fear conditioning circuits. Mol Neurobiol 13, 122.
  • Matsuyama, S., Namgung, U. & Routtenberg, A. (1997) Long-term potentiation persistence greater in C57BL/6 than DBA/2 mice: predicted on basis of protein kinase C levels and learning performance. Brain Res 763, 127130.
  • Mattson, M.P., Culmsee, C., Yu, Z. & Camandola, S. (2000) Roles of nuclear factor kappaB in neuronal survival and plasticity.J Neurochem 74, 443456.
  • McEwen, B.S., Eiland, L., Hunter, R.G. & Miller, M.M. (2011). Stress and anxiety: structural plasticity and epigenetic regulation as a consequence of stress. Neuropharmacology 62, 312.
  • Memet, S. (2006) NF-kappaB functions in the nervous system: from development to disease. Biochem Pharmacol 72, 11801195.
  • Mikics, E., Baranyi, J. & Haller, J. (2008) Rats exposed to traumatic stress bury unfamiliar objects--a novel measure of hyper-vigilance in PTSD models? Physiol Behav 94, 341348.
  • Milner, L.C. & Crabbe, J.C. (2008) Three murine anxiety models: results from multiple inbred strain comparisons. Genes Brain Behav 7, 496505.
  • Moy, S.S., Nadler, J.J., Young, N.B., Nonneman, R.J., Segall, S.K., Andrade, G.M., Crawley, J.N. & Magnuson, T.R. (2008) Social approach and repetitive behavior in eleven inbred mouse strains. Behav Brain Res 191, 118129.
  • Nemeroff, C.B., Bremner, J.D., Foa, E.B., Mayberg, H.S., North, C.S. & Stein, M.B. (2006) Posttraumatic stress disorder: a state-of-the-science review. J Psychiatr Res 40, 121.
  • Olff, M., Langeland, W. & Gersons, B.P. (2005) The psychobiology of PTSD: coping with trauma. Psychoneuroendocrinology 30, 974982.
  • Ottoni, E.B. (2000) EthoLog 2.2: a tool for the transcription and timing of behavior observation sessions. Behav Res Methods Instrum Comput 32, 446449.
  • Pang, P.T., Teng, H.K., Zaitsev, E., Woo, N.T., Sakata, K., Zhen, S., Teng, K.K., Yung, W.H., Hempstead, B.L. & Lu, B. (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306, 487491.
  • Paulus, M.P., Dulawa, S.C., Ralph, R.J. & Mark, A.G. (1999) Behavioral organization is independent of locomotor activity in 129 and C57 mouse strains. Brain Res 835, 2736.
  • Pawlak, R., Magarinos, A.M., Melchor, J., McEwen, B. & Strickland, S. (2003) Tissue plasminogen activator in the amygdala is critical for stress-induced anxiety-like behavior. Nat Neurosci 6, 168174.
  • Philbert, J., Pichat, P., Beeske, S., Decobert, M., Belzung, C. & Griebel, G. (2011) Acute inescapable stress exposure induces long-term sleep disturbances and avoidance behavior: a mouse model of post-traumatic stress disorder (PTSD). Behav Brain Res 221, 149154.
  • Piechota, M., Korostynski, M., Solecki, W., Gieryk, A., Slezak, M.,Bilecki, W., Ziolkowska, B., Kostrzewa, E., Cymerman, I., Swiech, L., Jaworski, J. & Przewlocki, R. (2010) The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum. Genome Biol 11, R48.
  • Pittenger, C. & Duman, R.S. (2008) Stress, depression, and neuroplasticity: a convergence of mechanisms. Neuropsychopharmacology 33, 88109.
  • Radley, J.J., Kabbaj, M., Jacobson, L., Heydendael, W., Yehuda, R. & Herman, J.P. (2011) Stress risk factors and stress-related pathology: Neuroplasticity, epigenetics and endophenotypes. Stress 14, 481497.
  • Radulovic, J., Kammermeier, J. & Spiess, J. (1998) Generalization of fear responses in C57BL/6 N mice subjected to one-trial foreground contextual fear conditioning. Behav Brain Res 95, 179189.
  • Romano, A., Freudenthal, R., Merlo, E. & Routtenberg, A. (2006) Evolutionarily-conserved role of the NF-kappaB transcription factor in neural plasticity and memory. Eur J Neurosci 24, 15071516.
  • Rozeske, R.R., Der-Avakian, A., Bland, S.T., Beckley, J.T., Watkins, L.R. & Maier, S.F. (2009) The medial prefrontal cortex regulates the differential expression of morphine-conditioned place preference following a single exposure to controllable or uncontrollable stress. Neuropsychopharmacology 34, 834843.
  • Salomons, A.R., van Luijk, J.A., Reinders, N.R., Kirchhoff, S., Arndt, S.S. & Ohl, F. (2010) Identifying emotional adaptation: behavioural habituation to novelty and immediate early gene expression in two inbred mouse strains. Genes Brain Behav 9, 110.
  • Schimanski, L.A., Ali, D.W., Baker, G.B. & Nguyen, P.V. (2007) Impaired hippocampal LTP in inbred mouse strains can be rescued by beta-adrenergic receptor activation. Eur J Neurosci 25, 15891598.
  • Shibuya, N., Suzuki, A., Sadahiro, R., Kamata, M., Matsumoto, Y., Goto, K., Hozumi, Y. & Otani, K. (2010) Association study between a functional polymorphism of FK506-binding protein 51 (FKBP5) gene and personality traits in healthy subjects. Neurosci Lett 485, 194197.
  • Shiosaka, S. (2004) Serine proteases regulating synaptic plasticity. Anat Sci Int 79, 137144.
  • Siegmund, A. & Wotjak, C.T. (2006) Toward an animal model of posttraumatic stress disorder. Ann N Y Acad Sci 1071, 324334.
  • Siegmund, A. & Wotjak, C.T. (2007a) Hyperarousal does not depend on trauma-related contextual memory in an animal model of posttraumatic stress disorder. Physiol Behav 90, 103107.
  • Siegmund, A. & Wotjak, C.T. (2007b) A mouse model of posttraumatic stress disorder that distinguishes between conditioned and sensitised fear. J Psychiatr Res 41, 848860.
  • Siegmund, A., Langnaese, K. & Wotjak, C.T. (2005) Differences in extinction of conditioned fear in C57BL/6 substrains are unrelated to expression of alpha-synuclein. Behav Brain Res 157, 291298.
  • Skrzypiec, A.E., Buczko, W. & Pawlak, R. (2008) Tissue plasminogen activator in the amygdala: a new role for an old protease. J Physiol Pharmacol 59 (Suppl. 8), 135146.
  • Solecki, W., Turek, A., Kubik, J. & Przewlocki, R. (2009) Motivational effects of opiates in conditioned place preference and aversion paradigm--a study in three inbred strains of mice. Psychopharmacology (Berl) 207, 245255.
  • Tang, X., Orchard, S.M. & Sanford, L.D. (2002) Home cage activity and behavioral performance in inbred and hybrid mice. Behav Brain Res 136, 555569.
  • Thoeringer, C.K., Henes, K., Eder, M., Dahlhoff, M., Wurst, W., Holsboer, F., Deussing, J.M., Moosmang, S. & Wotjak, C.T. (2012) Consolidation of remote fear memories involves Corticotropin-Releasing Hormone (CRH) receptor type 1-mediated enhancement of AMPA receptor GluR1 signaling in the dentate gyrus. Neuropsychopharmacology 37, 787796.
  • Touma, C., Gassen, N.C., Herrmann, L., Cheung-Flynn, J., Bull, D.R., Ionescu, I.A., Heinzmann, J.M., Knapman, A., Siebertz, A., Depping, A.M., Hartmann, J., Hausch, F., Schmidt, M.V., Holsboer, F., Ising, M., Cox, M.B., Schmidt, U. & Rein, T. (2011) FK506 binding protein 5 shapes stress responsiveness: modulation of neuroendocrine reactivity and coping behavior. Biol Psychiatry 70, 928936.
  • Tzschentke, T.M. (2007) Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade. Addict Biol 12, 227462.
  • Uchiumi, K., Aoki, M., Kikusui, T., Takeuchi, Y. & Mori, Y. (2008) Wheel-running activity increases with social stress in male DBA mice. Physiol Behav 93, 17.
  • Wang, X.D., Rammes, G., Kraev, I., Wolf, M., Liebl, C., Scharf, S.H., Rice, C.J., Wurst, W., Holsboer, F., Deussing, J.M., Baram, T.Z., Stewart, M.G., Muller, M.B. & Schmidt, M.V. (2011) Forebrain CRF(1) modulates early-life stress-programmed cognitive deficits. J Neurosci 31, 1362513634.
  • Wang, X.D., Labermaier, C., Holsboer, F., Wurst, W., Deussing, J.M., Muller, M.B. & Schmidt, M.V. (2012). Early-life stress-induced anxiety-related behavior in adult mice partially requires forebrain corticotropin-releasing hormone receptor 1. Eur J Neurosci 36, 23602367.
  • Wittmann, W., Schunk, E., Rosskothen, I., Gaburro, S., Singewald, N., Herzog, H. & Schwarzer, C. (2009) Prodynorphin-derived peptides are critical modulators of anxiety and regulate neurochemistry and corticosterone. Neuropsychopharmacology 34, 775785.
  • Yachi, K., Inoue, K., Tanaka, H., Yoshikawa, H. & Tohyama, M. (2007) Localization of glucocorticoid-induced leucine zipper (GILZ) expressing neurons in the central nervous system and its relationship to the stress response. Brain Res 1159, 141147.
  • Yang, N., Zhang, W. & Shi, X.M. (2008) Glucocorticoid-induced leucine zipper (GILZ) mediates glucocorticoid action and inhibits inflammatory cytokine-induced COX-2 expression. J Cell Biochem 103, 17601771.
  • Yeh, S.H., Lin, C.H. & Gean, P.W. (2004) Acetylation of nuclear factor-kappaB in rat amygdala improves long-term but not short-term retention of fear memory. Mol Pharmacol 65, 12861292.
  • Yehuda, R. & LeDoux, J. (2007) Response variation following trauma: a translational neuroscience approach to understanding PTSD. Neuron 56, 1932.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

This work was supported by Polish NSC grant 2011/03/B/NZ2/02479, Polish MSHE grant NN405 274137 and POIG De-Me-Ter 3.1, IUVENTUS Plus. Authors declare no conflict of interest.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
FilenameFormatSizeDescription
gbb850-sup-0001-FigureS1.docWord document59KFigure S1: Stress-induced regulation of the expression of selected genes in amygdala and hippocampus 4 h after the foot shock. Results are presented separately for each strain (a, b, c and d) as the fold change over the control group with standard errors (n = 4–8). Two-way anova with strain (C57BL/6J, DBA/2J, SWR/J and 129P3/J) and treatment (control, shocked group) as factors did not reveal any significant alternations.
gbb850-sup-0002-TableS1.docWord document169KTable S1: Analysis of variance for gene expression in different strains and brain samples collected 1 h after the foot shock.
gbb850-sup-0003-TableS2.docWord document166KTable S2: Analysis of variance for gene expression in different strains and brain samples collected 4 h after the foot shock.
gbb850-sup-0004-TableS3.docWord document63KTable S3: Analysis of variance for foot shock sensitivity and shock-induced different types of behaviour.
gbb850-sup-0005-TableS4.docWord document38KTable S4: Statistical analyses of freezing responses to shock chamber, pre-tone and tone period in a new context in four inbred mice strains.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.