Reduced hippocampus volume in the mouse model of Posttraumatic Stress Disorder
Introduction
Numerous clinical studies reported functional and volumetric changes in the hippocampus (HPC) formation of posttraumatic stress disorder (PTSD) patients, such as lower levels of N-acetylaspartate (a marker of neuronal integrity) (Schuff et al., 2001, Mohanakrishnan et al., 2003), smaller HPC volume, impaired performance in HPC-dependent memory tasks and altered activity in hippocampal/parahippocampal regions during memory processing (Gilbertson et al., 2007, Bremner et al., 2008, Geuze et al., 2008, Werner et al., 2009). Long-term SSRI treatment not only improved verbal declarative memory, but also increased HPC volume in PTSD patients (Vermetten et al., 2003). However, not all studies observed changes in HPC volume (e.g. De Bellis et al., 2001; Carrion et al., 2001, Fennema-Notestine et al., 2002). In particular two longitudinal studies with a 6-months or 2-years follow-up period failed to reveal reductions in HPC volume in the aftermath of a traumatic event (Bonne et al., 2001, De Bellis et al., 2001). In addition, it remains unclear whether volumetric and functional changes of HPC are related to PTSD symptomatology or to mere trauma exposure. For instance, Winter and Irle, (2004) failed to find a difference between healthy trauma-exposed individuals and trauma-exposed PTSD patients, arguing that smaller HPC size is a result of traumatic incidence and not a specific condition of PTSD. However, a challenging argument against trauma-related volume loss of the HPC came from a study with monozygotic twins, where the authors observed a strong association between HPC volume and the prevalence of PTSD in genetically identical twins discordant for Vietnam combat exposure and PTSD, suggesting that smaller HPC volume may represent a risk factor for development of PTSD rather than a marker of pathophysiology per se (Gilbertson et al., 2002).
The controversies of clinical studies might be explained by a large number of limitations immanent to such investigations, such as small sample sizes with subsequent low statistical power, methodological heterogeneity and sample heterogeneity in terms of type and severity of the trauma, incubation time, psychiatric co-morbidities and medical treatment. These constrains can be avoided in animal studies, where genetically identical animals can be exposed to the same traumatic stimulus, and volumetric changes can be measured at the same time point. In addition, in vivo volumetric assessments by magnetic resonance imaging can be confirmed by ex vivo investigations. Therefore, we employed our recently established animal model of PTSD (Siegmund and Wotjak, 2007) in order to investigate (i) whether a traumatic incident leads to volume reduction of the HPC, (ii) whether volume changes are modifiable by environmental conditions, and (iii) whether molecular markers of anatomical plasticity can mirror these volumetric alterations. As described in detail elsewhere (Siegmund and Wotjak, 2007, Golub et al., 2009), mice received a brief inescapable foot shock, and were tested for three clusters of PTSD-like symptoms one month later: (a) intensity of associative fear (freezing response to the shock context), (b) levels of non-associative fear (hyperarousal as assessed by acoustic startle responses) and (c) specificity of fear (i.e. context discrimination). In vivo Manganese Enhanced Magnetic Resonance Imaging (MEMRI) (Natt et al., 2002, Silva and Bock, 2008) and ex vivo three-dimensional ultramicroscopic visualization (Dodt et al., 2007) were employed in order to assess long-term consequences of the trauma on HPC volume. Furthermore, Western blot analysis was performed to study the expression levels of growth-associated protein-43 (GAP43), a phosphoprotein exclusively expressed in the cell body and axonal processes. GAP43 plays a role in neurite outgrowth and is thought to be involved in synaptogenesis (Goslin et al., 1988, Yankner et al., 1990, Aigner et al., 1995). Studies were performed with mice housed either under standard or enriched conditions in order to investigate the influence of environmental factors on trauma-related changes of behavioural and morphological parameters.
Section snippets
Methods
All experimental procedures were approved by the Committee on Animal Health and Care of the State of Upper Bavaria (Regierung von Oberbayern, Germany; Az. 55.2-1-54-2531-41-09) and performed in strict compliance with the European Union Directive for the care and use of laboratory animals (86/609/EEC).
Trauma-related fear and HPC volume loss in standard-housed mice
Experience of the inescapable foot shock led to the development of contextual fear, fear generalisation and hyperarousal in standard-housed mice of Experiment 1 one month after trauma. Specifically, shocked mice showed increased contextual fear (t = 8.3, df = 30, p < 0.001; Fig. 1A), increased fear to a single context reminder (t = 17.2, df = 30, p < 0.001; Fig. 1B) and increased acoustic startle responses (ASR) [2 (shock) × 5 (INT) ANOVA: F1,28 shock = 11.6, p = 0.002; F1.130, 31.643
Discussion
In the present study we demonstrate by ultramicroscopy that a traumatic experience, such as exposure to a brief inescapable foot shock, led to a decrease in HPC volume. MEMRI measurements confirmed the volume reduction for the left, but not the right, HPC of shocked standard-housed mice. A laterality effect could also be observed in terms of volume reduction of the right central amygdala. Moreover, we show a decrease in the expression levels of GAP43, an axonal marker implied in neurite
Role of the funding
This study has been supported in part by the Hübner Foundation (to C.T.W.). Hübner Foundation had no further role in study design, in the collection, analysis and interpretation of the data; in the writing of the report and in the decision to submit the paper for publication.
Contributors
Yulia Golub designed experiments, prepared experimental protocols, conducted all behavioural experiments and part of MEMRI measurements, analysed behavioural data, carried out the statistical analysis, participated in the data interpretation, wrote the manuscript.
Sebastian F. Kaltwasser conducted MEMRI measurements, processed MEMRI data, undertook the statistical analysis for the MEMRI results, participated in data interpretation, contributed to the preparation of the manuscript.
Christoph P.
Conflicts of interest
All authors disclose no competing interests including financial, personal or other relationships with other people or organisations within three years of beginning the work submitted that could inappropriately influence the present work.
Acknowledgements
We thank Albin Varga (MPI) for his support with enriched housing, Klaus Becker (Technical University Vienna, Austria) for his help with analysis of ultramicroscopic measurements and Barbara Grünecker for her help with the analysis of MRI measurements. This study has been supported in part by the Hübner Foundation (to C.T.W.).
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Current address: Department of Child and Adolescent Mental Health, University of Erlangen-Nürnberg, Germany.