Reduced hippocampus volume in the mouse model of Posttraumatic Stress Disorder

Abstract

Some, but not all studies in patients with posttraumatic stress disorder (PTSD), report reduced hippocampus (HPC) volume. In particular it is unclear, whether smaller hippocampal volume represents a susceptibility factor for PTSD rather than a consequence of the trauma. To gain insight into the relationship of brain morphology and trauma exposure, we investigated volumetric and molecular changes of the HPC in a mouse model of PTSD by means of in vivo Manganese Enhanced Magnetic Resonance Imaging (MEMRI) and ex vivo ultramicroscopic measurements. Exposure to a brief inescapable foot shock led to a volume reduction in both left HPC and right central amygdala two months later. This volume loss was mirrored by a down-regulation of growth-associated protein-43 (GAP43) in the HPC. Enriched housing decreased the intensity of trauma-associated contextual fear, independently of whether it was provided before or after the shock. Beyond that, enriched housing led to an increase in intracranial volume, including the lateral ventricles and the hippocampus, and to an up-regulation of GAP43 as revealed by MEMRI and Western blot analysis, thus partially compensating for trauma-related HPC volume loss and down-regulation of GAP43 expression. Together these data demonstrate that traumatic experience in mice causes a reduction in HPC and central amygdala volume possibly due to a shrinkage of axonal protrusions. Enriched housing might induce trophic changes, which may contribute to the amelioration of trauma-associated PTSD-like symptoms at behavioural, morphological and molecular levels.

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.