Christoph P. Mauch

Three-dimensional imaging of solvent-cleared organs using 3DISCO.

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in ∼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.

Reduced hippocampus volume in the mouse model of Posttraumatic Stress Disorder

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.

Consequences of extinction training on associative and non-associative fear in a mouse model of Posttraumatic Stress Disorder (PTSD)

A common approach to the clinical treatment of Posttraumatic Stress Disorder (PTSD) has focused on the facilitation of fear extinction through cognitive behavioural therapy that involves both safe exposure to the trauma-related cues and subsequent changes in conditioned stimulus-unconditioned stimulus (CS-US) contingency expectations. PTSD symptoms can be tracked back to pathologically modified associative fear, hyperarousal and a time-dependent fear generalization. We have used a mouse model of PTSD that is based on a brief exposure to an inescapable foot shock in order to investigate the influence of early (starting 1 day after the shock) and late (starting 1 month after the shock) extinction training. Both early and late extinction training led to a long-lasting reduction of contextual and generalized fear, but only early extinction caused an amelioration of hyperarousal. Consequently, our results suggest early post-shock intervention as a successful strategy for reducing hyperarousal in the aftermath of a trauma.

Prolonged fear incubation leads to generalized avoidance behavior in mice

Long-lasting presence of avoidance and emotional numbing are reliable behavioral markers for PTSD, but little is known about its psychological and biological underpinnings. We employed our recently established mouse model of PTSD (i) to study the emergence of avoidance behavior in the aftermath of a trauma, (ii) to disentangle the impact of context generalization vs. lack of motivation vs. novelty fear and (iii) to assess the therapeutic value of benzodiazepines and selective serotonin reuptake inhibitors (SSRIs). Specific conditioned avoidance to shock-paired odor turned into generalized avoidance after 28 days of fear incubation. Combination of habituation to the novel environment and extinction of contextual fear abolished both generalized and specific avoidance behavior. Chronic fluoxetine treatment partially reversed the phenotype, whereas acute treatment with diazepam did not. Our animal model may help understanding the mechanisms underlying psychological and biological mechanisms of PTSD for the benefit of developing pharmacotherapeutic strategies, which specifically address generalized avoidance.

Increased levels of conditioned fear and avoidance behavior coincide with changes in phosphorylation of the protein kinase B (AKT) within the amygdala in a mouse model of extremes in trait anxiety

Patients diagnosed for anxiety disorders often display faster acquisition and slower extinction of learned fear. To gain further insights into the mechanisms underlying these phenomenona, we studied conditioned fear in mice originating form a bi-directional selective breeding approach, which is based on elevated plus-maze behavior and results in CD1-derived high (HAB), normal (NAB), and low (LAB) anxiety-related behavior mice. HAB mice displayed pronounced cued-conditioned fear compared to NAB/CD1 and LAB mice that coincided with increased phosphorylation of the protein kinase B (AKT) in the basolateral amygdala 45 min after conditioning. No similar changes were observed after non-associative immediate shock presentations. Fear extinction of recent but not older fear memories was preserved. However, HAB mice were more prone to relapse of conditioned fear with the passage of time. HAB mice also displayed higher levels of contextual fear compared to NAB and LAB mice and exaggerated avoidance following step-down avoidance training. Interestingly, HAB mice showed lower and LAB mice higher levels of acoustic startle responses compared to NAB controls. The increase in arousal observed in LAB mice coincided with the general absence of conditioned freezing. Taken together, our results suggest that the genetic predisposition to high anxiety-related behavior may increase the risk of forming traumatic memories, phobic-like fear and avoidance behavior following aversive encounters, with a clear bias towards passive coping styles. In contrast, genetic predisposition to low anxiety-related and high risk-taking behavior seems to be associated with an increase in active coping styles. Our data imply changes in AKT phosphorylation as a therapeutic target for the prevention of exaggerated fear memories.

Biomimetic screening of class B G protein-coupled receptors

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF(1)R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF(1) receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure-activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC(50) = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.

P 227. Ultramicroscopy (UM) in neurobiology

UM is a prime example for an interdisciplinary research field invention. It enables researchers to obtain high-resolution 3D-reconstructions of organ systems in different animal models. Knowledge about the interconnections of the neuronal network to the vascular system is often essential neurobiological research. Ultramicroscopy (UM) is a light sheet based fluorescence microscopy. This novel bioimaging technique allows the 3D-visualization of cm-sized biological specimens with μm-resolution ( [Dodt et al., 2007] , [Jahrling, 2011] ). Artifacts of conventional microscopy such as distortion of the tissue are avoided. It is due to optical sectioning instead of mechanical sclicing ( [Jahrling and Saghafi, 2011] , [Jahrling, 2011] ). In UM, a specimen is illuminated by a thin sheet of laser light, formed by one or more cylindrical lenses ( [Jahrling and Saghafi, 2011] , [Jahrling, 2011] ). To generate a light sheet distribution in the standard UM, we basically employ a single cylindrical lens placed in front of a variable rectangular slit aperture ( [Jahrling and Saghafi, 2011] , [Jahrling, 2011] ). Lectinis are proteins that bind to sugar complexes, which are attached to proteins and lipids. We employed an approach using fluorescent conjugated lectins during the transcardial perfusion of mice to contrast the endothelium building up the vascular system ( [Jahrling, 2011] , [Jahrling et al., 2009] ). Biological samples are prepared chemically to become as transparent as possible ( Jahrling, 2011 ). In this study the architecture of the blood vessel system of whole organs of animal models is visualized ( [Jahrling, 2011] , [Jahrling et al., 2009] ). By combining light sheet based UM with lectin-labelling, 3D reconstructions of vascular structures of the mouse spinal cord can be generated. Alteration in optics, morphological analysis of neurobiological disorders, multiple labeling, improving histology of clearing procedure, the analysis of brain tumors are subjects of interests are subjects of interests in our future work.