Oliver Schmitt

Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells

Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore, there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl, sodium-valproate, kenpaullone, indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly, kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone, without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications.

Image Registration of Sectioned Brains

The physical (microtomy), optical (microscopy), and radiologic (tomography) sectioning of biological objects and their digitization lead to stacks of images. Due to the sectioning process and disturbances, movement of objects during imaging for example, adjacent images of the image stack are not optimally aligned to each other. Such mismatches have to be corrected automatically by suitable registration methods.Here, a whole brain of a Sprague Dawley rat was serially sectioned and stained followed by digitizing the 20 μm thin histologic sections. We describe how to prepare the images for subsequent automatic intensity based registration. Different registration schemes are presented and their results compared to each other from an anatomical and mathematical perspective. In the first part we concentrate on rigid and affine linear methods and deal only with linear mismatches of the images. Digitized images of stained histologic sections often exhibit inhomogenities of the gray level distribution coming from staining and/or sectioning variations. Therefore, a method is developed that is robust with respect to inhomogenities and artifacts. Furthermore we combined this approach by minimizing a suitable distance measure for shear and rotation mismatches of foreground objects after applying the principal axes transform. As a consequence of our investigations, we must emphasize that the combination of a robust principal axes based registration in combination with optimizing translation, rotation and shearing errors gives rise to the best reconstruction results from the mathematical and anatomical view point.Because the sectioning process introduces nonlinear deformations to the relative thin histologic sections as well, an elastic registration has to be applied to correct these deformations.In the second part of the study a detailed description of the advances of an elastic registration after affine linear registration of the rat brain is given. We found quantitative evidence that affine linear registration is a suitable starting point for the alignment of histologic sections but elastic registration must be performed to improve significantly the registration result. A strategy is presented that enables to register elastically the affine linear preregistered~ rat brain sections and the first one hundred images of serial histologic sections through both occipital lobes of a human brain (6112 images). Additionally, we will describe how a parallel implementation of the elastic registration was realized. Finally, the computed force fields have been applied here for the first time to the morphometrized data of cells determined automatically by an image analytic framework.

Orexinergic innervation of the extended amygdala and basal ganglia in the rat

The orexinergic system interacts with several functional states of emotions, stress, hunger, wakefulness and behavioral arousal through four pathways originating in the lateral hypothalamus (LH). Hundreds of orexinergic efferents have been described by tracing studies and direct immunohistochemistry of orexin in the forebrain, olfactory regions, hippocampus, amygdala, septum, basal ganglia, thalamus, hypothalamus, brain stem and spinal cord. Most of these tracing studies investigated the whole orexinergic projection to all regions of the intracranial part of the CNS. To identify the orexinergic efferents at the subnuclear level of resolution, we focussed on the orexinergic target in the amygdala, which is substantially involved in the LH output and contributes mostly to the functional outcome of the orexinergic system and the basal ganglia. Immunohistochemical identification of axonal orexin A and orexin B in male adult rats has been performed on serial sections. In the extended amygdala many new orexinergic targets were found in the anterior amygdaloid area (dense), anterior cortical nucleus (moderate), amygdalostriatal transition region (moderate), basolateral regions (moderate), basomedial nucleus (moderate), several bed nucleus of the stria terminals regions (few to dense), central amygdaloid subdivisions (dense), posteromedial cortical nucleus (moderate) and medial amygdaloid subnuclei (dense). Furthermore, the entopeduncular nucleus has been newly identified as another target for orexinergic fibers with a high density. These results suggest that subdivisions and subnuclei of the extended amygdala are specific targets of the orexinergic system.

Radial symmetries based decomposition of cell clusters in binary and gray level images

The segmentation of structures of complex cytological and histological images is a necessary intermediate step for image analysis that give rise to binary images. In many cases these binary images can be rather far away from a subsequent object specific quantification because biological structures digitized by optoelectronic devices may situated close together so that they appear as one fused object in the projective image. Such fusions of objects may become complex so that large clusters of biological structures emerge. To quantify individual objects of a cluster they must be separated. The shape, size and intensity variation of cells in complex organs like the brain may breed planar configurations that can be splitted only inadequate by common techniques, e.g., watershed separation or basic morphological processing of images. Considering iteratively object contours suitable features of saliency can be accumulated that give rise to markers of singular objects. Such significant markers may drive a separation process more effective than common approaches. The determination of markers by an iterative method should be scale, translation and rotation invariant and robust with regard to noise due to the variability of biological specimen. We realize a technique that splits cell clumps consisting of different cell sizes and shapes into meaningful parts. The multiscale method applied here is based on the analysis of the contour shape and the object area by iterative voting using oriented kernels. These cone-shaped kernels vote iteratively for the local center of mass of the components of an aggregation. The voting is performed along the gradient of the distance transformation of the binarized image of aggregates. Iterative voting is initialized by voting along the gradient direction where at each iteration the voting direction and shape of the kernel is refined, resp. the kernel topography is refined and reoriented iteratively. It turned out that the kernel topography is unique because it votes for the most likely set of grid points where the gravity center of an individual cluster component may be located. Furthermore, a new procedure is realized to use the local intensities of aggregations for kernel voting. The last voted iteration provides gravitation centers, resp. centers of mass of the clumped cells. These are extracted and used as markers to determine individual cell boundaries by a marker based watershed postprocessing. The subject of this paper is to highlight the basic algorithm of iterative kernel voting and expanding it to process intensities within clusters as well as contour information. The approach is applied to synthetic images that were modified systematically with regard to object topology. Natural aggregates of cells at the light microscopic level and cell clusters derived from high resolution flat bed scanning were splitted. In addition to these examples images from a benchmark databases were investigated. The splittings generated by the iterative voting approach were compared with expected splittings of test persons and with results of the watershed method. Especially the gray level based iterative voting method provides superior results for cell cluster separation in comparison to the watershed procedure.

Morphological multiscale decomposition of connected regions with emphasis on cell clusters

After binarization of cells in complex cytological and histological images the segmented structures can be rather far away from a final quantification of features of single cells since cells may overlap and cluster strongly. Separating optically, partially or totally fused entities like cells is a problem which frequently cannot be solved by a watershed segmentation or a basic morphological processing of images. However, considering different morphological scales after iterative erosion gives rise to dominant markers of singular objects. Performing a reconstruction by iterative dilation yields a scale-independent decomposition of multiple disjointed cell clumps of different sizes within an image. Accordingly we developed a technique that splits cell clumps into meaningful parts. Since this method is based on the analysis of the morphological-scale space, generated by iterative erosion, it is independent on the size of cell clusters. The detection of dominant points within the eroded scales are cell-specific markers. The converse integration of markers at different scales is obtained by a successive reconstruction based on constrained dilation of the original cell shape. The advantages of this approach are the independence of cell shapes which are clumped, the consideration of holes or background intensities within objects and the robustness with regard to convergence. An important benefit is the fitting of the operation time to the size of clusters by the size of the morphological structuring element. This means, that this approach requires only one parameter. Finally, a better match of the morphological scale space approach was found and compared with the ground truth as well as the results of the watershed technique. The primary object of this paper is to highlight the algorithm and its results by using different examples from benchmark databases, self generated images that exhibit different topological features and complex configurations of cells within histological images.

Efferent Projections of the Anterior and Posterodorsal Regions of the Medial Nucleus of the Amygdala in the Mouse

The efferent projections of the anterior and posterodorsal part of the medial nucleus (MePD) in the mouse were studied by means of anterograde axonal tracing using biotinylated dextran amine. The MePD axons ran mainly via the stria terminalis and to a lesser extent via the ventral amygdalofugal pathway. The projections to the forebrain were broadly distributed and varied from very strong to scant. The most significant connections were destined to the bed nucleus of the stria terminalis in which all parts of the medial division were innervated by MePD neurons. Moderate projections reached the limbic striatum (nucleus accumbens), olfactory tubercle and the lateral septal nucleus. The substantia innominata was also innervated by the MePD, and especially the projection to its ventral portion was substantial. The profuse innervation of the medial preoptic nucleus and medial preoptic area indicated significant involvement of the MePD in sexual behavior. Many hypothalamic nuclei were innervated but to a different extent. The very strong innervation of the ventral premammillary nucleus further indicated the involvement of the MePD in the neuronal circuitry for sexual behavior. Substantial projections also reached the anterior hypothalamus and tuber cinereum, while the connections to the lateral hypothalamus were widespread but showed moderate density. MePD strongly innervated the ventrolateral part of the ventromedial hypothalamic nucleus and moderately its remaining parts. The neurosecretory hypothalamic nuclei and the arcuate nucleus contained only a few MePD terminals. The thalamic innervation was very scant and reached the lateral habenular nucleus and the nuclei of the midline. The mesencephalic connections were moderate to sparse and projected to the mesolimbic dopaminergic groups in the ventral tegmental area, the pars lateralis and the dorsal tier of the substantia nigra pars compacta, the periaqueductal gray and the dorsal raphe nucleus. The present results principally resembled data known in other rodent species; however, the efferents of the MePD often differed in extent and/or topical distribution.

Differential proteome of the striatum from hemiparkinsonian rats displays vivid structural remodeling processes.

Parkinsons disease is a multifactorial, neurodegenerative disease where etiopathogenetic mechanisms are not fully understood. Animal models like the neurotoxic 6-OHDA-hemiparkinsonian rat model are used for standardized experiments. Here, we analyzed proteome changes of the striatum three months after 6-OHDA lesions of the nigral dopaminergic cell population. Striata were removed and proteins were separated by 2DE followed by differential spot analysis. Proteins in spots were identified by MALDI-TOF-MS. Most up-regulations of proteins were concerning energy metabolism in mitochondria. Proteins of calcium homeostasis like annexin A3, annexin A7, calbindin, calmodulin, calreticulin, and reticulocalbin 1 also were differentially regulated. Moreover, proteins involved in antioxidative mechanisms like superoxide dismutase, protein disulfide isomerase 1 and 3, N(G),N(G)-dimethylarginindimethyl-aminotransferase 2, and thioredoxin-dependent peroxide reductase were up-regulated. Interestingly, most cytoskeletal proteins belonging to the axon cytoskeleton and synapse were up-regulated pointing to long-distance axon remodeling. In addition, transcription factors, proteins of nucleic acid metabolism, chaperones, and degrading proteins (UCHL1) were up-regulated as well. In conclusion, the neurotoxin-induced proteome alterations indicate vivid long-distance remodeling processes of dendrites, axons, and synapses that are still ongoing even three months after perturbation, indicating a high plasticity and regeneration potential in the adult rat brain.

Brain stem afferent connections of the amygdala in the rat with special references to a projection from the parabigeminal nucleus: a fluorescent retrograde tracing study

A recently revealed important function of the amygdala (Am) is that it acts as the brain’s “lighthouse”, which constantly monitors the environment for stimuli which signal a threat to the organism. The data from patients with extensive lesions of the striate cortex indicate that “unseen” fearful and fear-conditioned faces elicit increased Am responses. Thus, also extrageniculostriate pathways are involved. A multisynaptic pathway from the retina to the Am via the superior colliculus (SC) and the pulvinar was recently suggested. We here present data based on retrograde neuronal labeling following injection of the fluorescent tracer Fluoro-Gold in the rat Am that the parabigeminal nucleus (Pbg) emits a substantial, bilateral projection to the Am. This small cholinergic nucleus (Ch8 group) in the midbrain tegmentum is a subcortical relay visual center that is reciprocally connected with the SC. We suggest the existence of a second extrageniculo-striate multisynaptic connection to Am: retina–SC–Pbg–Am, that might be very effective since all tracts listed above are bilateral. In addition, we present hodological details on other brainstem afferent connections of the Am, some of which are only recently described, and some others that still remain equivocal. Following selective injections of Fluoro-Gold in the Am, retrogradely labeled neurons were observed in parasubthalamic nucleus, peripeduncular nucleus, periaqueductal gray, dopaminergic nuclear complex (substantia nigra pars lateralis and pars compacta, paranigral, parabrachial pigmented and interfascicular nuclei, rostral and caudal linear nuclei, retrorubral area), deep mesencephalic nucleus, serotoninergic structures (dorsal, median and pontine raphe nuclei), laterodorsal and pedunculopontine tegmental nuclei (Ch6 and Ch5 groups), parabrachial nuclear complex, locus coeruleus, nucleus incertus, ventrolateral pontine tegmentum (A5 group), dorsomedial medulla (nucleus of the solitary tract, A2 group), ventrolateral medulla (A1/C1 group), and pars caudalis of the spinal trigeminal nucleus. A bilateral labeling of the upper cervical spinal cord was also observed.

A Robust Transcortical Profile Scanner for Generating 2-D Traverses in Histological Sections of Richly Curved Cortical Courses

Quantitative analysis of the cerebral cortex has become more important since neuroimaging methods have revealed many subfunctions of cortical regions that were thought to be typical for only one specific function. Furthermore, it is often unknown if a certain area may be subdivided observer independently into subareas. These questions lead to an analytical problem. How can we analyze the cytoarchitecture of the human cerebral cortex in a quantitative manner in order to confirm classical transition regions between distinct areas and to detect new ones. Scanning the cerebral cortex is difficult because it presents a richly curved course and sectioning always leads to partially nonperpendicular sectioned regions of the tissue. Therefore, different methods were tested to determine which of them are most reliable with respect to generating perpendicular testlines in the cerebral cortex. We introduce a new technique based on electrical field theory. The results of this technique are compared with those of conventional techniques. It was found that straight traverses generated by the electrodynamic model present significantly smaller intertraversal differences than the conventional approaches.

Intrastriatal botulinum toxin abolishes pathologic rotational behaviour and induces axonal varicosities in the 6-OHDA rat model of Parkinson's disease.

Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of dopaminergic and cholinergic circuits. In Parkinsons disease (PD) imbalanced cholinergic hyperactivity prevails in the striatum. Interruption of acetylcholine (ACh) release in the striatum by locally injected botulinum neurotoxin A (BoNT-A) has been studied in the rat 6-hydroxydopamine (6-OHDA) model of PD (hemi-PD). The hemi-PD was induced by injection of 6-OHDA into the right medial forebrain bundle. Motor dysfunction provoked by apomorphine-induced contralateral rotation was completely reversed for more than 3 months by ipsilateral intrastriatal application of 1-2 ng BoNT-A. Interestingly, BoNT-A injected alone into the right striatum of naïve rats caused a slight transient ipsilateral apomorphine-induced rotation, which lasted only for about one month. Immunohistochemically, large axonal swellings appeared within the striatum injected with BoNT-A, which we tentatively named BoNT-A-induced varicosities. They contained either choline acetyltransferase or tyrosine hydroxylase. These findings suggest a selective inhibition of evoked release of ACh by locally applied BoNT-A. Intrastriatal application of BoNT-A may antagonize localized relative functional disinhibited hypercholinergic activity in neurodegenerative diseases such as PD avoiding side effects of systemic anti-cholinergic treatment.