Lut Arckens

CALCIUM BINDING PROTEINS AND NEUROPEPTIDES AS MOLECULAR MARKERS OF GABAERGIC INTERNEURONS IN THE CAT VISUAL CORTEX

SummaryIn the cat visual cortex, almost all parvalbumin-positive cells are GABAergic, and about 80% of the calbindin D-28K-positive neurons are also GABA-immunoreactive. About 37% of the GABAergic neurons contain parvalbumin and a smaller fraction (about 18%) contains calbindin. Furthermore, parvalbumin and calbindin are localized in two separate neuronal populations in the cat visual cortex, suggesting that two GABAergic populations can be distinguished, one containing parvalbumin and one containing calbindin. Double staining for parvalbumin and neuropeptides (CCK, SRIF and NPY), revealed no double-labeled cells, with the exception of a few SRIF- and parvalbumin-positive neurons. These results show that cortical GABAergic cells can be differentiated on basis of their calcium binding protein and neuropeptide immunoreactivity.

Proteomics Analysis of Cytokine-induced Dysfunction and Death in Insulin-producing INS-1E Cells New Insights into the Pathways Involved

Cytokines released by islet-infiltrating immune cells play a crucial role in β-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this β-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 ± 164 (pH 4–7) and 1641 ± 73 (pH 6–9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p ≤ 0.01) by a combination of interleukin-1β and interferon-γ, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e−05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a β-cell dysfunction/apoptotic β-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to β-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of β-cell loss in type 1 diabetes.

Alterations in GAP-43 and synapsin immunoreactivity provide evidence for synaptic reorganization in adult cat dorsal lateral geniculate nucleus following retinal lesions.

Growth‐associated protein‐43 (GAP‐43) and synapsin were used as molecular markers for synaptic reorganization in the adult cat visual system following sensory deprivation. Small binocular retinal lesions (central 10°) were made with a xenon light photocoagulator in adult cats. One, 3, 5 and 7 weeks after induction of the lesion, the neuropil levels of synapsin and GAP‐43 in the dorsal lateral geniculate nucleus (dLGN) and area 17 were determined by immunocytochemistry. GAP‐43 displayed a moderately low basal level in the dLGN of normal adult cats. The parvocellular C layers and the interlaminar plexi were characterized by higher immunoreactivity for GAP‐43. Lesion‐induced alterations were observed in all layers: GAP‐43 immunoreactivity increased in the part of the dLGN representing central vision. This increase was maximal 3 weeks after the lesion. Under our experimental conditions, sensory deprivation did not significantly alter GAP‐43 levels in the visual cortex. The changes in synapsin immunoreactivity were also restricted to the dLGN. In this nucleus, synapsin immunoreactivity decreased in all layers in the part subserving central vision 1 week after lesion. By 3 weeks after lesion, the level of synapsin had already returned to normal. This study provides evidence for a capacity for structural remodelling in primary sensory brain areas such as the dLGN throughout adult life. The observed changes in GAP‐43 and synapsin in the dLGN suggest that synaptic reorganization is induced by retinal lesions. Normalization of synaptic density and activity could be important for the survival of the partially deafferented geniculate neurons.

Calcium binding proteins as molecular markers for cat geniculate neurons.

SummaryImmunocytochemistry revealed that in the cat dorsal lateral geniculate nucleus (dLGN) almost all parvalbumin-positive cells are GABAergic and about 56% of the calbindin D-28K calbindin-immunoreactive neurons are also GABA-positive. On the other hand, in the same nucleus, almost all GABAergic neurons contain parvalbumin, and about 89% of the GABA-immunoreactive neurons contain calbindin. Double-labeling with calbindin and parvalbumin revealed that approximately 50% of the immunoreactive neurons are doublestained. In the PGN, virtually all neurons are GABA and parvalbuminpositive. Only a few scattered cells were also calbindin-immunoreactive. These results show that GABAergic geniculate cells can be differentiated on the basis of their calcium-binding protein immunoreactivity. Four types of immunoreactive cells are described here: (1) cells positive for GABA, parvalbumin and calbindin, (2) cells positive for GABA and parvalbumin, but negative for calbindin, (3) cells negative for GABA and parvalbumin, but positive for calbindin, (4) cells negative for GABA, parvalbumin and calbindin.

Effect of sensory deafferentation on the GABAergic circuitry of the adult cat visual system

The effect of bilateral central retinal lesions on the GAD67 and GAD65 messenger RNA levels in the dorsal lateral geniculate nucleus, the perigeniculate nucleus and the visual cortex of the adult cat was investigated by in situ hybridization. Three days post-lesion, a decrease in the number of GAD67-expressing cells was apparent in the deafferented dorsal lateral geniculate nucleus. This decrease persisted until 7.5 months post-lesion and was more pronounced with longer survival times. The decrease in GAD67 mRNA was mirrored by a decrease in glutamate decarboxylase-immunoreactive cells. GAD65 messenger RNA expression levels were low in the dorsal lateral geniculate nucleus of both control and retinally-lesioned cats. In the perigeniculate nucleus the messenger RNA levels of both glutamate decarboxylase isoforms were clearly decreased over a restricted region. In the lesion-affected visual cortex, no changes at the messenger RNA level were observed for either GAD67 or GAD65 although changes in glutamate decarboxylase immunoreactivity have been previously described. Hence, in the dorsal lateral geniculate nucleus, the perigeniculate nuclcus and the visual cortex, different intracellular mechanisms seem to lead to decreased GABAergic inhibition in response to sensory deafferentation.

Applications and current challenges of proteomic approaches, focusing on two-dimensional electrophoresis.

Summary.Since the formulation of the concept of “proteomics” in 1995, a plethora of proteomic technologies have been developed in order to study proteomes of tissues, cells and organelles. The powerful new technologies enabled by proteomic approaches have lead to the application of these methods to an exponentially increasing variety of biological questions for highly complex protein mixtures. Continuous technical optimization allows for an ever-increasing sensitivity of proteomic techniques. In this review, a brief overview of currently available proteomic techniques and their applications is given, followed by a more detailed description of advantages and technical challenges of two-dimensional electrophoresis (2-DE). Some solutions to circumvent currently encountered technical difficulties for 2-DE analyses are proposed.

Cloning and expression of PKG, a candidate foraging regulating gene in Vespula vulgaris

In honey bees, enhancement of cGMP-dependent protein kinase (PKG) expression accompanies a behavioural transition from in-hive working nursing bees towards outdoors foraging worker bees. Accordingly this gene was named amfor or Apis mellifera foraging gene. In the red harvester ant Pogonomyrmex barbatus a gene homologue aff ected food seeking behaviour as well, but in this species PKG expression decreased from the onset of foraging behaviour. Since the wasp Vespula vulgaris is phylogenetically positioned between ants and bees, in this paper we tried to elucidate whether the involvement of PKG in foraging behaviour can be extended to this species and if so, whether its expression is enhanced or decreased by the transition from nursing to foraging. To enable this candidate gene approach, we fi rst had to clone the PKG homologue from the common wasp. QPCR indicated a relevantly higher expression of Vvfor in nursing versus foraging wasps although interpretation of the results was hampered by a remarkable degree of variation as could be predicted from wasps captured in the wild as a source for mRNA extraction and quantifi cation.

Thymosin beta 4 mRNA and peptide expression in phagocytic cells of different mouse tissues

Thymosin beta 4 (Tbeta4) is a peptide of 43 amino acids, mainly recognized as a regulator of actin polymerization by sequestering G-actin. Meanwhile, the peptide has been implicated in lymphocyte maturation, carcinogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. The peptide is also involved in lesion-induced neuroplasticity through microglia upregulation and it participates in the growth of neuronal processes. However, its precise cellular localization throughout the entire body of the mouse has not been documented. We therefore initiated a detailed investigation of the tissue distribution and cellular expression of the Tbeta4 peptide and its precursor mRNA by immunocytochemistry and in situ hybridization, respectively. In the brain, Tbeta4 was clearly present in neurons of the olfactory bulb, neocortex, hippocampus, striatum, amygdala, piriform cortex and cerebellum, and in microglia across the entire brain. We further localized Tbeta4 in cells, typically with many processes, inside thymus, spleen, lung, kidney, liver, adrenal gland, stomach and intestine. Remarkably, Tbeta4 was thus associated with microglia and macrophages, the differentiated phagocytic cells residing in every tissue. Motility and phagocytosis, two important activities of macrophages, depend on actin, which can explain the presence of Tbeta4 in these cells.

Immunocytochemical detection of astrocyte GABAA receptors in cat visual cortex.

A nine amino-acid peptide derived from the beta 1-subunit of the bovine GABAA receptor was used for immunization of mice and subsequent production of monoclonal antibodies (MAb). In view of the later immunocytochemical application of the MAb to sections of cat visual cortex, the MAb were characterized on similar tissue. The GABAA receptor was isolated by affinity chromatography of protein material obtained from cat cortical gray matter. The antibodies were characterized by SDS-PAGE, followed by immunoblotting and immunoadsorption. Immunocytochemical staining with the MAb revealed labeled cells throughout all layers of the cat visual cortex as well as within the white matter. The morphology of most stained cells in the white matter and of some cells in the cortical gray matter closely resembled that of astrocytes. Double immunocytochemical staining with an antiserum against glial fibrillary acidic protein (GFAP), followed by light microscopic examination, indeed confirmed that astrocytes in both white and gray matter in the cat cortex possess GABAA receptors.

Localization of the two protein kinase Cβ-mRNA subtypes in cat visual system

Abstract Protein kinase C (PKC) consists of a family of different subtypes encoded by different PKC genes. We investigated the distribution of PKCβ1 and PKCβ2 in the visual system of the adult cat by in situ hybridization using oligonucleotide probes complementary to the PKCβ1 and PKCβ2 mRNAs, two splicing variants of the same gene transcript. In the primary visual cortex PKCβ1 and PKCβ2 were both present. The laminar distribution patterns found for the two PKC subtypes were identical. A remarkable finding was the difference between the laminar distribution of the PKCβs in areas 17 and 18 when compared with area 19. In all three areas the highest expression levels were found in layer VI, moderately high levels were found in layers II, III and V, while layer I was devoid of signal. In area 17 and 18 layer IV stood out by its low PKCβ signal. In sharp contrast, layer IV of area 19 was indiscernible from the superficial layers because of an evenly high signal. In the dLGN of the adult cat PKCβ1 and PKCβ2 mRNAs were distributed rather homogeneously over the different layers, but the expression levels for PKCβ1 were clearly higher than those for PKCβ2.