Lieselotte Cnops

Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat

The recent introduction of fluorescent two‐dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30‐day‐old kittens, in order to identify proteins expressed in an age‐related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two‐dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.

Hepatitis B virus replication causes oxidative stress in HepAD38 liver cells

We used human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promotor, to investigate changes induced in the host cell by HBV replication that could contribute to malignant transformation. Parameters of oxidative stress (malondialdehyde, glutathione) and cell proliferation were determined at different times after induction (0–96 h). In HBV-producing cells, the redox status peaked at 72 h. cDNA micro array analysis at 72 h post induction revealed 3 groups of genes that were up-regulated by HBV: (i) heat shock proteins, (ii) oxidative and metabolic stress and (iii) growth and apoptosis related genes. Continuous HBV production did not accelerate karyotypic changes in cells cultured for 4 months (18 passages). In conclusion: HBV replication modulates host gene expression and induces oxidative stress. In this HepAD38 model early events (0–4 days) in the host cell after induction of HBV replication can be studied under strictly defined conditions.

High-affinity Na+/K+-dependent glutamate transporter EAAT4 is expressed throughout the rat fore- and midbrain.

Excitatory amino acid transporter 4 (EAAT4), a member of the high‐affinity Na+/K+‐dependent glutamate transporter family, is highly enriched in Purkinje cells of the cerebellum, although it is not restricted to these cells. The detailed expression of EAAT4 protein in different adult rat fore‐ and midbrain regions was examined. Despite moderate expression levels compared with the cerebellum, EAAT4 protein was omnipresent throughout the fore‐ and midbrain. With antibodies raised against the N‐terminal mouse EAAT4 sequence, the highest protein expression levels were observed in the substantia nigra pars compacta, ventral tegmental area, paranigral nucleus, habenulo‐interpeduncular system, supraoptic nucleus, lateral posterior thalamic nucleus, subiculum, and superficial layers of the superior colliculus. Relatively high levels of EAAT4 protein were also detected in the hippocampal principal cells, in the glutamatergic, γ‐aminobutyric acid (GABA)ergic, dopaminergic and most likely cholinergic cells of all nuclei of the basal ganglia, and in neurons of layers II/III and V of the cerebral cortex. The expression of EAAT4 was confirmed at the mRNA level in some important fore‐ and midbrain structures by in situ hybridization and reverse transcriptase‐polymerase chain reaction (RT‐PCR) and estimated to range from 6.7 to 1.6% of the amount in the cerebellum as measured by real‐time PCR. J. Comp. Neurol. 511:155–172, 2008.

Extracellular GABA concentrations in area 17 of cat visual cortex during topographic map reorganization following binocular central retinal lesioning

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of mammals, plays an important role in cortical reorganization following sensory deprivation, by regulating the level of cortical inhibition and gating changes in receptive field size and synaptic efficacy. In cats it has been shown that 2 weeks after the induction of binocular retinal lesions, GABAergic inhibition, as determined by immunocytochemistry, is decreased in the deafferented region of area 17, whereas 3 months post-lesion, normal GABAergic control is restored within the cortical scotoma. In this study we used in vivo microdialysis to investigate the extracellular GABA concentrations 1-2 months post-lesion, in the sensory-deprived and remote, non-deprived region of area 17. Data were collected at those sample times and sites for which the extracellular glutamate concentrations had been determined in a previous investigation to elucidate the role of this excitatory neurotransmitter in cortical reorganization. As for glutamate, we observed significantly increased extracellular GABA concentrations in non-deprived area 17, whereas in deafferented area 17, extracellular GABA concentrations were comparable to those observed in normal, control subjects. These data suggest that 1-2 months post-lesion the deafferented cortex behaves like normal visual cortex, in contrast to remote, non-deprived cortex. Notwithstanding the increase in extracellular GABA concentration of 134%, the parallel increase in glutamate concentration of 269% could give rise to a net increase in excitability in remote area 17. We therefore suggest that LTP-like mechanisms, and thereby cortical reorganization, might still be facilitated, while possible excessive hyperexcitability is balanced by the moderately increased GABAergic control.

Analysis of c-fos and zif268 Expression Reveals Time-Dependent Changes in Activity Inside and Outside the Lesion Projection Zone in Adult Cat Area 17 after Retinal Lesions

Retinal lesions induce a topographic reorganization in the corresponding lesion projection zone (LPZ) in the visual cortex of adult cats. To gain a better insight into the reactivation dynamics, we investigated the alterations in cortical activity throughout area 17. We implemented in situ hybridization and real-time polymerase chain reaction to analyze the spatiotemporal expression patterns of the activity marker genes zif268 and c-fos. The immediate early gene (IEG) data confirmed a strong and permanent activity decrease in the center of the LPZ as previously described by electrophysiology. A recovery of IEG expression was clearly measured in the border of the LPZ. We were able to register reorganization over 2.5-6 mm. We also present evidence that the central retinal lesions concomitantly influence the activity in far peripheral parts of area 17. Its IEG expression levels appeared dependent of time and distance from the LPZ. We therefore propose that coupled changes in activity occur inside and outside the LPZ. In conclusion, alterations in activity reporter gene expression throughout area 17 contribute to the lesion-induced functional reorganization.

Retinal lesions affect extracellular glutamate levels in sensory-deprived and remote non-deprived regions of cat area 17 as revealed by in vivo microdialysis

This study aimed at gaining insight into the role of the excitatory neurotransmitter glutamate in topographic map reorganization in the sensory systems of adult mammals after restricted deafferentations. Hereto, in vivo microdialysis was used to sample extracellular glutamate from sensory-deprived and non-deprived visual cortex of adult awake cats 18 to 53 days after the induction of restricted binocular retinal lesions, and in topographically corresponding cortical regions of control animals. A microbore HPLC-ED method was applied for the analysis of the microdialysates. In normal subjects, the visual cortex subserving central and peripheral vision showed similar extracellular fluid glutamate concentrations. In contrast, in animals with homonymous central retinal lesions, the extracellular glutamate concentration was significantly lower in central, sensory-deprived cortex compared to peripheral, non-deprived cortex. Compared to control regions in normal subjects, glutamate decreased in the extracellular fluid of deprived cortex but increased significantly in remote non-deprived visual cortex. These results not only suggest an activity-dependent regulation of the glutamate levels in visual cortex but also imply a role for perilesional cortical regions in topographic map reorganization following sensory deafferentation.

Age-dependent alterations in CRMP2 and CRMP4 protein expression profiles in cat visual cortex.

We monitored the protein expression profiles of collapsin response mediator protein 2 and 4 (CRMP2 and CRMP4) throughout cat primary visual area 17 at different postnatal ages. Single immunocytochemical stainings revealed a clear effect of cortical maturation on the spatial and laminar distribution profile of CRMP2 and CRMP4. In kittens of postnatal day 10 (P10) and 30 (P30), CRMP2 and CRMP4 immunoreactivity was exclusively present in fibers running perpendicular to the cortical surface and crossing all cortical layers, but was never found in neuronal cell bodies. The immunoreactive fibers were embedded in an intensely and homogeneously stained neuropil. In contrast, mature visual cortex immunocytochemistry located CRMP2 and CRMP4 in the somatodendritic compartment of neurons with a clear CRMP-specific lamination pattern. Similar to kitten, neuropil staining was clearly observed but showed a decreasing gradient from layer I to VI in adult area 17. Detailed analysis of cellular morphology and size classified the CRMP2- and CRMP4-immunopositive cells in distinct neuronal populations. Double labeling of CRMP2 or CRMP4 with the typical interneuron marker parvalbumin (PV) showed many double-labeled cells immunoreactive for CRMP4 and PV, but not for CRMP2 and PV, corroborating the cell type-specific character of each CRMP. Our present results clearly illustrate that CRMP2 and CRMP4 may play an important role in visual cortex, possibly providing different classes of neurons with the potential to form a functionally meaningful network, not only during development, but also in adulthood, coincident with the belief that CRMPs are involved in neurite growth and guidance.

Age-dependent expression of collapsin response mediator proteins (CRMPs) in cat visual cortex

The functional properties and anatomical organization of the mammalian visual cortex are immature at birth and develop gradually during the first postnatal weeks. There is a ‘critical period’ where the cortex is plastic and susceptible to changes in visual input. Knowledge of proteins with a high expression during this period has great importance for the understanding of activity‐driven maturation of the brain. The collapsin response mediator protein family consists of five cytosolic phosphoproteins (CRMP1–5) that are involved in neuronal differentiation during the development of the nervous system. They have been implicated in axon guidance and growth cone collapse through their action in the signalling pathway of collapsin/semaphorin. We examined the distribution of the CRMPs throughout the visual cortex of kitten and adult cat by in situ hybridization. While CRMP3 could not be detected in cat forebrain, the other CRMPs showed a higher expression in the immature brain compared to the adult state. Western blotting allowed the quantification of the observed age‐dependent differences in the expression of CRMP2, 4 and 5. Moreover, for CRMP2 and 5 we observed a number of development‐dependent post‐translational modifications. We thus conclude that CRMPs might be important during the normal postnatal development of the visual cortex possibly for the fine‐tuning of the specific connections in the brain.

Effect of binocular retinal lesions on CRMP2 and CRMP4 but not Dyn I and Syt I expression in adult cat area 17.

Removal of retinal input from a restricted region of adult cat visual cortex leads to a substantial reorganization of the retinotopy within the sensory‐deprived cortical lesion projection zone (LPZ). Still little is known about the molecular mechanisms underlying this cortical map reorganization. We chose two members of the collapsin response mediator protein (CRMP) family, CRMP2 and CRMP4, because of their involvement in neurite growth, and compared gene and protein expression levels between normal control and reorganizing visual cortex upon induction of central retinal lesions. Parallel analysis of Dynamin I (Dyn I) and Synaptotagmin I (Syt I), two molecules implicated in the exocytosis–endocytosis cycle, was performed because changes in neurotransmitter release have been implicated in cortical plasticity. Western blotting and real‐time polymerase chain reaction revealed a clear time‐dependent effect of retinal lesioning on CRMP2 and CRMP4 expression, with maximal impact 2 weeks post‐lesion. Altered CRMP levels were not a direct consequence of decreased visual activity in the LPZ as complete surgical removal of retinal input to one hemisphere had no effect on CRMP2 or CRMP4 expression. Thus, CRMP expression is correlated to cortical reorganization following partial deafferentation of adult visual cortex. In contrast, Dyn I and Syt I were not influenced and thereby do not promote exocytosis–endocytosis cycle modifications in adult cat cortical plasticity.

Age- and experience-dependent expression of Dynamin I and Synaptotagmin I in cat visual system

Dynamin I (Dyn I) and Synaptotagmin I (Syt I) are essential for endocytosis‐exocytosis processes, thus for neurotransmission. Despite their related function at presynaptic terminals, Dyn I and Syt I displayed opposite expression patterns during visual cortex maturation in the cat. Dyn I was more abundantly expressed in adults, while Syt I exhibited higher levels in kittens of postnatal day 30 (P30). In area 17 this developmental difference was most obvious in layers II/III. Layer VI displayed a strong hybridization signal for both molecules, independent of age. In addition, Syt I levels were higher in posterior compared to anterior area 17 in adult subjects. Moreover, in higher‐order visual areas Syt I was unevenly distributed over the cortical layers, thereby setting clear areal boundaries in mature cortex. In contrast, Dyn I was rather homogeneously distributed over extrastriate areas at both ages. Both molecules thus demonstrated a widespread but different distribution and an opposite temporal expression pattern during visual system development. Notably, monocular deprivation during the critical period of ocular dominance plasticity significantly decreased Syt I expression levels in area 17 ipsilateral to the deprived eye, while no effect was observed on Dyn I expression. We therefore conclude that visual experience induces changes in Syt I expression that may reflect changes in constitutive exocytosis involved in postnatal structural refinements of the visual cortex. On the other hand, the spatial and temporal expression patterns of Dyn I correlate with the establishment and maintenance of the mature neuronal structure rather than neurite remodeling. J. Comp. Neurol. 504:254–264, 2007.