Biomedical subjects

Gvido Cebers

Publications and source records attributed to Gvido Cebers.

12 recordsLinked to original sources

PubMed2006

Prodynorphin storage and processing in axon terminals and dendrites.

The classical view postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans-Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN-containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and striatum, PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium-induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission.

Animals↗

PubMed2006

Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue.

BACKGROUND: Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. METHODS: Uterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. RESULTS: In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to non-pregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy. CONCLUSION: The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility.

Adult↗

PubMed2005

Impaired long-term habituation is dissociated from increased locomotor activity after sensorimotor cortex compression.

Behavioural habituation to a novel environment is a simple form of learning in rodents. We studied the habituation and locomotor activity (LMA) of Wistar rats subjected to unilateral, transient (30min) extradural compression (EC) of the right sensorimotor cortex. One group of rats was tested every 24h during the first 5 days (D1-D5) post-EC. Two other groups were tested for the first time in the LMA boxes on D3 and D6 post-EC and their performance was compared with the group tested on D1 (activity in a novel environment). Total and center locomotion, vertical activity and time spent in the center of the LMA box were reduced on D1 post-EC and normalized by D2. The EC-induced motor paresis was undetectable on the rotarod by D2 and on the beam-walking by D3. Total locomotion, vertical activity and time spent in the center of EC-rats significantly increased from D1 to D3. EC caused neurodegeneration in the cortex, caudate putamen and thalamus as detected by Fluoro-Jade staining. The size of the cortical damage decreased from D2 to D5 in the medial and caudal regions of the compressed hemisphere, in accordance with recovery of motor function. LMA provided additional information in the follow-up of recovery from brain injury and habituation to the environment. Thus, long-term, inter-session habituation was impaired from D1 to D3 but dissociated from increased LMA intra-session on D3, when the motor deficits provoked by EC were already undetectable in the rotarod and beam-walking tests.

Analysis of Variance↗

PubMed2005

Extradural compression of the sensorimotor cortex delays the acquisition but not the recalling of a lever-pressing task in Wistar rats.

The learning and recalling of a lever-press task (LPT) after brief unilateral extradural compression (EC) of the right sensorimotor cortex was studied in Wistar rats. All rats, regardless of the time-point for EC, were trained to lever press for food from D(day)1 to D6. On D8, the position of the active lever was changed to the right side of the operant box and performance was tested until D14. Total and active lever presses, as well as % errors were used to analyse the performance. Rats submitted to EC 24 h before initiating the LPT schedule (naïve-compressed group) showed delayed task acquisition and impaired performance until D10. No significant impairments were detected by D3 on a beam-walking test, excluding paresis as the cause to the delay. Rats submitted to EC after they learned the LPT (trained-compressed group) showed only mildly impaired post-compression performance with no effects on the recalling of the task. Using a progressive ratio LPT, the maximum number of presses to obtain a food-pellet (breaking point) was significantly reduced 24h after EC suggesting reduced motivation for the task early after brain injury. The delayed acquisition of the LPT in naïve-compressed rats was accompanied by consistent cortical, striatal and thalamic degeneration detected by Fluoro-Jade and anti-glial fibrillary acidic protein (GFAP) staining, whereas the improvement in the performance of this group was accompanied by a reduction of the cortical damage on D10. Recall of the LPT in trained-compressed rats was not altered by EC, suggesting the contribution of compensatory mechanisms.

Analysis of Variance↗

PubMed2005

Prodynorphin transcripts and proteins differentially expressed and regulated in the adult human brain.

Transcription from multiple promoters along with alternative mRNA splicing constitutes the basis for cell-specific gene expression and mRNA and protein diversity. The prodynorphin gene (PDYN) gives rise to prodynorphin (PDYN), precursor to dynorphin opioid peptides that regulate diverse physiological functions and are implicated in various neuropsychiatric disorders. Here, we characterized PDYN transcripts and proteins in the adult human brain and studied PDYN processing and intracellular localization in model cell lines. Seven PDYN mRNAs were identified in the human brain; two of the transcripts, FL1 and FL2, encode the full-length PDYN. The dominant, FL1 transcript shows high expression in limbic-related structures such as the nucleus accumbens and amygdala. The second, FL2 transcript is only expressed in few brain structures such as the claustrum and hypothalamus. FL-PDYN was identified for the first time in the brain as the dominant PDYN protein product. Three novel PDYNs expressed from spliced or truncated PDYN transcripts either lack a central segment but are still processed into dynorphins, or are translated into N-terminally truncated proteins. One truncated PDYN is located in the cell nucleus, suggesting a novel nonopioid function for this protein. The complexity of PDYN expression and diversity of its protein products may be relevant for diverse levels of plasticity in adaptive responses for the dynorphin system.

Adult↗

PubMed2005

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission.

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

Animals↗

PubMed2004

Nicotine-induced dopamine release in the nucleus accumbens is inhibited by the novel AMPA antagonist ZK200775 and the NMDA antagonist CGP39551.

RATIONALE: Accumulated data suggest that N-methyl-D-aspartate (NMDA) receptors are involved in the reinforcing properties of nicotine. However, less is known about the role of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors in this context. OBJECTIVES: To study the effect of the novel systemically administered AMPA receptor antagonist ZK200775 ([1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl) quinoxalin-1-yl] methylphosphonate) on nicotine-induced dopamine (DA) release in the nucleus accumbens (NAcc) and nicotine-stimulated locomotor activity (LMA) and particularly the relative role of NMDA and AMPA receptors in nicotine-stimulated DA release and LMA. METHODS: Male Wistar rats were administered ZK200775, CGP39551 or NBQX 30 min prior to nicotine and DA release and LMA was measured using in vivo microdialysis or photocell equipped activity boxes. Glutamate-produced neurotoxicity in cultured brain cells and binding assays were performed to determine the glutamate receptor subtype selectivity and affinity to nicotine receptors of ZK200775, respectively. RESULTS: ZK200775 (3.0 but not 1.5 or 6.0 mg/kg) significantly decreased the nicotine-induced (0.6 mg/kg) DA release in the NAcc and nicotine-stimulated LMA. ZK200775 (1.5, 3.0, 6.0 mg/kg) alone influenced neither DA release nor LMA. ZK200775 showed 34-fold selectivity for AMPA receptors compared to NMDA receptors and no affinity to nicotine receptors. The NMDA receptor antagonist CGP39551 (10 mg/kg) significantly decreased both the nicotine-induced DA release and nicotine-stimulated LMA whereas the AMPA receptor antagonist NBQX (10 mg/kg) had no effect. Notably, CGP39551 and ZK200775 (3.0 mg/kg) displayed a different pattern in inhibition of nicotine-induced DA release. CONCLUSIONS: Both NMDA- and AMPA receptors are involved in nicotine's dependence-producing properties, although in a spatiotemporally differential manner.

2-Amino-5-phosphonovalerate↗

PubMed2004

The NMDA NR2B subunit-selective receptor antagonist, CP-101,606, enhances the functional recovery the NMDA NR2B subunit-selective receptor and reduces brain damage after cortical compression-induced brain ischemia.

Using a novel in vivo model for cerebral ischemia produced by short-lasting compression of a well-defined brain area of sensorimotor cortex we studied neuroprotective effects of the NMDA NR2B subunit selective antagonist, CP-101,606, in Sprague-Dawley rats. Cortical compression for 30 min produced a consistent and highly reproducible functional impairment, that is paresis of contralateral hind and fore limbs. The neurological deficit was accompanied by marked brain damage in cerebral cortex, hippocampus and thalamus as identified by Fluoro-Jade, a marker of general neuronal cell death. Using a daily performed beam walking test it was shown that untreated animals recovered from their functional impairment within 5-7 days following surgery. Intravenous administration of increasing doses (1, 5, 10, 20 mg/kg) of the NMDA NR2B subunit receptor specific antagonist, CP-101,606, dose-dependently improved the rate of functional recovery and protected against the ischemic brain damage in cerebral cortex, hippocampus, and thalamus as identified 2 days after the ischemic insult. Based upon these results, we conclude that NMDA NR2B receptor subunits represent potential targets to reduce not only the functional deficits, but also neuronal death in cortex and several midbrain regions produced by moderate, transient, cerebral ischemia.

Animals↗

PubMed2003

Loss of GABAergic neuronal phenotype in primary cerebellar cultures following blockade of glutamate reuptake.

Prolonged inhibition of glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a specific glutamate transporter blocker, reduced the number of GABA positive neurons in a primary cerebellar culture by 54%. The disappearance of immunostaining for GABA was gradual and was partially prevented by the N-methyl-D-aspartate (NMDA) receptor blocker, MK-801, and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, NBQX. Combined blockade of NMDA and AMPA receptors restored the original proportion of GABAergic neurons observed in control cultures. Following the PDC exposure, expression of other GABAergic markers, such as glutamic acid decarboxylase (GAD) and vesicular GABA transporter (VGAT) was also dramatically decreased in an AMPA receptor-dependent manner. Loss of GABA or GAD immunostaining is commonly regarded as a sign of degeneration of GABAergic neurons. However, none of the GABAergic neurons were positive for propidium iodide uptake or showed abnormal nuclear morphology. Based on the above data we conclude that prolonged activation of ionotropic glutamate receptors by endogenously released glutamate was not toxic to cerebellar GABAergic neurons, but lead to the loss of their characteristic neurotransmitter phenotype.

Animals↗

PubMed2002

Spermidine attenuates the inhibitory effect of ethanol on NMDA-induced neurotoxicity.

The exact site(s) and the molecular mechanism(s) by which ethanol inhibits the activity of NMDA receptors in the brain have so far not been identified although the involvement of several NMDA receptor modulatory sites activated by glycine, Mg2+, Zn2+, polyamines and red-ox agents has been suggested. In this study we investigated the effects of spermidine, a polyamine site agonist, on NMDA-induced neurotoxicity and its ability to modulate the inhibitory action of ethanol on neurotoxicity produced by the maximal neurotoxic concentration of NMDA as measured by the MTT assay in rat cerebellar granule cells. This assay measures the enzymatic activity in mitochondria and/or endosome/lysosome compartment that closely correlates with the cell viability. Spermidine dramatically potentiated NMDA-induced responses both at nontoxic and maximally neurotoxic NMDA concentrations. Ethanol, as expected, concentration-dependently inhibited the maximal neurotoxicity produced by NMDA. The potentiating effect of spermidine observed at nontoxic concentrations of NMDA was not altered by ethanol evidenced by the fact that the EC(50) value for spermidine was not significantly changed in the presence of ethanol. This suggests that ethanol and spermidine produce their effects by acting at different sites within the NMDA receptor complex. In contrast, the inhibitory effect of ethanol on the maximally neurotoxic action of NMDA was significantly reduced by spermidine in a concentration-dependent manner, suggesting that the spermidine enhancement of NMDA receptor function in this situation is more potent and is able mask the inhibitory action of ethanol on other sites within the NMDA receptor.

Analysis of Variance↗

PubMed2002

Selective and AMPA receptor-dependent astrocyte death following prolonged blockade of glutamate reuptake in rat cerebellar cultures.

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 microM), whereas MK-801 (10 microM), a noncompetitive blocker of N-methyl-D-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1-4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.

Animals↗

PubMed2002

Expression and regulation of the pattern recognition receptors Toll-like receptor-2 and Toll-like receptor-4 in the human placenta.

The placenta constitutes a physical and immunological barrier against invading infectious agents and has been suggested to be a pregnancy-specific component of the innate immune system. The aim of this study was to investigate the presence and regulation of Toll-like receptors-2 and -4 (TLR2 and TLR4) in the human placenta, because these receptors are believed to be important for immune responses against pathogens. Twenty-eight placentas from normal term pregnancies were analysed with immunohistochemistry, which showed a strong immunoreactivity for TLR2 and TLR4 in the villous and the intermediate trophoblasts. The regulation of TLR2 and TLR4 by microbial stimulus was assessed by incubating explants of term chorionic villi with zymosan or lipopolysaccharide (LPS) and analysed with real-time reverse transcriptase-polymerase chain reaction. Stimulation with zymosan and LPS readily induced interleukin (IL)-6 and IL-8 cytokine production in the placenta cultures, whereas TLR2 and TLR4 mRNA and protein expression remained at the same high level as in unstimulated explants. These data suggests a novel mechanism for the fetoplacental unit to interact with micro-organisms.

Chorionic Villi↗