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The effect of mild chronic renal failure (CRF) induced by 4/6-nephrectomy (4/6NX) on central neuronal activations was investigated by c-Fos immunohistochemistry staining and compared to sham-operated rats. In the 4/6 NX rats also the effect of the angiotensin receptor blocker, losartan, and the central sympatholyticum moxonidine was studied for two months. In serial brain sections Fos-immunoreactive neurons were localized and classified semiquantitatively. In 37 brain areas/nuclei several neurons with different functional properties were strongly affected in 4/6NX. It elicited a moderate to high Fos-activity in areas responsible for the monoaminergic innervation of the cerebral cortex, the limbic system, the thalamus and hypothalamus (e.g. noradrenergic neurons of the locus coeruleus, serotonergic neurons in dorsal raphe, histaminergic neurons in the tuberomamillary nucleus). Other monoaminergic cell groups (A5 noradrenaline, C1 adrenaline, medullary raphe serotonin neurons) and neurons in the hypothalamic paraventricular nucleus (innervating the sympathetic preganglionic neurons and affecting the peripheral sympathetic outflow) did not show Fos-activity. Stress- and pain-sensitive cortical/subcortical areas, neurons in the limbic system, the hypothalamus and the circumventricular organs were also affected by 4/6NX. Administration of losartan and more strongly moxonidine modulated most effects and particularly inhibited Fos-activity in locus coeruleus neurons. In conclusion, 4/6NX elicits high activity in central sympathetic, stress- and pain-related brain areas as well as in the limbic system, which can be ameliorated by losartan and particularly by moxonidine. These changes indicate a high sensitivity of CNS in initial stages of CKD which could be causative in clinical disturbances.
Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.
To trigger innate behavior, sensory neural networks are pre-tuned to extract biologically relevant stimuli. Many male-female or insect-plant interactions depend on this phenomenon. Especially communication among individuals within social groups depends on innate behaviors. One example is the efficient recruitment of nest mates by successful bumblebee foragers. Returning foragers release a recruitment pheromone in the nest while they perform a ‘dance’ behavior to activate unemployed nest mates. A major component of this pheromone is the sesquiterpenoid farnesol. How farnesol is processed and perceived by the olfactory system, has not yet been identified. It is much likely that processing farnesol involves an innate mechanism for the extraction of relevant information to trigger a fast and reliable behavioral response. To test this hypothesis, we used population response analyses of 100 antennal lobe (AL) neurons recorded in alive bumblebee workers under repeated stimulation with four behaviorally different, but chemically related odorants (geraniol, citronellol, citronellal and farnesol). The analysis identified a unique neural representation of the recruitment pheromone component compared to the other odorants that are predominantly emitted by flowers. The farnesol induced population activity in the AL allowed a reliable separation of farnesol from all other chemically related odor stimuli we tested. We conclude that the farnesol induced population activity may reflect a predetermined representation within the AL-neural network allowing efficient and fast extraction of a behaviorally relevant stimulus. Furthermore, the results show that population response analyses of multiple single AL-units may provide a powerful tool to identify distinct representations of behaviorally relevant odors.
TrkB mediates the effects of brain-derived neurotrophic factor (BDNF) in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR) signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC). TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.
Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.
Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.