Imaging cellular network dynamics in three dimensions using fast 3D laser scanning

Spatiotemporal activity patterns in three-dimensionally organized cellular networks are fundamental to the function of the nervous system. Despite advances in functional imaging of cell populations, a method to resolve local network activity in three dimensions has been lacking. Here we introduce a three-dimensional (3D) line-scan technology for two-photon microscopy that permits fast fluorescence measurements from several hundred cells distributed in 3D space. We combined sinusoidal vibration of the microscope objective at 10 Hz with 'smart' movements of galvanometric x-y scanners to repeatedly scan the laser focus along a closed 3D trajectory. More than 90% of cell somata were sampled by the scan line within volumes of 250 microm side length. Using bulk-loading of calcium indicator, we applied this method to reveal spatiotemporal activity patterns in neuronal and astrocytic networks in the rat neocortex in vivo. Two-photon population imaging using 3D scanning opens the field for comprehensive studies of local network dynamics in intact tissue.     

Renal IL-17 expression in human ANCA-associated glomerulonephritis

Interleukin-17A (IL-17) promotes inflammatory renal tissue damage in mouse models of crescentic glomerulonephritis, including murine experimental autoimmune anti-myeloperoxidase glomerulonephritis, which most likely depends on IL-17-producing Th17 cells. In human anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, however, the cellular sources of IL-17 remain to be elucidated. Therefore, we analyzed human kidney biopsies of active necrotizing and crescentic ANCA-associated glomerulonephritis by immunohistochemistry using an IL-17-specific antibody and by immunofluorescent colocalization with cell type markers. We detected numerous IL-17-expressing (IL-17(+)) cells in the glomeruli and in the tubulointerstitium. Unexpectedly, most of these IL-17(+) cells were polymorphonuclear neutrophilic granulocytes, while IL-17(+) T cells and IL-17(+) mast cells were present at significantly lower frequencies. IL-17 was not detected in other infiltrating or resident kidney cells. In those patients who had not received immunosuppressive treatment before biopsy, serum creatinine levels were positively correlated with tubulointerstitial IL-17(+) neutrophils as well as IL-17(+) T cells. Furthermore, we could demonstrate that purified human blood neutrophils expressed IL-17 protein and released it upon stimulation in vitro. In conclusion, these results support a pathogenic role for IL-17 in human ANCA-associated glomerulonephritis. Our data suggest that in the acute stage of the disease neutrophils may act as an important immediate-early innate source of IL-17 and may thereby initiate and promote ongoing renal inflammation. IL-17 may thus be a target for treating acute ANCA-associated glomerulonephritis.     

Differential effects of the brefeldin A analogue (6R)-hydroxy-BFA in tobacco and Arabidopsis

The effects of two brefeldin A (BFA) analogues (BFA lactam; 6(R)-hydroxy-BFA) on plant cells were tested. Although these two compounds elicited BFA-like effects in mammalian cells, the lactam analogue failed to elicit a response in plant cells. By contrast, while the 6(R)-hydroxy-BFA analogue gave rise to a classic BFA response in tobacco mesophyll protoplasts and true leaves of Arabidopsis (redistribution of Golgi enzymes into the ER), it failed to cause the formation of BFA-compartments in Arabidopsis root cells and cotyledonary leaves. Even when the GNL1-LM mutant of Arabidopsis, which has a cis-Golgi located BFA-sensitive ARF-GEF, was used, the 6(R)-hydroxy analogue failed to elicit a response at conventional BFA concentrations. Only at concentrations of over 200 μM did 6(R)-hydroxy-BFA elicit a BFA-like effect. These differences are interpreted in terms of the different properties of the respective TGN- (Arabidopsis roots) and cis-Golgi- (tobacco mesophyll) localized BFA-sensitive ARF-GEFs.     

Expression pattern of cell cycle-related gene products in synovial stroma and synovial lining in active and quiescent stages of rheumatoid arthritis

OBJECTIVE: To investigate the expression pattern of cell cycle related gene products in active and quiescent Rheumatoid arthritis (RA). METHODS: Synovial tissue from 20 patients with active proliferative RA and 28 patients with RA in remission was immunohistochemically examined for expression of p53, p63, p21, p27, p16, cyclin D1, CDK4, RB, E2F, Ki-67 on tissue microarrays and by DNA flow cytometry for cell cycle phases. RESULTS: Elevated expression of p53 and p27 was found in synovial lining and in stromal cells in proliferative active RA. In the remission stage this finding was confined to the synovial lining. Most of the cells were in the G0-phase. Ki-67 proliferation index was maximum 10% in synovial cells. CONCLUSION: The p53 pathway is activated in synovial cells in active RA as well as in quiescent stage of disease. Differences in the spatial expression pattern of proteins involved in the p53 pathway in RA in remission compared to actively proliferating RA reflect the phasic nature of the disease and support in our opinion the concept of adaptive role of p53 pathway in RA.     

Activity in a Premotor Cortical Nucleus of Zebra Finches Is Locally Organized and Exhibits Auditory Selectivity in Neurons but Not in Glia

Motor functions are often guided by sensory experience, most convincingly illustrated by complex learned behaviors. Key to sensory guidance in motor areas may be the structural and functional organization of sensory inputs and their evoked responses. We study sensory responses in large populations of neurons and neuron-assistive cells in the songbird motor area HVC, an auditory-vocal brain area involved in sensory learning and in adult song production. HVC spike responses to auditory stimulation display remarkable preference for the bird''s own song (BOS) compared to other stimuli. Using two-photon calcium imaging in anesthetized zebra finches we measure the spatio-temporal structure of baseline activity and of auditory evoked responses in identified populations of HVC cells. We find strong correlations between calcium signal fluctuations in nearby cells of a given type, both in identified neurons and in astroglia. In identified HVC neurons only, auditory stimulation decorrelates ongoing calcium signals, less for BOS than for other sound stimuli. Overall, calcium transients show strong preference for BOS in identified HVC neurons but not in astroglia, showing diversity in local functional organization among identified neuron and astroglia populations.     

From rio to johannesburg — a business perspective the 7th international business forum

Conclusions  When we take stock of developments in the decade since the 1992 United Nations Conference on Environment and Development in Rio de Janeiro, we see a diverse picture with hopeful and promising steps in the right direction, but also reasons to be concerned.     

Competitive Calcium Binding: Implications for Dendritic Calcium Signaling

Action potentials evoke calcium transients in dendrites of neocortical pyramidal neurons with time constants of <100 ms at physiological temperature. This time period may not be sufficient for inflowing calcium ions to equilibrate with all present Ca²⁺-binding molecules. We therefore explored nonequilibrium dynamics of Ca²⁺ binding to numerous Ca²⁺ reaction partners within a dendritelike compartment using numerical simulations. After a brief Ca²⁺ influx, the reaction partner with the fastest Ca²⁺ binding kinetics initially binds more Ca²⁺ than predicted from chemical equilibrium, while companion reaction partners bind less. This difference is consolidated and may result in bypassing of slow reaction partners if a Ca²⁺ clearance mechanism is active. On the other hand, slower reaction partners effectively bind Ca²⁺ during repetitive calcium current pulses or during slower Ca²⁺ influx. Nonequilibrium Ca²⁺ distribution can further be enhanced through strategic placement of the reaction partners within the compartment. Using the Ca²⁺ buffer EGTA as a competitor of fluo-3, we demonstrate competitive Ca²⁺ binding within dendrites experimentally. Nonequilibrium calcium dynamics is proposed as a potential mechanism for differential and conditional activation of intradendritic targets.     

Itch Relief by Mirror Scratching. A Psychophysical Study

Objective

The goal of this study was to test whether central mechanisms of scratching-induced itch attenuation can be activated by scratching the limb contralateral to the itching limb when the participant is made to visually perceive the non-itching limb as the itching limb by means of mirror images.

Methods

Healthy participants were asked to assess the intensity of an experimentally induced itch at their right forearm while they observed externally guided scratch movements either at their right (itching) or left (non-itching) forearm which were either mirrored or not mirrored. In the first experiment, a mirror placed between the participant’s forearms was used to create the visual illusion that the participant’s itching (right) forearm was being scratched while in fact the non-itching (left) forearm was scratched. To control visibility of the left (non-mirrored) forearm, a second experiment was performed in which unflipped and flipped real-time video displays of the participant’s forearms were used to create experimental conditions in which the participant visually perceived scratching either on one forearm only, on both forearms, or no scratching at all.

Results

In both experiments, scratching the non-itching limb attenuated perceived itch intensity significantly and selectively in the mirror condition, i.e., when the non-itching forearm was visually perceived as the itching limb.

Discussion

These data provide evidence that the visual illusion that an itching limb is being scratched while in fact the non-itching limb contralateral to the itching limb is scratched, can lead to significant itch relief. This effect might be due to a transient illusionary intersensory perceptual congruency of visual, tactile and pruriceptive signals. “Mirror scratching” might provide an alternative treatment to reduce itch perception in focal skin diseases with persistent pruritus without causing additional harm to the affected skin and might therefore have significant clinical impact.