Imaging cytoplasmic cAMP in mouse brainstem neurons

<p>Abstract</p> <p>Background</p> <p>cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca²⁺levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before.</p> <p>Results</p> <p>Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Bötzinger complex, constituting the rhythm-generating part of the respiratory network. Fluorescent cAMP sensor Epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to physiological stimuli. We recorded [cAMP]_ichanges in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP]_ilevels increased after membrane depolarisation and release of Ca²⁺from internal stores. The effects developed slowly and reached their maximum after transient [Ca²⁺]_ielevations subsided. Ca²⁺-dependent [cAMP]_itransients were suppressed after blockade of adenylate cyclase with 0.1 mM adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. During paired stimulations, the second depolarisation and Ca²⁺release evoked bigger cAMP responses. These effects were abolished after inhibition of protein kinase A with H-89 pointing to the important role of phosphorylation of calcium channels in the potentiation of [cAMP]_itransients.</p> <p>Conclusion</p> <p>We constructed and characterized a neuron-specific cAMP probe based on Epac1-camps. Using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. Strong fluorescence, resistance to photobleaching and possibility of direct estimation of [cAMP] levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. Epac1-camps was applied to examine the crosstalk between Ca²⁺and cAMP signalling and revealed a synergism of actions of these two second messengers.</p

Dynamic assembly of ribbon synapses and circuit maintenance in a vertebrate sensory system

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity

Crosswell traveltime tomography in three dimensions

Conventional crosswell direct-arrival traveltime tomography solves for velocity in a 2-D slice of the subsurface joining two wells. Many 3-D aspects of real crosswell surveys, including well deviations and out-of-well-plane structure, are ignored in 2-D models. We present a 3-D approach to crosswell tomography that is capable of handling severe well deviations and multiple-profile datasets. Three-dimensional pixelized models would be even more seriously underdetermined than the pixelized models that have been used in 2-D tomography. We, therefore, employ a thinly layered, vertically discontinuous 3-D velocity model that greatly reduces the number of model parameters. The layers are separated by 2-D interfaces represented as 2-D Chebyshev polynomials that are determined using a priori structural information and remain fixed in the traveltime inversion. The velocity in each layer is also represented as a 2-D Chebyshev polynomial. Unlike pixelized models that provide limited vertical resolution and may be overparameterized horizontally, this 3-D model provides vertical resolution comparable to the scale of wireline logs, and reduces the degrees of freedom in the horizontal parameterization to the expected in-line and out-of-well-plane horizontal resolution available in crosswell traveltime data. Ray tracing for the nonlinear traveltime inversion is performed in three dimensions. The 3-D tomography problem is regularized using penalty constraints with a continuation strategy that allows us to extrapolate the velocity field to a 3-D region containing the 2-D crosswell profile. Although this velocity field cannot be expected to be accurate throughout the 3-D region, it is at least as accurate as 2-D tomograms near the well plane of each 2-D crosswell profile. Futhermore, multiple-profile crosswell data can be inverted simultaneously to resolve better the 3-D distribution of velocity near the profiles. Our velocity parameterization is quite different from pixelized models, so resolution properties will be different. Using wave-modeled synthetic data, we find that near horizontal raypaths have the largest mismatch between ray-traced traveltimes and traveltimes estimated from the data. In conventional tomography, horizontal raypaths are essential for high vertical resolution. With our model, however, the highest resolution and most accurate inversions are achieved by excluding raypaths that travel nearly parallel to the geologic layering. We perform this exclusion in both a static and model-based manner. We apply our 3-D method to a multiple-profile crosswell survey at the Cymric oil field in California, an area of very steep structural dips and significant well trajectory deviations. Results of this multiple-profile 3-D tomography correlate very well with the independently-processed single profile results, with the advantage of an improved tie at the common well