Correction to: BIOMEMBRANES 2018 : Evaluation of the possibility of simultaneous using membrane biosensors and vital dyes in determining the sensitivity of 2D and 2.5D systems based on cancer cell lines to oncotoxic drugs.

This research was supported by RSF grant (No. 18-15-00391).

Epac-mediated cAMP-signalling in the mouse model of Rett Syndrome.

Rett Syndrome (RTT) is a neurodevelopmental disease thought to be caused by deficits in synaptogenesis and neuronal circuitry. cAMP is one of the key factors for neuronal outgrowth, plasticity and regeneration. We examined its homeostasis in RTT during early postnatal development of the essential part of the respiratory network, pre-Bötzinger complex. Using targeted expression of Epac1-camps sensor in neurons we quantified cAMP levels and their fluctuations in MeCP2-/y mice, an established model of RTT. Resting cAMP levels in the mutant were smaller than in the wild-type. cAMP transients elicited by depolarisation and stimulation of adenylate cyclase had also smaller amplitudes and faster time-courses. The anomalies in MeCP2 -/y mice were removed after inhibition of phosphodiesterase PDE4 with rolipram. Brief cAMP elevations triggered elongation of neuronal processes that was significantly bigger in the wild-type. The effects were observed after inhibition of protein kinase A and mimicked by activation of a guanine nucleotide exchange factor, Epac, with 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pCPT). The agonist reinforced bursting in preBötC neurons in the mutant and converted it to the wild-type. All actions of 8-pCPT were not reproduced by its non-active analogue and abolished by Epac signalling inhibitor Brefeldin A. We propose that disturbances in cAMP homeostasis in MeCP2 -/y mice can lead to inadequate Epac signalling. Concomitant defective development of respiratory circuits may be responsible for irregular breathing activity in RTT.

Imaging cytoplasmic cAMP in mouse brainstem neurons.

BACKGROUND: 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. RESULTS: 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]i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP]i levels increased after membrane depolarisation and release of Ca2+ from internal stores. The effects developed slowly and reached their maximum after transient [Ca2+]i elevations subsided. Ca2+-dependent [cAMP]i transients 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]i transients. CONCLUSION: 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.

Imaging of respiratory network topology in living brainstem slices.

Topology of neuronal networks contributes to their functioning but the structure-function relationships are not yet understood. In order to reveal the spatial organisation of the respiratory network, we expressed enhanced green fluorescent proteins in neurons in brainstem slices containing the respiratory kernel (pre-Bötzinger complex). The expression was neuron specific due to use of adeno-associated viral vector driving transgene expression from synapsin 1 promoter. Both neuronal cell bodies and their dendrites were labelled with high efficacy. This labelling allowed for enhanced spatial resolution as compared to conventional calcium-sensitive dyes. Neurons occupied about 10% of tissue volume and formed an interconnected network. Using custom-developed software, we quantified the network structure that had a modular structure consisting of clusters having transverse (dorso-ventral) orientation. They contained in average seven neurons and connections between the cells in different clusters were less frequent. This novel in situ imaging technique is promising to gain new knowledge about the fine structure and function of neuronal networks in living slice preparations.