Distinct mechanisms of signal processing by lamina I spino-parabrachial neurons

Lamina I spino-parabrachial neurons (SPNs) receive peripheral nociceptive input, process it and transmit to the supraspinal centres. Although responses of SPNs to cutaneous receptive field stimulations have been intensively studied, the mechanisms of signal processing in these neurons are poorly understood. Therefore, we used an ex-vivo spinal cord preparation to examine synaptic and cellular mechanisms determining specific input-output characteristics of the neurons. The vast majority of the SPNs received a few direct nociceptive C-fiber inputs and generated one spike in response to saturating afferent stimulation, thus functioning as simple transducers of painful stimulus. However, 69% of afferent stimulation-induced action potentials in the entire SPN population originated from a small fraction (19%) of high-output neurons. These neurons received a larger number of direct Ad- and C-fiber inputs, generated intrinsic bursts and efficiently integrated a local network activity via NMDA-receptor-dependent mechanisms. The high-output SPNs amplified and integrated the nociceptive input gradually encoding its intensity into the number of generated spikes. Thus, different mechanisms of signal processing allow lamina I SPNs to play distinct roles in nociception.The authors thank Mr. Andrew Dromaretsky for the technical assistance. P.B. was supported by the National Academy of Sciences of Ukraine (NASU), grant NASU # 0116U004470, grant NASU#67/15-Н. N.V. was supported by the NASU Biotechnology and NASU-KNU grants; NIH 1R01NS113189-01. B.V.S. was supported by the FEDER funds through the COMPETE 2020 (POCI), Portugal 2020, and by the FCT project PTDC/NEU-NMC/1259/2014 (POCI-01-0145-FEDER-016588

Measurement of intracellular concentration of fluorescently-labeled targets in living cells

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells