3 research outputs found
Functional Characterization of Lamina X Neurons in ex-Vivo Spinal Cord Preparation
Functional properties of lamina X neurons in the spinal cord remain unknown despite
the established role of this area for somatosensory integration, visceral nociception,
autonomic regulation and motoneuron output modulation. Investigations of neuronal
functioning in the lamina X have been hampered by technical challenges. Here we
introduce an ex-vivo spinal cord preparation with both dorsal and ventral roots still
attached for functional studies of the lamina X neurons and their connectivity using an
oblique LED illumination for resolved visualization of lamina X neurons in a thick tissue.
With the elaborated approach, we demonstrate electrophysiological characteristics
of lamina X neurons by their membrane properties, firing pattern discharge and
fiber innervation (either afferent or efferent). The tissue preparation has been also
probed using Ca2+ imaging with fluorescent Ca2+ dyes (membrane-impermeable or
-permeable) to demonstrate the depolarization-induced changes in intracellular calcium
concentration in lamina X neurons. Finally, we performed visualization of subpopulations
of lamina X neurons stained by retrograde labeling with aminostilbamidine dye to
identify sympathetic preganglionic and projection neurons in the lamina X. Thus, the
elaborated approach provides a reliable tool for investigation of functional properties
and connectivity in specific neuronal subpopulations, boosting research of lamina X of
the spinal cord
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