Calcium sensitive dye. Uploading in vitro and in vivo, biological application and achievements.
One pf the common indicator of the intracellular Calcium - Fura-2
One pf the common indicator of the intracellular Calcium - Fura-2
A: schematic showing the transverse plane of the spinal cord together with different sites for dye injection.
B: schematic showing the injection site for subdural dye injections and location of electroporation electrodes.
C: high power view of a cell located dorsally in L1 following electroporation of calcium green salt (T12–L3).
D: labeling pattern on a transverse section at L6 following electroporation of the calcium green salt (L4–S2).
E: transverse section at L5 following dye injection in the ventral subdural space but no electroporation.
F: midline sagittal section showing the labeling following electroporation of the calcium green salt (L5–S2).
Vertical dotted lines indicate the approximate
border between spinal segments. In all figures, dorsal is up and
ventral is down.
Electroporation Loading of Calcium-Sensitive Dyes Into the CNS
Agne`s Bonnot, George Z. Mentis, Jesse Skoch, and Michael J. O’Donovan; Laboratory of Neural Control, Section on Developmental Neurobiology, National Institutes of Health, Bethesda, Maryland
Calcium transients recorded from electroporated cells in response to a single stimulus to dorsal root afferents.
A: fluorescence microgram of the L5–L6 area of the cord following subdural application of the calcium green salt and electroporation (L4–S4) and viewed through the lateral white matter. Vertical dotted line is the border between L5 and L6.
B: difference image obtained by subtracting the control fluorescence (image A) from an average generated during the peak response to a single stimulus applied to the ipsilateral L6 dorsal root.
C: optical (top trace measured from red boxed area shown in A) and electrical responses
(bottom trace, L6 ventral root) following a single stimulus to the dorsal root. Arrowhead indicates stimulus. Note that the optical trace is derived from dorsally located interneurons, while the electrical trace corresponds to activity of ventrally located motoneurons. D: 16 frame average (7.8 s/frame) from dorsal horn cells located at a depth of 170 m from the lateral surface.
E:
active difference image obtained by subtracting the control
fluorescence from an average generated during a single stimulus
applied to the L6 dorsal root.
F: calcium transients corresponding to the 3 regions of interest drawn in E. Bottom trace is the simultaneously recorded ventral root L6 electrical response (DC to 3 kHz). Dotted line at top of panel indicates the period over which active frames were averaged to generate the difference image in E.