Calcium activity of upper thoracic dorsal root ganglion neurons in zucker diabetic Fatty rats

Abstract

The aim of the present study was to examine the calcium activity of C8-T5 dorsal root ganglion (DRG) neurons from Zucker diabetic fatty rats. In total, 8 diabetic ZDF fatty animals and 8 age-matched control ZDF lean rats were employed in the study. C8-T5 dorsal root ganglia were isolated bilaterally from 14 to 18 weeks old rats, and a primary culture was prepared. Calcium activity was measured ratiometrically using the fluorescent Ca(2+)-indicator Fura-2 acetoxymethyl ester. All neurons were stimulated twice with 20 mM K(+), followed by stimulation with either 0.3 or 0.5 μ M Capsaicin, alone or in combination with algogenic chemicals (bradykinin, serotonin, prostaglandin E2 (all 10(-5) M), and adenosine (10(-3) M)) at pH 7.4 and 6.0. Neurons from diabetic animals exhibited an overall increased response to stimulation with 20 mM K(+) compared to neurons from control. Stimulation with Capsaicin alone caused an augmented response in neurons from diabetic animals compared to control animals. When stimulated with a combination of Capsaicin and algogenic chemicals, no differences between the two groups of neurons were measured, neither at pH 7.4 nor 6.0. In conclusion, diabetes-induced alterations in calcium activity of the DRG neurons were found, potentially indicating altered neuronal responses during myocardial ischemia.

Figure 1

Distribution of neuronal cell diameter. The neurons were divided into 5 μm bins. Small-diameter neurons had diameters < 30 μm, medium-sized neurons had diameters between 30–39 μm, and large-diameter neurons had diameters of ≥ 40 μm. The majority of tested neurons were small-sized. n (control) = 552, n (diabetic) = 586.

Figure 2

Example of calcium activity measurement. The figure depicts the trace of two cells obtained during a single experiment: a capsaicin-responsive (dark purple) and a capsaicin-unresponsive (light purple) neuron. The three small photomicrographs show the same neurons, at different stages of the experiment: (1) before stimulation, (2) during stimulation with 20 mM K⁺, and (3) during stimulation with 0.5 μM Capsaicin + algogenic chemicals. Black arrows beneath the trace indicate the beginning of stimulation. Time scale bar; 120 s. Cap; capsaicin.

Figure 3

Example of time-[Ca²⁺]_i ratio development in dorsal root ganglion neurons from control and diabetic rats. The neurons were stimulated with 20 mM K⁺. For clarity in the illustration, the traces were background corrected. Arbitrary scale; vertical scale: 0.20 (340/380 nm light intensity ratio), and horizontal scale: 20 s.

Figure 4

Traces and box plots from capsaicin experiments. Panels (a), (d), (g), and (j) show traces (examples) of the time-[Ca²⁺]_i development for control and diabetic dorsal root ganglion neurons. The arrows indicate the onset of 10 sec stimulation with (a) capsaicin, (d) combination of Capsaicin and algogenic chemicals (AC); (g) combination of 0.3 μM Capsaicin and AC at pH 6.0, and (j) 0.5 μM Capsaicin and AC at pH 6.0. The traces were background corrected. As a measure of [Ca²⁺]_i, the vertical axis is shown as the 340/380 nm light intensity ratio. (a) Vertical scale: 0.10; horizontal scale: 20 s, (d) vertical scale: 0.20; horizontal scale: 20 s.; (g) vertical scale: 0.10; horizontal scale: 20 s., and (j) vertical scale: 0.20; horizontal scale: 20 sec. Panels (b), (e), (h), and (k) display box plots on the absolute area under the curve (AUC) derived from stimulation with the in (a), (d), (g), and (j) mentioned capsaicin-containing solutions. Panels (c), (f), (i), and (l) display box plots with median, 25th and 75th percentiles on the relative AUC of the capsaicin-containing solution-derived [Ca²⁺]_i increase and the preceding 20 mM K⁺-derived [Ca²⁺]_i increase. The error bars indicate the 10th and 90th percentiles. Values below the 10th and above the 90th percentiles are shown as dots. + indicates the mean. *P < 0.05.

Figure 5

Scatter plots displaying AUC for neurons from control and diabetic animals derived from stimulation with (a) capsaicin, (b) combination of Capsaicin and algogenic chemicals (AC); (c) combination of 0.3 μM Capsaicin and AC at pH 6.0, and (d) 0.5 μM Capsaicin and AC at pH 6.0.