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. 2014:2014:938235.
doi: 10.1155/2014/938235. Epub 2014 Feb 18.

Nociceptive neurons differentially express fast and slow T-type Ca²⁺ currents in different types of diabetic neuropathy

Eugen V Khomula  1 , Anya L Borisyuk  2 , Viacheslav Y Viatchenko-Karpinski  2 , Andrea Briede  2 , Pavel V Belan  1 , Nana V Voitenko  1
Affiliations

Nociceptive neurons differentially express fast and slow T-type Ca²⁺ currents in different types of diabetic neuropathy

Eugen V Khomula et al. Neural Plast. 2014.

Abstract

T-type Ca²⁺ channels are known as important participants of nociception and their remodeling contributes to diabetes-induced alterations of pain sensation. In this work we have established that about 30% of rat nonpeptidergic thermal C-type nociceptive (NTCN) neurons of segments L4-L6 express a slow T-type Ca²⁺ current (T-current) while a fast T-current is expressed in the other 70% of these neurons. Streptozotocin-induced diabetes in young rats resulted in thermal hyperalgesia, hypoalgesia, or normalgesia 5-6 weeks after the induction. Our results show that NTCN neurons obtained from hyperalgesic animals do not express the slow T-current. Meanwhile, the fraction of neurons expressing the slow T-current did not significantly change in the hypo- and normalgesic diabetic groups. Moreover, the peak current density of fast T-current was significantly increased only in the neurons of hyperalgesic group. In contrast, the peak current density of slow T-current was significantly decreased in the hypo- and normalgesic groups. Experimental diabetes also resulted in a depolarizing shift of steady-state inactivation of fast T-current in the hyperalgesic group and slow T-current in the hypo- and normalgesic groups. We suggest that the observed changes may contribute to expression of different types of peripheral diabetic neuropathy occurring during the development of diabetes mellitus.

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Figures

Figure 1
Figure 1
NTCN neurons express both fast and slow T-currents. (a) Identification of NTCN neurons. (A) A typical fluorescent image of an IB4-positive small-size DRG neuron. Note the intensive fluorescent ring associated with the neuronal plasma membrane. Scale bar, 20 μm. (B) A typical trace of transmembrane current induced by application of capsaicin (2 μM) in IB4-positive small-sized DRG neuron. IB4-positive capsaicin-sensitive small size DRG neurons were further considered as nonpeptidergic thermal C-type nociceptive (NTCN) neurons. (b) Representative current traces illustrate expression of T-currents with fast and slow kinetics of inactivation in different NTCN neurons. Currents were elicited using a 0.5 s voltage step to −45 mV after preconditioning at −95 mV for 3 s. A grey inset shows the same currents normalized by amplitude to underline a difference in kinetics of current inactivation. (c) A histogram demonstrates a pooled distribution of inactivation time constants of T-currents recorded from 85 neurons of control and PDN groups. The time constants were calculated from a single-exponential fit of current decay. A smooth curve is a fit of the distribution by a sum of two Gaussians. According to this fit T-currents were divided into fast (τ in < 50 ms; white bars) and slow (τ in > 50 ms; black bars) subtypes. (d) Kinetics of inactivation of fast and slow T-currents in control and PDN groups. Each column is the mean and SEM from the number of neurons specified in Figure 2(a). No significant difference compared to control was revealed under PDN conditions in kinetics of inactivation for both fast and slow T-currents. (e) Peak current density (PCD) of fast and slow T-currents under the control conditions. The columns are the mean and SEM calculated from 31 fast and 12 slow T-currents. ***P < 0.001. (f) PCD plotted versus inactivation time constant for fast and slow T-currents recorded under the control conditions. No significant correlations were found for both current types indicating that the difference in inactivation between fast and slow T-currents was not due to voltage clamp problems. Lines were liner fits of the dependencies; R 2 as a measure of correlation is shown in the plot.
Figure 2
Figure 2
Fast and slow T-currents expressed by NTCN neurons reveal different sensitivity to low Ni2+ concentration. (a) Representative current traces illustrate effect of Ni2+ application to NTCN neurons of naive rats expressing fast (left) and slow (right) T-currents. Initial (total) T-current traces are shown in black while grey traces represent a residual Ni2+-insensitive component of T-current persisted during Ni2+ application. Note the considerably larger blocking effect of Ni2+ application on the fast compared to slow T-currents. Insets show the total and Ni2+-insensitive currents normalized by their amplitudes in order to directly compare their inactivation kinetics further shown in (c). Note the slower inactivation of Ni2+-insensitive component compared to the total current for the case of slow T-current. Scale bars shown in (a) are applicable to all current traces in (a) and (b). (b) Representative traces for a Ni2+-sensitive component of fast (left) and slow (right) T-currents were obtained by digital subtraction of the Ni2+-insensitive component from the total T-current for traces shown in (a). Insets demonstrate normalized Ni2+-sensitive (gray) and Ni2+-insensitive (black) components. Note the absence of visible difference in kinetics of inactivation between these components of the fast T-current and a substantially slower Ni2+-insensitive component as compared to the Ni2+-sensitive one for the case of slow T-current. (c) Fractions of Ni2+-insensitive component in the fast and slow T-currents were significantly different. *-P < 0.05. (d) A ratio of inactivation kinetics of Ni2+-insensitive component and the total T-current for NTCN neurons expressing the fast and slow T-currents. There were no significant changes observed in the case of fast T-current (P > 0.4), while the inactivation kinetics of Ni2+-insensitive component of slow T-current was significantly slower compared to the inactivation kinetics of the total current. **-P < 0.01. (e) Inactivation kinetics of Ni2+-sensitive components of fast and slow T-currents. n.s.: no significant difference was revealed between the inactivation kinetics of Ni2+-sensitive components of the fast and slow T-currents (P > 0.3). Each column in (c), (d), and (e) is the mean and SEM from 6 fast and 3 slow T-currents.
Figure 3
Figure 3
Functional expression of fast and slow T-currents in NTCN neurons under different PDN conditions. (a) Percentage of NTCN neurons revealing the slow T-current in control (C), hyper- (D+), hypo- (D−), and normalgesic (Dn) groups. The slow T-current was not observed under hyperalgesic conditions, *P < 0.05 (Fisher's exact test). The numbers above the columns indicate the number of NTCN neurons expressing the slow T-current of the total number of tested neurons in the respective group. (b) PCD of fast and slow T-currents under the control and PDN conditions. It is interesting to note that the fast T-current was upregulated in hyperalgesic conditions while the slow T-current was strongly downregulated in norm- and hypoalgesia. Each column was the mean and SEM from number of neurons specified in (a). **P < 0.01 (ANOVA). n.s.: not significant (ANOVA).
Figure 4
Figure 4
PDN-specific changes in steady-state inactivation of T-currents in NTCN neurons. Each column demonstrates the mean and SEM of half-inactivation potential of steady-state inactivation (SSI) calculated for 13 “fast” and 7 “slow” neurons of control group (C), 7 neurons of hyperalgesic group (D+), 5 “fast” and 3 “slow” neurons of hypoalgesic group (D−), and 8 “fast” and 4 “slow” neurons of normalgesic group (Dn). The results demonstrate that a depolarization shift in SSI was observed for the fast T-current in a case of hyperalgesia and for the slow T-current in norm- and hypoalgesia. ANOVA between all columns produced P < 0.02. ***P < 0.001 (t-test for merged “C, fast,” “C, slow,” and “D−, fast,” “Dn, fast” versus merged “D+, fast,” “D−, slow,” and “Dn, slow”). n.s.: not significant (ANOVA).
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