This study is devoted to revealing of mechanisms underlying development of different types of peripheral diabetic neuropathy manifested by presence of thermal hyperalgesia, hypoalgesia and normalgesia (unchanged pain sensitivity). Using rats of the same age with thermal hyper-, hypo- and normalgesia developed at the same duration of streptozotocin-induced diabetes (STZ-diabetes) it has been established that differences in thermal pain sensitivity are associated with specific remodeling of TRPV1 channels and T-type voltage-gated calcium channels (T-channels) and their Ca2+ signaling in small IB4-positive capsaicin-sensitive DRG neurons known to be nonpeptidergic thermal C-type nociceptive neurons involved in development and maintenance of neuropathic pain. In the current study it is found that hyperalgesic (18%), hypoalgesic (25%) and normalgesic (57%) animals are simultaneously present in the same population of rats with 6-7 weeks of STZ-diabetes. No difference is observed in blood glucose levels and weight between diabetic rats with different thermal pain sensitivity suggesting that this difference is unlikely due to a different metabolic state of the experimental animals. Using isolectin B4 (IB4) for in vitro labeling of nonpeptidergic neurons, it is shown that there is no significant redistribution between C-type nociceptors of strongly IB4-positive, weakly IB4-positive and IB4-negative classes between hyperalgesic, hypoalgesic and normalgesic animals. The IB4-positive small DRG neurons are the most abundant and constitute 73 ± 6 %, 76 ± 7 %, 78 ± 8 %, 76 ± 7 % of control, hyperalgesic, hypoalgesic and normalgesic groups, correspondingly. Thus observed differences in thermal pain sensitivity are unlikely due to elimination of this specific neuronal class or redistribution between nonpeptidergic and peptidergic primary nociceptors. It is demonstrated in small IB4-positive capsaicin-sensitive DRG neurons (that are the focus of this study) that TRPV1-mediated currents and respective [Ca2+]i transients are significantly (p < 0.01) decreased under hypoalgesia and normalgesia (correspondingly, by 43 ± 15 % and 48 ± 12 % for peak current density and by 67 ± 8% and 67 ± 7% for amplitude of transients) and significantly increased under hyperalgesia (by 45 ± 11% for peak current density and by 112 ± 11 % for amplitude of transients) as compared to control. Thus TRPV1-mediated signaling is significantly and oppositely altered under hyperalgesia versus hypoalgesia and normalgesia implying TRPV1 channels in differences in thermal pain sensitivity. It is revealed in the same small IB4-positive capsaicin-sensitive DRG neurons that peak current density via T-channels is significantly (p < 0.05) decreased under hypoalgesia and normalgesia (correspondingly, by 43 ± 15 % and 48 ± 12 %) and significantly increased under hyperalgesia (by 45 ± 11%) as compared to control. Amplitudes of respective [Ca2+]i transients are significantly (p < 0.05) increased in all diabetic groups: by 66 ± 24 % under hyperalgesia, by 55 ± 18% under hypoalgesia and by 88 ± 18% under normalgesia as compared to control indicating differential changes of Ca2+-regulating systems associated with hyperalgesic, hypoalgesic and normalgesic peripheral diabetic neuropathy. The voltage dependence and kinetics of activation as well as kinetics of inactivation of T-channels are found unaffected in diabetic rats. At the same time, there is ~7 mV shift in steady state inactivation of T-channels towards depolarization in hyperalgesic, hypoalgesic and normalgesic groups as compared to control. This shift underlies T-channels' upregulation under hyperalgesia and normalgesia and partially compensate their downregulation under hypoalgesia in the range of resting membrane potentials (~-60 mV) despite of unchanged T-channels' functional expression under hyperalgesia and almost twofold lower functional expression under hyperalgesia and normalgesia as compared to control. T-channels window current is increased up to 0.5 pA under hyperalgesia versus 0.1 pA in control at the resting membrane potentials contributing up to 50 nM to increased resting [Ca2+]i level (230 ± 20 nM versus 72 ± 11 nM in control). Pharmacological properties of T-channels in the studied neurons, specifically strong blocking effects of amiloride (500 M), nickel (50 M) as well as potentiation by L-cysteine (100 M), are not different between control and neuropathic groups, suggesting that Cav3.2 isoform majorly (~80% of current amplitude) contributes in control as well as hyperalgesic, hypoalgesic and normalgesic animals. All in all, in this study it is revealed that under peripheral diabetic neuropathy in rats with 6-7 weeks of STZ-diabetes: (1) thermal hyperalgesia is associated with cooperative upregulation of Cav3.2 channels and TRPV1; (2) thermal normalgesia is associated with upregulation of Cav3.2 channels and downregulation of TRPV1; (3) thermal hypoalgesia was associated with cooperative downregulation of Cav3.2 channels and TRPV1. In summary, our results demonstrate that specific and simultaneous alterations in functioning of Cav3.2 T-type and TRPV1 channels in nonpeptidergic thermal C-type nociceptive neurons are associated with different types of peripheral diabetic neuropathy. Our results provide a better understanding of potential molecular mechanisms underlying the variety of pain syndromes induced by type 1 diabetes. Keywords: streptozotocin-induced diabetes (STZ-diabetes), dorsal root ganglion (DRG), patch clamp, calcium imaging, calcium transients, Cav3.2, hypoalgesia, normalgesia, hyperalgesia.
