Streptozotocin (STZ)-induced type 1 diabetes in rats leads to the development of peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia at early stages (4th week) followed by hypoalgesia after 8weeks of diabetes development. Here we found that 6-7 week STZ-diabetic rats developed either thermal hyper- (18%), hypo- (25%) or normalgesic (57%) types of PDN. These developmentally similar diabetic rats were studied in order to analyze mechanisms potentially underlying different thermal nociception. The proportion of IB4-positive capsaicin-sensitive small DRG neurons, strongly involved in thermal nociception, was not altered under different types of PDN implying differential changes at cellular and molecular level. We further focused on properties of T-type calcium and TRPV1 channels, which are known to be involved in Ca(2+) signaling and pathological nociception. Indeed, TRPV1-mediated signaling in these neurons was downregulated under hypo- and normalgesia and upregulated under hyperalgesia. A complex interplay between diabetes-induced changes in functional expression of Cav3.2 T-type calcium channels and depolarizing shift of their steady-state inactivation resulted in upregulation of these channels under hyper- and normalgesia and their downregulation under hypoalgesia. As a result, T-type window current was increased by several times under hyperalgesia partially underlying the increased resting [Ca(2+)]i observed in the hyperalgesic rats. At the same time Cav3.2-dependent Ca(2+) signaling was upregulated in all types of PDN. These findings indicate that alterations in functioning of Cav3.2 T-type and TRPV1 channels, specific for each type of PDN, may underlie the variety of pain syndromes induced by type 1 diabetes.

BackgroundPrevious studies have shown that increased excitability of capsaicin-sensitive DRG neurons and thermal hyperalgesia in rats with short-term (2–4 weeks) streptozotocin-induced diabetes is mediated by upregulation of T-type Ca2+ current. In longer–term diabetes (after the 8th week) thermal hyperalgesia is changed to hypoalgesia that is accompanied by downregulation of T-type current in capsaicin-sensitive small-sized nociceptors. At the same time pain symptoms of diabetic neuropathy other than thermal persist in STZ-diabetic animals and patients during progression of diabetes into later stages suggesting that other types of DRG neurons may be sensitized and contribute to pain. In this study, we examined functional expression of T-type Ca2+ channels in capsaicin-insensitive DRG neurons and excitability of these neurons in longer-term diabetic rats and in thermally hypoalgesic diabetic rats.ResultsHere we have demonstrated that in STZ-diabetes T-type current was upregulated in capsaicin-insensitive low-pH-sensitive small-sized nociceptive DRG neurons of longer-term diabetic rats and thermally hypoalgesic diabetic rats. This upregulation was not accompanied by significant changes in biophysical properties of T-type channels suggesting that a density of functionally active channels was increased. Sensitivity of T-type current to amiloride (1 mM) and low concentration of Ni2+ (50 μM) implicates prevalence of Cav3.2 subtype of T-type channels in the capsaicin-insensitive low-pH-sensitive neurons of both naïve and diabetic rats. The upregulation of T-type channels resulted in the increased neuronal excitability of these nociceptive neurons revealed by a lower threshold for action potential initiation, prominent afterdepolarizing potentials and burst firing. Sodium current was not significantly changed in these neurons during long-term diabetes and could not contribute to the diabetes-induced increase of neuronal excitability.ConclusionsCapsaicin-insensitive low-pH-sensitive type of DRG neurons shows diabetes-induced upregulation of Cav3.2 subtype of T-type channels. This upregulation results in the increased excitability of these neurons and may contribute to nonthermal nociception at a later-stage diabetes.

The Piezo2 channel is a newly identified mammalian mechanical transducer that confers rapidly adapting mechanically activated (RA-MA) currents in primary afferent neurons. The Piezo2 channels sense rapid membrane displacement, but it is not clear whether they are sensitive to osmotic swelling, which slowly increases static plasma membrane tension (SPMT). Here, we show that SPMT exerts a profound impact on the mechanical sensitivity of RA-MA channels in primary afferent neurons. RA-MA currents are greatly enhanced, and the mechanical threshold was reduced in both primary afferent neurons of rat dorsal root ganglia (DRG) and HEK293 cells heterologously expressing Piezo2 when these cells undergo osmotic swelling to increase SPMT. Osmotic swelling switches the kinetics of RA-MA currents to the slowly adapting type in both cultured DRG neurons and HEK293 cells heterologously expressing Piezo2. The potentiation of RA-MA currents is abolished when cultured DRG neurons are treated with cytochalasin D, an actin filament disruptor that prevents SPMT of cultured DRG neurons from an increase by osmotic swelling. Osmotic swelling significantly increases DRG neuron mechano-excitability such that a subthreshold mechanical stimulus can result in action potential firing. Behaviorally, the mechanical hind paw withdrawal threshold in rats is reduced following the injection of a hypotonic solution, but this osmotic effect is abolished when cytochalasin D or Gd 3؉ is co-administered with the hypoosmotic solution. Taken together, our findings suggest that Piezo2-mediated mechanotransduction is regulated by SPMT in primary afferent neurons. Because SPMT can be changed by multiple biological factors, our findings may have broad implications in mechanical sensitivity under physiological and pathological conditions.

Persistent inflammation promotes internalization of synaptic GluR2-containing Ca2+-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing Ca2+-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires an activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are still unclear. By utilizing the antisence oligodeoxynucleotides that specifically knockdown PKCα, we have found that a decrease in dorsal horn PKCα expression prevents complete Freund’s adjuvant (CFA)-induced increase in a functional expression of extrasynaptic Ca2+-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. This was manifested as an abolishment of augmented AMPA-induced currents and associated [Ca2+]i transients, and as a reverse of the current rectification 1 d post-CFA. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibiting strong adaptation. Finally, dorsal horn PKCα knockdown produced anti-nociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Altogether, our results indicate that inflammation-induced trafficking of extrasynaptic Ca2+-permeable AMPARs in tonically firing SG neurons depends on PKCα, and suggest that this PKCα-dependent trafficking may contribute to the persistent inflammatory pain maintenance.

Chemotherapy drugs such as oxaliplatin can increase nociceptive neuron excitability to result in neuropathic pain in orofacial and other regions in patients following chemotherapy. However, mechanisms underlying chemotherapy-induced increases of nociceptive neuron excitability are not fully understood. Kv4.3 channels are voltage-gated K+ channels mediating A-type K+ (IA) currents to control neuronal excitability. In the present study, we examined Kv4.3 channel expression on trigeminal neurons that innervate orofacial regions (V2 TG neurons) of rats using immunostaining method. We showed that strong Kv4.3 immunoreactivity (Kv4.3-ir) was present mainly in small-sized V2 TG neurons. The numbers of Kv4.3-ir positive V2 TG neurons were significantly reduced in oxaliplatin-treated rats, suggesting down-regulation of Kv4.3 channel expression on V2 TG neurons by the chemotherapy drug. Patch-clamp recordings from acutely dissociated rat V2 TG neurons showed that almost all nociceptive-like V2 TG neurons displayed IA currents with slow inactivation kinetics. The amplitudes of IA currents were significantly reduced in these nociceptive-like V2 TG neurons of oxaliplatin-treated group. Furthermore, we found that the excitability of nociceptive-like V2 TG neurons was significantly higher in the oxaliplatin-treated group than in the control group. These findings raise a possibility that down-regulation of Kv4.3 channels and IA currents in nociceptive V2 TG neurons is an underlying mechanism of oxaliplatin-induced orofacial neuropathic pain.