The expression of Oxtr in DRG sensory neurons increased after PTX treatment
To examinetheOXTR expression of DRG sensory neurons in PTX-induced neuropathic pain, wefirstdetermined its mRNAlevel in the DRGs after intraperitoneal administration of PTX (2 mg·kg−1, i.p., repeated on days1, 3, 5, 7). In quantitative terms, the qRT-PCR analysis in Fig. 1a (right) revealedthatPTX increased the relative expression of Oxtr mRNA in the DRGs compared with vehicle treatment at day 10, but not day 1, after the onset of PTX application. To verify the qRT-PCR results and determine the distribution of Oxtr mRNA in neurons or non-neuronal cells in DRGs after PTX treatment, we usedFISHtechnology. The results show that the red puncta of OxtrmRNA signal was restricted within DRG neuronsthatwere classified by Nissl (Fig. 1b-c). Additionally, the expression of Oxtr mRNA in DRG neurons wasextremelylow in vehicle group but upregulated greatly at day 10 after PTX administration (Fig. 1d). Itwasnoted that Oxtr mRNAwasdistributed mainly in small and medium DRG neurons (Fig. 1e), which are predominant nociceptors involved in pain processing.
In accordance with the change at the mRNA level, the expression ofOXTRprotein in DRGs increased significantly on day 10 after initial PTX treatment as compared with the vehicle group, in whichOXTR protein was barely detectable (Fig. 2a). In addition, we performed immunostaining in cultured DRG neurons in each group. As shown in Fig. 2b, OXTR immunoreactivity was almost undetectable in cultured DRG neurons from micereceiving thevehicle. In comparison, DRG neurons from mice treated with PTX at day 10 showed strong OXTR immunoreactivity (Fig. 2c-e). Based on these observations, we concluded that the expression of OXTR in DRG sensory neuronsis markedly augmentedin PTX-treated mice.
Intravenous injection of OXT exhibitedanalgesic effects via activation of OXTR after PTX treatment
To examinewhether OXTR expression in DRG sensory neurons mediates peripheral analgesia of OXT, we evaluated peripheral analgesic effects of OXT by intravenous injection (i.v.),as OXTcannot cross the blood–brain barrier (BBB)[25].The micepre-treatedwith PTX were given a single dose of OXT (0.12 mg·kg−1, i.v.) or its vehicle (Fig. 3a and d), and then their nociceptive behaviour was tracked every 20 min. OXT showed a considerable increase in boththemechanical withdrawal threshold (Fig. 3b) and thermal withdrawal latency (Fig. 3c) versusthesaline injection. The duration of analgesia sustained for 40 min and disappeared at 60 min after injection (Fig. 3b-c). In contrast, OXT in different doses (0.12, 0.4, 1.2 mg·kg−1, i.v.) did not modify the mechanical paw withdrawal thresholdorthe thermal paw withdrawal latency (Fig. 3e-f) in micepre-treatedwith multiple vehicleinjection (i.p.). As shown in Fig. S1, therewasno gender difference in the above behaviouralexperiments.
Our above results demonstrate thatPTXpre-treatmentsignificantly up-regulatedexpression of OXTR in DRG neurons,and intravenous application of OXT relieved PTX-induced peripheral neuropathic pain. To further assesswhetherthese findings were mediated by peripheral OXTR activation, we tested the effects of the OXTR selective agonist TC OT 39 (0.12 mg·kg−1, i.v.). We found that TC OT39 produced equipotent analgesia of OXT in PTX-treated mice (Fig. 4b-c). The effect of TC OT 39 appearedto betime-dependent, lastingfor 40minafter injection. In contrast, the analgesic effect of OXT in mice was completely blocked whentheOXTR antagonist dVOT (0.12 mg·kg−1, i.v.) , which applied 15 min prior to the OXTinjection(0.12 mg·kg−1, i.v.)(Fig. 4e-f).These results suggest that the analgesic effect of OXT systemic administrationmaybe mediated by the OXTR located on DRG neurons in PTX-treated mice.
OXT suppressed the activity of small-sized DRG neurons in PTX-treated mice via OXTR activation
Having identified the increased OXTR expression in DRG neurons,whichmaycontribute to the analgesic effect of systemic OXT application after PTX treatment, we then investigatedwhether the activation of OXTR in DRG neurons alters neuronal activity. Hence, we evaluatedthe effects of OXT on the excitability of cultured small-diameter neurons (< 25 µm in diameter, presumably nociceptors) byusingpatch clamp recordings.First, we appliedthecurrent injection stepped from 0–130 pA,withincrementsof 10 pAover600 ms,to evoke APs in the recorded neurons. As shown in Fig. 5aand b, the firing rate of APs in DRG neurons from PTX-treated mice increased significantlyin contrast to thevehicle-treated mice at day 10 after the onset of injection. OXT perfusion (0.5 μM, 2 min) eliminated the increased firing rate of APs (Fig. 5a-b), but OXT did not change the firing rate in micethat hadreceivedthevehicle injection (Fig.5c-d). Consistent with the effect of OXT, TC OT 39 (0.5 μM, 2 min) also significantly attenuated the firing rate of APs in the neurons from PTX-treated mice (Fig. 5e-f). In contrast, in the presence ofdVOT (1 μM, 4 min prior to the OXT),the effect of OT on the firing rate of APs was abolished (Fig. 5g-h).Together, both gain- and loss-of-function pharmacological approaches proved that the activation of OXTR suppressed the excitability of nociceptive neurons.
Multiple PTX injection increased Na+ current insmall-sizedDRG neurons,and OXT perfusion reduced the Na+ current, especially Nav1.7 current
Nav channels serve as the basis for the generation and conduction of APs. Thus, we examined the effect of OXT/OXTR on Nav channels in cultured small neurons usingthewhole-cell patch clamp. We observedthatthe Na+ current amplitude in the PTX groupwasgreater thanthatin the vehicle-treated group(Fig. 6a-b).As shown in Fig. 6cand 6d, OXT perfusion (0.5 μM, 2 min) significantly diminished the Na+ current amplitude recorded from small DRG neurons in the PTX group, which lasted for about 4 min from the beginning of OXT perfusion.
GiventhatPTX enhances Nav1.7 currents in DRG neurons[15],we tested the effect of OXT on Nav1.7 currents in cultured DRG neurons from PTX-treated mice.The total Na+ currents were recordedfirstusingwhole-cell patch-clamp recording(Fig. 6e, the red line), and then ProTx II (10 nM, 5 min) was applied to selectively inhibittheNav1.7 channel in the same recorded neuron.The blue trace in Fig. 6erepresentsNa+ currents after ProTx II perfusion. Nav1.7-mediated currents were isolated by subtraction of the ProTx II-resistant Na+ currents from total Na+ currents. As shown in Fig. 6eand 6f, OXT perfusion (0.5 μM, 2 min) significantly reduced the Nav1.7 current amplitudes in cultured DRG neurons from PTX-treated mice at day 10 after the initial treatment (Fig. 6f).
The activation of OXTR decreased the Na+ current and relieved PTX-induced pain via PLC-PKC pathway after PTX treatment
AsOXTR isaG-protein coupled receptor, we investigated the intracellular mechanisms of how OXTRaffects theNa+ current in nociceptors after PTX treatment. First, the effect of OXT on the Na+ current was completely blocked bytheG-protein antagonist GDP-βS (1 mM),which was added in the patch-pipette solution (Fig. 7a). Next, we foundthatthe effect of OXT ontheNa+ current disappeared in the presence ofthePLC antagonist U73122 (10 μM, Fig. 7b) and PKC antagonist Chelerythrine (10 μM, Fig. 7c).In line with the findings in electrophysiological experiments, the effect of OXT on alleviating mechanical allodynia induced by PTX was inhibited by Chelerythrine (Fig. 7d-e), as well. These datasuggestthat G-protein, PLC,andPKC play a critical role in OXTR peripheral analgesia.
OXT decreased glutamatergic spontaneous and dorsal root-evoked monosynaptic excitatory transmission in spinal lamina II from PTX-treated mice
GiventhatOXT impaired DRG neuron excitability in PTX-treated mice, we investigated the excitatory transmission in spinal lamina II, whichis a key area for the nociceptive transmission fromtheprimary afferent. As shown in Fig. 8a,theperfusion of OXT (0.5 μM, 2 min) decreased spontaneous excitatory transmission in spinal lamina II. The frequency ofthespontaneous excitatory postsynaptic current (sEPSC) decreased gradually over time, peaking 1 min after the onset oftheOXT addition; this reduction was accompanied by an impairment inthesEPSC amplitude (Fig. 8a-b).
Next, we examined the effects of OXT (0.5 μM, 2 min) on monosynaptic Aδ-fibre and C-fibre EPSCs in spinal lamina II. As shown in Fig. 8c-d, monosynaptic A-fibre and C-fibre EPSC amplitudes were reduced by OXT as well. However, OXT did not change the frequency and amplitude of sEPSC or the amplitude of evoked EPSC in the vehicle-treated mice (Fig. 8g-l).