Potentiation of glutamatergic synaptic transmission by protein kinase C-mediated sensitization of TRPV1 at the first sensory synapse

Abstract

Sensory input from the periphery to the CNS is critically dependent on the strength of synaptic transmission at the first sensory synapse formed between primary afferent dorsal root ganglion (DRG) and superficial dorsal horn (DH) neurons of the spinal cord. Transient receptor potential vanilloid 1 (TRPV1) expressed on a subset of sensory neurons plays an important role in chronic inflammatory thermal nociception. Activation of protein kinase C (PKC) sensitizes TRPV1, which may contribute to the pathophysiology of chronic pain conditions. In this study, we have examined the modulation of TRPV1-mediated enhancement of excitatory synaptic transmission in response to PKC activation. Miniature excitatory postsynaptic currents (mEPSCs) from embryonic rat DRG-DH neuronal cocultures were recorded by patch clamping DH neurons. Capsaicin potently increased the frequency but not the amplitude of mEPSCs in a calcium-dependent manner, suggesting TRPV1-mediated glutamate release from presynaptic terminals of sensory neurons. Continued or repeated applications of capsaicin reduced the frequency of mEPSCs over time. The PKC activator phorbol 12,13-dibutyrate (PDBu) alone increased mEPSC events to a certain extent in a reversible manner but capsaicin further synergistically enhanced the frequency of mEPSCs. The PKC inhibitor bisindolylmaleimide (BIM) abolished PDBu-mediated potentiation of TRPV1-dependent increases in mEPSC frequency, suggesting modulation of TRPV1 by PKC-induced phosphorylation. In addition, at normal body temperatures ( approximately 37 degrees C) PKC-mediated enhancement of mEPSC frequency is significantly decreased by a specific TRPV1 antagonist, suggesting a physiological role of TRPV1 at the central terminals. Furthermore, bradykinin (BK) significantly potentiated TRPV1-modulated synaptic responses by activating the PLC-PKC pathway. Our results indicate that TRPV1 activation can modulate excitatory synaptic transmission at the first sensory synapse and its effects can further be augmented by activation of PKC. Increased gain of sensory input by TRPV1-induced enhancement of glutamate release and its potentiation by various inflammatory mediators may contribute to persistent pain conditions. Selective targeting of TRPV1 expressed on the central terminals of sensory neurons may serve as a strategy to alleviate chronic intractable pain conditions.

Figure 1. Enhancement of synaptic transmission by activation of TRPV1 by capsaicin at the first sensory synapse

A, application of capsaicin (10 nm) increases the frequency of mEPSCs in a reversible manner. The synaptic events are shown in higher time resolution below. B, cumulative probability plot showing decreased interevent intervals representing increased frequency of mEPSCs (P < 0.0001, KS test). C, the increase in frequency was not accompanied by a change in the amplitude (P = 0.159, KS test). D, summary graph showing capsaicin-mediated increases in the frequency of mEPSCs in a dose-dependent manner (10 nm, n = 17, P < 0.05; 100 nm, n = 11, P < 0.05). The increases in mEPSC events are dependent on extracellular calcium (n = 3). Furthermore, the increase in capsaicin-induced synaptic events are blocked by 10 μm capsazepine (n = 5, P < 0.05). The augmented synaptic transmission upon capsaicin application is only observed in DRG–DH cocultures and not in DH–DH monocultures (n = 3).

Figure 2. TRPV1-mediated enhancement of synaptic transmission shows run-down upon continuous and tachyphylaxis upon repeated capsaicin application

A, capsaicin (10 nm)-induced enhancement of mEPSCs progressively decreases with repeated capsaicin application. Synaptic currents are shown in a higher time resolution below. B, cumulative probability plots showing decreased mEPSC frequency as indicated by a progressive increase in the interevent intervals with each subsequent capsaicin application. C, summary graph representing a progressive decline in mEPSC frequency with repeated capsaicin application. A significant decrease in frequency is observed only between the first and second capsaicin application (first application, n = 17; second application, n = 8, P < 0.05). D, summary graph representing a progressive decrease in the number of mEPSC events analysed in 5 s epochs immediately following capsaicin (10 nm) application (581.65 ± 111.95%, n = 8; 644.45 ± 103.95%, n = 8; 447.7 ± 57.88%, n = 8; 328.5 ± 36.73%, n = 6)

Figure 3. Modulation of synaptic transmission by PDBu

A, PDBu increases the frequency of mEPSCs in a reversible manner. B, pretreatment with PKC inhibitor BIM (500 nm) does not inhibit PDBu-mediated increases in the synaptic transmission. Synaptic currents are shown in higher resolution below. C and D, cumulative probability graphs showing enhanced frequency of mEPSCs (P < 0.001, KS test) in response to PDBu without change in their amplitudes (P = 0.48, KS test). E, summary graphs showing that PDBu-mediated increase in mEPSCs are not inhibited by BIM (10 nm, n = 6; 100 nm, n = 5; 1 μm, n = 10, P < 0.05, n = 7).

Figure 4. Enhancement of capsaicin-induced changes in synaptic transmission by the PKC-activating phorbol ester PDBu

A, capsaicin-induced increases in mEPSCs were enhanced by pretreatment with PDBu. Synaptic currents are shown in higher time resolution below. B, cumulative probability graphs indicating an increase in the frequency of synaptic events with capsaicin (100 nm, P < 0.02, KS test), which were further significantly enhanced following PDBu (1 μm) pretreatment (P < 0.0001, KS test). C, the increase in frequency of events is not accompanied by a change in their amplitude (P = 0.68, KS test). D, summary graphs showing potentiation of capsaicin-induced increase in mEPSC frequency by PDBu (n = 6, P < 0.05).

Figure 5. BIM inhibits PDBu-induced potentiation of TRPV1-modulated synaptic responses

A, the specific PKC inhibitor BIM inhibited PDBu-induced potentiation of capsaicin-mediated synaptic activity, without inhibiting the direct effects of PDBu on synaptic transmission. Synaptic currents are shown in higher time resolution. B and C, cumulative probability curves for the inter-event intervals (P = 0.23, KS test) and amplitude (P = 0.18, KS test) of mEPSC events are similar with capsaicin before and after PDBu in presence of BIM. D, summary graph demonstrating that BIM attenuates PKC-induced potentiation of TRPV1-modulated synaptic transmission (n = 5, P < 0.05).

Figure 6. Modulation of synaptic transmission by phosphorylated TRPV1 at normal body temperature

A, PDBu-mediated enhancement in synaptic transmission at 37°C can be partially blocked by the TRPV1 antagonist BCTC (100 nm). B, cumulative probability curves demonstrating that PDBu-mediated increases in the mEPSC events (P < 0.0001, KS test) are inhibited by BCTC application at 37°C. C, cumulative probability graph showing that PDBu and BCTC application does not affect the amplitude of mEPSC events as compared to the control (P = 0.13, KS test). D, summary graph demonstrating TRPV1-mediated component that is responsible for the increase in the frequency of mEPSC events (n = 5, P < 0.05).

Figure 7. Influence of BK on TRPV1-modulated synaptic transmission

A, capsaicin-induced increases in mEPSCs were enhanced by pretreatment with BK (1 μm). Synaptic currents are shown in higher time resolution below. B, cumulative probability curves showing a decrease in inter-event intervals, indicating an increase in the frequency of synaptic events, which were significantly enhanced after treatment with BK (P < 0.0001, KS test). C, the increase in frequency of mEPSCs is not accompanied by an increase in amplitude (P = 0.92, KS test). D, summary graph showing capsaicin-induced increase in mEPSC frequency is potentiated (n = 5, P < 0.05) by BK.

Figure 8. BK potentiates TRPV1-modulated responses by activating PKC

A, traces representing potentiation of capsaicin responses by BK (1 μm). B, pretreatment with BIM (500 nm) inhibits BK-mediated potentiation of capsaicin-induced responses. Synaptic currents are shown in a higher resolution below. C, decrease in inter-event intervals indicates increase in the frequency of synaptic events with capsaicin after BK. This effect is significantly reduced after application of BIM (P < 0.0001, KS test). D, the changes in the frequency of events are not accompanied by a change in amplitude (P = 0.21, KS test). E, summary graph showing that BK-mediated potentiation of capsaicin-induced increases in glutamatergic transmission is abolished by treatment with BIM (n = 4, P < 0.05).

Figure 9. BK-induced sensitization of TRPV1-mediated changes in synaptic transmission involves PLC pathway

A, traces of mEPSCs representing potentiation of capsaicin-mediated synaptic events by BK pretreatment. B, the PLC inhibitor U73122 (2 μm) inhibited BK-mediated potentiation of capsaicin responses. Traces are represented in a higher time resolution below. C and D, graphs showing cumulative probability of inter-event intervals (P < 0.0001, KS test) and amplitude (P = 0.96, KS test), indicating that U73122 prevents BK induced sensitization of capsaicin responses. E, summary graph showing that BK-induced sensitization of TRPV1 is inhibited by the PLC inhibitor U73122 (n = 6, P < 0.05) but not its inactive analogue, U73343 (2 μm; n = 3, P < 0.05).