Osmolality-induced tuning of action potentials in trigeminal ganglion neurons

Abstract

The present study explored the effect of anisotonicity on action potential (AP) in cultured trigeminal ganglion (TG) neurons. We demonstrate that the number of evoked APs was increased by both hypo- and hypertonic treatment. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased the number of APs, but only hypotonic-response was markedly blocked in TRPV4-/- mice. Additionally, inhibition of PKC attenuated hypotonicity-induced increase, whereas antagonism of PKA attenuated hypertonicity-response. We conclude that anisotonicity increases excitability of nociceptors, which might be involved in anisotonicity-induced nociception. The increase of APs by hypo- and hypertonicity is mediated through different receptor and intracellular signaling pathways.

Figure 1. Effect of anisotonicity on APs in TG neurons

A. The number of evoked APs was increased from 9.3±3.1 to 17.2±3.4 (n=30, paired t test, P<0.01) by hypotonic treatment (260mOsm) and recovered to 13.5±3.0 after washout. Typical recordings show that the number of APs triggered by ramp protocol was 7, 17 and 10 before, during and after hypotonic treatment. Capsacin- and 4α-PDD-induced currents were recorded in the same TG neuron. B. The number of evoked APs was increased from 8.8±4.1 to 16.7±2.1 (n=22, paired t test, P<0.01) by hypertonic treatment (330mOsm). After washout, the number of APs was 15.7±1.3. Typical recordings show that the number of APs was 10, 19 and 18 before, during and after hypertonic treatment. Capsacin- and 4α-PDD-induced currents were recorded in the same TG neuron. C. Plot of the percentage in increased APs as a function of osmolality shows that more effect is produced with larger osmotic gradient.

Figure 2. Involvement of TRPV4 receptor in hypotonic modulation

A. After exposed to 10 μM 4α-PDD, the number of APs was markedly increased from 7.3±2.1 to 18.1±4.9. **P<0.01 vs. 300mOsm, paired t test. B. After hypotonic treatment, the number of APs was reversibly increased by 98.0±11.3% (from 5.4±1.5 to 10.7±2.0, n=26, paired t test, P<0.01) in TRPV4^+/+ mice TG neurons, but only by 11.1±3.4% (from 6.2±1.5 and 6.9±2.0, n=23, paired t test, P>0.05) in TRPV4^−/− mice TG neurons. However, the number of APs was irreversibly increased by 120.5±15.2% (from 6.1±2.8 to 13.5±3.4, n=25, paired t test, P<0.01) and 123.0±10.8% (from 5.9±2.2 to 13.1±1.4, n=23, paired t test, P<0.01) in TRPV4^+/+ and TRPV4^−/− mice TG neurons, respectively. **P<0.01 vs. TRPV4^+/+ mice, unpaired t test.

Figure 3. Involvement of intracellular signaling pathway in hypo- versus hypertonic modulation

A. For PKA system, hypotonicity-induced increase of APs was not affected by pre-incubation of H-89 and the number of APs was increased by 78.7±10.3%from 8.5±1.6 to 15.1±2.1 after exposed to hypotonicity (n=20, paired t test, P<0.01). By contrast, hypertonicity-induced modulation was markedly attenuated and the number of APs was increased only by 31.5±7.1% from 9.3±1.5 to 12.1±1.0 after hypertonic treatment (n=20, paired t test, P<0.05). *P<0.01 vs. 330mOsm, unpaired t test. B. For PKC system, hypotonicity-induced increase of APs was selectively attenuated in the presence of BIM and the number of APs was increased only by 41.3±9.0% from 9.1±1.6 to 13.0±0.8 after exposed to hypotonicity (n=28, paired t test, P<0.05). However, hypertonic-response was not unaffected by BIM and the number of APs was increased by 90.3±12.3% from 8.7±2.0 to 15.6±1.3 after hypertonic treatment (n=21, paired t test, P<0.01). *P<0.01 vs. 260mOsm, unpaired t test.

Figure A