Melatonin Reduces Excitability in Dorsal Root Ganglia Neurons with Inflection on the Repolarization Phase of the Action Potential

Abstract

Melatonin is a neurohormone produced and secreted at night by pineal gland. Many effects of melatonin have already been described, for example: Activation of potassium channels in the suprachiasmatic nucleus and inhibition of excitability of a sub-population of neurons of the dorsal root ganglia (DRG). The DRG is described as a structure with several neuronal populations. One classification, based on the repolarizing phase of the action potential (AP), divides DRG neurons into two types: Without (N₀) and with (N_inf) inflection on the repolarization phase of the action potential. We have previously demonstrated that melatonin inhibits excitability in N₀ neurons, and in the present work, we aimed to investigate the melatonin effects on the other neurons (N_inf) of the DRG neuronal population. This investigation was done using sharp microelectrode technique in the current clamp mode. Melatonin (0.01-1000.0 nM) showed inhibitory activity on neuronal excitability, which can be observed by the blockade of the AP and by the increase in rheobase. However, we observed that, while some neurons were sensitive to melatonin effect on excitability (excitability melatonin sensitive-EMS), other neurons were not sensitive to melatonin effect on excitability (excitability melatonin not sensitive-EMNS). Concerning the passive electrophysiological properties of the neurons, melatonin caused a hyperpolarization of the resting membrane potential in both cell types. Regarding the input resistance (R_in), melatonin did not change this parameter in the EMS cells, but increased its values in the EMNS cells. Melatonin also altered several AP parameters in EMS cells, the most conspicuously changed was the (dV/dt)_max of AP depolarization, which is in coherence with melatonin effects on excitability. Otherwise, in EMNS cells, melatonin (0.1-1000.0 nM) induced no alteration of (dV/dt)_max of AP depolarization. Thus, taking these data together, and the data of previous publication on melatonin effect on N₀ neurons shows that this substance has a greater pharmacological potency on N_inf neurons. We suggest that melatonin has important physiological function related to N_inf neurons and this is likely to bear a potential relevant therapeutic use, since N_inf neurons are related to nociception.

Keywords: DRG; action potential; dorsal root ganglion; excitability; melatonin; passive electric properties.

Figure 1

Melatonin-induced AP blockade in N_inf neurons. Panel A shows representative traces illustrating melatonin effect on the AP of N_inf neurons. Traces represent: upper row–current pulses; lower row–left, control AP; right–exposure to melatonin (1000.0 nM). Insert–dV/dt of control AP. Broken line is zero voltage reference. Panel B shows the percentage of neurons with AP triggering blocked (ordinate) by melatonin 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM (abscissa). These data, expressed in percentage values, are presented as means ± SEM.

Figure 2

Effect of melatonin on the rheobase of N_inf excitability melatonin sensitive (EMS) and excitability melatonin not sensitive (EMNS) cells. These data, expressed in normalized values, are presented as means ± SEM and rheobase absolute control values are found in Table 1. The number of experiments of N_inf EMS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 1, 4, 6, 10, 3, and, 10, respectively. The number of experiments of N_inf EMNS neurons at concentrations of 0.01, 0,1, 1.0, 10.0, 100.0, and 1000.0 nM were 13, 7, 12, 15, 10, and 18, respectively. Ordinate, percentage of own control value. Abscissa, melatonin concentration (control, only vehicle). The symbol * indicates statistical difference compared to control (p < 0.05, paired Student’s t-test).

Figure 3

Effect of melatonin on the resting membrane potential (RMP) of N_inf excitability melatonin sensitive (EMS) and excitability melatonin not sensitive (EMNS) cells. These data, expressed in normalized values, are presented as means ± SEM and RMP absolute control values are found in Table 1. The number of experiments of N_inf EMS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 1, 4, 6, 10, 3, and, 10, respectively. The number of experiments of N_inf EMNS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 13, 7, 12, 15, 10, and 18, respectively. The symbol * indicates statistically significant difference as compared to control (p < 0.05, paired Student’s t-test).

Figure 4

Effect of melatonin on the input resistance (R_in) of N_inf excitability melatonin sensitive (EMS) and excitability melatonin not sensitive (EMNS) cells. These data, expressed in normalized values, are presented as means ± SEM and R_in absolute control values are found in Table 1. The number of experiments of N_inf EMS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 1, 4, 6, 10, 3, and, 10, respectively. The number of experiments of N_inf EMNS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 13, 7, 12, 15, 10, and 18, respectively. The symbol * indicates statistically significant difference as compared to control (p < 0.05, paired Student’s t-test).

Figure 5

Effect of melatonin on the amplitude (A), duration (B), maximum ascendant inclination (C), and maximum descendant inclination (D) of N_inf excitability melatonin sensitive (EMS) and excitability melatonin not sensitive (EMNS) cells. These data, expressed in normalized values, are presented as means ± SEM and absolute control values for these parameters are found in Table 1. The number of experiments of N_inf EMS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 1, 4, 6, 10, 3, and, 10, respectively. The number of experiments of N_inf EMNS neurons at concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, and 1000.0 nM were 13, 7, 12, 15, 10, and 18, respectively. The symbol * indicates statistically significant difference as compared to control (p < 0.05, paired Student’s t-test).

Figure 6

Effect of melatonin (100.0 nM) on Na⁺K⁺-ATPase activity. Activities expressed as paired measurements of individual increases related to individual controls normalized as percentage of control activity (means ± SEM). Na⁺K⁺-ATPase activity is expressed as nmol (Pi)/µg protein/hour. The symbol * indicates statistically significant difference as compared to control (p < 0.05, paired Student’s t-test).