Ionic Mechanisms Underlying the Excitatory Effect of Orexin on Rat Subthalamic Nucleus Neurons

Abstract

Central orexinergic system deficiency results in cataplexy, a motor deficit characterized with a sudden loss of muscle tone, highlighting a direct modulatory role of orexin in motor control. However, the neural mechanisms underlying the regulation of orexin on motor function are still largely unknown. The subthalamic nucleus (STN), the only excitatory structure of the basal ganglia, holds a key position in the basal ganglia circuitry and motor control. Previous study has revealed a wide distribution of orexinergic fibers as well as orexin receptors in the basal ganglia including the STN. Therefore, in the present study, by using whole-cell patch clamp recording and immunostaining techniques, the direct effect of orexin on the STN neurons in brain slices, especially the underlying receptor and ionic mechanisms, were investigated. Our results show that orexin-A elicits an excitatory effect on STN neurons in rats. Tetrodotoxin (TTX) does not block the orexin-induced excitation on STN neurons, suggesting a direct postsynaptic action of the neuropeptide. The orexin-A-induced inward current on STN neurons is mediated by the activation of both OX1 and OX2 receptors. Immunofluorescence result shows that OX1 and OX2 receptors are co-expressed and co-localized in STN neurons. Furthermore, Na⁺-Ca²⁺ exchangers (NCXs) and inward rectifier K⁺ channels co-mediate the excitatory effect of orexin-A on STN neurons. These results demonstrate a dual receptor in conjunction with the downstream ionic mechanisms underlying the excitatory action of orexin on STN neurons, suggesting a potential modulation of the central orexinergic system on basal ganglia circuitry as well as its related motor control and motor diseases.

Keywords: ionic mechanisms; motor control; neuronal excitability; orexin; subthalamic nucleus.

Figure 1

The direct excitatory effect of orexin on the subthalamic nucleus (STN) neurons. (A) Microscope image of a STN which centrally located in a 300 μm thick brain sagittal slice (observed with Olympus BX51WI, using a 40× water immersed objective) and a glutamatergic STN neuron labeled with biocytin after patch-clamp recording. (B) Orexin-A (300 nM) excited a STN spontaneous firing neuron in current clamp recording. (C) Orexin changed the distribution of inter-spike intervals (the red curve is Gaussian fit to the data) and increased firing rate of the STN neuron presented in (B). (D) Group data of the effect of orexin-A on firing rate of STN neurons (n = 8). (E) Orexin-A concentration-dependently elicited the inward current and increased time to peak and duration of response of the recorded STN neuron. (F) A group of data recorded from 10 STN neurons. (G) Concentration-response curve for orexin-A on STN neurons show mean EC50 value of 29.0 ± 14.3 nM (n = 8). Data are presented as mean ± SEM; **P < 0.01. In this and the following figures, the short horizontal bars above the experimental records indicate the 1 min period of application of orexin-A, and the long horizontal bars indicate the exposure of the slice to tetrodotoxin (TTX), antagonists or blockers of receptors, ion exchangers or channels.

Figure 2

Orexin-A excited the recorded STN neurons with a postsynaptic manner. (A) TTX, NBQX, D-AP5 and gabazine did not block the inward currents induced by orexin-A on a recorded STN neuron. (B) Group data of the recorded STN neurons (n = 8). Data are presented as mean ± SEM; n.s., no statistical difference.

Figure 3

OX1 and OX2 receptors co-mediate the excitation of orexin on STN neurons. (A) Orexin-A (300 nM) elicited an inward current in a STN neuron, SB334867 (10 μM), a selective antagonist for OX1 receptor, partly blocked the current induced by orexin-A and SB334867 combined with JNJ10397049, a selective antagonist for OX2 totally abolished the orexin-A-induced inward current. (B) Orexin-A (300 nM) elicited an inward current in a STN neuron, JNJ10397049 (10 μM) partly blocked the current induced by orexin-A and JNJ10397049 combined with SB334867 totally abolished the orexin-A-induced inward current. (C) Group data of the tested STN neurons under orexin-A induced inward current as present in (A, n = 8) and (B, n = 8). Data are presented as mean ± SEM, **P < 0.01, ***P < 0.001.

Figure 4

Double-labeled immunofluorescence staining for OX1 (green) and OX2 (red) receptors in rat STN. (A1–A3) OX1 receptor staining. (B1–B3) OX2 receptor staining. (C1–C3) Merged images showing colocalization of OX1 and OX2 receptors in the same STN neurons. STN, subthalamic nucleus; ZI, zona incerta; 3V, 3th ventricle; 4V, 4th ventricle; cp, cerebral peduncle; ic, internal capsule; mt, mammillothalamic tract; PLH, peduncular part of the lateral hypothalamus.

Figure 5

Na⁺-Ca²⁺ exchangers (NCXs) and K⁺ channels co-mediate the excitation of orexin on STN neurons. (A1–A3) I-V relationships of STN neurons in the absence and presence of orexin. In 63.8% of the neurons tested, the orexin A-induced inward current was larger at the more hyperpolarized potential of −130 mV than at −55 mV (A1); in 22.4% of these neurons tested, the orexin A-induced inward current reversed near the calculated Ek of −105 mV (A2); in 13.8% neurons, the orexin A-induced inward current first decreased then increase amplitude along with the holding potential hyperpolarization, and was similar in magnitude at −55 and −130 mV (A3). (B) Orexin-A (300 nM) elicited an inward current in a STN neuron. BaCl₂, a broad spectrum blocker of K⁺ channels, partly blocked the effect of orexin-A on STN neurons and combined application of the NCX blocker KB-R7943 totally abolished the orexin-A-induced inward current (n = 8). (C) Orexin-A (300 nM) elicited an inward current in a STN neuron. KB-R7943 partly blocked the effect of orexin-A on STN neurons and combined application of the BaCl₂ totally abolished the orexin-A-induced inward current (n = 8). (D) Group data of the 16 tested STN neurons under orexin-A induced inward current as present in (B,C). Data are presented as mean ± SEM, **P < 0.01, ***P < 0.001.

Figure 6

Inward rectifier K⁺ channels and NCXs contribute to the excitatory effect of orexin on STN neurons. (A1,A2) I-V relationship shows an outward rectifier K⁺ current was exposed after KB-R7943 inhibited the activation of the NCX. (B) Orexin-A (300 nM) elicited an inward current in a STN neuron. KB-R7943 partly blocked the effect of orexin-A on STN neurons and combined application of the inward rectifier K⁺ channel antagonist tertiapin-Q totally abolished the orexin-A-induced inward current. (C) Group data of the 10 tested STN neurons under orexin-A induced inward current as present in (B). Data are presented as mean ± SEM, **P < 0.01, ***P < 0.001.