| Central motor control is coordinated by diverse groups of motor-related neuronal populations in the central nervous system(CNS). Among these separated groups, the reticulospinal neurons in the caudal pontine reticular nucleus(Pn C) are regarded as a bridge that connects diverse motor structures with the spinal cord. Interestingly, the Pn C reticulospinal neurons are especially important in posture and muscle tone regulation, which is obviously a prerequisite for the execution of any other forms of motor behaviors. In accordance, lesions of the Pn C largely impaired the normal posture and muscle tone maintenance and consequently affected a variety of motor functions.It has been reported that sleep deprivation led to impairment in normal posture and muscle tone maintenance, suggesting normal maintenance of posture and muscle tone depends on a good arousal state. The maintenance of central arousal state relies on several subcortical arousal promoting systems, of which the hypothalamus orexinergic system and the brainstem noradrenergic system are the most robust. Intriguingly, several lines of evidence have shown that orexin or noradrenaline(NA) can directly act on central motor structures and thereby participate in central posture and muscle tone regulations, such as in the lateral vestibular nucleus(LVN) or the spinal cord. More importantly, cataplexy that refers to the sudden uncontrollable loss of skeletal muscle tone is directly caused by the orexin deficiency and the central noradrenergic neurons also ceased firing during the episode of cataplexy, further suggesting a co-operative role of them in posture and muscle tone regulations.Interestingly, both the orexinergic and noradrenergic fibers have been reported to distribute in Pn C. However the functions of orexin and NA in Pn C remain elusive. Here, we first used a combination of electrophysiological and morphological methods to determine the modulatory effects and mechanisms of orexin and NA on the activity of Pn C reticulospinal neurons, respectively. Intriguingly, orexin and NA post-synaptically affect the activities of Pn C reticulospinal neurons in different manners, suggesting distinctive roles for orexin and NA in Pn C, respectively. We further examined the modulatory effects and mechanisms of orexin on neurotransmissions to Pn C reticulospinal neurons. After revealing the pre- and post-synaptic modulations of orexin in Pn C reticulospinal neurons, we further revealed how these modulations participate in the the control of Pn C-mediated postural motor activities at the behavioral level.The main findings in the present study are as follows,1. The distribution of orexinergic and noradrenergic receptors in Pn C.We first employed immunohistochemical and western blot methods to explore the expression of orexinergic and noradrenergic receptors in Pn C. The orexin-2 receptors have been proved to distribute in Pn C. In the present study, our immunofluorescence results further demonstrated that the orexin-1 receptors were also expressed in Pn C. Besides, our western blot and immunoenzyme results revealed that diverse subtypes of adrenoceptors were also expressed in Pn C. These results thus suggest possible modulatory roles for orexin and NA in Pn C.2. The modulatory effects of orexin and NA on the whole cell currents of Pn C reticulospinal neurons.To directly examine and compare the modulatory effects of orexin and NA in the Pn C reticulospinal neurons, we employed the whole-cell patch clamp methods and directly patched the Pn C reticulospinal neurons according to their large soma diameters(> 35 μm) and electrophysiological features.(1) Orexin induced inward currents in Pn C reticulospinal neurons.At the holding potential of-70 m V, bath application of orexin-A(30-300 n M) induced an inward current in all(107/107) identified Pn C reticulospinal neurons. Through utilizing different concentrations of orexin-A(30-300 n M) in the above test, a concentrationdependent response pattern of orexin-A in Pn C reticulospinal neurons has also been revealed. In 11 tested Pn C reticulospinal neurons, 30, 100, and 300 n M orexin-A induced an inward current of 29.8 ± 9.1 p A(n = 4), 39.8 ± 4.5 p A(n = 11), and 50.6 ± 5.8 p A(n = 11), respectively.(2) NA induced inward currents in some Pn C reticulospinal neurons, but outward currents in others.Using the same methods, the effects of NA on the whole cell currents in Pn C reticulospinal neurons were also observed. Interestingly, different from orexin, NA(1-10 μM) induced an inward current in some(33/51, 64.7 %) of the tested Pn C reticulospinal neurons,while an outward current in the remaining(18/51, 35.3 %). Furthermore, NA induced inward or outward currents in Pn C reticulospinal neurons in a concentrationdependent manner. In nine Pn C reticulospinal neurons, 1, 3, and 10 μM NA elicited inward currents of-20.8 ± 3.8 p A(n=8),-34.9 ± 6.2 p A(n=9), and-46.5 ± 7.3 p A(n=7), respectively. In another nine Pn C reticulospinal neurons, 1, 3, and 10 μM NA elicited outward currents of 21.3 ± 6.4 p A(n = 8), 30.9 ± 6.8 p A(n = 9), and 34.3 ± 6.7 p A(n = 9), respectively.These results demonstrate that orexin and NA both can modulate the activity of Pn C reticulospinal neurons. However, different from the unique excitatory effects of orexin, NA exerts a more complex modulatory effect in Pn C reticulospinal neurons.3. The mechanisms underlying the modulatory effects of orexin and NA in Pn C reticulospinal neurons.(1) The orexin-induced inward currents are mediated by post-synaptic orexin-1 and orexin-2 receptors.The orexin-induced inward currents remained unaffected(n = 6, P = 0.995) in the presence of 1 μM tetrodotoxin(TTX), indicating that the orexin-induced inward current in Pn C reticulospinal neurons is mediated via a post-synaptic mechanism. In the presence of the orexin-1 receptor antagonist SB 334867(10 μM), the orexin-induced inward current was only partially blocked(n = 4, P < 0.01), indicating the orexin-induced inward current in Pn C reticulospinal neurons is partially mediated by the post-synaptic orexin-1 receptors. Moreover, bath application of the dual orexin receptor antagonist TCS 1102(1 μM) totally blocked the orexin-induced inward current in Pn C reticulospinal neurons(n = 6, P < 0.01),demonstrating that orexin-1 and orexin-2 receptors are indeed expressed in the post-synaptic part of Pn C reticulospinal neurons and mediate the orexin-induced inward current upon activation.(2) The opening of the non-selective cation channels(NSCCs) is coupled to the activation of the post-synaptic orexinergic receptors.In the CNS, the K+ channels, Na+-Ca2+ exchangers, and NSCCs have been reported to be coupled to the activation of orexinergic receptors. To test whether the orexin-induced inward current reverses at the equilibrium potential for K+(-102.3 m V under our conditions calculated by the Nernst equation). We applied slow(7 s) voltage ramps from-125 m V to-55 m V before and during the application of orexin and compared the response in baseline from that in orexin. However, these responses did not cross in any of the 8 tested Pn C reticulospinal neurons. In 5 of these neurons, the curves remained parallel in the tested voltage range. In the remaining neurons, the current difference between orexin application and control converged as the voltage became less negative, suggesting a reversal at more positive potentials. As a result, a decrease in K+ conductance is not the major mediator of the orexin-induced inward current in Pn C reticulospinal neurons.To directly determine the equilibrium potential of the orexin-induced inward current, we further applied slow(13 s) ramps from-125 to +5 m V in TTX-containing ACSF. In this circumstances, in the majority of tested Pn C reticulospinal neurons(9/10), the orexin-induced inward current reversed at-27.2 ± 2.7 m V(n = 9), near the equilibrium potential of the conductances for the NSCCs. Only in one neuron, the orexin-induced inward current reversed at-99.7 m V, near the equilibrium potential of K+. These data indicates that in most Pn C reticulospinal neurons, orexin opens the NSCCs permeable to both Na+ and K+. However, closure of K+ channels may also be involved in a small population of Pn C reticulospinal neurons, but is obviously not a major component.If Na+ is the main ion carrying the orexin-induced inward current in most Pn C reticulospinal neurons, removing extracellular Na+ should strongly reduce the orexininduced inward current. To test this hypothesis, we reduced the extracellular Na+ concentration from 152.25 to 27 m M by replacing Na Cl by equimolar NMDG chloride. Indeed, switching the bath solution from normal ACSF to the low-Na+ ACSF largely reduced the orexin-induced inward current(n = 7, P < 0.01). More importantly, the NSCCs blocker flufenamic acid(100 μM) also largely blocked the orexin-induced inward current in Pn C reticulospinal neurons(n = 5, P < 0.01). Besides, the Na+-Ca2+ exchangers blocker SN-6(10 μM) had no effects on the orexin-induced inward current in Pn C reticulospinal neurons(n = 4, P = 0.746). Together, these results thus strongly indicate that the orexin-induced inward current in Pn C reticulospinal neurons is mainly mediated by opening the downstream NSCCs.(3) The α1- and α2-adrenoceptors are co-expressed in a single reticulospinal neuron and mediate the post-synaptic effects of NA.Using the same methods, the mechanisms underlying the effects of NA on the whole cell currents in Pn C reticulospinal neurons were also revealed, respectively. Both the NA-induced inward(n = 6, P = 0.364) and outward currents(n = 6, P = 0.511) were in a TTX-resistant manner, suggesting that NA also exerts complex modulatory effects in Pn C reticulospinal neurons via post-synaptic mechanisms.In all seven tested neurons exhibiting an inward current response to 3 μM NA, 0.01 μM prazosin(Pra, a selective α1-adrenoceptor antagonist) partially blocked the NA-elicited inward current(P < 0.05), indicating the involvement of α1-adrenoceptors. We further employed a higher concentration of Pra(10 μM) for confirmation. Indeed, the inward current elicited by 3 μM NA was totally abolished in this condition. But intriguingly, an NA-elicited stable outward current was observed in the same seven neurons(23.5 ± 5.1 p A). Moreover, this reversed outward current could be totally blocked by the application of 10 μM yohimbine(Yh, a selective α2-adrenoceptor antagonist) and 10 μM Pra(P < 0.01). Therefore, the overall NA-elicited inward current in Pn C reticulospinal neurons actually consistes of a mixture of both the α1-adrenoceptor-mediated inward and α2- adrenoceptormediated outward currents.Considering the above results, we next examined whether the observed NA-elicited outward current in the Pn C reticulospinal neurons at the holding potential of-70 m V was similarly composed of both outward and inward components. In all six of the tested Pn C reticulospinal neurons exhibiting the outward current response to 3 μM NA, 0.1 μM Yh partially blocked the NA-elicited outward current(P < 0.01). Next, in the presence of 10 μM Yh, 3 μM NA elicited an inward current in the same six neurons(-13.7 ± 2.3 p A). This NA-elicited inward current was totally abolished by further co-application of 10 μM Pra with 10 μM Yh(P < 0.01).All of the above results strongly indicate that the α1- and α2-adrenoceptors are functionally co-expressed on the postsynaptic membrane of a single reticulospinal neuron to mediate the inward and outward current components, respectively, which are superposed to produce the overall NA-elicited current. Consistently, our double immunofluorescence staining revealed that α1A- and α2A-adrenoceptors were indeed co-localized in individual Pn C reticulospinal neurons.4. The modulatory effects of orexin on the neurotransmissions to Pn C reticulospinal neurons.We next isolated the m EPSCs and m IPSCs of Pn C reticulospinal neurons to test whether orexin could also modulate neurotransmissions to these neurons.(1) Orexin inhibits the frequency of m EPSC, but did not influence the m IPSC in Pn C reticulospinal neurons.In 9 tested Pn C reticulospinal neurons, bath application of orexin-A(300 n M) neither influenced the m IPSCs frequency(n = 9, P = 0.469) nor the amplitude(n = 9, P = 0.653) in these neurons. Interestingly, bath application of orexin-A(300 n M) inhibited the m EPSC frequency(n = 8, P < 0.01) but not the m EPSC amplitude(n = 8, P = 0.501). These results suggest that orexin inhibits excitatory release in Pn C by a pre-synaptic mechanism.(2). Orexin inhibits excitatory release by mobilizing the endocannabinoid system and subsequently retrogradely activating pre-synaptic cannabinoid type 1 receptors(CB1R).In the presence of 1 μM AM 251(cannabinoid type 1 receptor antagonist), the inhibition of the m EPSC frequency induced by orexin-A was completely blocked(n = 8, P = 0.521). The m EPSC amplitude was also unaffected(n = 8, P = 0.114). These results demonstrate that the orexin-induced inhibitory effects over the excitatory release onto Pn C reticulospinal neurons through mobilizing the endocannabinoid system. Consistently, our immunofluorescence results revealed that the endocannabinoid synthesizing enzyme diacylglycerol lipase α(DAGLα) was highly expressed in Pn C reticulospinal neurons.5. The amplitude of the CRN-evoked EPSCs onto Pn C reticulospinal neurons is attenuated by the pre-synaptic effect of orexin.To further determine whether the inhibitory effect on excitatory release mediated by the orexin-endocanabinoid system ultimately participated in functionally modulating the functionally excitatory input onto Pn C reticulospinal neurons, we evoked the EPSCs in Pn C reticulospinal neurons by stimulating the input fibers arising from the cochlear root nucleus(CRN). Bath application of orexin-A(300 n M) decreased the CRN-e EPSCs amplitude by 23.5 ± 0.4 %(n = 10, P < 0.01). Meanwhile, this depression was accompanied by an increase in the PPR(e EPSC2/e EPSC1)(n = 10, P < 0.01). Moreover, in the presence of 1 μM AM 251, the orexin-induced depressive modulation of the CRN-e EPSCs were nearly totally blocked(n = 6, P = 0.203). An accompanying increase of the PPR in ACSF was also not observed in the presence of 1 μM AM 251(n = 6, P = 0.841). Together, these results demonstrate that the pre-synaptic inhibitory effect of the orexin-endocanabinoid system on excitatory release indeed participates in functionally modulating the functionally excitatory input onto Pn C reticulospinal neurons.6. The post- and pre-synaptic modulations by orexin in Pn C reticulospinal neurons participate in the regulation of ASR activity.After revealing the post- and pre-synaptic modulations by orexin in Pn C reticulospinal neurons, we further explored how these modulations participate in Pn C-mediated ASR activity at the behavioral level. Bilateral microinjection of 30 μM orexin-A into Pn C significantly increased the ASR amplitude in all tested sound intensities(from 94 to 112 d B). This suggests that orexin increased the ASR amplitude by post-synaptic excitatory modulations in Pn C reticulospinal neurons. However, bilateral microinjection of 3 μM orexin-A into Pn C significantly decreased the ASR amplitude at 94 d B(P < 0.01, n = 13). Microinjection of 3 μM orexin-A with AM 251(1 m M) blocked the inhibitory effect of 3 μM orexin-A on the ASR amplitude at 94 d B. These results thus indicate that in lower concentrations of orexin and weaker stimulus, the pre-synaptic inhibitions by orexin on Pn C reticulospinal spinal neurons is the leading force. Consistently, 30 μM orexin-A had no effect on the ASR threshold(n = 9, P = 0.347) while 3 μM orexin-A significantly increased the ASR threshold(n = 9, P = 0.347). Together, these results suggest that both the pre- and post-synaptic modulations by orexin in Pn C finally participate in the modulation of the ASR activity. The possible physiological significance for the above pattern is to promote the motor response to salient stimulus by post-synaptic excitatory modulations and avoid the interference from irrelevant noises by pre-synaptic inhibitory modulations.In summary, the present study has revealed orexin and NA both directly modulate the activities of Pn C reticulospinal neurons via different post-synaptic mechanisms. While orexin exerts unique excitatory modulatory effects, the modulatory effects of NA are more complex. At a first step towards understanding the different roles for orexin and NA in Pn C, we at the first step focused on the role of orexin in Pn C. We have further revealed orexin inhibits the excitatory input to Pn C. Finally, both the pre- and post-synaptic modulations by orexin finally participate in the regulation of Pn C-mediated ASR activities. These findings suggest that through modulating the activities of Pn C reticulospinal neurons. Orexin may promote motor responses to salient stimulus and inhibit motor responses to irrelevant noises. However, the role of NA in Pn C may still need further investigations in the future work. |
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