| 5-HT and histamine are two important monoaminergic neurotransmitters in central nervous system. It has long been focused on their non-somatic modulatory functions, however, the serotonergic fibers, derived from the raphe nuclei as well as the medullary/pontine reticular formations, and the histaminergic fibers originated from tuberomammillary nucleus of the hypothalamus, also directly innervate some subcortical motor structures, such as cerebellum, basal ganglia, red nucleus and vestibular nuclei. These fibers release5-HT or histamine through varicosities, which modulates neuronal activities mainly via the correspondingly metabotropic receptors and subsequently influences animal’s motor abilities, such as motor balance and coordination.In addition to the cerebral cortex, brainstem, and spinal cord in the three motor control hierarchies, the cerebellum and the basal ganglia constitute two important subcortical side loops responsible for the motor control. The cerebellum, the largest subcortical brain region, holds a key position in the control of balance, equilibrium, muscle tone, and the coordination of voluntary movement. Cerebellar damage could cause a variety of motor dysfunctions, such as cerebellar ataxia. Basal ganglia principally accounts for the initiation of voluntary movement, and its disorders could lead to a series of movement disorders, including Parkinson’s disease (PD) and Huntington’s disease (HD).However, the role and the underlying mechanism of central serotonergic/histaminergic inputs on cerebellum-/STN-mediated motor behaviors are still largely unknown. In this study, by using RT-PCR, immunofluorescence, electrophysiological, and behavioral methods, the effects of5-HT/histamine on cerebellar/STN neuronal activities, and especially, their motor behavioral influences were investigated.1. Modulation of5-HT on cerebellar FN-mediated motor behavior and the underlying mechanismThe serotonergic fibers are the largest afferent population except the mossy fibers and climbing fibers, innervating not only the cerebellar cortex but also the cerebellar nuclei. Correspondingly, radioligand binding, in situ hybridization, neuropharmacological and immunohistochemical studies have revealed an existence of several subtypes of5-HT receptors including5-HT2receptors in the cerebellum. However, effect mediated by the activation of5-HT2receptors on cerebellar neurons, particularly on cerebellar motor behaviors, still remains enigmatic.In the cerebellar circuitry, the cerebellar nuclei are the final integrative processing unit rather than a relay station just receiving projections from Purkinje cells (the principal neurons) in the cerebellar cortex. The cerebellar nuclei integrate information from the cerebellar cortex, signals from the mossy fibers and climbing fibers, as well as complicated modulatory messages from the third type of inputs including the serotonergic to make the ultimate outputs of the cerebellum together with the flocculonodular node. Among the cerebellar nuclei, the fastigial nucleus (FN) is the phylogenetically oldest nucleus, and holds a key position in fine-tuning body and proximal limb movements. Therefore, in the present study, we investigated role of5-HT2receptors in neuronal activity of the cerebellar FN and in FN-mediated motor behaviors.Real-time RT-PCR and immunostaining results demonstrated that only5-HT2A receptors rather than5-HT2B or5-HT2C receptors in5-HT2receptor subfamily were expressed and localized in the cerebellar FN. From in vitro extracellular recordings, we found that the majority of the recorded FN neurons (92/116,79.3%) responded to5-HT simulation with a unique excitatory effect in a concentration-dependent manner. Importantly, low-Ca2+/high-Mg2+medium did not block the5-HT-evoked concentration-dependent excitations on FN neurons(n=9), indicating that the excitatory effect of5-HT on FN neurons is primarily postsynaptic. Furthermore, selective5-HT2A receptor agonist TCB-2mimicked the5-HT-induced excitations (n=42), and selective5-HT2A receptor antagonist M100907significantly blocked the excitatory responses induced not only by TCB-2but also by5-HT (n=32), suggesting postsynaptic5-HT2A receptors contribute greatly to the excitatory effect of5-HT on rat FN neurons.Next, we microinjected serotonergic reagents to bilateral cerebellar FNs to evaluate the effect of5-HT/serotonergic inputs on FN mediated motor behaviors. Although microinjection of serotonergic compounds into cerebellar FNs did not influence the overground locomotion in an open field (n=12), microinjection of5-HT into FNs remarkably promoted motor performances on rota-rod and balance beam (n=11), and narrowed stride width in footprint tests (n=12). This facilitation effect of5-HT was mimicked by5-HT2A receptor agonist TCB-2(n=12or n=13). Moreover, microinjection of5-HT2A receptor antagonist M100907to block endogenous serotonergic inputs in FNs significantly attenuated motor balance and coordination and enlarged stride width (n=12). These results strongly suggest that5-HT/serotonergic inputs improve motor behaviors through modulation of FNs, to which5-HT2A receptors are greatly contributed. Furthermore, microinjection of5-HT into FNs did not affect the stride length, nor did it influence grip strength, demonstrating that5-HT/serotonergic inputs in FNs may precisely modulate trunk and proximal musculature rather than distal muscles. Thus, we speculate that serotonergic afferent inputs may actively regulate final cerebellar outputs by modulation of cerebellar nuclear neurons, and subsequently influence the cerebellum-mediated ongoing motor balance and coordination.These results demonstrate that central serotonergic system could influence crebellar FN neuronal activities via5-HT2A receptors, and consequently participate in modulation of cerebellum-mediated motor control.2. Histamine excites rat subthalamic nucleus neurons and restores motor behavior in animal models of Parkinson’s diseaseThe subthalamic nucleus (STN) is the only excitatory glutamatergic nucleus in the basal ganglia motor circuit, and its widespread projections to other basal ganglia regions lead it to be viewed as a ’driving force’ or ’control structure’ in the integrative function of the basal ganglia circuitry. Numerous studies show that in PD patients and PD animal models the spontaneous firing rate of STN neurons increased with irregular β-synchronized oscillatory. Thus, the STN has long been clinically regarded as an important target in deep brain stimulation for PD.A series of autoradiography, immunohistochemistry and in situ hybridization studies showed that the STN of squirrels received the hypothalamic histaminergic innervations, and histamine H1receptors and H2receptors were present in the STN of human and guinea pigs. Other studies demonstrated that the density of histaminergic fibers in the brain of PD patients and the concentration of histamine in their basal ganglia and the blood were significantly higher. However, research about effect of histamine on STN neuron activities and role of the central histaminergic nervous system in PD is relatively rare. Thus, in this study, the effect and the underlying mechanism of histamine on STN-mediated motor performances of normal and Parkinsonian rats were investigated. Behavioral tests demonstrated that microinjection of histamergic reagents to STN ipslateral to6-OHDA lesion considerably influenced apomorphine-induced rotation in PD rat models. Compared with the saline treatment (234.1±12.5in30min, n-12), Parkinsonian rats microinjected with histamine (125.7±7.6, n=12; P<0.01), H2receptor agonist dimaprit (174.9±8.5, n=12; P<0.01) and high K+(176.9±10.4, n=12; P<0.01) significantly reduced the rotation; Parkinsonian rats microinjected with H1receptor agonist2-pyridylethylamine (228.5±12.9, n=12; P>0.05), H1receptor antagonist mepyramine (243.1±15.3, n=12; P>0.05), H4receptor agonist VUF8430(240.2±13.3, n=12; P>0.05) and H4receptor antagonist JNJ7777120(236.6±12.3, n=12; P>0.05) had no significant effects on rotation; while Parkinsonian rats microinjected with H2receptor antagonist ranitidine (337.6±13.7, n=12; P<0.01) and HCN channel antagonist ZD7288(334.9±18.7, n=12;P <0.01) significant increased the rotation. These results indicated that histaminergic activities on STN improved the motor behaviors of PD rat models, which was mediated by H2receptors and their downstream HCN channels. Furthermore, we found that histamine improved Parkinsonian rat’s motor initiation and motor coordination through H2receptor in adhesive removal and footprint tests.Extracellular recording demonstrated that histamine postsynaptically excited STN neurons via H2receptor, which is consistent with our subsequent immunofluorescence and RT-PCR results that only H2recptor was found in STN. Moreover, using whole-cell patch clamp recordings, we found that histamine-elicited excitation on STN neuron was mediated through HCN channels coupled to H2receptors. Furthermore, our extracellular recordings showed that histamine not only increased the firing rate in STN neurons, but also markedly reduced the coefficient of variation in interspike interval (n=30), while high K+only showed an increase in the firing rate (n=16). Considering that histamine also excites STN neurons in normal rats, we subsequently used rota-rod and balance beam tests to evaluate histaminergic effects on the STN-mediated motor behaviors, and the results confirmed histamine promoted STN-mediated motor balance and motor coordination via H2receptor in normal rats. These results demonstrate that histamine restores motor performances of PD rat models by activation of H2receptors and the HCN channels. Considering histamine also excites the substantia nigra, neostriatum and globus pallidus, we speculate that the central histaminergic system may actively modulate the balance between direct and indirect pathways in the basal ganglia through its direct parallel innervations of those structures as well as the STN, and consequently regulate motor initiation and execution. Presumably, histamine H2receptor and the HCN channel in the basal ganglia is a potential target for clinical treatment of PD. |
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