HistamineexcitesneuronsoftheinferiorvestibularnucleusinratsbyactivationofH1andH2receptors【推薦論文】.doc

精品論文histamine excites neurons of the inferior vestibular nucleus in rats by activation of h1 and h2 receptorspeng shiyu, zhuang qianxing, he yecheng, zhu jingning, wang jianjun5(school of life sciences, nanjing university, nanjing 210093)abstract: by using brain slice preparations and extracellular recordings, the effect of histamine on spontaneous firing activities of neurons in the inferior vestibular nucleus (ivn), a key structure responsible for integration of vestibular, multisensory, and cerebellar inputs, in rats was investigated. perfusing slices with histamine (1-10 m) elicited an excitatory response on ivn neurons. the10responses were not blocked by low ca2+/high mg2+ medium, indicating a direct postsynaptic effect of the amine. furthermore, the histamine-induced excitation was partially blocked by selective histamineh1 receptor antagonist mepyramine (1 m) and h2 receptor antagonist ranitidine (1 m), respectively. co-application of mepyramine and ranitidine nearly totally antagonized the histamine-induced excitation. additionally, both selective h1 receptor agonist 2-pyridylethylamine (30-300 m) and h215receptor agonist dimaprit (10-100 m) effectively mimicked the excitatory action of histamine on ivn neurons. moreover, selective h4 antagonist jnj7777120 (10 m) and agonist vuf8430 (30-300 m) had no effect on ivn neurons. these results demonstrate that histamine excites ivn neurons via postsynaptic h1 and h2 rather than h4 receptors, and suggest that the central histaminergic systemactively modulate all four major vestibular nuclei including the ivn and may subsequently influence20the vestibular nuclei-related reflexes and functions.keywords: histamine; inferior vestibular nucleus; histamine receptors; vestibular reflexes0introductionthe inferior vestibular nucleus (ivn, also called the descending or spinal vestibular nucleus)25is the largest nucleus in the central vestibular nuclear complex in brainstem. the ivn receives predominantly otolith inputs and afferents from the semicircular canals and vermis of the cerebellum, and projects to the contralateral vestibular nuclei, cerebellum, reticular formation, as well as to the spinal cord 1,9,16. therefore, unlike the other three vestibular nuclei, the ivn is generally considered as a key structure integrating vestibular, multisensory, and cerebellar signals30in the vestibular nuclear complex.interestingly, neuroanatomical and immunohistochemical studies have revealed a moderate dense of histaminergic projections from the hypothalamus to the vestibular nuclei including the ivn in various species, such as rats, rabbits, and cats 18,19,26,31. accordingly, molecular, autoradiographic and electrophysiological studies have showed that the medial (mvn), lateral35(lvn) and superior (svn) vestibular nuclei are all endowed with histamine receptors with diverse subtypes 33,35,36,40. however, the role of histamine/the histaminergic afferent inputs in the ivn, an important integrative center for information implicated in the control of body posture, is still unknown. thus, in the present study, by using brain slice preparations and extracellular recordings, the direct action of histamine on neurons in the ivn and the underlying receptor40mechanism is investigated and concentrated on. the results demonstrate that histamine excitesivn neurons via activation of both postsynaptic h1 and h2 receptors.foundations: this work was supported by grants 31070959, 31071021, and 31171050 nsfc/rgc joint research scheme 30931160433 from the national natural science foundation of china; rfdp grant 20100091110016, ncet program, and fundamental research funds for the central universities 1094020806 and 1095020821 from the state educational ministry of china; grant bk2011014 from the natural science foundation of jiangsu province, china.brief author introduction:peng shiyu, (1986-), male, phd, neuroscience. e-mail: - 9 -1materials and methods1.1 animals and brain slice preparationscoronal brain slices (400 m in thickness) containing the ivn were prepared from45sprague-dawley rats (150-250 g) of either sex. under sodium pentobarbital (40 mg/kg) anesthesia, rats were decapitated. after carefully removing the skull, the brainstem extending from obex to the superior colliculi was rapidly removed into ice-cold artificial cerebrospinal fluid (acsf, composition in mm: 124 nacl, 5 kcl, 1.2 nah2po4, 1.3 mgso4, 26 nahco3, 2 cacl2 and 10d-glucose) equilibrated with 95% o2/5% co2. according to the rat brain atlas of paxinos and50watson 25, coronal slices containing the ivn were cut (fig. 1a) with a vibroslicer (vt 1200s, leica, germany) at 4 c 10. the slices were subsequently transferred into a recording chamber, which was continuously perfused with 95% o2/5% co2 oxygenated acsf (ph 7.4, 33 0.2 c, flow rate 2-3 ml/min). all slices were incubated for at least 40 min before neuronal electrophysiological recordings. all experiments completely conformed to the u.s. national55institutes of health guide for the care and use of laboratory animals (nih publication 80-23, revised 1996). all efforts were made to minimize the number of animals used and their suffering.1.2 electrophysiological recordings, data acquisition and statistical analysisspontaneous unitary activity of ivn neurons was recorded extracellularly from the slices by using glass microelectrodes filled with 2 m nacl (resistance 5-10 m). before bath application of60histaminergic compounds at known concentrations, the discharge rate of the recorded neuron wasobserved for at least 40 min to assure stability. in some experiments, low-ca2+/high-mg2+ medium was used to decrease presynaptic neurotransmitter release 7,15,34. histamine or histamine receptor agonist was added to the perfusing acsf to stimulate the recorded ivn neuron for a test period of 1 min. if ivn neuron responded to the stimulation, the perfusing medium was switched65from normal acsf to the acsf containing histamine receptor antagonist(s). after the slice was equilibrated with the acsf containing the antagonist(s) for at least 15 min, histamine or histamine receptor agonist was re-applied and the effect of antagonist(s) on the response of ivn neuron to histamine or histamine receptor agonist was observed. histamine and 2-pyridylethylamine (2-pyea) used in this study were purchased from sigma, usa, and mepyramine, ranitidine and70dimaprit were from tocris, uk. jnj7777120 and vuf8430 were kind gifts from dr. rob leurs(vu university amsterdam, amsterdam, the netherlands).the neuronal discharges of single unit were amplified and displayed conventionally, and fed into a window discriminator simultaneously. the standard rectangle pulses (5 v, 1 ms) triggered from the spikes were sent through an interface (1404 plus, ced, uk) to a laboratory computer,75which was used to analyze the discharge rate online by the software spike 2 (ced, uk).peri-stimulus time histograms (psths, sampling interval = 1 s) and the interspike intervals (isis, sampling interval = 1 ms) distributions of neuronal discharges were generated by the computer to assess the effects of histamine and histamine receptor agonists on ivn neurons. drug-induced effects on spontaneous unitary activity of ivn neurons were considered to be substance specific80provided they were reversible and reproducible. the response magnitude of a neuron to the stimulation of histamine or histamine receptor agonist was calculated as the percentage change in the cells peak discharge rate following stimulation with respect to its basal firing rate. all data were expressed as means sem. students t-test was employed for statistical analysis of the data and p-values of 0.05; fig. 1e vs c), although the spontaneous firing rates of some tested neurons were slightly decreased, which might be related to a disturbance of normal ca2+ concentration in local milieu and/or to actions of mg2+ on intracellular ca2+-dependent processes 11,15,34. this result suggests a direct postsynaptic excitatory effect of histamine on ivn neurons.115120125fig. 1. the direct postsynaptic excitatory effect of histamine on ivn neurons. (a) diagram of coronal brainstemsections shows the area of the ivn investigated in this study. 4v, 4th ventricle; dc, dorsal cochlear nucleus; icp, inferior cerebellar peduncle; ivn, inferior vestibular nucleus; mvn, medial vestibular nucleus. (b) oscilloscope traces show the action potentials of a recorded ivn neuron and the neurons response to 10 m histamine. the mean firing rate of the neuron before, during, and after application of histamine was given below each recording. (c) psths show that the same ivn neuron exhibited a concentration-dependent excitatory response to 1, 3 and 10 m histamine stimulation. (d) isis show that histamine shortened the intervals of spikes of the ivn neuron. (e) histograms show the histamine-induced excitation on the same neuron when the slice was equilibrated withlow-ca2+/high-mg2+ medium. in this and the following figures, the short horizontal bars above the data indicate the1 min period of application of histamine or histamine receptor agonists, and the long horizontal bars denote the exposure of the slice to the low-ca2+/high-mg2+ medium or histamine receptor antagonists.it has been well known that histamine exerts its action via four distinct receptor subtypes, in which the histamine h1, h2 and h4 receptors are postsynaptic whereas h3 receptors are presynaptic 13,14. therefore, in the present study, we further used histamine h1, h2 and h4130135140145150receptor antagonists to examine which postsynaptic histamine receptor(s) mediated the histamine-induced excitation on ivn neurons (figs. 2 and 3). the results showed that both mepyramine (1 m; fig. 2b), a highly selective histamine h1 receptor antagonist, and ranitidine (1 m; fig. 2c), a highly selective histamine h2 receptor antagonist, effectively blocked the excitatory responses of ivn neurons (n = 31 and 29, respectively) to histamine (1-10 m); but highly selective histamine h4 receptor antagonist jnj7777120 (10 m; fig. 2d) did not influence the excitatory effects of histamine on the neurons (n = 25). furthermore, mepyramine (1 m) combined with ranitidine (1 m) blocked the histamine-induced excitation almost totally (n = 17; fig. 2e). on the other hand, the effect of histamine receptor agonist-mimicked responses was further examined. the results showed that both the highly selective h1 receptor agonist 2-pyea (30-300 m) and highly selective h2 receptor agonist dimaprit (10-100 m) mimicked the excitatory effect of histamine on ivn neurons (n = 49 and 44, respectively; fig. 2f), whereas the highly selective h4 receptor agonist vuf8430 (30-300 m) did not elicit any response in ivn neurons (n = 37; fig. 2f). these data strongly suggest that histamine excites ivn neurons through activation of both histamine h1 and h2 receptors rather than h4 receptors.fig. 2. effects of histamine receptor antagonists on the histamine-induced excitation and effects of histamine receptor agonists on the ivn neurons. (a) concentration-related excitatory responses of a recorded ivn neuron to histamine. (b-d) the effects of selective histamine h1 receptor antagonist mepyramine, h2 receptor antagonist ranitidine and h4 receptor antagonist jnj7777120 on the histamine-induced excitations on the same ivn neuron. note that mepyramine combined with ranitidine blocked the histamine-induced excitation nearly totally (e). (f) the effects of selective histamine h1 receptor agonist 2-pyea, h2 receptor agonist dimaprit and h4 receptor agonist vuf8430 on the same cell.155160as summarized in figure 3, ivn neurons exhibited a concentration-dependent excitatory response to the stimulation of histamine. mepyramine, ranitidine and a combination of both rather than jnj7777120 pushed the concentration-response curve of histamine down to the lower level. in addition, 2-pyea and dimaprit rather than vuf8430 mimicked the excitatory response of histamine. furthermore, double immunostaining results showed that histamine h1 and h2 receptors were not only present in the rat ivn but also co-localized in the same ivn neurons (fig.4a-c) and histamine h4 receptors were not detected in the ivn (fig. 4d and e). taking all these described results together, the data indicate that a co-mediation mechanism of both h1 and h2receptors underlying the histamine-induced excitation on ivn neurons.165170175fig. 3. the averaged concentration-response curves at level of group data showing the effects of histamine,selective histamine h1, h2 and h4 receptor agonists (2-pyea, dimaprit, and vuf8430) on the ivn neurons, and the effects of selective histamine h1, h2 and h4 receptor antagonists (mepyramine, ranitidine, and jnj7777120) on the histamine-induced excitation. numbers in the parentheses denote the number of cells tested in each experiment. data are presented as mean sem.fig. 4. immunostaining results showing that histamine h1 (a1-a3) and h2 (b1-b3) receptors were not only present in the rat ivn but also co-localized in the same ivn neurons (c1-c3), but histamine h4receptor-immunolabeled neurons were not detected (d1-d3). negative staining controls (e1-e3). scale bars: (a1), (b1), (c1), (d1) and (e1), 550 m; (a2), (b2), (c2), (d2) and (e2), 100 m; (a3), (b3), (c3), (d3) and (e3), 20 m.1801851901952002052102152203discussionanti-histaminergic drugs have traditionally been used for clinical treatment of balance disorders and symptoms, such as vertigo, motion sickness, nausea and nystagmus 12,20,22,32. besides the peripheral vestibular apparatus in the inner ear 5,29, the central vestibular nuclear complex in brainstem is also a critical target for those anti-histaminergic drugs. actually, histamine not only plays a large role in allergies in periphery, but also acts as a neurotransmitter in the brain and functions as a general modulator for whole brain activity. although the central histaminergic system strictly originates from the tuberomammillary nucleus of the hypothalamus, it extensively innervates almost the whole brain including the vestibular nuclear complex 4,13,14. thus, via widely modulating various brain areas involved in different functions, histamine/histaminergic system holds a key position in the regulation of multiple physiological processes, including the sleep-wake cycle, energy and endocrine homeostasis, synaptic plasticity and learning, as well as motor control 4,13,14,39.among the central motor structures, the vestibular nuclei in brainstem constitute a sensorimotor complex that integrates vestibular, visual and motor signals to make compensatory eye and head movements as well as postural adjustments possible 16,30. studies from our and other laboratories have documented that histamine/central histaminergic system may actively modulate subcortical motor structures such as the cerebellum 15, basal ganglia 27,38, red nucleus 6, as well as the lvn 36, mvn 33 and svn 40 in the vestibular nuclear complex. the present study demon