组胺通过突触后H1和H2受体兴奋大鼠前庭上核神经元

组胺通过突触后H1和H2受体兴奋大鼠前庭上核神经元 Histamine excites rat superior vestibular nuclear neurons via postsynaptic H1 and H2 receptors in vitro ZHUANG Qianxing, WU Yonghui, WU Guanyi, ZHU Jingning, WANG Jianjun 5 (School of Life Sciences, Nanjing University, NanJing 210093) Abstract: The superior vestibular nucleus (SVN), which holds a key position in vestibulo-ocular reflexes and nystagmus, receives direct hypothalamic histaminergic innervations. By using rat brainstem slice preparations and extracellular unitary recordings, we investigated the effect of histamine on SVN neurons and the underlying receptor mechanisms. Bath application of histamine 2+/high-Mg2+ evoked an excitatory response of the SVN neurons, which was not blocked by the low-Ca10 medium, indicating a direct postsynaptic effect of the amine. Selective histamine H1 receptor agonist 2-pyridylethylamine (2-PyEA) and H2 receptor agonist dimaprit, rather than VUF8430, a selective H4 receptor agonist, mimicked the excitation of histamine on SVN neurons. In addition, selective H1 receptor antagonist mepyramine and H2 receptor antagonist ranitidine, but not JNJ7777120, a selective 15 H4 receptor antagonist, partially blocked the excitatory response of SVN neurons to histamine. Moreover, mepyramine together with ranitidine nearly totally blocked the histamine-induced excitation. Immunostainings further showed that histamine H1 and H2 instead of H4 receptors existed in the SVN. These results demonstrate that histamine excites the SVN neurons via post-synaptic histamine H1 and H2 receptors, and suggest that the central histaminergic innervation from the hypothalamus may actively bias the SVN neuronal activity and subsequently modulate the SVN-mediated vestibular 20 functions and gaze control. Keywords: Histamine; Superior vestibular nucleus; Histamine receptors; Vestibular reflexes 0 Introduction Among the vestibular disorders, pathologic nystagmus is a symptom which often 25 accompanies vertigo and motion sickness. Interestingly, a recent investigation reported an efficacy of the histaminergic drug, betahistine dihydrochloride, in not only antivertigo and anti-motion sickness but also improving oculomotor activity, including increased gain during pursuit movements and faster and more accurate saccades [1]. In fact, anti-histaminergic drugs have been used for clinical treatment of vestibular related diseases, including vertigo, emesis and motion 30 sickness, for almost a century. The therapeutic mechanisms are involved not only in peripheral vestibular system, such as labyrinth in the inner ear [2-3], but also in central vestibular nuclear complex, including at least the medial (MVN) and lateral (LVN) vestibular nucleus [4-6]. Immunohistochemical studies have already revealed a moderately dense direct histaminergic projection from the tuberomammillary nucleus of the hypothalamus to the vestibular nuclei in 35 many species including rat, cat and pigeons [7-9]. Nevertheless, histaminergic fiber distribution in the vestibular nuclei shows spatial variations, with significantly heavier labelling in the superior vestibular nucleus (SVN) and MVN than in the LVN and descending vestibular nucleus [9]. On the other hand, molecular, autoradiographic and pharmacological studies have also demonstrated that the LVN and MVN are both endowed with histamine H1, H2 and/or H3 receptors [10-11]. 40 Among the four nuclei in the central vestibular nuclear complex, the SVN, together with the MVN, receives fibers predominantly from the semicircular canals and sends fibers through the medial longitudinal fasciculus rostrally to oculomotor centers and caudally to the spinal cord [12-13]. Foundations: This work was supported by grants 31070959, 31071021, 31171050, J1103512 and 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. This work was also supported by a grant 9112020802 of Brief author introduction:ZHUANG Qianxing (1980-), male, PhD, Neuroscience Correspondance author: WANG Jianjun, male, PhD, Neuroscience. E-mail: jjwang@nju.edu.cn -1- Both of these two nuclei actually hold a key position in vestibulo-ocular reflexes and gaze control 45 [12, 14-16] and are closely related to nystagmus [17-18]. In recent years, role of histamine/hypothalamic histaminergic innervation in the MVN neuronal activity and the MVN-mediated vestibular compensation has been received increasing attention [4, 19-20], however, the action of histamine on the SVN, another key vestibular nucleus primarily involved in reflexes controlling gaze, still remains enigmatic. Thus, by using extracellular recordings and immunostainings, this work was designed to investigate the effect of 50 histamine on neurons in the SVN and the underlying receptor mechanism. The results 1 and demonstrated that histamine excited the SVN neurons via activation of both postsynaptic H H2 receptors. 1 Material and methods 55 1.1 Slice preparations and incubations Sagittal brain slices (400 μM thickness) containing the SVN were prepared from Sprague-Dawley rats (120-250 g) of either sex. Under sodium pentobarbital (40 mg/kg) anesthesia, rats were decapitated. After carefully removing the skull, the brain 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 10 60 D-glucose) equilibrated with 95% O2/5% CO2. According to the rat brain atlas of Paxinos and Watson [21], the sagittal slices [22] containing the SVN, cerebellum and part of midbrain (including colliculi) were cut (Fig. 1A) with a vibroslicer (VT 1200S, Leica, Germany) at 4 ?C. 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 1.5-2 ml/min). 65 All slices were incubated for at least 40 min before neuronal electrophysiological recordings. All experiments completely conformed to the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications 80-23, revised 1996). All efforts were made to minimize the number of animals used and their suffering. 70 1.2 Electrophysiological recordings, data acquisition and statistical analysis The SVN was visually identified with the aid of a stereomicroscope (SD-3045F, Olympus, Japan) and spontaneous unitary activity of the SVN neurons was recorded extracellularly from the slices by using glass microelectrodes filled with 2 M NaCl (resistance 5-10 M?). Before bath application of histaminergic compounds at known concentrations, the discharge rate of the recorded neuron was observed for at least 40 min to assure stability. In some experiments, 75 low-Ca2+/high-Mg2+ medium was used to decrease presynaptic neurotransmitter release. In these cases, the concentration of Ca2+ was lowered to 0.3 mM and Mg2+ was raised to 9.0 mM [23-27]. Histamine or histamine receptor agonist was added to the perfusing ACSF to stimulate the recorded SVN neuron for a test period of 1 min. After each stimulation, cells were given at least 20 min for recovery and preventing receptors from desensitization. If the SVN neuron responded 80 to the stimulation, the perfusing medium was switched from 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 SVN neuron to histamine or histamine receptor agonist was observed. 85 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 -2- from the spikes were sent through an interface (1404 Plus, CED, UK) to a laboratory computer, which was used to analyze the discharge rate online by the software Spike 2 (CED, UK). 90 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 the SVN neurons. Drug-induced effects on spontaneous unitary activity of SVN neurons were considered to be substance specific provided 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 95 the cell’s peak discharge rate following stimulation with respect to its basal firing rate. All data were expressed as means ? S.E.M.s. Student’s t-test was employed for statistical analysis of the data and P-values of < 0.05 were considered to be significant. 1.3 Immunofluorescence The experimental procedures for immunostaining followed our previous work [28]. Rats 100 (weighing 150-200 g) were deeply anesthetized with sodium pentobarbital and perfused transcardially with 100 ml normal saline, followed by 450-500 ml 4% paraformaldehyde in 0.1 M phosphate buffer. Subsequently, the brain was removed, trimmed, and post-fixed in the same fixative for 12 hours at 4 ?C and then cryoprotected with 30% sucrose for 48 hours. Frozen coronal sections (25 μm thickness) containing the SVN were obtained by using a freezing 105 microtome (CM 3050S, Leica, Germany) and mounted on gelatin-coated slides. The slices were rinsed in phosphate buffered saline containing 0.1% Triton X-100 (PBST) and then incubated in 10% normal bovine serum in PBST for 30 min. Sections were incubated overnight at 4 ?C with primary antibodies to histamine H1, H2 or H4 receptor, respectively: a rabbit anti-H1 receptor polyclonal antibody (1:50, Santa Cruz, USA), a rabbit anti-histamine H2 receptor polyclonal 110 antibody (1:50, Santa Cruz, USA), or a rabbit anti-histamine H4 receptor polyclonal antibody (1:50, Santa Cruz, USA). After a complete wash in PBS, these sections were incubated in the Alexa 488-conjugated secondary antibody solutions (goat anti-rabbit 1:2000, Invitrogen, USA) for 2 h at room temperature in dark. The slides were washed and mounted in UltraCruz mounting medium (Santa Cruz, USA). All micrographs were taken with an inverted laser scanning confocal 115 microscope (FV1000, Olympus, Japan). Incubations replacing the primary antiserum with control immunoglobulins and/or omitting the primary antiserum were used as negative controls. 1.4 Histaminergic reagents Stocks solutions of histaminergic compounds were made in distilled water, and dilutions were freshly prepared in ACSF and equilibrated with 95% O2/5% CO2 before superfusing the 120 slices. The histaminergic compounds used in this experiment included histamine (Sigma, USA), highly selective histamine H1 receptor antagonist mepyramine (Tocris, UK), highly selective histamine H2 receptor antagonist ranitidine (Tocris, UK), highly selective histamine H1 receptor agonist 2-pyridylethylamine (2-PyEA; Tocris, UK), and highly selective histamine H2 receptor agonist dimaprit (Tocris, UK). JNJ7777120, a highly selective histamine H4 receptor antagonist, 125 and VUF8430, a highly selective histamine H4 receptor agonist, were kind gifts from Dr. Rob Leurs (VU University Amsterdam, Amsterdam, The Netherlands). The concentrations of histaminergic reagents were chosen according to the previous reports [4, 6, 23-27, 29-30]. 130 -3- 2 Results 2.1 Histamine directly excites the SVN neurons in a concentration-dependent manner One hundred and twenty SVN neurons with tonic spontaneous firing activity were 135 extracellularly recorded from 55 brainstem slices of rats in the present study. The spontaneous firing rate of the SVN neurons ranged from 5 to 80 spikes/s and the mean firing rate was 25.32 ? 0.86 spikes/s, which are similar to the previous study on the SVN neurons in vivo [31] and slightly higher than MVN neuronal spontaneous firing rate in vitro [4, 32-33]. All of the 120 recorded SVN neurons (120/120, 100%) sampled from various sub-regions of the SVN (Fig. 1A) were excited by the histamine stimulation (1-30 μM; Fig. 1B). As illustrated in 140 Fig. 1C (see also Fig. 3), the recorded SVN neuron showed a concentration-dependent excitation to 1, 3, and 10 μM histamine with peak firing values of 24.5, 26.8 and 29.8 spikes/s, i.e. 8.4%, 18.6% and 31.9% increment in the peak discharge rate compared with its basal firing rate of 22.6 spikes/s (P < 0.05 or 0.01), respectively. Furthermore, ISIs revealed that during the period of histamine-induced excitation, the interspike intervals of SVN neurons were significantly shortened 145 (P < 0.05 or 0.01, Fig. 1D). The results indicate that histamine is capable of exciting SVN neurons. To exclude the possibility that the excitatory response of SVN neurons was indirectly induced by the effect of histamine on presynaptic elements, we tested the effects of histamine on 2+/high-Mg2+ medium (n = the SVN cells when the normal ACSF had been replaced with a low-Ca150 5). The results showed that the low-Ca2+/high-Mg2+ medium did not block the histamine-induced excitation (P > 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 [23, 32, 34-35] This result suggests a direct postsynaptic excitatory effect of histamine on the SVN neurons. 155 2.2 The histamine-induced postsynaptic excitation on SVN neurons is co-mediated by histamine H1 and H2 receptors Since the histamine H1, H2 and H4 receptors are all postsynaptic whereas H3 receptors are presynaptic [36-37], we further used histamine receptor agonists and antagonists to examine which postsynaptic histamine receptor(s) mediated the histamine-induced excitation on SVN neurons. 160 The results showed that both the selective H1 receptor agonist 2-PyEA (3-100 μM) and selective H2 receptor agonist dimaprit (3-100 μM) mimicked the excitatory effect of histamine on SVN neurons (n = 49 and 56, respectively, Fig. 2B and C, see also Fig. 3), whereas selective H4 receptor agonist VUF8430 (3-100 μM) did not elicit any response in SVN neurons (n = 12, Fig. 2D, see also Fig. 3). As shown in Fig. 2, the recorded SVN neurons exhibited 165 concentration-related excitatory responses to histamine (1, 3, and 10 μM, with 23.6%, 35.1% and 48.7% increase in the peak discharge rate compared with its basal firing rate, P < 0.05 or 0.01, Fig. 2A), 2-PyEA (3, 10, and 30 μM, with 11.1%, 21.5% and 33.4% increase in the peak discharge rate compared with its basal firing rate, P < 0.05 or 0.01, Fig. 2B) and dimaprit (3, 10, and 30 μM, with 3.5%, 17.2% and 29.0% increase in the peak discharge rate compared with its basal firing 170 rate, P < 0.05 or 0.01, Fig. 2C) but not VUF8430 (3, 10, and 30 μM, P > 0.05, Fig. 2D). -4- Fig. 1. Histamine directly/post-synaptically excited the SVN neurons. (A) Diagram of sagittal brainstem sections shows the area of the SVN investigated in this study. 4V, 4th ventricle; IN, interpositus nucleus; IVN, inferior vestibular nucleus; LVN, lateral 175 vestibular nucleus; MVN, medial vestibular nucleus; SVN, superior vestibular nucleus; scp, superior cerebellar peduncle. (B) Oscilloscope traces show the action potentials of a recorded SVN neuron and the neuron’s response to 10 μM histamine. The mean firing rate of the neuron before, during, and after application of histamine was 22.6, 29.8, and 22.9 spikes/s, respectively. (C) Histograms show that the SVN neuron exhibited a concentration-dependent excitatory response to 1, 3 and 10 μM histamine stimulation. (D) Interspike intervals (ISIs) show that histamine shortened the intervals of spikes of the SVN neuron. (E) Histograms show the histamine-induced excitation on the same neuron when the slice was equilibrated with low-Ca2+/high-Mg2+ 180 medium. In this and the following figures, the short horizontal bars above the data indicate the 1 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. -5- Fig. 2. Effects of histamine receptor agonists on the SVN neurons and effects of histamine receptor antagonists on 185 the histamine-induced excitation. (A) Concentration-related excitatory responses of a recorded SVN neuron to histamine. (B-D) The effects of selective histamine H1 receptor agonist 2-PyEA, H2 receptor agonist dimaprit and H4 receptor agonist VUF8430 on the same cell. (E-H) The effects of selective histamine H1 receptor antagonist mepyramine, H2 receptor antagonist ranitidine and H4 receptor antagonist JNJ777120 on the histamine-induced 190 excitations on the same SVN neuron. Note that mepyramine combined with ranitidine blocked the histamine-induced excitation nearly totally. On the other hand, mepyramine (1 µM), a selective histamine H1 receptor antagonist, and ranitidine (1 µM), a selective histamine H2 receptor antagonist, effectively blocked the excitatory responses of SVN neurons (n = 20 and 12, respectively) to histamine (1-30 µM, Fig. 2E and F), 195 but selective histamine H4 receptor antagonist JNJ7777120 (1 µM) did not influence the excitatory effects of histamine on the neurons (n = 10, Fig. 2G). As illustrated in Fig. 2, on the same recorded SVN neuron, mepyramine (1 µM) and ranitidine (1 µM) significantly reduced the 23.6%, 35.1% and 48.7% increase in the peak firing rate evoked by 1, 3, and 10 μM histamine to 8.3%, 14.9% and 22.7% (P < 0.05 or 0.01, Fig. 2E vs 2A) and 7.1%, 13.7% and 18.3% (P < 0.05 or 0.01, Fig. 200 2F vs 2A), respectively; however, JNJ7777120 (1 µM) did not influence the histamine-induced excitatory responses, i.e., the cell’s responses to histamine remained at the same levels with the control experiments (22.2%, 34.5% and 47.3% increase in the peak firing rate, P > 0.05, Fig. 2G). Furthermore, mepyramine (1 µM) combined with ranitidine (1 µM) blocked the histamine-induced excitation nearly totally (n = 8, Fig. 2H). 205 As summarized in Fig. 3, the SVN neurons exhibited a concentration-dependent excitatory response to the stimulation of histamine, 2-PyEA, and dimaprit rather than VUF8430. In addition, mepyramine, ranitidine and a combination of both rather than JNJ7777120 pushed the concentration-response curve of histamine down to the lower level, indicating a co-mediation mechanism of both H1 and H2 receptors. 210 -6- Fig. 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 SVN neurons, and 215 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 ? S.E.M. To confirm the receptor mechanisms underlying the histamine-induced excitation on SVN neurons, the effect of histamine receptor antagonists on agonist-mimicked responses was further 220 examined. The results showed that mepyramine (1 µM) significantly blocked the concentration-related excitation mimicked by 10, 30 and 100 μM 2-PyEA (Fig. 4C vs B), and ranitidine (1 µM) remarkably antagonized the concentration-related excitation mimicked by 10, 30 and 100 μM dimaprit (Fig. 4E vs D). Furthermore, the additive effects of co-application of 2-PyEA and dimaprit on the SVN neurons were showed in Fig. 5. The averaged 225 concentration-response curves were plotted in Fig. 6. -7- Fig. 4. The blocking effect of histamine H1 receptor antagonist mepyramine and H2 receptor antagonist ranitidine on selective histamine H1 and H2 receptor agonists 2-PyEA- and dimaprit-mimicked excitations on the SVN neurons. (A) A concentration-dependent excitation induced by 1, 3, 10 μM histamine on a recorded SVN neuron. 230 (B-C) The excitatory response of the neuron to 2-PyEA and the blocking effect of mepyramine on 2-PyEA-induced excitations. (D-E) The excitatory response of the same neuron to dimaprit and the blocking effect of ranitidine on the dimaprit-induced excitations. -8- 235 Fig. 5. The additive excitatory effect of co-application of histamine H1 and H2 receptor agonists 2-PyEA and dimaprit on a single SVN neuron (A-D). 240 Fig. 6. The averaged concentration-response curves at level of group data showing the effects of histamine, 2-PyEA, dimaprit, and co-application of 2-PyEA and dimaprit on the SVN neurons, and the effects of mepyramine (1 μM) and ranitidine (1 μM) on 2-PyEA- and dimaprit-induced excitation, respectively. Numbers in the parentheses denote the number of cells tested in each case. Data are presented as mean ? S.E.M. 245 Table 1 summarized the effects of histamine and histamine receptor agonists on SVN neurons and the effects of histamine receptor antagonists on histamine- or agonist-induced excitations. The data strongly suggest that it is H1 and H2 receptors, instead of H4 receptors, mediate histamine-induced excitatory responses on SVN neurons. 250 -9- Table 1 Comparison of the effects of histamine and histamine receptor agonists on SVN neurons, and the effects of histamine receptor antagonists on histamine- or agonist-induced excitation on the SVN neurons. No. of cells % of response Cells excited by histamine 120 100% (120/120) Cells excited by 2-PyEA 49 100% (49/49) Cells excited by dimaprit 56 100% (56/56) Cells responded to VUF8430 12 0% (0/12) Cells excited by co-application of 2-PyEA and dimaprit 10 100% (10/10) Histamine-induced excitation blocked by mepyramine 20 100% (20/20) Histamine-induced excitation blocked by ranitidine 12 100% (12/12) Histamine-induced excitation blocked by JNJ7777120 10 0% (0/10) 2-PyEA-induced excitation blocked by mepyramine 10 100% (10/10) Dimaprit-induced excitation blocked by ranitidine 10 100% (10/10) 255 2.3 Both histamine H1 and H2 receptors are expressed in the SVN To map the distribution of histamine receptors in the SVN, we performed immunostaining of the rat brainstem slices containing the SVN with an antibody against histamine H1, H2 and H4 receptors, respectively. We found that histamine H1 and H2 rather than H4 receptors were localized in the SVN (Fig. 7). These observations are consistent with the above-mentioned electrophysiological results and prove that histamine excites the SVN neurons through activation 260 of both histamine H1 and H2 receptors. Fig. 7. Confocal photomicrographs showing histamine receptor immunoreactivity in the SVN in rats. Histamine H1 receptor- (A1, A2, A3) and H2 receptor- (B1, B2, B3) immunolabeled neurons were detected in the SVN, but H4 receptor- (C1, C2, C3) immunolabeled neurons were not detected. (D1, D2, D3) Negative staining controls. A2, 265 B2, C2 and D2 are higher magnification of the square in A1, B1, C1 and D1. A3, B3, C3 and D3 are higher magnification of the square in A2, B2, C2 and D2. Scale bars: A1, B1, C1, D1 = 990 ,m; A2, B2, C2, D2 = 270 ,m; A3, B3, C3, D3 = 20 ,m. LR4V, lateral recess of the 4th ventricle; MVN, medial vestibular nucleus; SVN, superior vestibular nucleus. 270 3 Discussion Central histaminergic system solely originates from the tuberomammillary nucleus of the hypothalamus but extensively innervates almost the whole brain including various subcortical motor structures [36-38]. It holds a key position in the regulation of many basic physiological 275 functions, including the sleep-wake cycle, energy and endocrine homeostasis, synaptic plasticity - 10 - and learning, as well as motor control [36-39]. Thus, the central histaminergic system/histamine has been considered as a general modulator for whole brain activity. Intriguingly, neuroanatomical and immunohistochemical studies have also revealed a direct histaminergic innervation on the central vestibular nuclear complex [7-9], and the 280 electrophysiological studies further reported an excitatory effect of histamine on neurons in both the MVN and LVN [4, 6]. However, these studies on role of histamine/histaminergic system in vestibular neurons and vestibular-related functions neglect the SVN, an important sub-nucleus in the vestibular nuclear complex and playing a critical role in the vestibulo-ocular reflexes and gaze control. In the present study, we demonstrated that histamine excited the rat SVN neurons by 1 and H2 receptors. mediation of both histamine H285 Although histamine exerts a uniform excitatory effect on neurons in all of the LVN, MVN, and SVN, the receptor mechanisms underlying the histamine-induced excitation on neurons in the three vestibular nuclei are different. In the LVN, histamine depolarizes the neurons via only postsynaptic histamine H2 receptors [6], whereas in the MVN, not only postsynaptic histamine H1 and H2 receptors but also presynaptic histamine H3 receptors are involved in the action of 290 histamine on the neurons [4, 19]. In this study, low-Ca2+/high-Mg2+ medium did not affect the excitatory response of SVN neurons to histamine (Fig. 1E), suggesting that the histamine-induced excitation was due to a direct postsynaptic effect of the amine on the cells, but not indirect effect induced by presynaptic neural events. Therefore, selective agonists and antagonists for postsynaptic histamine H1, H2 and H4 receptors were applied to determine the receptor 295 mechanisms on the SVN neurons. Mepyramine and ranitidine, selective antagonists for histamine H1 and H2 receptors, separately blocked the histamine-induced excitation in part and together antagonized the excitation nearly totally (Figs. 2 and 3). Moreover, 2-PyEA and dimaprit, selective agonists for histamine H1 and H2 receptors, separately mimicked the histamine-induced excitation in a concentration dependent manner (Figs. 2 and 3), and co-application of 2-PyEA and 300 dimaprit evoked an additive excitatory effect (Figs. 5 and 6). And the 2-PyEA- and dimaprit-mimicked excitations on SVN neurons were blocked by mepyramine and ranitidine, respectively (Figs. 4 and 6). On the other hand, VUF8430 and JNJ7777120, selective agonist and antagonist for histamine H4 receptors, had no effect on the SVN neurons and histamine-induced excitation (Figs. 2 and 3). Furthermore, immunostaining results revealed that only histamine H1 305 and H2 receptors instead of H4 receptors were expressed in the SVN (Fig. 7). These results clearly demonstrate that histamine H1 and H2 receptors rather than H3 and H4 receptors co-mediate the histamine-induced excitatory effect on rat SVN neurons. The vestibular nuclei in the brainstem are a sensorimotor complex that integrates vestibular, visual and motor signals to make compensatory eye and head movements as well as postural 310 adjustments [13]. Among the four major vestibular nuclei, the SVN receives inputs predominantly from the semicircular canals and sends axons to the oculomotor nucleus, the cerebellar nodulus, uvula and flocculus, and the ventral posterior nuclear complex of the thalamus, which in turn projects to the cortical areas relevant to vestibular sensations [12]. Consequently, slightly different from the other vestibular nuclei which are also essential for posture adjustments of the head and/or 315 body, the SVN primarily participates in vertical vestibulo-ocular reflexes and holds a key position in gaze control [12, 14-16], together with the MVN. Therefore, the present results that histamine directly excites the SVN neurons suggest that the histaminergic innervation from the hypothalamus on vestibular nuclei may actively participate in stabilizing not only head and posture through the MVN and LVN but also gaze through the MVN as well as the SVN. 320 Besides the vestibular nuclear complex in the brainstem, various important subcortical motor - 11 - structures have been found to receive and be modulated by direct hypothalamic histaminergic projections, including the cerebellum [23, 26], the red nucleus [24], and the substantia nigra [40-41], neostriatum [42], and globus pallidus [25] in the basal ganglia. Interestingly, without 325 exception, neurons in all these subcortical motor centers are uniformly excited by histamine. Since the most histaminergic endings (varicosities) do not in typical form synaptic specializations and all of the histamine receptors are metabotropic [36-37, 43], we suggest that histamine or histaminergic inputs from the hypothalamus to these central motor structures may not transmit fast concrete signals, but act as a biasing force to influence electrophysiological properties of these motor neurons and hold their excitability and responsiveness to an appropriate level. Consequently, 330 the central histaminergic system may ultimately regulate movements. In fact, in the cerebellum, histamine/the histaminergic afferents regulates the cerebellar nuclear neuronal activity and improves motor balance and motor coordination during ongoing movements [26, 44]. Also, depletion of brain histamine or knockout of histamine receptors alters ambulatory activity and reduces exploratory behavior [45-47]. Thus, we speculate that hypothalamic histaminergic 335 system/histamine may actively participate in central motor control by extensive modulation of the sensorimotor integration in circuits through various subcortical motor structures including the SVN. 4 Conclusion In conclusion, we found a direct excitatory action of histamine on the SVN neurons, which is 340 mediated by both postsynaptic histamine H1 and H2 receptors. Through biasing neuronal activity in the SVN, the histaminergic system may be involved in the gaze control and contribute to generate an appropriate compensatory eye movements and stable vision during head rotations. The modulation of hypothalamic histaminergic system on the SVN may constitute an important and functional component of the vestibular motor control, including not only postural stability of both 345 the head and body but also compensatory eye movements. On the other hand, the modulation of histaminergic system on the SVN cannot be ignored in the symptomatic treatment of vestibular-related diseases (including nystagmus), and the postsynaptic histamine receptors in central vestibular nuclei may be potential targets for the clinic therapy. 350 Acknowledgements We thank Dr. Rob Leurs (VU University Amsterdam, Amsterdam, The Netherlands) for his generous gifts of histamine H4 receptor agonist and antagonist. References [1] Matsnev EI, Sigaleva EE: Efficacy of histaminergic drugs in experimental motion sickness. J Vestib Res 355 2007;17:313-321. [2] Soto E, Chávez H, Valli P, Benvenuti C, Vega R: Betahistine produces post-synaptic inhibition of the excitability of the primary afferent neurons in the vestibular endorgans. Acta Otolaryngol 2001;545(Suppl):19-24. [3] Chávez H, Vega R, Soto E: Histamine H3 receptors modulate the excitatory amino acid receptor response of the vestibular afferents. 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Mol Pharmacol 2002;62:389-397. 450 组胺通过突触后 H1 和 H2 受体兴奋大鼠前庭上核神 经元 庄乾兴，伍永辉，伍冠一，朱景宁，王建军 (南京大学生命科学学院，南京 210093) 455 摘要:我们采用大鼠脑干切片和细胞外 记录 混凝土 养护记录下载土方回填监理旁站记录免费下载集备记录下载集备记录下载集备记录下载 的方法，研究组胺对前庭上核(superior vestibular nucleus, SVN)神经元的作用及其受体机制。结果 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 明，组胺(1-30 μM)可以兴奋 SVN 神 经元，且这种兴奋性效应不能被低 Ca 2+/高 Mg2+阻断，表明组胺对 SVN 神经元的这种效应 是直接的突触后效应。选择性组胺 H1 受体激动剂 2-pyridylethylamine (2-PyEA, 3-100 μM) 和选择性组胺 H2 受体激动剂 dimaprit(3-100 μM)均可以模拟组胺对 SVN 神经元的兴奋 460 性效应，但是选择性组胺 H4 受体激动剂 VUF8430(3-100 μM)则不能模拟组胺引起的兴奋 性效应。另一方面，选择性组胺 H1 受体阻断剂 mepyramine(1 μM)和选择性组胺 H2 受 体阻断剂 ranitidine(1 μM)可以部分阻断组胺诱导的 SVN 神经元的兴奋性反应，但是选 择性组胺 H4 受体阻断剂 JNJ7777120(10 μM)则不能影响组胺引起的神经元兴奋性效应。 同时应用 mepyramine(1 μM)和 ranitidine(1 μM)，则几乎可以完全阻断组胺引起的兴奋 465 性效应。并且 mepyramine 和 ranitidine 能够分别有效地阻断 2-PyEA 和 dimaprit 诱导的兴 奋性反应。免疫组化的结果进一步表明了 H1 和 H2 受体均存在 SVN 中。以上结果表明组胺 可以通过突触后组胺 H1 和 H2 受体兴奋 SVN 神经元，提示源自下丘脑的中枢组胺能神经系 统能够通过影响 SVN 神经元的活动调节经 SVN 介导的前庭反射和眼动调控。 关键词:组胺;前庭上核;组胺受体;前庭反射 470 中图分类号:Q424 - 14 -