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Histamine evokes excitatory response of neurons in the cerebellar dentate nucleus via H2 receptors
Neuroscience Letters 502 (2011) 133–137
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Histamine evokes excitatory response of neurons in the cerebellar dentate nucleus via H2 receptors Yong-Ting Qin a,1 , Song-Hua Ma a,1 , Qian-Xing Zhuang b , Yi-Hua Qiu a , Bing Li a , Yu-Ping Peng a,∗ , Jian-Jun Wang b,∗∗ a
Department of Physiology, School of Medicine, Nantong University, 19 Qixiu Road, Nantong 226001, China Department of Biological Science and Technology and State Key Laboratory of Pharmaceutical Biotechnology, Mailbox 426, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China b
a r t i c l e
i n f o
Article history: Received 8 February 2011 Accepted 31 May 2011 Keywords: Histamine Histamine receptors Cerebellar dentate nucleus Hypothalamocerebellar histaminergic projections
a b s t r a c t Previous studies have shown an excitatory effect of histamine on neurons in two cerebellar nuclei, the fastigial nucleus and the interposed nucleus. Here we investigated action of histamine on the dentate nucleus (DN), another nucleus of the cerebellum, and provided more evidence for motor control by histamine via the cerebellum. Spontaneous unitary discharge of neurons in the DN was extracellularly recorded by use of cerebellar slice preparations. In total 79-recorded neurons, which were from 53 cerebellar slices, 67 neurons (84.8%) had an excitatory response to histamine stimulation, and the rest (15.2%) were not reactive. The histamine-induced excitation of the DN neurons was not blocked by low-Ca2+ /highMg2+ medium, demonstrating that this effect of histamine was postsynaptic. Triprolidine, an antagonist of histamine H1 receptors, did not block the excitatory effect of histamine, but ranitidine, an antagonist for H2 receptors, blocked the excitatory response to histamine in a concentration-dependent manner. Further, histamine H1 receptor agonist 2-pyridylethylamine did not elicit any response of DN neurons, but H2 receptor agonist dimaprit had an excitatory action on the DN cells and this action was blocked by ranitidine. These results indicate that histamine excites cerebellar DN neurons via histamine H2 receptors. Since the DN receives hypothalamocerebellar histaminergic projections and plays a role in initiation and planning of somatic movement, the postsynaptic excitation of the DN neurons by histamine suggests the possibility that the initiation and planning of movement may be modulated by the histaminergic projections. © 2011 Elsevier Ireland Ltd. All rights reserved.
Histamine is a transmitter in the nervous system and a signaling molecule in the gut, the skin, and the immune system [7]. Histaminergic neurons in mammalian brain are located exclusively in the tuberomamillary nucleus of the posterior hypothalamus and send their axons all over the central nervous system [7]. The central histaminergic system holds a key position in the regulation of basic body functions, including the sleep-waking cycle, energy and endocrine homeostasis, synaptic plasticity and learning [6]. In addition to these classical functions, the motor modulation by the system is also paid an attention because of the existence of histaminergic projections from the hypothalamus to the structures related to motor control such as cerebellum, basal ganglia, and red nucleus [2–4,10,15,18,20]. The cerebellum, a largest subcor-
∗ Corresponding author. Tel.: +86 513 85051714; fax: +86 513 85051506. ∗∗ Corresponding author. Tel.: +86 25 83592714; fax: +86 25 83592705. E-mail addresses: [email protected] (Y.-P. Peng), [email protected] (J.-J. Wang). 1 These authors contributed equally to this study. 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.05.241
tical motor center, exports its modulatory information principally through the three deep nuclei, fastigial nucleus, interposed nucleus and dentate nucleus (DN) [8]. The hypothalamocerebellar projections also terminate in the cerebellar nuclei and they are, at least in part, histaminergic [5]. On the basis of these reported facts, we hypothesized that the hypothalamocerebellar histaminergic system affects activities of the cerebellar nuclei and subsequently modulates somatic movement by the cerebellar output. In our previous work, we have shown that the central histaminergic system modulates motor balance and coordination by activating neurons in the interposed nucleus of the cerebellum [16]. Here, we provide further evidence for the involvement of the central histaminergic system in motor control at the profile of cerebellar DN neuronal activity. Histamine acts in the brain via three receptors, H1 , H2 and H3 [11]. H1 receptors are mainly postsynaptically located and have high densities especially in the hypothalamus and other limbic regions; H2 receptors are also mainly postsynaptically located and have high densities in hippocampus, amygdala and basal ganglia; and H3 receptors are exclusively presynaptically located and have
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high densities in the basal ganglia [2]. Moreover, several studies have also documented the presence of histamine H1 and H2 receptors in the cerebellar nuclei of rat [1,9]. The previous work in vivo and in vitro from our laboratory has indicated that via H2 receptors, histamine enhances rat motor balance and coordination [16], and also excites neurons in the cerebellar interposed nucleus and fastigial nucleus [14,17]. However, in the cerebellar DN, the functional significance of the histaminergic input and its receptor pathway remain less clear. The experiments were carried out on cerebellar slices from 57 Sprague-Dawley rats (150–250 g). Under ether anesthesia, the animals were decapitated. The brain was immediately removed and placed in ice-cold oxygenated artificial cerebrospinal fluid (ACSF, composition in mM: NaCl 124, KCl 5, MgSO4 1.3, KH2 PO4 1.2, NaHCO3 26, CaCl2 2.4 and d-glucose 10). The cerebellum was cut vertically into two parts along the midline of the vermis, and one of the hemispheres was chosen to make slices. Cemented onto the stage of a vibratome (VT1000S, Leica, Germany), the cerebellar hemisphere was cut in the sagittal plane (300–400 m thick), and the slices containing the DN were transferred to a recording chamber which was continuously perfused with ACSF equilibrated with 95% O2 /5% CO2 (pH 7.4, 33 ± 0.2 ◦ C, flow rate 2–3 ml/min). All slices were incubated for about 60 min before recording. In some experiments, a 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 [14]. Spontaneous unitary activity of DN neurons in the cerebellar slices was extracellularly recorded by use of glass microelectrodes filled with 0.5 M NaAc (impedance 5–10 M). Before bath application of histaminergic compounds, the discharge frequency of the recorded neuron was observed for at least 20 min to assure stability. Solutions of the drugs were freshly prepared in the perfusing ACSF. Histamine (Sigma, 5, 15 or 45 M), selective histamine H1 receptor agonist 2-pyridylethylamine (Sigma, 100 or 300 M) or H2 receptor agonist dimaprit (Sigma, 30 or 100 M) was added to the perfusing ACSF for a test period of 1 min. If the recorded DN neuron responded to the histamine, or the histamine receptor agonists, the perfusing medium was switched from the normal ACSF to the ACSF containing histamine receptor antagonist. After the slice was equilibrated with the ACSF containing selective histamine H1 receptor antagonist triprolidine (Sigma, 1 or 10 M) or selective H2 receptor
antagonist ranitidine (Sigma, 1, 3 or 10 M) for 15 min, histamine or histamine receptor agonist was re-applied to the ACSF for 1 min. The neuronal discharges were amplified and displayed on the oscilloscope conventionally. The spikes were sent through an interface (Micro1401, CED, UK) to a laboratory microcomputer, which was used to analyze the discharge rate online by the software Spike 2 (CED, UK). Peri-stimulus time histograms (sampling interval = 1 s) of neuronal discharges were generated by the computer to assess effects of histaminergic drugs on DN neurons. Drug-induced neuronal responses were considered substance-specific provided they were reversible and reproducible. Data were expressed as mean and standard deviation of percentage of change in peak discharge rate with respect to basal firing rate. Student’s t-test or one-way analysis of variance was employed to statistically analyze the data and differences were considered significant at p < 0.05. Seventy-nine DN neurons with a tonic discharge were recorded from 53 cerebellar slices in this study. The spontaneous firing rate of the DN neurons ranged from 16 to 83 spikes/s and the mean firing rate was 50.2 ± 13.8 spikes/s. Of the 79 recorded DN neurons, 67 (84.8%) were excited by histamine and the remaining 12 (15.2%) had no response to the histamine stimulation. As illustrated in Fig. 1A, the peak discharge rate of the DN neurons after the stimulation with 5, 15 and 45 M of histamine increased by 11.1%, 19.9% and 33.2%, respectively, with a significant difference from the basal firing rate (p < 0.05 or 0.01, n = 23). Between the three concentrations of histamine, the difference in the increased firing rate was also statistically significant (p < 0.01), showing that the excitatory response to histamine was concentration-dependent. While slices were perfused with low-Ca2+ /high-Mg2+ medium (n = 8), the histamine still elicited an increase in the firing rate of the DN neurons, similar to those with the normal ACSF perfusion except the decrease in the spontaneous basal firing rate (Fig. 1B). The result indicated that the low-Ca2+ /high-Mg2+ perfusion did not block the excitatory response of DN cells to histamine, although the decreased spontaneous firing rate occurred due to the possible inhibition of neuronal functional activities by the low Ca2+ environment. Effects of antagonists for histamine H1 and H2 receptors on histamine-evoked excitation were examined. While the cerebellar slices were perfused with ACSF containing triprolidine (1 or 10 M), a selective H1 receptor antagonist, the excitatory response of the DN neurons to histamine (15 M) still occurred. There was no sig-
Fig. 1. Effect of histamine on spontaneous discharge rate of a DN neuron under the perfusion of normal ACSF (A) or low-Ca2+ /high-Mg2+ medium (B). The cerebellar slice was successively equilibrated with the normal ACSF or the low-Ca2+ /high-Mg2+ medium. Histamine was added to the perfusion media and acted on the neuron for 1 min. The short scale bars denote the period of histamine application and the long scale bar is the period of the slice exposure to the low-Ca2+ /high-Mg2+ medium.
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Fig. 2. Influences of antagonists for histamine H1 and H2 receptors on histamine-induced excitatory response of DN neurons. The cerebellar slice was successively perfused with the normal ACSF containing histamine H1 receptor antagonist triprolidine (A) or H2 receptor antagonist ranitidine (B–D) for 15 min. Histamine was added to the perfusion and acted on the neuron for 1 min. The short scale bars denote the period of histamine application and the long scale bars point out the period of the slice exposure to the antagonists of histamine H1 or H2 receptors. Statistical diagram showing that a dose-dependent relationship between histamine and H2 receptor antagonist ranitidine is exhibited in (E). Each point is the mean and standard deviation of percentage of change in the peak firing rate relative to the basal discharge rate. The data of control or various concentrations of ranitidine are from 12 neurons. *p < 0.05, **p < 0.01, compared with the basal firing rate; #p < 0.05, ##p < 0.01, compared with histamine in the normal ACSF without the antagonists.
nificant difference in the increased peak discharge rate between triprolidine plus histamine (20.5 ± 1.3% and 20.6 ± 1.2% for 1 and 10 M of triprolidine, respectively) and histamine (20.6 ± 1.3%, n = 16, Fig. 2A). However, while the slices were equilibrated with ACSF containing ranitidine (1, 3 or 10 M), a selective H2 receptor antagonist, the excitatory effect of histamine (5, 15 or 45 M) on the DN neurons was remarkably reduced (Fig. 2). This reduction in histamine effect by ranitidine was dependent on both histamine and ranitidine concentrations. With the increase in ranitidine concentrations from 1 to 10 M, the effect of reducing the
histamine-evoked excitation increased. As far as the same concentration of ranitidine was concerned, its effect of antagonizing the lower dose of histamine was larger than that against the higher dose of histamine (Fig. 2). The concentration-dependent relationship between histamine and ranitidine displayed that the dose-response curves of the DN neurons to histamine stimulation were gradually shifted to the right with the increase in ranitidine concentrations (Fig. 2E). These data demonstrated that histamine H2 receptor antagonist ranitidine blocked the excitatory response of DN neurons to histamine.
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Fig. 3. Effects of agonists for histamine H1 and H2 receptors on spontaneous firing rate of DN neurons. The histamine H1 receptor agonist 2-pyridylethylamine (A) or H2 receptor agonist dimaprit (B) was applied to the neuron for 1 min. The short scale bars represent the period of the agonist action and the long scale bar indicates the period of the antagonist exposure.
Further, effects of H1 and H2 receptor agonists on DN neurons were observed. The selective H1 receptor agonist 2pyridylethylamine (100 or 300 M) did not elicit any excitatory response in the DN neurons (n = 16, Fig. 3A). However, the selective H2 receptor agonist dimaprit (30 or 100 M) had an excitatory effect on the DN cells (Fig. 3B). The peak firing rate increased by 13.1% and 29.4% for 30 and 100 M of dimaprit, respectively, relative to the basal discharge rate (n = 16, both p < 0.05). Between the two concentrations of dimaprit, the increase in the firing rate was significantly different (p < 0.05), indicating a concentrationdependent excitation by the dimaprit. Furthermore, the perfusion with the selective H2 receptor antagonist ranitidine (0.6 M) obviously reduced the 100 M of dimaprit-induced excitatory response (n = 16, p < 0.05, Fig. 3B), showing a blockage of dimaprit effect by ranitidine. Histamine has been shown to excite neuronal activities in the two cerebellar nuclei, interposed nucleus and fastigial nucleus [14,17]. In this study, we indicated that histamine excited the cells in another cerebellar nucleus, the DN. The excitatory effect of histamine was not blocked by low-Ca2+ /high-Mg2+ perfusion, suggesting that the action of histamine on the DN neurons is postsynaptic. The result demonstrates that besides the interposed nucleus and the fastigial nucleus (outputs for the spinocerebellum), the DN, an output for the corticocerebellum, is also adjusted by histamine. Thus, the present data provide further evidence for the involvement of histamine in the modulation of cerebellar functional activities. Although the three cerebellar nuclei are all related to the motor control, they send their outputs to different parts of the brain and perform distinct functions. For example, the fastigial nucleus sends its output to medial systems (vestibular nucleus, reticular formation and motor cortex), and executes axial and proximal motor control; the interposed nucleus sends its output to lateral systems (magnocellular part of the red nucleus and distal regions of motor cortex), and executes distal motor control; and the DN projects its axons to integration areas (parvocellular part of the red nucleus and premotor cortex), and plays a role in initiation, planning and timing of movement [8]. Therefore, the current results suggest that in addition to modulation of the ongoing execution of axial, proximal and distal movement, histamine is also implicated in the control of initiation, planning and timing of movement. Bilateral microinjection of histamine in the cerebellar interposed nucleus of rats led to a prolong of the animals remaining on rota-rod and a shortening of their passing through balance beam, suggesting that histamine enhances motor balance and coordination through affecting cerebellar motor control function [16]. The study in vivo supports our hypothesis that histaminergic innervations to the cerebellum play a role in motor control. The hypothesis
is also advocated by the reports on histamine modulating neuronal activities in the other subcortical motor control structures, such as the red nucleus, a cerebellar output destination, and the globus pallidus, a substructure of the basal ganglia [3,4]. Collectively, histamine is actively involved in the motor regulation by postsynaptically exciting neurons of the subcortical motor control centers. Since the histaminergic innervations to the subcortical motor control structures originate exclusively from the hypothalamus [2,7], a center for regulating visceral activities, the excitatory response of cerebellar motor efferent neurons to histamine implies a visceral-somatic integration by the hypothalamocerebellar histaminergic projections. The DN has been reported to have histamine H1 and H2 receptors, but scarcity of histamine H3 receptors [1,12]. In the present study, the histamine-induced DN neuronal excitation was blocked by highly selective histamine H2 receptor antagonist ranitidine, but not by selective histamine H1 receptor antagonist triprolidine. Furthermore, a highly selective histamine H2 receptor agonist dimaprit mimicked the excitatory action of histamine on the DN cells and the dimaprit-induced excitation was diminished by ranitidine. In contrast, 2-pyridylethylamine, a histamine H1 receptor agonist, had no effect on the DN cells. These results revealed that the histamine-induced excitation of DN neurons was mediated by histamine H2 receptors rather than by H1 receptors. This is compatible with those data obtained from cerebellar interposed nuclear and fastigial nuclear cells [14,17], as well as from the neurons of the globus pallidus [3] and the red nucleus [4]. Although both histamine H1 and H2 receptors are mainly postsynaptically located and their mediated actions are mostly excitatory, distribution of H2 receptors in the rodent brain is widespread but more consistent than that of H1 receptors with histaminergic projections, indicating that H2 receptors mediate a larger number of postsynaptic actions of histamine [7,13,19]. Our previous and present findings that between the two types of postsynaptic histamine receptors, only histamine H2 receptors mediate the excitatory effect of histamine on neurons in these subcortical motor control structures suggest that the motor regulation by the hypothalamocerebellar histaminergic projections is implemented by the activation of histamine H2 receptors. The previous study in vivo from our laboratory supports this point. Histamine injection into the interposed nuclei increased motor performance, balance and coordination; injection of H2 receptor antagonist ranitidine in the nuclei gave rise to an effect opposite to the histamine injection; but H1 receptor antagonist triprolidine did not influence the motor performance, balance and coordination [16]. These highlight the functional importance of histamine H2 receptors in the motor control by hypothalamocerebellar histaminergic projections.
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Taken together, these results show that histamine has a postsynaptically excitatory effect on neurons of the DN, and that this effect is mediated by histamine H2 receptors. The modulation of neuronal activities of the DN, a cerebellar output nucleus related to initiation and planning of movement, by histamine implies that the central histaminergic nervous system regulates the initiation and planning of somatic movement. Acknowledgements This work was supported by grants 30870819, 30870929, 31070959 and NSFC/RGC Joint Research Scheme 30931160433 from the National Natural Science Foundation of China, BK2010278 from the Natural Science Foundation of Jiangsu Province of China, RFDP 20100091110016 from the State Educational Ministry of China, PAPD, and K2008019 from the Nantong Applied Research Program of China. References [1] J.M. Arrang, G. Drutel, M. Garbarg, M. Ruat, E. Traiffort, J.C. Schwartz, Molecular and functional diversity of histamine receptor subtypes, Ann. N. Y. Acad. Sci. 757 (1995) 314–323. [2] R.E. Brown, D.R. Stevens, H.L. Haas, The physiology of brain histamine, Prog. Neurobiol. 63 (2001) 637–672. [3] K. Chen, W.H. Yung, Y.S. Chan, B.K.-C. Chow, J.J. Wang, Excitatory effect of histamine on neuronal activity of rat globus pallidus by activation of H2 receptors in vitro, Neurosci. Res. 53 (2005) 288–297. [4] K. Chen, J.N. Zhu, H.Z. Li, J.J. Wang, Histamine elicits neuronal excitatory response of red nucleus in the rat via H2 receptors in vitro, Neurosci. Lett. 351 (2003) 25–28. [5] E. Dietrichs, D.E. Haines, G.K. Roste, L.S. Roste, Hypothalamocerebellar and cerebellohypothalamic projections – circuits for regulating nonsomatic cerebellar activity? Histol. Histopathol. 9 (1994) 603–614.
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[6] H. Haas, P. Panula, The role of histamine and the tuberomamillary nucleus in the nervous system, Nat. Rev. Neurosci. 4 (2003) 121–130. [7] H.L. Haas, O.A. Sergeeva, O. Selbach, Histamine in the nervous system, Physiol. Rev. 88 (2008) 1183–1241. [8] M. Ito, The Cerebellum and Neural Control, Raven Press, NewYork, 1984. [9] K. Karlstedt, A. Senkas, M. Ahman, P. Panula, Regional expression of the histamine H2 receptor in adult and developing rat brain, Neuroscience 102 (2001) 201–208. [10] W.C. Li, X.H. Tang, H.Z. Li, J.J. Wang, Histamine excites rat cerebellar granule cells in vitro through H1 and H2 receptors, J. Physiol. (Paris) 93 (1999) 239– 244. [11] P. Lieberman, Histamine, antihistamines, and the central nervous system, Allergy Asthma Proc. 30 (2009) 482–486. [12] H. Pollard, J. Moreau, J.M. Arrang, J.C. Schwartz, A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas, Neuroscience 52 (1993) 169–189. [13] M. Ruat, E. Traiffort, M.L. Bouthenet, J.C. Schwartz, J. Hirschfeld, A. Buschauer, W. Schunack, Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using [125I] iodinated probes, Proc. Natl. Acad. Sci. U.S.A 87 (1990) 1658–1662. [14] B. Shen, H.Z. Li, J.J. Wang, Excitatory effects of histamine on cerebellar interpositus nuclear cells of rats through H2 receptors in vitro, Brain Res. 948 (2002) 64–71. [15] N. Sittig, H. Davidowa, Histamine reduces firing and bursting of anterior and intralaminar thalamic neurons and activates striatal cells in anesthetized rats, Behav. Brain Res. 124 (2001) 137–143. [16] Y.N. Song, H.Z. Li, J.N. Zhu, C.L. Guo, J.J. Wang, Histamine improves rat rotarod and balance beam performances through H2 receptors in the cerebellar interpositus nucleus, Neuroscience 140 (2006) 33–43. [17] B. Tang, J. Zhang, H.Z. Li, J.N. Zhu, J.J. Wang, Excitatory effect of histamine on neuronal activity of rat cerebellar fastigial nucleus in vitro, Chin. Sci. Bull. 52 (2007) 497–503. [18] L. Tian, Y.Q. Wen, H.Z. Li, C.C. Zuo, J.J. Wang, Histamine excites rat cerebellar Purkinje cells via H2 receptors in vitro, Neurosci. Res. 36 (2000) 61–66. [19] M.L. Vizuete, E. Traiffort, M.L. Bouthenet, M. Ruat, E. Souil, J. Tardivel-Lacombe, J.C. Schwartz, Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain, Neuroscience 80 (1997) 321–343. [20] F.W. Zhou, J.J. Xu, Y. Zhao, M.S. LeDoux, F.M. Zhou, Opposite functions of histamine H1 and H2 receptors and H3 receptor in substantia nigra pars reticulata, J. Neurophysiol. 96 (2006) 1581–1591.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Histamine evokes excitatory response of neurons in the cerebellar dentate nucleus via H2 receptors Yong-Ting Qin a,1 , Song-Hua Ma a,1 , Qian-Xing Zhuang b , Yi-Hua Qiu a , Bing Li a , Yu-Ping Peng a,∗ , Jian-Jun Wang b,∗∗ a
Department of Physiology, School of Medicine, Nantong University, 19 Qixiu Road, Nantong 226001, China Department of Biological Science and Technology and State Key Laboratory of Pharmaceutical Biotechnology, Mailbox 426, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China b
a r t i c l e
i n f o
Article history: Received 8 February 2011 Accepted 31 May 2011 Keywords: Histamine Histamine receptors Cerebellar dentate nucleus Hypothalamocerebellar histaminergic projections
a b s t r a c t Previous studies have shown an excitatory effect of histamine on neurons in two cerebellar nuclei, the fastigial nucleus and the interposed nucleus. Here we investigated action of histamine on the dentate nucleus (DN), another nucleus of the cerebellum, and provided more evidence for motor control by histamine via the cerebellum. Spontaneous unitary discharge of neurons in the DN was extracellularly recorded by use of cerebellar slice preparations. In total 79-recorded neurons, which were from 53 cerebellar slices, 67 neurons (84.8%) had an excitatory response to histamine stimulation, and the rest (15.2%) were not reactive. The histamine-induced excitation of the DN neurons was not blocked by low-Ca2+ /highMg2+ medium, demonstrating that this effect of histamine was postsynaptic. Triprolidine, an antagonist of histamine H1 receptors, did not block the excitatory effect of histamine, but ranitidine, an antagonist for H2 receptors, blocked the excitatory response to histamine in a concentration-dependent manner. Further, histamine H1 receptor agonist 2-pyridylethylamine did not elicit any response of DN neurons, but H2 receptor agonist dimaprit had an excitatory action on the DN cells and this action was blocked by ranitidine. These results indicate that histamine excites cerebellar DN neurons via histamine H2 receptors. Since the DN receives hypothalamocerebellar histaminergic projections and plays a role in initiation and planning of somatic movement, the postsynaptic excitation of the DN neurons by histamine suggests the possibility that the initiation and planning of movement may be modulated by the histaminergic projections. © 2011 Elsevier Ireland Ltd. All rights reserved.
Histamine is a transmitter in the nervous system and a signaling molecule in the gut, the skin, and the immune system [7]. Histaminergic neurons in mammalian brain are located exclusively in the tuberomamillary nucleus of the posterior hypothalamus and send their axons all over the central nervous system [7]. The central histaminergic system holds a key position in the regulation of basic body functions, including the sleep-waking cycle, energy and endocrine homeostasis, synaptic plasticity and learning [6]. In addition to these classical functions, the motor modulation by the system is also paid an attention because of the existence of histaminergic projections from the hypothalamus to the structures related to motor control such as cerebellum, basal ganglia, and red nucleus [2–4,10,15,18,20]. The cerebellum, a largest subcor-
∗ Corresponding author. Tel.: +86 513 85051714; fax: +86 513 85051506. ∗∗ Corresponding author. Tel.: +86 25 83592714; fax: +86 25 83592705. E-mail addresses: [email protected] (Y.-P. Peng), [email protected] (J.-J. Wang). 1 These authors contributed equally to this study. 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.05.241
tical motor center, exports its modulatory information principally through the three deep nuclei, fastigial nucleus, interposed nucleus and dentate nucleus (DN) [8]. The hypothalamocerebellar projections also terminate in the cerebellar nuclei and they are, at least in part, histaminergic [5]. On the basis of these reported facts, we hypothesized that the hypothalamocerebellar histaminergic system affects activities of the cerebellar nuclei and subsequently modulates somatic movement by the cerebellar output. In our previous work, we have shown that the central histaminergic system modulates motor balance and coordination by activating neurons in the interposed nucleus of the cerebellum [16]. Here, we provide further evidence for the involvement of the central histaminergic system in motor control at the profile of cerebellar DN neuronal activity. Histamine acts in the brain via three receptors, H1 , H2 and H3 [11]. H1 receptors are mainly postsynaptically located and have high densities especially in the hypothalamus and other limbic regions; H2 receptors are also mainly postsynaptically located and have high densities in hippocampus, amygdala and basal ganglia; and H3 receptors are exclusively presynaptically located and have
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high densities in the basal ganglia [2]. Moreover, several studies have also documented the presence of histamine H1 and H2 receptors in the cerebellar nuclei of rat [1,9]. The previous work in vivo and in vitro from our laboratory has indicated that via H2 receptors, histamine enhances rat motor balance and coordination [16], and also excites neurons in the cerebellar interposed nucleus and fastigial nucleus [14,17]. However, in the cerebellar DN, the functional significance of the histaminergic input and its receptor pathway remain less clear. The experiments were carried out on cerebellar slices from 57 Sprague-Dawley rats (150–250 g). Under ether anesthesia, the animals were decapitated. The brain was immediately removed and placed in ice-cold oxygenated artificial cerebrospinal fluid (ACSF, composition in mM: NaCl 124, KCl 5, MgSO4 1.3, KH2 PO4 1.2, NaHCO3 26, CaCl2 2.4 and d-glucose 10). The cerebellum was cut vertically into two parts along the midline of the vermis, and one of the hemispheres was chosen to make slices. Cemented onto the stage of a vibratome (VT1000S, Leica, Germany), the cerebellar hemisphere was cut in the sagittal plane (300–400 m thick), and the slices containing the DN were transferred to a recording chamber which was continuously perfused with ACSF equilibrated with 95% O2 /5% CO2 (pH 7.4, 33 ± 0.2 ◦ C, flow rate 2–3 ml/min). All slices were incubated for about 60 min before recording. In some experiments, a 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 [14]. Spontaneous unitary activity of DN neurons in the cerebellar slices was extracellularly recorded by use of glass microelectrodes filled with 0.5 M NaAc (impedance 5–10 M). Before bath application of histaminergic compounds, the discharge frequency of the recorded neuron was observed for at least 20 min to assure stability. Solutions of the drugs were freshly prepared in the perfusing ACSF. Histamine (Sigma, 5, 15 or 45 M), selective histamine H1 receptor agonist 2-pyridylethylamine (Sigma, 100 or 300 M) or H2 receptor agonist dimaprit (Sigma, 30 or 100 M) was added to the perfusing ACSF for a test period of 1 min. If the recorded DN neuron responded to the histamine, or the histamine receptor agonists, the perfusing medium was switched from the normal ACSF to the ACSF containing histamine receptor antagonist. After the slice was equilibrated with the ACSF containing selective histamine H1 receptor antagonist triprolidine (Sigma, 1 or 10 M) or selective H2 receptor
antagonist ranitidine (Sigma, 1, 3 or 10 M) for 15 min, histamine or histamine receptor agonist was re-applied to the ACSF for 1 min. The neuronal discharges were amplified and displayed on the oscilloscope conventionally. The spikes were sent through an interface (Micro1401, CED, UK) to a laboratory microcomputer, which was used to analyze the discharge rate online by the software Spike 2 (CED, UK). Peri-stimulus time histograms (sampling interval = 1 s) of neuronal discharges were generated by the computer to assess effects of histaminergic drugs on DN neurons. Drug-induced neuronal responses were considered substance-specific provided they were reversible and reproducible. Data were expressed as mean and standard deviation of percentage of change in peak discharge rate with respect to basal firing rate. Student’s t-test or one-way analysis of variance was employed to statistically analyze the data and differences were considered significant at p < 0.05. Seventy-nine DN neurons with a tonic discharge were recorded from 53 cerebellar slices in this study. The spontaneous firing rate of the DN neurons ranged from 16 to 83 spikes/s and the mean firing rate was 50.2 ± 13.8 spikes/s. Of the 79 recorded DN neurons, 67 (84.8%) were excited by histamine and the remaining 12 (15.2%) had no response to the histamine stimulation. As illustrated in Fig. 1A, the peak discharge rate of the DN neurons after the stimulation with 5, 15 and 45 M of histamine increased by 11.1%, 19.9% and 33.2%, respectively, with a significant difference from the basal firing rate (p < 0.05 or 0.01, n = 23). Between the three concentrations of histamine, the difference in the increased firing rate was also statistically significant (p < 0.01), showing that the excitatory response to histamine was concentration-dependent. While slices were perfused with low-Ca2+ /high-Mg2+ medium (n = 8), the histamine still elicited an increase in the firing rate of the DN neurons, similar to those with the normal ACSF perfusion except the decrease in the spontaneous basal firing rate (Fig. 1B). The result indicated that the low-Ca2+ /high-Mg2+ perfusion did not block the excitatory response of DN cells to histamine, although the decreased spontaneous firing rate occurred due to the possible inhibition of neuronal functional activities by the low Ca2+ environment. Effects of antagonists for histamine H1 and H2 receptors on histamine-evoked excitation were examined. While the cerebellar slices were perfused with ACSF containing triprolidine (1 or 10 M), a selective H1 receptor antagonist, the excitatory response of the DN neurons to histamine (15 M) still occurred. There was no sig-
Fig. 1. Effect of histamine on spontaneous discharge rate of a DN neuron under the perfusion of normal ACSF (A) or low-Ca2+ /high-Mg2+ medium (B). The cerebellar slice was successively equilibrated with the normal ACSF or the low-Ca2+ /high-Mg2+ medium. Histamine was added to the perfusion media and acted on the neuron for 1 min. The short scale bars denote the period of histamine application and the long scale bar is the period of the slice exposure to the low-Ca2+ /high-Mg2+ medium.
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Fig. 2. Influences of antagonists for histamine H1 and H2 receptors on histamine-induced excitatory response of DN neurons. The cerebellar slice was successively perfused with the normal ACSF containing histamine H1 receptor antagonist triprolidine (A) or H2 receptor antagonist ranitidine (B–D) for 15 min. Histamine was added to the perfusion and acted on the neuron for 1 min. The short scale bars denote the period of histamine application and the long scale bars point out the period of the slice exposure to the antagonists of histamine H1 or H2 receptors. Statistical diagram showing that a dose-dependent relationship between histamine and H2 receptor antagonist ranitidine is exhibited in (E). Each point is the mean and standard deviation of percentage of change in the peak firing rate relative to the basal discharge rate. The data of control or various concentrations of ranitidine are from 12 neurons. *p < 0.05, **p < 0.01, compared with the basal firing rate; #p < 0.05, ##p < 0.01, compared with histamine in the normal ACSF without the antagonists.
nificant difference in the increased peak discharge rate between triprolidine plus histamine (20.5 ± 1.3% and 20.6 ± 1.2% for 1 and 10 M of triprolidine, respectively) and histamine (20.6 ± 1.3%, n = 16, Fig. 2A). However, while the slices were equilibrated with ACSF containing ranitidine (1, 3 or 10 M), a selective H2 receptor antagonist, the excitatory effect of histamine (5, 15 or 45 M) on the DN neurons was remarkably reduced (Fig. 2). This reduction in histamine effect by ranitidine was dependent on both histamine and ranitidine concentrations. With the increase in ranitidine concentrations from 1 to 10 M, the effect of reducing the
histamine-evoked excitation increased. As far as the same concentration of ranitidine was concerned, its effect of antagonizing the lower dose of histamine was larger than that against the higher dose of histamine (Fig. 2). The concentration-dependent relationship between histamine and ranitidine displayed that the dose-response curves of the DN neurons to histamine stimulation were gradually shifted to the right with the increase in ranitidine concentrations (Fig. 2E). These data demonstrated that histamine H2 receptor antagonist ranitidine blocked the excitatory response of DN neurons to histamine.
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Fig. 3. Effects of agonists for histamine H1 and H2 receptors on spontaneous firing rate of DN neurons. The histamine H1 receptor agonist 2-pyridylethylamine (A) or H2 receptor agonist dimaprit (B) was applied to the neuron for 1 min. The short scale bars represent the period of the agonist action and the long scale bar indicates the period of the antagonist exposure.
Further, effects of H1 and H2 receptor agonists on DN neurons were observed. The selective H1 receptor agonist 2pyridylethylamine (100 or 300 M) did not elicit any excitatory response in the DN neurons (n = 16, Fig. 3A). However, the selective H2 receptor agonist dimaprit (30 or 100 M) had an excitatory effect on the DN cells (Fig. 3B). The peak firing rate increased by 13.1% and 29.4% for 30 and 100 M of dimaprit, respectively, relative to the basal discharge rate (n = 16, both p < 0.05). Between the two concentrations of dimaprit, the increase in the firing rate was significantly different (p < 0.05), indicating a concentrationdependent excitation by the dimaprit. Furthermore, the perfusion with the selective H2 receptor antagonist ranitidine (0.6 M) obviously reduced the 100 M of dimaprit-induced excitatory response (n = 16, p < 0.05, Fig. 3B), showing a blockage of dimaprit effect by ranitidine. Histamine has been shown to excite neuronal activities in the two cerebellar nuclei, interposed nucleus and fastigial nucleus [14,17]. In this study, we indicated that histamine excited the cells in another cerebellar nucleus, the DN. The excitatory effect of histamine was not blocked by low-Ca2+ /high-Mg2+ perfusion, suggesting that the action of histamine on the DN neurons is postsynaptic. The result demonstrates that besides the interposed nucleus and the fastigial nucleus (outputs for the spinocerebellum), the DN, an output for the corticocerebellum, is also adjusted by histamine. Thus, the present data provide further evidence for the involvement of histamine in the modulation of cerebellar functional activities. Although the three cerebellar nuclei are all related to the motor control, they send their outputs to different parts of the brain and perform distinct functions. For example, the fastigial nucleus sends its output to medial systems (vestibular nucleus, reticular formation and motor cortex), and executes axial and proximal motor control; the interposed nucleus sends its output to lateral systems (magnocellular part of the red nucleus and distal regions of motor cortex), and executes distal motor control; and the DN projects its axons to integration areas (parvocellular part of the red nucleus and premotor cortex), and plays a role in initiation, planning and timing of movement [8]. Therefore, the current results suggest that in addition to modulation of the ongoing execution of axial, proximal and distal movement, histamine is also implicated in the control of initiation, planning and timing of movement. Bilateral microinjection of histamine in the cerebellar interposed nucleus of rats led to a prolong of the animals remaining on rota-rod and a shortening of their passing through balance beam, suggesting that histamine enhances motor balance and coordination through affecting cerebellar motor control function [16]. The study in vivo supports our hypothesis that histaminergic innervations to the cerebellum play a role in motor control. The hypothesis
is also advocated by the reports on histamine modulating neuronal activities in the other subcortical motor control structures, such as the red nucleus, a cerebellar output destination, and the globus pallidus, a substructure of the basal ganglia [3,4]. Collectively, histamine is actively involved in the motor regulation by postsynaptically exciting neurons of the subcortical motor control centers. Since the histaminergic innervations to the subcortical motor control structures originate exclusively from the hypothalamus [2,7], a center for regulating visceral activities, the excitatory response of cerebellar motor efferent neurons to histamine implies a visceral-somatic integration by the hypothalamocerebellar histaminergic projections. The DN has been reported to have histamine H1 and H2 receptors, but scarcity of histamine H3 receptors [1,12]. In the present study, the histamine-induced DN neuronal excitation was blocked by highly selective histamine H2 receptor antagonist ranitidine, but not by selective histamine H1 receptor antagonist triprolidine. Furthermore, a highly selective histamine H2 receptor agonist dimaprit mimicked the excitatory action of histamine on the DN cells and the dimaprit-induced excitation was diminished by ranitidine. In contrast, 2-pyridylethylamine, a histamine H1 receptor agonist, had no effect on the DN cells. These results revealed that the histamine-induced excitation of DN neurons was mediated by histamine H2 receptors rather than by H1 receptors. This is compatible with those data obtained from cerebellar interposed nuclear and fastigial nuclear cells [14,17], as well as from the neurons of the globus pallidus [3] and the red nucleus [4]. Although both histamine H1 and H2 receptors are mainly postsynaptically located and their mediated actions are mostly excitatory, distribution of H2 receptors in the rodent brain is widespread but more consistent than that of H1 receptors with histaminergic projections, indicating that H2 receptors mediate a larger number of postsynaptic actions of histamine [7,13,19]. Our previous and present findings that between the two types of postsynaptic histamine receptors, only histamine H2 receptors mediate the excitatory effect of histamine on neurons in these subcortical motor control structures suggest that the motor regulation by the hypothalamocerebellar histaminergic projections is implemented by the activation of histamine H2 receptors. The previous study in vivo from our laboratory supports this point. Histamine injection into the interposed nuclei increased motor performance, balance and coordination; injection of H2 receptor antagonist ranitidine in the nuclei gave rise to an effect opposite to the histamine injection; but H1 receptor antagonist triprolidine did not influence the motor performance, balance and coordination [16]. These highlight the functional importance of histamine H2 receptors in the motor control by hypothalamocerebellar histaminergic projections.
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Taken together, these results show that histamine has a postsynaptically excitatory effect on neurons of the DN, and that this effect is mediated by histamine H2 receptors. The modulation of neuronal activities of the DN, a cerebellar output nucleus related to initiation and planning of movement, by histamine implies that the central histaminergic nervous system regulates the initiation and planning of somatic movement. Acknowledgements This work was supported by grants 30870819, 30870929, 31070959 and NSFC/RGC Joint Research Scheme 30931160433 from the National Natural Science Foundation of China, BK2010278 from the Natural Science Foundation of Jiangsu Province of China, RFDP 20100091110016 from the State Educational Ministry of China, PAPD, and K2008019 from the Nantong Applied Research Program of China. References [1] J.M. Arrang, G. Drutel, M. Garbarg, M. Ruat, E. Traiffort, J.C. Schwartz, Molecular and functional diversity of histamine receptor subtypes, Ann. N. Y. Acad. Sci. 757 (1995) 314–323. [2] R.E. Brown, D.R. Stevens, H.L. Haas, The physiology of brain histamine, Prog. Neurobiol. 63 (2001) 637–672. [3] K. Chen, W.H. Yung, Y.S. Chan, B.K.-C. Chow, J.J. Wang, Excitatory effect of histamine on neuronal activity of rat globus pallidus by activation of H2 receptors in vitro, Neurosci. Res. 53 (2005) 288–297. [4] K. Chen, J.N. Zhu, H.Z. Li, J.J. Wang, Histamine elicits neuronal excitatory response of red nucleus in the rat via H2 receptors in vitro, Neurosci. Lett. 351 (2003) 25–28. [5] E. Dietrichs, D.E. Haines, G.K. Roste, L.S. Roste, Hypothalamocerebellar and cerebellohypothalamic projections – circuits for regulating nonsomatic cerebellar activity? Histol. Histopathol. 9 (1994) 603–614.
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