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Chewing rescues stress-suppressed hippocampal long-term potentiation via activation of histamine H1 receptor
Neuroscience Research 64 (2009) 385–390
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Chewing rescues stress-suppressed hippocampal long-term potentiation via activation of histamine H1 receptor Yumie Ono a,1,*, Tsuyoshi Kataoka b,1, Shinjiro Miyake b, Kenichi Sasaguri b, Sadao Sato b, Minoru Onozuka a a b
Department of Physiology and Neuroscience, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka Kanagawa, 238-8580, Japan Department of Craniofacial Growth and Developmental Dentistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka Kanagawa, 238-8580, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 January 2009 Received in revised form 19 March 2009 Accepted 13 April 2009 Available online 22 April 2009
We have previously found in rats that chewing, an active behavioral strategy to cope with a stressful situation, rescues long-term potentiation (LTP) in the hippocampus through activating stresssuppressed N-methyl-D-aspartate (NMDA) receptor function. To further examine the mechanisms underlying this ameliorative effect of chewing, we studied the involvement of the histaminergic system, which has been shown to be activated by mastication, in the LTP of hippocampal slices of rats that were allowed to chew a wooden stick during exposure to immobilization stress. Chewing failed to rescue stress-suppressed LTP in the rats treated with histamine H1 receptor (H1R) antagonist pyrilamine (5 mg/ kg, i.p.) before exposure to stress, although administration of pyrilamine did not affect LTP in naive rats and in stressed rats that did not chew. However, when pyrilamine was administrated immediately after exposure to stress, chewing rescued LTP whose magnitude was statistically comparable to that in the rats that chewed without drug treatment. These results suggest that chewing-induced histamine release in the hippocampus and the subsequent H1 receptor activation may be essential to rescue stresssuppressed synaptic plasticity. ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Chewing Coping Histamine H1 receptor Hippocampus Long-term potentiation Pyrilamine Stress Tuberomammillary nucleus
1. Introduction In general, exposure to severe stress causes prolonged activation of the hypothalamic–pituitary–adrenal (HPA) axis, which in turn causes the adrenal cortex to secrete corticosterone, which finally disrupts fundamental cellular processes in the central nervous system. Especially in the hippocampus, an excessive corticosterone level causes a Ca2+ influx into neurons via activation of glucocorticoid receptors (GR) (Elliott and Sapolsky, 1993) and attenuates cellular excitability (Joe¨ls and de Kloet, 1991), which selectively weakens the long-term potentiation (LTP) of hippocampal neurons that depends on the N-methylD-aspartate receptor (NMDAR) (Foy et al., 1987; Wiegert et al., 2005). We have recently found that chewing during a stressful event ameliorates the stress-induced impairment of LTP in hippocampal CA1 neurons by rescuing NMDAR function (Ono et al., 2008). However, the rescue mechanism remains to be elucidated.
* Corresponding author at: Department of Physiology and Neuroscience, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka Kanagawa, 238-8580, Japan. Tel.: +81 46 822 8869; fax: +81 46 822 9522. E-mail address: [email protected] (Y. Ono). 1 Equally contributed.
The mesencephalic trigeminal nucleus (Me5) receives chewingrelated proprioceptive sensory afferents of the trigeminal nerve from the jaw-closing muscle spindle and the periodontal ligaments (Luschei, 1987). A subpopulation of the Me5 neurons projects its fibers into the tuberomammillary nuclei (TMN) of the posterior hypothalamus where cell bodies of histaminergic neurons are localized (Ericson et al., 1989, 1991). Indeed, activation of the Me5 by mastication increases extracellular concentration of histamine (HA) in the hypothalamus to control satiety (Fujise et al., 1998; Sakata et al., 2003), suggesting the facilitative effect of chewing on histaminergic neurons in the TMN. The axons of histaminergic neurons in the TMN innervate practically the entire brain, including the hippocampus (Ko¨hler et al., 1985; Inagaki et al., 1988; Panula et al., 1989; Brown et al., 2001) and the electrical stimulation of the TMN facilitates extracellular concentration of HA in the hippocampus (Mochizuki et al., 1994). Thus, a chewinginduced increase of the HA level in the hippocampus probably restores the stress-attenuated NMDAR to normal function and thereby rescues LTP. At the present time, there are three known mechanisms by which HA facilitates NMDAR function in vitro. First, HA can stimulate the activity of phospholipase C through histamine H1 receptor (H1R) which is associated with the Gq/11 protein, followed by intracellular production of two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3)
0168-0102/$ – see front matter ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2009.04.011
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(Leurs et al., 1994). DAG and IP3 further potentiate the activity of protein kinase C and increase intracellular Ca2+, respectively, both of which increase the excitability of NMDAR (MacDonald et al., 2001; Markram and Segal, 1992). Payne and Neuman (1997) have shown that applying HA increases NMDA current in the hippocampal CA1 neurons, an effect that is reduced in a concentration-dependent manner by H1R antagonists but neither by histamine H2 receptor (H2R) nor by histamine H3 receptor (H3R) antagonist. Second, activation of H2R, which is associated with the Gs protein, stimulates adenylate cyclase to enhance the production of second messenger cyclic adenosine 3’,5’-monophosphate (cAMP) (Selbach et al., 1997). An elevation in intracellular cAMP concentrations further activates protein kinase A, increasing charge transfer and Ca2+ influx through NMDARs (Raman et al., 1996). Third, HA acts directly on the polyamine binding site of NMDAR to enhance its function (Bekkers, 1993; Vorobjev et al., 1993). Taken together, these reports further strengthen the hypothesis that HA participates in the rescue of stress-attenuated NMDAR function. In behaving animals, NMDAR-dependent LTP in the neurons of the CA1 area in the hippocampus plays an essential role in learning and memory (Whitlock et al., 2006). HA is also one of the possible facilitators of the hippocampal memory function. Interestingly, in vivo, H1R-dependent enhancement of NMDAR is more likely to facilitate the hippocampal-dependent learning and memory process, rather than the H2R-dependent mechanism or the direct action on NMDAR that are also well confirmed in vitro. Kamei and Tasaka (1993) have reported that intracerebroventricular administration of HA or intraperitoneal administration of HA precursor histidine improved the response latency in an active avoidance task via H1R activation in old rats. Moreover, antagonism of H1R causes memory deficit in a radial maze task, which can be restored by facilitating the NMDAR function by activating either the glycine binding site or the polyamine binding site of NMDAR (Masuoka et al., 2008) and vice versa (Huang et al., 2003). These findings show that there is H1R-mediated facilitation of NMDAR function in the hippocampus, suggesting a dominant role of H1R in modulating synaptic plasticity in the hippocampus of behaving animals. Therefore, to elucidate the involvement of H1R in the chewinginduced amelioration of hippocampal synaptic plasticity, we have examined the effect of H1R antagonism on LTP of the rats that underwent immobilization stress with chewing. We also tested H1R antagonism with two different time points of pre- and poststress to determine how activation of the histaminergic system during chewing contributes to ameliorating the stress-suppressed LTP.
2.1.2. Drug administration and stress protocol We dissolved pyrilamine (5 mg/kg; Sigma–Aldrich Japan, Tokyo, Japan) in saline and intraperitoneally injected it into all of the 18 rats tested. The dose of pyrilamine was set to the amount that sufficiently inhibits H1Rs in the hippocampus (Kamei and Tasaka, 1991) but that does not affect the arousal level (Saitou et al., 1999). Fifteen minutes after the injection, we produced immobilization stress in 12 of the rats by fixing their limbs to a wooden board for 30 min in a spread-eagle, supine position. We left half of them alone for the entire immobilization period (pyrilamine administration followed by stress; STp). For the other half, we placed a wooden stick (diameter, 5 mm) near the immobilized animal’s mouth (pyrilamine administration followed by stress and chewing; SCp). All the rats given a wooden stick except one responded to it by chewing on it with a rapid and repetitive sequence of jaw opening and closing movements for at least two-thirds of the total restraint period. The one rat that did not touch the offered stick was included in group STp. We returned the remaining six rats to their home cage without subjecting them to immobilization stress (control of pyrilamine administration only; CTp). We performed all drug administration and stress inducement between 10:00 a.m. and 11:30 a.m. 2.1.3. Electrophysiology We anesthetized rats with 2-bromo-2-chloro-1,1,1-trifluoroethane (2 ml/kg; Takeda Chemical Industries, Osaka, Japan) at 24 h after the immobilization stress, the time at which the ameliorative effect of chewing on hippocampal LTP was the most prominent in our previous study (Ono et al., 2008). We decapitated and removed their brains to make transverse hippocampal slices (450 mm thick). As to the rats in group CTp that did not experience stress, we set post-injection survival time to be the same as that in the other groups (Fig. 1). We maintained the slices in a holding chamber filled with artificial cerebrospinal fluid (ACSF) containing 119 NaCl, 2.5 KCl, 26.2 NaHCO3, 1 NaH2PO4, 1.3 MgSO4, 2.5 CaCl2, and 11 glucose (in mM, bubbled with 95% O2–5% CO2) until use. After at least 1 h of recovery time, we transferred one of the slices to an immersion-type recording chamber perfused at 1 ml/min
2. Materials and methods 2.1. Experiment 1: administration of pyrilamine and chewing effects on stress-suppressed hippocampal LTP The goal of experiment 1 was to determine whether activation of H1R is crucial for ameliorative effect of chewing on hippocampal LTP in the stressed rats. To test this, we used H1R antagonist pyrilamine to block histaminergic signal transduction through H1R in the hippocampal neurons before stress. 2.1.1. Animals We used 10-week-old male Sprague-Dawley rats in all the experiments reported here. The rats took water and food freely in a temperature-controlled room (22 3 8C) with a 12 h light/dark cycle (lights on at 7:00 a.m.). All our experiments accorded with the guidelines for Animal Experimentation of Kanagawa Dental College. All efforts were made to minimize the number of animals used and their suffering.
Fig. 1. Time intervals from intraperitoneal pyrilamine administration to stress exposure and from stress exposure to hippocampal slice preparation in experiments 1 and 2.
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with ACSF containing 0.1 mM picrotoxin (Sigma–Aldrich, Tokyo, Japan). A glass pipette filled with 3 M NaCl and positioned in the stratum radiatum of the CA1 area recorded the field excitatory postsynaptic potential (fEPSP). Bipolar stainless-steel electrodes (Unique Medical, Tokyo, Japan) placed in the stratum radiatum on opposite sides of the recording pipette stimulated the Schaffer collateral branches. We adjusted the stimulus intensity to around 30% of the maximal response and then recorded stable baseline fEPSP activity at the determined intensity every 30 s for at least 10 min. High frequency stimulation (HFS; 1 s, 100 Hz), applied nine times at intervals of 90 s, induced LTP. Since the general LTP protocol of applying single HFS was not sufficient to produce LTP in some stressed rats in our preliminary experiment, repetitive HFS was adopted to obtain large and stable LTP. All signals were filtered at 2 kHz using a low-pass Bessel filter and digitized at 5 kHz using a MultiClamp 700A interface running pCLAMP software (Axon Instruments, Union City, CA, USA). We measured the initial slopes of the fEPSP and normalized them to the average of the values measured over the baseline period. We used a single slice from a single animal for subsequent analysis.
signals was integrated with every 100 ms of time window, and the time windows whose integrated value was larger than 5% of the maximum value through the measurement were determined to be the active state of the masseter muscle. Consecutive time windows of active states were regarded as a single event of the masseter muscle activation. The total number of masseter muscle activations during 30 min of immobilization was defined to be the frequency of chewing.
2.2. Experiment 2: pre-stress and post-stress administration of pyrilamine and chewing effects on stress-suppressed hippocampal LTP
3. Results
Experiment 1 showed that administering pyrilamine before stress exposure effectively prevents an ameliorative effect of chewing on stress-suppressed hippocampal LTP. However it was still unclear what time period from stress exposure until the end of the 24 h of post-stress survival is critical for pyrilamine to counteract the effect of chewing. Therefore, we next tested whether administering pyrilamine after stress can likewise prevent chewing effects on the LTP. We subjected six rats to the same immobilization stress environment that the rats in group SCp in experiment 1 had been subjected to, but we administered pyrilamine (same dosage as in the earlier experiment) immediately after exposure to stress (pyrilamine administration after stress and chewing; SCpa). After immobilization stress, we returned the rats to their home cages, prepared hippocampal slices 24 h later, and measured LTP as in experiment 1 (Fig. 1).
2.4. Statistical analysis All values shown are mean S.E.M. The magnitude of LTP, the average value of the slope of the fEPSP recorded between 30 and 40 min after the end of HFS, provided the basis for our statistical comparisons. We compared the magnitudes of LTP among groups using the analysis of variance (ANOVA) test and the post hoc Tukey’s multiple comparison. We used Student’s t-test to compare the magnitudes of LTP in group SCpa with that in group SCp in experiment 2 and the number of chewing events in group SCp with that in group SC in the EMG experiment.
3.1. Chewing behavior is not affected by administering 5 mg/kg of pyrilamine Every rat in the chewing group showed robust chewing behavior that continued during most of the period of immobilization (Fig. 2A).
2.3. Effect of pyrilamine on chewing behavior Even though the dose of pyrilamine administered in this study was less than that required to produce a sedative effect (Saitou et al., 1999), its involvement in chewing behavior has not been reported so far. Therefore we recorded an electromyogram (EMG) of the activity of the masseter muscle during the period of immobilization and chewing to study the effect of pyrilamine on the chewing behavior. Six other rats were subjected to immobilization stress with chewing. We administered pyrilamine to half of them before immobilization as described in experiment 1 (group SCp), while we simply handled the other half of the rats without administering anything (manipulation same as in our previous study without any drug application; labeled group SC in Ono et al., 2008). Immediately after fixing a rat onto a board, a bipolar needle electrode (UI2-513, Unique Medical, Tokyo, Japan) was inserted into the left masseter muscle. After the electrode was inserted, we offered a wooden stick to the rat for chewing. We also measured EMG of two other rats, treated the same as in group STp and stressed only without pyrilamine administration (manipulation same as group ST in Ono et al., 2008), as a control for group SCp and group SC, respectively (n = 1 each). All signals were recorded using Bagnoli-2 EMG system (Delsys, MA, USA) and frequency of chewing behavior was analyzed using Masseter Muscle Analysis Software (G1 Systems, Aichi, Japan). In brief, the power of EMG
Fig. 2. Effects of pyrilamine on chewing behavior during immobilization stress. (A) Representative EMG waveforms from the masseter muscle of rats that were subjected to stress (ST), pyrilamine injection followed by stress (STp), stress with chewing a wooden stick (SC), and pyrilamine injection followed by stress with chewing a wooden stick (SCp); (B) comparison of mean values of frequency of chewing between group SC and group SCp (n = 3 each).
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The frequency of chewing was not affected by pyrilamine administration (1060 439 times and 1232 377 times in group SC and group SCp, respectively; n = 3, p = 0.634; Fig. 2B). 3.2. Experiment 1: H1R blocker prevents ameliorative effect of chewing on hippocampal LTP We have previously found that chewing counteracts stresssuppressed LTP of hippocampal CA1 neurons (Ono et al., 2008). The present result in experiment 1 further showed that the ameliorative effect of chewing is completely abolished when rats are treated with pyrilamine before exposure to stress (Fig. 3). A oneway ANOVA showed a significant group difference in the magnitude of LTP (F(2,15) = 6.18; p < 0.05) (Fig. 3D). Post-hoc tests showed statistically significant impairment of synaptic plasticity in both group STp (154.0 11% of baseline, n = 7, p < 0.01) and group SCp (149.7 2.4% of baseline, n = 5, p < 0.01) compared with group CTp (203.1 16% of baseline, n = 6). There was no significant difference between the values of group STp and group SCp (p = 0.965). When we compare these values with those obtained without pyrilamine administration in our previous study (control without pyrilamine administration (group CT) = 206.6 11%, stressed without pyrilamine administration (group ST 24 h) = 141.0 11%, stressed and chewing without pyrilamine administration (group SC
Fig. 3. Effects of pyrilamine and stress on hippocampal CA1 LTP. (A-C) Time-course of mean LTP magnitudes for the control (CTp; A) group and for the stressed (STp; B) and chewing (SCp; C) groups. CT, ST, and SC refer to rats in the control, stressed, and stressed-with-chewing groups, respectively, without pyrilamine administration: These values were taken from our previous study (CT, ST 24 h, and SC 24 h; Ono et al., 2008, with permission) and superimposed to the respective figures for comparison. Insets show representative fEPSP waveforms before (gray) and after (black) HFS; (D) comparison of mean values of fEPSP slope. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01).
Fig. 4. Effects of pre-stress and immediate post-stress administration of pyrilamine on hippocampal CA1 LTP in rats that chewed during stress. (A) Time-course of mean LTP magnitude for rats given pyrilamine before (SCp) and immediately after (SCpa) stress exposure with chewing. Insets show representative fEPSP waveforms before (gray) and after (black) HFS. (B) comparison of mean values of fEPSP slope. Asterisk indicates a statistically significant difference (p < 0.05). Horizontal dotted lines in A and B indicate the mean value of LTP magnitude obtained from rats that were treated in the same way but without pyrilamine administration (189.6%, group SC 24 h; Ono et al., 2008).
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24 h) = 189.6 7.8% of baseline; taken from Fig. 1B in Ono et al., 2008, with permission), there was a significant main effect of task (two-way ANOVA, F(2,30) = 14.3; p < 0.001). Post hoc tests indicated that the magnitude of LTP in chewing rats with pyrilamine administration (group SCp) was significantly lower than that without pyrilamine administration (group SC 24 h; Fig. 3C and D, p < 0.05), although for the pair of groups CTp and CT (p = 0.822) and for the pair of groups STp and ST 24 h (p = 0.400), the administration of pyrilamine caused no statistically significant change in the magnitude of LTP (Fig. 3A, B, and D). 3.3. Experiment 2: activation of H1R associated with chewing is essential to rescue stress-suppressed hippocampal LTP Administering pyrilamine immediately after stress had less effect on preventing chewing-induced recovery of LTP (Fig. 4). The mean magnitude of LTP for group SCpa was significantly larger (170.3 7.5% of baseline, n = 6, p < 0.05) than that for group SCp. There was no statistical difference between the magnitude of LTP for groups SC 24 h and SCpa (p = 0.103), but there was a statistically significant difference for groups SC 24 h and SCp (p < 0.05; in post hoc comparison after one-way ANOVA, with significant group difference F(2,14) = 6.11; p < 0.05). 4. Discussion We found that inhibiting histamine H1R during chewing prevents the ameliorative effect of chewing on the stresssuppressed LTP in the hippocampal CA1 neurons. Blocking H1R before the chewing and stress session prevents chewing from rescuing LTP, although blocking H1R immediately after the session has no effect on chewing-related rescue of LTP. These results suggest that chewing activates the histaminergic nervous system to facilitate the extracellular concentration of HA in the hippocampus, restoring stress-attenuated NMDAR function of CA1 neurons via H1R activation. The effect of pyrilamine in modulating LTP was specific in the rats that were administrated pyrilamine in advance and that chewed during stress (group SCp; Fig. 3C). This suppression of LTP in group SCp was not caused by the insufficiency of chewing behavior, since the sedative effect of pyrilamine in chewing behavior was negligible with the dose we used (Fig. 2B). Moreover, pyrilamine affected the magnitude of LTP neither in rats that did not chew (group CTp and group STp; Fig. 3A and B) nor in rats that chewed but whose H1Rs were blocked after the stress exposure (group SCpa; Fig. 4). We, thus, suggest that chewing raises the HA level in the hippocampus, possibly through activating histaminergic neurons in the TMN in the same way that mastication during food intake increases the extracellular concentration of HA in the ventromedial hypothalamus and in the Me5 (Fujise et al., 1998; Sakata et al., 2003). We also found that the activation of H1R in the hippocampus is essential to ameliorate stress-suppressed LTP in the chewing rats. This chewing-induced increase of brain HA level and the subsequent amelioration of LTP via activation of H1R is possibly one of the mechanisms by which chewing effectively maintains the learning and memory functions in the hippocampus (Onozuka et al., 1999; Tsutsui et al., 2007; Hirano et al., 2008). Further experiments with locally applied H1R agonist or H1R antagonist in the hippocampus of stressed or stressed-withchewing rats would confirm our current hypothesis of H1Rmediated amelioration on hippocampal memory process. There are several ways (H1R-mediated, H2R-mediated, and directly acting on the polyamine binding site) by which HA facilitates the excitability of NMDARs in the hippocampal CA1 neurons (Bekkers, 1993; Vorobjev et al., 1993; Payne and Neuman, 1997; Selbach et al., 1997). However, in the present study,
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inhibition of H1R completely prevented the recovery of stresssuppressed LTP in rats that chewed. LTP magnitudes for these rats were comparable to those for rats that did not chew (Fig. 3D). These results imply that H1R-mediated recovery of NMDAR function, rather than H2R-mediated or direct HA binding-mediated, might be a dominant pathway by which HA ameliorates stresssuppressed LTP in the hippocampal CA1 neurons. In addition, there remain other possible explanations of how chewing rescues NMDAR function in the hippocampal CA1 neurons, such as by suppressing HPA-axis responses (Hori et al., 2004; Ono et al., 2008), which would themselves suggest that chewing involves both direct hormonal effects and the indirect neuronal mechanisms such as histaminergic system to rescue NMDAR functions. However in the current study, the magnitude of LTP in group SCpa did not completely recover to the level of that of rats treated in the same way but without pyrilamine administration. Even when we administered pyrilamine to rats after the chewing session, there was still a small, not statistically significant, decrement in LTP of these rats compared to those without drug treatment (Fig. 4). The decrement possibly is caused by a prolonged elevation of HA level in the hippocampus after chewing and the cessation of H1R-mediated rescue of NMDARs. Yoshitake et al. (2003) reported that the elevated HA concentration in the hippocampus takes 70 min to return to the basal values once the TMN is behaviorally stimulated by 20 min of forced swimming. Together with our current results, we hypothesize that the higher HA level was kept in the hippocampus for several tens of minutes after the chewing session, possibly due to the prolonged activation of the TMN. Thus, administering pyrilamine immediately after the chewing and stress session might interfere with HA, being abundant after a certain period of time, to bind to H1R and partially suppress the H1R-mediated amelioration of LTP. Nevertheless, the magnitudes of LTP were not statistically different between rats with post-stress pyrilamine administration and those without drug treatment, suggesting that release of HA during chewing is one of the possible mechanisms for the amelioration of NMDARs via H1R activation and the subsequent recovery of LTP. Systemically inhibiting H1R by intraperitoneally administering pyrilamine might affect other parts of the brain and modulate the hippocampal LTP indirectly. Indeed, H1Rs abundantly exist in cholinergic, noradrenergic, and serotonergic neurons of the basal forebrain, the locus ceruleus, and the raphe nuclei, respectively (Brown et al., 2001), densely projecting their axons into the hippocampus. Among these neurons, cholinergic and noradrenergic neurons release acetylcholine (ACh) and noradrenaline (NA), respectively, both of which are potent facilitators of NMDARdependent CA1-LTP (Huerta and Lisman, 1995; Lin et al., 2003; Shinoe et al., 2005). In particular, the electrical stimulation of TMN has been reported to induce synchronous facilitation of extracellular concentration of HA in the diagonal band and that of ACh in the hippocampus, suggesting a stimulatory effect of HA on the Ach release in the hippocampus. However, the ACh release in the hippocampus is not inhibited by application of pyrilamine but by zolantidine, an H2R antagonist (Mochizuki et al., 1994). On the other hand, Korotkova et al. (2005) reported that bath application of pyrilamine inhibits HA-mediated facilitation of neuron firing in the locus ceruleus, suggesting an inhibitory effect of pyrilamine in NA release in the hippocampus. Furthermore, pyrilamine might modulate serotonin (5-HT) release from the dorsal raphe nucleus and modulate CA1 LTP in the hippocampus since HA has been reported to increase serotonergic neuron firing in the dorsal raphe nucleus via activation of H1R (Ba´rbara et al., 2002; Brown et al., 2002). However, these modulatory effects of NA or 5-HT might also be excluded, since extracellular concentrations of NA and 5-HT in the hippocampus during immobilization stress are virtually identical in rats in group ST and in group SC when they are
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measured by in vivo microdialysis (Ono, unpublished observation). Thus, we strongly suggest that the direct activation of H1Rs in the hippocampal CA1 neurons mediates the ameliorative effect of chewing in stress-suppressed LTP. In conclusion, activation of H1R mediates the recovery process of stress-suppressed hippocampal synaptic plasticity by chewing. The HA release from the TMN, possibly induced by chewing during a condition of stress, may contribute to rescuing impaired NMDAR function via activation of H1R. These results further suggest a novel role of chewing as an active coping strategy to ameliorate the stress-attenuated hippocampal memory process via activation of the brain histaminergic system. Acknowledgement The Ministry of Education, Culture, Sports, Science and Technology (MEXT) supported this work with an Open Research Center subsidy for the Research Center of Brain and Oral Science, Kanagawa Dental College. References ˜ o, J.A., 2002. Histamine H1 receptors in rat Ba´rbara, A., Aceves, J., Arias-Montan dorsal raphe nucleus: pharmacological characterisation and linking to increased neuronal activity. Brain Res. 954 (2), 247–255. Bekkers, J.M., 1993. Enhancement by histamine of NMDA-mediated synaptic transmission in the hippocampus. Science 261 (5117), 104–106. Brown, R.E., Stevens, D.R., Haas, H.L., 2001. The physiology of brain histamine. Prog. Neurobiol. 63 (6), 637–672. Brown, R.E., Sergeeva, O.A., Eriksson, K.S., Haas, H.L., 2002. Convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems (orexin/hypocretin, histamine and noradrenaline). J. Neurosci. 22 (20), 8850–8859. Elliott, E., Sapolsky, R., 1993. Corticosterone impairs hippocampal neuronal calcium regulation: possible mediating mechanisms. Brain Res. 602, 84–90. Ericson, H., Blomqvist, A., Ko¨hler, C., 1989. Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation. J. Comp. Neurol. 281 (2), 169–192. Ericson, H., Blomqvist, A., Ko¨hler, C., 1991. Origin of neuronal inputs to the region of the tuberomammillary nucleus of the rat brain. J. Comp. Neurol. 311 (1), 45–64. Foy, M.R., Stanton, M.E., Levine, S., Thompson, R.F., 1987. Behavioral stress impairs long-term potentiation in rodent hippocampus. Behav. Neural. Biol. 48, 138– 149. Fujise, T., Yoshimatsu, H., Kurokawa, M., Oohara, A., Kang, M., Nakata, M., Sakata, T., 1998. Satiation and masticatory function modulated by brain histamine in rats. Proc. Soc. Exp. Biol. Med. 217 (2), 228–234. Huang, Y.W., Chen, Z., Hu, W.W., Zhang, L.S., Wu, W., Ying, L.Y., Wei, E.Q., 2003. Facilitating effect of histamine on spatial memory deficits induced by dizocilpine as evaluated by 8-arm radial maze in SD rats. Acta Pharmacol. Sin. 24 (12), 1270–1276. Hirano, Y., Obata, T., Kashikura, K., Nonaka, H., Tachibana, A., Ikehira, H., Onozuka, M., 2008. Effects of chewing in working memory processing. Neurosci. Lett. 436 (2), 189–192. Hori, N., Yuyama, N., Tamura, K., 2004. Chewing suppresses stress-induced expression of corticotropin-releasing factor (CRF) in the rat hypothalamus. J. Dent. Res. 83 (2), 124–128. Huerta, P.T., Lisman, J.E., 1995. Bidirectional synaptic plasticity induced by a single burst during cholinergic theta oscillation in CA1 in vitro. Neuron 15 (5), 1053– 1063. Inagaki, N., Yamatodani, A., Ando-Yamamoto, M., Tohyama, M., Watanabe, T., Wada, H., 1988. Organization of histaminergic fibers in the rat brain. J. Comp. Neurol. 273 (3), 283–300. Joe¨ls, M., de Kloet, E.R., 1991. Effect of corticosteroid hormones on electrical activity in rat hippocampus. J. Steroid. Biochem. Mol. Biol. 40 (1–3), 83–86. Kamei, C., Tasaka, K., 1991. Participation of histamine in the step-through active avoidance response and its inhibition by H1-blockers. Jpn. J. Pharmacol. 57, 473–482.
Kamei, C., Tasaka, K., 1993. Effect of histamine on memory retrieval in old rats. Biol. Pharm. Bull. 16 (2), 128–132. Ko¨hler, C., Swanson, L.W., Haglund, L., Wu, J.Y., 1985. The cytoarchitecture, histochemistry and projections of the tuberomammillary nucleus in the rat. Neuroscience 16 (1), 85–110. Korotkova, T.M., Sergeeva, O.A., Ponomarenko, A.A., Haas, H.L., 2005. Histamine excites noradrenergic neurons in locus coeruleus in rats. Neuropharmacology 49 (1), 129–134. Leurs, R., Traiffort, E., Arrang, J.M., Tardivel-Lacombe, J., Ruat, M., Schwartz, J.C., 1994. Guinea pig histamine H1 receptor. II. Stable expression in Chinese hamster ovary cells reveals the interaction with three major signal transduction pathways. J. Neurochem. 62 (2), 519–527. Lin, Y.W., Min, M.Y., Chiu, T.H., Yang, H.W., 2003. Enhancement of associative longterm potentiation by activation of beta-adrenergic receptors at CA1 synapses in rat hippocampal slices. J. Neurosci. 23 (10), 4173–4181. Luschei, E.S., 1987. Central projections of the mesencephalic nucleus of the fifth nerve: an autoradiographic study. J. Comp. Neurol. 263 (1), 137–145. MacDonald, J.F., Kotecha, S.A., Lu, W.Y., Jackson, M.F., 2001. Convergence of PKCdependent kinase signal cascades on NMDA receptors. Curr. Drug Targets 2 (3), 299–312. Markram, H., Segal, M., 1992. The inositol 1,4,5-trisphosphate pathway mediates cholinergic potentiation of rat hippocampal neuronal responses to NMDA. J. Physiol. 447, 513–533. Masuoka, T., Saito, S., Kamei, C., 2008. Participation of hippocampal ionotropic glutamate receptors in histamine H(1) antagonist-induced memory deficit in rats. Psychopharmacology (Berl) 197 (1), 107–114. Mochizuki, T., Okakura-Mochizuki, K., Horii, A., Yamamoto, Y., Yamatodani, A., 1994. Histaminergic modulation of hippocampal acetylcholine release in vivo. J. Neurochem. 62 (6), 2275–2282. Ono, Y., Kataoka, T., Miyake, S., Cheng, S.J., Tachibana, A., Sasaguri, K.I., Onozuka, M., 2008. Chewing ameliorates stress-induced suppression of hippocampal longterm potentiation. Neuroscience 154 (4), 1352–1359. Onozuka, M., Watanabe, K., Mirbod, S.M., Ozono, S., Nishiyama, K., Karasawa, N., Nagatsu, I., 1999. Reduced mastication stimulates impairment of spatial memory and degeneration of hippocampal neurons in aged SAMP8 mice. Brain Res. 826 (1), 148–153. Panula, P., Pirvola, U., Auvinen, S., Airaksinen, M.S., 1989. Histamine-immunoreactive nerve fibers in the rat brain. Neuroscience 28 (3), 585–610. Payne, G.W., Neuman, R.S., 1997. Effects of hypomagnesia on histamine H1 receptor-mediated facilitation of NMDA responses. Br. J. Pharmacol. 121 (2), 199– 204. Raman, I.M., Tong, G., Jahr, C.E., 1996. Beta-adrenergic regulation of synaptic NMDA receptors by cAMP-dependent protein kinase. Neuron 16 (2), 415–421. Saitou, K., Kaneko, Y., Sugimoto, Y., Chen, Z., Kamei, C., 1999. Slow wave sleepinducing effects of first generation H1-antagonists. Biol. Pharm. Bull. 22 (10), 1079–1082. Sakata, T., Yoshimatsu, H., Masaki, T., Tsuda, K., 2003. Anti-obesity actions of mastication driven by histamine neurons in rats. Exp. Biol. Med. (Maywood) 228 (10), 1106–1110. Selbach, O., Brown, R.E., Haas, H.L., 1997. Long-term increase of hippocampal excitability by histamine and cyclic AMP. Neuropharmacology 36 (11–12), 1539–1548. Shinoe, T., Matsui, M., Taketo, M.M., Manabe, T., 2005. Modulation of synaptic plasticity by physiological activation of M1 muscarinic acetylcholine receptors in the mouse hippocampus. J. Neurosci. 25 (48), 11194–11200. Tsutsui, K., Kaku, M., Motokawa, M., Tohma, Y., Kawata, T., Fujita, T., Kohno, S., Ohtani, J., Tenjoh, K., Nakano, M., Kamada, H., Tanne, K., 2007. Influences of reduced masticatory sensory input from soft-diet feeding upon spatial memory/learning ability in mice. Biomed. Res. 28 (1), 1–7. Vorobjev, V.S., Sharonova, I.N., Walsh, I.B., Haas, H.L., 1993. Histamine potentiates N-methyl-D-aspartate responses in acutely isolated hippocampal neurons. Neuron 11 (5), 837–844. Whitlock, J.R., Heynen, A.J., Shuler, M.G., Bear, M.F., 2006. Learning induces longterm potentiation in the hippocampus. Science 313 (5790), 1093–1097. Wiegert, O., Pu, Z., Shor, S., Joe¨ls, M., Krugers, H., 2005. Glucocorticoid receptor activation selectively hampers N-methyl-D-aspartate receptor dependent hippocampal synaptic plasticity in vitro. Neuroscience 135 (2), 403–411. Yoshitake, T., Yamaguchi, M., Nohta, H., Ichinose, F., Yoshida, H., Yoshitake, S., Fuxe, K., Kehr, J., 2003. Determination of histamine in microdialysis samples from rat brain by microbore column liquid chromatography following intramolecular excimer-forming derivatization with pyrene-labeling reagent. J. Neurosci. Methods 127 (1), 11–17.
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Chewing rescues stress-suppressed hippocampal long-term potentiation via activation of histamine H1 receptor Yumie Ono a,1,*, Tsuyoshi Kataoka b,1, Shinjiro Miyake b, Kenichi Sasaguri b, Sadao Sato b, Minoru Onozuka a a b
Department of Physiology and Neuroscience, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka Kanagawa, 238-8580, Japan Department of Craniofacial Growth and Developmental Dentistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka Kanagawa, 238-8580, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 January 2009 Received in revised form 19 March 2009 Accepted 13 April 2009 Available online 22 April 2009
We have previously found in rats that chewing, an active behavioral strategy to cope with a stressful situation, rescues long-term potentiation (LTP) in the hippocampus through activating stresssuppressed N-methyl-D-aspartate (NMDA) receptor function. To further examine the mechanisms underlying this ameliorative effect of chewing, we studied the involvement of the histaminergic system, which has been shown to be activated by mastication, in the LTP of hippocampal slices of rats that were allowed to chew a wooden stick during exposure to immobilization stress. Chewing failed to rescue stress-suppressed LTP in the rats treated with histamine H1 receptor (H1R) antagonist pyrilamine (5 mg/ kg, i.p.) before exposure to stress, although administration of pyrilamine did not affect LTP in naive rats and in stressed rats that did not chew. However, when pyrilamine was administrated immediately after exposure to stress, chewing rescued LTP whose magnitude was statistically comparable to that in the rats that chewed without drug treatment. These results suggest that chewing-induced histamine release in the hippocampus and the subsequent H1 receptor activation may be essential to rescue stresssuppressed synaptic plasticity. ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Chewing Coping Histamine H1 receptor Hippocampus Long-term potentiation Pyrilamine Stress Tuberomammillary nucleus
1. Introduction In general, exposure to severe stress causes prolonged activation of the hypothalamic–pituitary–adrenal (HPA) axis, which in turn causes the adrenal cortex to secrete corticosterone, which finally disrupts fundamental cellular processes in the central nervous system. Especially in the hippocampus, an excessive corticosterone level causes a Ca2+ influx into neurons via activation of glucocorticoid receptors (GR) (Elliott and Sapolsky, 1993) and attenuates cellular excitability (Joe¨ls and de Kloet, 1991), which selectively weakens the long-term potentiation (LTP) of hippocampal neurons that depends on the N-methylD-aspartate receptor (NMDAR) (Foy et al., 1987; Wiegert et al., 2005). We have recently found that chewing during a stressful event ameliorates the stress-induced impairment of LTP in hippocampal CA1 neurons by rescuing NMDAR function (Ono et al., 2008). However, the rescue mechanism remains to be elucidated.
* Corresponding author at: Department of Physiology and Neuroscience, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka Kanagawa, 238-8580, Japan. Tel.: +81 46 822 8869; fax: +81 46 822 9522. E-mail address: [email protected] (Y. Ono). 1 Equally contributed.
The mesencephalic trigeminal nucleus (Me5) receives chewingrelated proprioceptive sensory afferents of the trigeminal nerve from the jaw-closing muscle spindle and the periodontal ligaments (Luschei, 1987). A subpopulation of the Me5 neurons projects its fibers into the tuberomammillary nuclei (TMN) of the posterior hypothalamus where cell bodies of histaminergic neurons are localized (Ericson et al., 1989, 1991). Indeed, activation of the Me5 by mastication increases extracellular concentration of histamine (HA) in the hypothalamus to control satiety (Fujise et al., 1998; Sakata et al., 2003), suggesting the facilitative effect of chewing on histaminergic neurons in the TMN. The axons of histaminergic neurons in the TMN innervate practically the entire brain, including the hippocampus (Ko¨hler et al., 1985; Inagaki et al., 1988; Panula et al., 1989; Brown et al., 2001) and the electrical stimulation of the TMN facilitates extracellular concentration of HA in the hippocampus (Mochizuki et al., 1994). Thus, a chewinginduced increase of the HA level in the hippocampus probably restores the stress-attenuated NMDAR to normal function and thereby rescues LTP. At the present time, there are three known mechanisms by which HA facilitates NMDAR function in vitro. First, HA can stimulate the activity of phospholipase C through histamine H1 receptor (H1R) which is associated with the Gq/11 protein, followed by intracellular production of two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3)
0168-0102/$ – see front matter ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2009.04.011
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(Leurs et al., 1994). DAG and IP3 further potentiate the activity of protein kinase C and increase intracellular Ca2+, respectively, both of which increase the excitability of NMDAR (MacDonald et al., 2001; Markram and Segal, 1992). Payne and Neuman (1997) have shown that applying HA increases NMDA current in the hippocampal CA1 neurons, an effect that is reduced in a concentration-dependent manner by H1R antagonists but neither by histamine H2 receptor (H2R) nor by histamine H3 receptor (H3R) antagonist. Second, activation of H2R, which is associated with the Gs protein, stimulates adenylate cyclase to enhance the production of second messenger cyclic adenosine 3’,5’-monophosphate (cAMP) (Selbach et al., 1997). An elevation in intracellular cAMP concentrations further activates protein kinase A, increasing charge transfer and Ca2+ influx through NMDARs (Raman et al., 1996). Third, HA acts directly on the polyamine binding site of NMDAR to enhance its function (Bekkers, 1993; Vorobjev et al., 1993). Taken together, these reports further strengthen the hypothesis that HA participates in the rescue of stress-attenuated NMDAR function. In behaving animals, NMDAR-dependent LTP in the neurons of the CA1 area in the hippocampus plays an essential role in learning and memory (Whitlock et al., 2006). HA is also one of the possible facilitators of the hippocampal memory function. Interestingly, in vivo, H1R-dependent enhancement of NMDAR is more likely to facilitate the hippocampal-dependent learning and memory process, rather than the H2R-dependent mechanism or the direct action on NMDAR that are also well confirmed in vitro. Kamei and Tasaka (1993) have reported that intracerebroventricular administration of HA or intraperitoneal administration of HA precursor histidine improved the response latency in an active avoidance task via H1R activation in old rats. Moreover, antagonism of H1R causes memory deficit in a radial maze task, which can be restored by facilitating the NMDAR function by activating either the glycine binding site or the polyamine binding site of NMDAR (Masuoka et al., 2008) and vice versa (Huang et al., 2003). These findings show that there is H1R-mediated facilitation of NMDAR function in the hippocampus, suggesting a dominant role of H1R in modulating synaptic plasticity in the hippocampus of behaving animals. Therefore, to elucidate the involvement of H1R in the chewinginduced amelioration of hippocampal synaptic plasticity, we have examined the effect of H1R antagonism on LTP of the rats that underwent immobilization stress with chewing. We also tested H1R antagonism with two different time points of pre- and poststress to determine how activation of the histaminergic system during chewing contributes to ameliorating the stress-suppressed LTP.
2.1.2. Drug administration and stress protocol We dissolved pyrilamine (5 mg/kg; Sigma–Aldrich Japan, Tokyo, Japan) in saline and intraperitoneally injected it into all of the 18 rats tested. The dose of pyrilamine was set to the amount that sufficiently inhibits H1Rs in the hippocampus (Kamei and Tasaka, 1991) but that does not affect the arousal level (Saitou et al., 1999). Fifteen minutes after the injection, we produced immobilization stress in 12 of the rats by fixing their limbs to a wooden board for 30 min in a spread-eagle, supine position. We left half of them alone for the entire immobilization period (pyrilamine administration followed by stress; STp). For the other half, we placed a wooden stick (diameter, 5 mm) near the immobilized animal’s mouth (pyrilamine administration followed by stress and chewing; SCp). All the rats given a wooden stick except one responded to it by chewing on it with a rapid and repetitive sequence of jaw opening and closing movements for at least two-thirds of the total restraint period. The one rat that did not touch the offered stick was included in group STp. We returned the remaining six rats to their home cage without subjecting them to immobilization stress (control of pyrilamine administration only; CTp). We performed all drug administration and stress inducement between 10:00 a.m. and 11:30 a.m. 2.1.3. Electrophysiology We anesthetized rats with 2-bromo-2-chloro-1,1,1-trifluoroethane (2 ml/kg; Takeda Chemical Industries, Osaka, Japan) at 24 h after the immobilization stress, the time at which the ameliorative effect of chewing on hippocampal LTP was the most prominent in our previous study (Ono et al., 2008). We decapitated and removed their brains to make transverse hippocampal slices (450 mm thick). As to the rats in group CTp that did not experience stress, we set post-injection survival time to be the same as that in the other groups (Fig. 1). We maintained the slices in a holding chamber filled with artificial cerebrospinal fluid (ACSF) containing 119 NaCl, 2.5 KCl, 26.2 NaHCO3, 1 NaH2PO4, 1.3 MgSO4, 2.5 CaCl2, and 11 glucose (in mM, bubbled with 95% O2–5% CO2) until use. After at least 1 h of recovery time, we transferred one of the slices to an immersion-type recording chamber perfused at 1 ml/min
2. Materials and methods 2.1. Experiment 1: administration of pyrilamine and chewing effects on stress-suppressed hippocampal LTP The goal of experiment 1 was to determine whether activation of H1R is crucial for ameliorative effect of chewing on hippocampal LTP in the stressed rats. To test this, we used H1R antagonist pyrilamine to block histaminergic signal transduction through H1R in the hippocampal neurons before stress. 2.1.1. Animals We used 10-week-old male Sprague-Dawley rats in all the experiments reported here. The rats took water and food freely in a temperature-controlled room (22 3 8C) with a 12 h light/dark cycle (lights on at 7:00 a.m.). All our experiments accorded with the guidelines for Animal Experimentation of Kanagawa Dental College. All efforts were made to minimize the number of animals used and their suffering.
Fig. 1. Time intervals from intraperitoneal pyrilamine administration to stress exposure and from stress exposure to hippocampal slice preparation in experiments 1 and 2.
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with ACSF containing 0.1 mM picrotoxin (Sigma–Aldrich, Tokyo, Japan). A glass pipette filled with 3 M NaCl and positioned in the stratum radiatum of the CA1 area recorded the field excitatory postsynaptic potential (fEPSP). Bipolar stainless-steel electrodes (Unique Medical, Tokyo, Japan) placed in the stratum radiatum on opposite sides of the recording pipette stimulated the Schaffer collateral branches. We adjusted the stimulus intensity to around 30% of the maximal response and then recorded stable baseline fEPSP activity at the determined intensity every 30 s for at least 10 min. High frequency stimulation (HFS; 1 s, 100 Hz), applied nine times at intervals of 90 s, induced LTP. Since the general LTP protocol of applying single HFS was not sufficient to produce LTP in some stressed rats in our preliminary experiment, repetitive HFS was adopted to obtain large and stable LTP. All signals were filtered at 2 kHz using a low-pass Bessel filter and digitized at 5 kHz using a MultiClamp 700A interface running pCLAMP software (Axon Instruments, Union City, CA, USA). We measured the initial slopes of the fEPSP and normalized them to the average of the values measured over the baseline period. We used a single slice from a single animal for subsequent analysis.
signals was integrated with every 100 ms of time window, and the time windows whose integrated value was larger than 5% of the maximum value through the measurement were determined to be the active state of the masseter muscle. Consecutive time windows of active states were regarded as a single event of the masseter muscle activation. The total number of masseter muscle activations during 30 min of immobilization was defined to be the frequency of chewing.
2.2. Experiment 2: pre-stress and post-stress administration of pyrilamine and chewing effects on stress-suppressed hippocampal LTP
3. Results
Experiment 1 showed that administering pyrilamine before stress exposure effectively prevents an ameliorative effect of chewing on stress-suppressed hippocampal LTP. However it was still unclear what time period from stress exposure until the end of the 24 h of post-stress survival is critical for pyrilamine to counteract the effect of chewing. Therefore, we next tested whether administering pyrilamine after stress can likewise prevent chewing effects on the LTP. We subjected six rats to the same immobilization stress environment that the rats in group SCp in experiment 1 had been subjected to, but we administered pyrilamine (same dosage as in the earlier experiment) immediately after exposure to stress (pyrilamine administration after stress and chewing; SCpa). After immobilization stress, we returned the rats to their home cages, prepared hippocampal slices 24 h later, and measured LTP as in experiment 1 (Fig. 1).
2.4. Statistical analysis All values shown are mean S.E.M. The magnitude of LTP, the average value of the slope of the fEPSP recorded between 30 and 40 min after the end of HFS, provided the basis for our statistical comparisons. We compared the magnitudes of LTP among groups using the analysis of variance (ANOVA) test and the post hoc Tukey’s multiple comparison. We used Student’s t-test to compare the magnitudes of LTP in group SCpa with that in group SCp in experiment 2 and the number of chewing events in group SCp with that in group SC in the EMG experiment.
3.1. Chewing behavior is not affected by administering 5 mg/kg of pyrilamine Every rat in the chewing group showed robust chewing behavior that continued during most of the period of immobilization (Fig. 2A).
2.3. Effect of pyrilamine on chewing behavior Even though the dose of pyrilamine administered in this study was less than that required to produce a sedative effect (Saitou et al., 1999), its involvement in chewing behavior has not been reported so far. Therefore we recorded an electromyogram (EMG) of the activity of the masseter muscle during the period of immobilization and chewing to study the effect of pyrilamine on the chewing behavior. Six other rats were subjected to immobilization stress with chewing. We administered pyrilamine to half of them before immobilization as described in experiment 1 (group SCp), while we simply handled the other half of the rats without administering anything (manipulation same as in our previous study without any drug application; labeled group SC in Ono et al., 2008). Immediately after fixing a rat onto a board, a bipolar needle electrode (UI2-513, Unique Medical, Tokyo, Japan) was inserted into the left masseter muscle. After the electrode was inserted, we offered a wooden stick to the rat for chewing. We also measured EMG of two other rats, treated the same as in group STp and stressed only without pyrilamine administration (manipulation same as group ST in Ono et al., 2008), as a control for group SCp and group SC, respectively (n = 1 each). All signals were recorded using Bagnoli-2 EMG system (Delsys, MA, USA) and frequency of chewing behavior was analyzed using Masseter Muscle Analysis Software (G1 Systems, Aichi, Japan). In brief, the power of EMG
Fig. 2. Effects of pyrilamine on chewing behavior during immobilization stress. (A) Representative EMG waveforms from the masseter muscle of rats that were subjected to stress (ST), pyrilamine injection followed by stress (STp), stress with chewing a wooden stick (SC), and pyrilamine injection followed by stress with chewing a wooden stick (SCp); (B) comparison of mean values of frequency of chewing between group SC and group SCp (n = 3 each).
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The frequency of chewing was not affected by pyrilamine administration (1060 439 times and 1232 377 times in group SC and group SCp, respectively; n = 3, p = 0.634; Fig. 2B). 3.2. Experiment 1: H1R blocker prevents ameliorative effect of chewing on hippocampal LTP We have previously found that chewing counteracts stresssuppressed LTP of hippocampal CA1 neurons (Ono et al., 2008). The present result in experiment 1 further showed that the ameliorative effect of chewing is completely abolished when rats are treated with pyrilamine before exposure to stress (Fig. 3). A oneway ANOVA showed a significant group difference in the magnitude of LTP (F(2,15) = 6.18; p < 0.05) (Fig. 3D). Post-hoc tests showed statistically significant impairment of synaptic plasticity in both group STp (154.0 11% of baseline, n = 7, p < 0.01) and group SCp (149.7 2.4% of baseline, n = 5, p < 0.01) compared with group CTp (203.1 16% of baseline, n = 6). There was no significant difference between the values of group STp and group SCp (p = 0.965). When we compare these values with those obtained without pyrilamine administration in our previous study (control without pyrilamine administration (group CT) = 206.6 11%, stressed without pyrilamine administration (group ST 24 h) = 141.0 11%, stressed and chewing without pyrilamine administration (group SC
Fig. 3. Effects of pyrilamine and stress on hippocampal CA1 LTP. (A-C) Time-course of mean LTP magnitudes for the control (CTp; A) group and for the stressed (STp; B) and chewing (SCp; C) groups. CT, ST, and SC refer to rats in the control, stressed, and stressed-with-chewing groups, respectively, without pyrilamine administration: These values were taken from our previous study (CT, ST 24 h, and SC 24 h; Ono et al., 2008, with permission) and superimposed to the respective figures for comparison. Insets show representative fEPSP waveforms before (gray) and after (black) HFS; (D) comparison of mean values of fEPSP slope. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01).
Fig. 4. Effects of pre-stress and immediate post-stress administration of pyrilamine on hippocampal CA1 LTP in rats that chewed during stress. (A) Time-course of mean LTP magnitude for rats given pyrilamine before (SCp) and immediately after (SCpa) stress exposure with chewing. Insets show representative fEPSP waveforms before (gray) and after (black) HFS. (B) comparison of mean values of fEPSP slope. Asterisk indicates a statistically significant difference (p < 0.05). Horizontal dotted lines in A and B indicate the mean value of LTP magnitude obtained from rats that were treated in the same way but without pyrilamine administration (189.6%, group SC 24 h; Ono et al., 2008).
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24 h) = 189.6 7.8% of baseline; taken from Fig. 1B in Ono et al., 2008, with permission), there was a significant main effect of task (two-way ANOVA, F(2,30) = 14.3; p < 0.001). Post hoc tests indicated that the magnitude of LTP in chewing rats with pyrilamine administration (group SCp) was significantly lower than that without pyrilamine administration (group SC 24 h; Fig. 3C and D, p < 0.05), although for the pair of groups CTp and CT (p = 0.822) and for the pair of groups STp and ST 24 h (p = 0.400), the administration of pyrilamine caused no statistically significant change in the magnitude of LTP (Fig. 3A, B, and D). 3.3. Experiment 2: activation of H1R associated with chewing is essential to rescue stress-suppressed hippocampal LTP Administering pyrilamine immediately after stress had less effect on preventing chewing-induced recovery of LTP (Fig. 4). The mean magnitude of LTP for group SCpa was significantly larger (170.3 7.5% of baseline, n = 6, p < 0.05) than that for group SCp. There was no statistical difference between the magnitude of LTP for groups SC 24 h and SCpa (p = 0.103), but there was a statistically significant difference for groups SC 24 h and SCp (p < 0.05; in post hoc comparison after one-way ANOVA, with significant group difference F(2,14) = 6.11; p < 0.05). 4. Discussion We found that inhibiting histamine H1R during chewing prevents the ameliorative effect of chewing on the stresssuppressed LTP in the hippocampal CA1 neurons. Blocking H1R before the chewing and stress session prevents chewing from rescuing LTP, although blocking H1R immediately after the session has no effect on chewing-related rescue of LTP. These results suggest that chewing activates the histaminergic nervous system to facilitate the extracellular concentration of HA in the hippocampus, restoring stress-attenuated NMDAR function of CA1 neurons via H1R activation. The effect of pyrilamine in modulating LTP was specific in the rats that were administrated pyrilamine in advance and that chewed during stress (group SCp; Fig. 3C). This suppression of LTP in group SCp was not caused by the insufficiency of chewing behavior, since the sedative effect of pyrilamine in chewing behavior was negligible with the dose we used (Fig. 2B). Moreover, pyrilamine affected the magnitude of LTP neither in rats that did not chew (group CTp and group STp; Fig. 3A and B) nor in rats that chewed but whose H1Rs were blocked after the stress exposure (group SCpa; Fig. 4). We, thus, suggest that chewing raises the HA level in the hippocampus, possibly through activating histaminergic neurons in the TMN in the same way that mastication during food intake increases the extracellular concentration of HA in the ventromedial hypothalamus and in the Me5 (Fujise et al., 1998; Sakata et al., 2003). We also found that the activation of H1R in the hippocampus is essential to ameliorate stress-suppressed LTP in the chewing rats. This chewing-induced increase of brain HA level and the subsequent amelioration of LTP via activation of H1R is possibly one of the mechanisms by which chewing effectively maintains the learning and memory functions in the hippocampus (Onozuka et al., 1999; Tsutsui et al., 2007; Hirano et al., 2008). Further experiments with locally applied H1R agonist or H1R antagonist in the hippocampus of stressed or stressed-withchewing rats would confirm our current hypothesis of H1Rmediated amelioration on hippocampal memory process. There are several ways (H1R-mediated, H2R-mediated, and directly acting on the polyamine binding site) by which HA facilitates the excitability of NMDARs in the hippocampal CA1 neurons (Bekkers, 1993; Vorobjev et al., 1993; Payne and Neuman, 1997; Selbach et al., 1997). However, in the present study,
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inhibition of H1R completely prevented the recovery of stresssuppressed LTP in rats that chewed. LTP magnitudes for these rats were comparable to those for rats that did not chew (Fig. 3D). These results imply that H1R-mediated recovery of NMDAR function, rather than H2R-mediated or direct HA binding-mediated, might be a dominant pathway by which HA ameliorates stresssuppressed LTP in the hippocampal CA1 neurons. In addition, there remain other possible explanations of how chewing rescues NMDAR function in the hippocampal CA1 neurons, such as by suppressing HPA-axis responses (Hori et al., 2004; Ono et al., 2008), which would themselves suggest that chewing involves both direct hormonal effects and the indirect neuronal mechanisms such as histaminergic system to rescue NMDAR functions. However in the current study, the magnitude of LTP in group SCpa did not completely recover to the level of that of rats treated in the same way but without pyrilamine administration. Even when we administered pyrilamine to rats after the chewing session, there was still a small, not statistically significant, decrement in LTP of these rats compared to those without drug treatment (Fig. 4). The decrement possibly is caused by a prolonged elevation of HA level in the hippocampus after chewing and the cessation of H1R-mediated rescue of NMDARs. Yoshitake et al. (2003) reported that the elevated HA concentration in the hippocampus takes 70 min to return to the basal values once the TMN is behaviorally stimulated by 20 min of forced swimming. Together with our current results, we hypothesize that the higher HA level was kept in the hippocampus for several tens of minutes after the chewing session, possibly due to the prolonged activation of the TMN. Thus, administering pyrilamine immediately after the chewing and stress session might interfere with HA, being abundant after a certain period of time, to bind to H1R and partially suppress the H1R-mediated amelioration of LTP. Nevertheless, the magnitudes of LTP were not statistically different between rats with post-stress pyrilamine administration and those without drug treatment, suggesting that release of HA during chewing is one of the possible mechanisms for the amelioration of NMDARs via H1R activation and the subsequent recovery of LTP. Systemically inhibiting H1R by intraperitoneally administering pyrilamine might affect other parts of the brain and modulate the hippocampal LTP indirectly. Indeed, H1Rs abundantly exist in cholinergic, noradrenergic, and serotonergic neurons of the basal forebrain, the locus ceruleus, and the raphe nuclei, respectively (Brown et al., 2001), densely projecting their axons into the hippocampus. Among these neurons, cholinergic and noradrenergic neurons release acetylcholine (ACh) and noradrenaline (NA), respectively, both of which are potent facilitators of NMDARdependent CA1-LTP (Huerta and Lisman, 1995; Lin et al., 2003; Shinoe et al., 2005). In particular, the electrical stimulation of TMN has been reported to induce synchronous facilitation of extracellular concentration of HA in the diagonal band and that of ACh in the hippocampus, suggesting a stimulatory effect of HA on the Ach release in the hippocampus. However, the ACh release in the hippocampus is not inhibited by application of pyrilamine but by zolantidine, an H2R antagonist (Mochizuki et al., 1994). On the other hand, Korotkova et al. (2005) reported that bath application of pyrilamine inhibits HA-mediated facilitation of neuron firing in the locus ceruleus, suggesting an inhibitory effect of pyrilamine in NA release in the hippocampus. Furthermore, pyrilamine might modulate serotonin (5-HT) release from the dorsal raphe nucleus and modulate CA1 LTP in the hippocampus since HA has been reported to increase serotonergic neuron firing in the dorsal raphe nucleus via activation of H1R (Ba´rbara et al., 2002; Brown et al., 2002). However, these modulatory effects of NA or 5-HT might also be excluded, since extracellular concentrations of NA and 5-HT in the hippocampus during immobilization stress are virtually identical in rats in group ST and in group SC when they are
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measured by in vivo microdialysis (Ono, unpublished observation). Thus, we strongly suggest that the direct activation of H1Rs in the hippocampal CA1 neurons mediates the ameliorative effect of chewing in stress-suppressed LTP. In conclusion, activation of H1R mediates the recovery process of stress-suppressed hippocampal synaptic plasticity by chewing. The HA release from the TMN, possibly induced by chewing during a condition of stress, may contribute to rescuing impaired NMDAR function via activation of H1R. These results further suggest a novel role of chewing as an active coping strategy to ameliorate the stress-attenuated hippocampal memory process via activation of the brain histaminergic system. Acknowledgement The Ministry of Education, Culture, Sports, Science and Technology (MEXT) supported this work with an Open Research Center subsidy for the Research Center of Brain and Oral Science, Kanagawa Dental College. References ˜ o, J.A., 2002. Histamine H1 receptors in rat Ba´rbara, A., Aceves, J., Arias-Montan dorsal raphe nucleus: pharmacological characterisation and linking to increased neuronal activity. Brain Res. 954 (2), 247–255. Bekkers, J.M., 1993. Enhancement by histamine of NMDA-mediated synaptic transmission in the hippocampus. Science 261 (5117), 104–106. Brown, R.E., Stevens, D.R., Haas, H.L., 2001. The physiology of brain histamine. Prog. Neurobiol. 63 (6), 637–672. Brown, R.E., Sergeeva, O.A., Eriksson, K.S., Haas, H.L., 2002. Convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems (orexin/hypocretin, histamine and noradrenaline). J. Neurosci. 22 (20), 8850–8859. Elliott, E., Sapolsky, R., 1993. Corticosterone impairs hippocampal neuronal calcium regulation: possible mediating mechanisms. Brain Res. 602, 84–90. Ericson, H., Blomqvist, A., Ko¨hler, C., 1989. Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation. J. Comp. Neurol. 281 (2), 169–192. Ericson, H., Blomqvist, A., Ko¨hler, C., 1991. Origin of neuronal inputs to the region of the tuberomammillary nucleus of the rat brain. J. Comp. Neurol. 311 (1), 45–64. Foy, M.R., Stanton, M.E., Levine, S., Thompson, R.F., 1987. Behavioral stress impairs long-term potentiation in rodent hippocampus. Behav. Neural. Biol. 48, 138– 149. Fujise, T., Yoshimatsu, H., Kurokawa, M., Oohara, A., Kang, M., Nakata, M., Sakata, T., 1998. Satiation and masticatory function modulated by brain histamine in rats. Proc. Soc. Exp. Biol. Med. 217 (2), 228–234. Huang, Y.W., Chen, Z., Hu, W.W., Zhang, L.S., Wu, W., Ying, L.Y., Wei, E.Q., 2003. Facilitating effect of histamine on spatial memory deficits induced by dizocilpine as evaluated by 8-arm radial maze in SD rats. Acta Pharmacol. Sin. 24 (12), 1270–1276. Hirano, Y., Obata, T., Kashikura, K., Nonaka, H., Tachibana, A., Ikehira, H., Onozuka, M., 2008. Effects of chewing in working memory processing. Neurosci. Lett. 436 (2), 189–192. Hori, N., Yuyama, N., Tamura, K., 2004. Chewing suppresses stress-induced expression of corticotropin-releasing factor (CRF) in the rat hypothalamus. J. Dent. Res. 83 (2), 124–128. Huerta, P.T., Lisman, J.E., 1995. Bidirectional synaptic plasticity induced by a single burst during cholinergic theta oscillation in CA1 in vitro. Neuron 15 (5), 1053– 1063. Inagaki, N., Yamatodani, A., Ando-Yamamoto, M., Tohyama, M., Watanabe, T., Wada, H., 1988. Organization of histaminergic fibers in the rat brain. J. Comp. Neurol. 273 (3), 283–300. Joe¨ls, M., de Kloet, E.R., 1991. Effect of corticosteroid hormones on electrical activity in rat hippocampus. J. Steroid. Biochem. Mol. Biol. 40 (1–3), 83–86. Kamei, C., Tasaka, K., 1991. Participation of histamine in the step-through active avoidance response and its inhibition by H1-blockers. Jpn. J. Pharmacol. 57, 473–482.
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